tr ;cr id ' CD r-=1 D im : o THIRD EDITION SEX AND INTERNAL SECRETIONS VOLUME II VOLUME II CONTRIBUTORS A. Albert David W. Bishop Richard J. Blandau R. K. Burns A. T. Cowie John W. Everett S. J. Folley Thomas R. Forbes J. W. Gowen Roy O. Greep A. M. Guhl Joan G. Hampson John L. Hampson Frederick L. Hisaw Frederick L. Hisaw, James H. Leathem Daniel S. Lehrman Margaret Mead John W. Money Jr. Helen Padykula Dorothy Price Herbert D. Purves Ari van Tienhoven Claude A. Villee H. Guy Williams- Ashman George B. Wislocki William C. Young M, X. Zarrow Baltimore • 1961 Y // S THIRD EDITION SEX AND ^ INTERNAL SECRETIONS Edited by William C. Young, Ph.D. Professor of Anatomy, University of Kansas, Lawrence Foreword by George W. Corner, M.D., D.Sc. Director Emeritus, Department of Embryology. Carnegie Institution of ^ ashington The \YiIIiains & Wilklns Co. Publication was supported in part by Public Health Service Research Grant M-4648 from the National Institute of Mental Health, Public Health Service. Copyright ©, 1961 The Williams & Wilkins Company Made in the United States of America Library of Congress Catalog Card Number 60-12279 COMPOSED AND PRINTED BY THE WAVERLY PRESS, INC. BALTIMORE 2, MARYLAND, U.S.A. fl ®/ CONTENTS Volume I Foreword. George JV. Corner ix Edgar Allen. William C. Young xiii Preface to Third Edition xxi Preface to First Edition xxiii section a Biologic Basis of Sex 1. Cytologic and Genetic Basis of Sex. J. W. Gowen 3 2. Role of Hormones in the Differentiation of Sex. R. K. Burns 76 section b The Hypophysis and the Gonadotrophic Hormones in Relation TO Reproduction 3. Morphology of the Hypophysis Related to Its Function. Herbert D. Purves. . . . 161 4. Physiology of the Anterior Hypophysis in Relation to Reproduction. Roy 0. Greep .240 section c Physiology of the Gonads and Accessory Organs 5. The Mammalian Testis. A. Albert 305 6. The Accessory Reproductive Glands of Mammals. Dorothy Price and H. Guy Williams- Ashman 366 7. The Mammalian Ovary. William C. Young 449 8. The Mammalian Female Reproductive Cycle and Its Controlling ]\Iechanisms. John W. Everett 497 9. Action of Estrogen and Progesterone on the Reproductive Ti-act of Lower Primates. Frederick L. Hisaw and Frederick L. Hisaw, Jr 55() 10. The Mammary Gland and Lactation. A. T. Cowie and S. J. Folley 590 11. Some Problems of the IVIetabolism and Mechanism of Action of Steroid Sex Hor- mones. Claude A . Villee 643 12. Nutritional Effects on Endocrine Secretions. James H. Leathem 666 Volume II SECTION D Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, and prec4nancy 13. Biology of Spermatozoa. David W. Bishop 707 14. Biology of Eggs and Implantation. Richard J. Blandau 797 15. Histochemistry and Electron Microscopy of the Placenta. George B. Wislocki and Helen Padykula 883 16. Gestation. M. X. Zarrow 958 section e Physiology of Reproduction in Submammalian Vertebrates 17. Endocrinology of Reproduction in Cold-blooded Vertebrates. Thomas R. Forbes. . 1035 18. Endocrinology of Reproduction in Birds. Ari van Tienhoven 1088 V vi CONTENTS section f Hormonal Regulation of Reproductive Behavior 19. The Hormones and Mating Behavior. William C. Young 1173 20. Gonadal Hormones and Social Behavior in Infrahuman Vertebrates. A. M. Guhl . . 1240 21. Gonadal Hormones and Parental Behavior in Birds and Infrahuman Mammals. Daniel S. Lehrman 1268 22. Sex Hormones and Other Variables in Human Eroticism. John W. Money. . . .1383 23. The Ontogenesis of Sexual Behavior in Man. John L. Hampson and Joan G. Hampson 1401 24. Cultural Determinants of Sexual Behavior. Margaret Mead 1433 Index 148 1 SECTION D Biology of Sperm and Ova. Fertilization^ Implanta- tion^ the Placenta^ and Pregnancy 13 BIOLOGY OF SPERMATOZOA David W. Bishop, Ph.D. STAFF MEMBER, DEPARTMENT OF EMBRYOLOGY, CARNEGIE INSTITUTION OF WASHINGTON. BALTIMORE. MARYLAND I. Introduction 707 II. Functional State of Gametes after Spermatogenesis 709 A. The ^Maturation of Spermatozoa . . 709 B. Cytogenetic Differences in Sperm 710 C. Requirements for Large Sperm Numbers 711 III. Sperm Transport and Storage in the Male Tract 711 A. Sperm Transport 711 B. Sperm Survival 713 C. The Functional Microanatomy of the Epididymis 714 I). The Epididymis in Relation to Sperm Physiology 7](i E. The Fate of the Nonutilized Sperm in the Male 720 F. Acquisition bv Sperm of the Capac- ity for Motility 721 IV. Insemination 722 A. Ejaculation 723 B. Collection of Seminal Components. 725 C. Seminal Volume and Successive Fractions 725 D. Effective Sperm Concentration. . . . 727 E. Site of Insemination 727 F. Artificial Insemination 727 V. Sperm Transport and Survival in THE Female Tr.\ct 729 A. Duration of Transport 729 B. Mechanism of Transport in the LTterus and Oviduct 731 C. Critical Regions of Sperm Trans- port 732 1. The cervix 734 2. The uterotubal junction 735 3. The isthmus 737 D. Number of Sperm at the Site of Fertilization 738 E;. Duration of Fertilizing Capacity. . . 738 F. Duration of Sperm Motility throughout the Tract 740 Ci. Sperm Viability in Relation to Tubal Physiology 740 H. The Fate of Nonfertilizing Sperma- tozoa 744 VI. Imminologic Problems Assoct.a.ted wuru Spermatozoa 745 A. Antigenicity of Sperm 745 B. Sperm-induced Immune Responses in the Male 746 C. Sperm-induced Immune Responses in the Female 747 VII. Morphology and Composition of Spermatozoa 750 A. Structural Features 750 B. Biochemical Features 754 C. The Localization of Enzymes 755 D. The Sperm Surface 75(1 nil. Sperm Metabolism 757 A. Sources of Energy 757 B. Invertebrate Sperm Metabolism. . . 758 C. Mammalian Sperm Metabolism. . . . 759 D. Epididymal Sperm and Metabolic Regulation 701 E. Human Sperm Metabolism 702 F. Metabolic-Thermodynamic Inter- relations 764 G. Biosynthetic Activity 764 IX. Sperm Flagellation 7(55 A. Wave Patterns 765 B. Sperm Velocit}' 766 C. Hydrodynamics 766 D. Mechanism of Motility 767 X. Fertilizing Capacity of Treated Spermatozoa 769 A. Dilution of the Sperm Suspension. . 770 B. Temperature Effects 770 C. Ionizing Radiation 770 D. Ionic and Osmotic Effects 771 E. Effects of Biologic Fluids 772 XI. Conclusion 775 XII. References 775 I. Introduction In no way is the present review intended to represent a renovation of the comparable section in the second edition of Sex and In- terjial Secretions (Hartman, 1939) . It would be both presumptions and impracticable to attempt to update Professor Hartman's dis- cussion of the physiologic role of spermato- zoa in reproduction; this stands as a land- 707 708 SPERM, OVA, AND PREGNANCY mark now two decades old. In his review many problems were noted, some since solved, others still in the course of solution, and many even yet ignored. The major advances in sperm biology during the intervening years have been world-wide and substantial. Stimulated in large measure by the exigencies of the ani- mal-breeding industry. Lardy and co-work- ers at Wisconsin developed biochemical methods and concepts pertaining to sperma- tozoa, particularly those of the bull. Mann and his many able Cambridge colleagues have elucidated major aspects of the meta- bolic and enzymatic activities of sperma- tozoa in several domestic species. Signifi- cant contributions have appeared from various laboratories, too numerous to desig- nate, from the basic demonstrations, by the Engelhardt school, of the role of adenosine triphosphate (ATP) and adenosinetriphos- phatase (ATPase) in the motility of sperm, to the apparently unique metabolic charac- teristics of human spermatozoa reported principally by MacLeod. A second major stride in the study of the male gamete has been provided by the de- velopment of the electron microscope. By virtue of the increase in magnification, up to 1000-fold, cells can be visually dissected down to elements on the order of 10 A in size. Not the least of its accomplishments, the electron microscope has made possible the demonstration that all sperm flagella and all cilia throughout the plant and ani- mal kingdoms possess the same basic pat- tern of longitudinal filaments, the well known 2x9 + 2 array. The full signifi- cance of this structural constancy is yet to be realized, but inasmuch as these filaments are generally assumed to represent the mo- tile organelles of the cell, the physicochemi- cal basis for motility may eventually be resolved. Likewise, the electron microscope has facilitated the study of spermatozoa during their maturation and in the initial stages of fertilization. Significant altera- tions in the acrosome, for example, seem to be related to the processes involved in the union of gametes. The sperm has, in fact, been more closely scrutinized and is now recognized as some- thing more than a uniform, finished product of the spermatogenic process. Mammalian spermatozoa from the same gonad may well differ with respect to phenotypic and anti- genic characteristics. They are, moreover, far from functionally mature as they leave the testis; structural and biochemical changes occur during their sojourn and transport through both the male and female genital tracts such that their capacity for fertilization is enhanced with time and mi- gration. Investigation of these processes constitutes a very active area of research in current studies of reproductive physiology. Another important advance in recent years, which may here be singled out for comment, concerns the mechanism of trans- port of spermatozoa through the female gen- ital tract. One of the earliest features of mammalian reproduction to be studied, only recently has the full weight of experimental attack demonstrated the important endo- crinologic role involved in the process. These, among other, developments in sperm biology are considered in some detail in the pages that follow. No attempt is made to survey completely the available litera- ture, which is enormous; rather, what seem to be significant current principles and proc- esses are discussed within the scope and space allotments of the present volume. The general characteristics of whole semen and its jiroduction, reviewed elsewhere (see Mann, 1954; chapters by Albert and by Price and Williams-Ashman) , are necessar- ily slighted in favor of a fuller discussion of the internal environment of the male and female genital tracts and the probable con- ditions surrounding the sperm in vivo. For- tunately, a number of recent reviews cover many of the principal, broad points noted above and serve as background for the ma- terial reported here (MacLeod, 1943b; Ivanov, 1945; Mann, 1949, 1954; Austin and Bishop, 1957; Colwin and Colwin, 1957; Fawcett, 1958; Mann and Lutwak-Mann, 1958; Bishop, 1961; Tyler and Bishop. 1961). The function of the male gamete is to serve as activator of the ovum and contribu- tor of paternal hereditary components to the zygote. The sperm thus stimulates an other- wise relatively inert egg and initiates a new course of development. In Weissmannian BIOLOGY OF SPERMATOZOA 709 terms, it represents a continuity, of part at least, of the germ plasm from one generation to the next. In a very real phylogenetic sense, the gamete is one haploid generation momentarily sandwiched between two ex- tended diploid generations. Sperm, unlike most cells, are designed to function outside of their native environ- ment. Where fertilization is external, sper- matozoa may be shed into an aqueous me- dium of different ionic strength which offers little shelter, scant buffering capacity, toxic ions, and a lack of energy substrate essential for extended metabolic activity. In the case of internal fertilization, on the other hand, these conditions are generally obviated, and the seminal plasma, the vehicle for trans- port, affords additional security features beneficial to sperm survival. However, the introduction of sperm into the female ani- mal places them, even here, in foreign sur- roundings which, although natural, may not always prove hospitable. There is evidence to indicate, for example, that the normal protective and immune responses of the fe- male against foreign invasion reach even to the oviduct and uterus and to their luminal secretions. The motility of typical spermatozoa is certainly their most striking characteristic. Indeed, the degree of motility is frequently equated to fertilizing capacity and survival. The sperm of many nonmammalian species, however, may appear quite immotile, al- though they are fully capable of fertilizing normal eggs. The sperm of the herring {Clu- pea) , for example, are immotile as shed and remain so until brought into the vicinity of homologous eggs (Yanagimachi, 1957). The giant sperm of the hemipteran insect, Xoto- necta glauca, show no movement until acti- vated by fluids from the female genital sys- tem (Pantel and de Sinety, 1906). The sperm of many invertebrate animals, more- over, are nonflagellated, a specialization particularly common among decapod Crus- tacea. Amoeboid spermatozoa are found among ascarids, and in the sponge, Grantia, it is claimed that the sperm lose their fla- gella and are engulfed by modified collar cells which transform into amoeboid forms and transport the parasite-like sperm to the oocytes (Gatenby, 1920). It thus appears that, whereas nature has endowed the male gametes with fiagella from the lowest pro- tistan to the highest mammal, she has de- veloped secondary modifications toward less specialized conditions among numerous or- ganisms between the evolutionary extremes. II. Functional State of Gametes after Spermatogenesis The gross structure and organization of sjiermatozoa are generally considered com- plete when the gametes leave the testis. The statement is frequently seen that spermato- zoa, as found in the male efferent ducts, are ready for fertilization, unlike the egg cells which often, and in all mammals, are ovu- lated in a cytogenetically incomplete state of development, later to be activated by the sperm. In a general way, this contrast in the functional activity of the gametes is real- ized, and the most cogent evidence in behalf of the fertilizing capacity (although less than normal) of testicular sperm is the rec- ord of conceptions resulting from insemina- tion by sperm removed from the gonads of chickens and men (Munro, 1938; Adler and Makris, 1951). Normally, however, the process of sperm maturescence is not com- plete until some time after sperm formation, and fertilizing capacity is fully realized only after a period of sojourn in the male and female genital tracts (Redenz, 1926; Young, 1929a, 1931; Munro, 1938; Bishop, 1955). A. THE MATURATION OF SPERMATOZOA A number of structural and physical changes, here only briefly noted, occur in spermatozoa during transit through the ducts. Morphologically, the most obvious modification is the loss of the "kinoplasmic droplet," a cytoplasmic residuum of dubious function characteristic of immature cells and only rarely found in sperm of a normal ejaculate (Merton, 1939a; Gresson and Zlotnik, 1945; Mukherjee and Bhattacha- rya, 1949). Less obvious changes are a con- comitant decrease in free-water content and an increase in specific gravity of sperm as they mature, as in the bull (Lindahl and Kihlstrom, 1952). Salisbury (1956) found evidence to indicate that changes occur in permeability to water and in intracellular 710 SPERM, OVA, AND PREGNANCY concentrations of monovalent cations (Na+ and K+), modifications which might ac- count for the increase in capacity for move- ment of sperm at this stage in their develop- mental history. Unpublished observations of electron micrographs of human sperm by Fawcett indicate that the midpiece may undergo further significant alterations after spermatogenesis, changes which involve par- ticularly the mitochondrial sheath and the annulus at the junction of the midpiece and principal piece of the flagellum. In the female genital tract, further sperm modifications occur which appear to be nec- essary for fertilization. A period of incuba- tion in the tubal fluids is required during which changes (capacitation) occur that seem to involve both enzymatic and struc- tural properties of the sperm (Austin and Bishop, 1958a; Chang, 1958; Noyes, 1959b t. During this 2- to 6-hour interval, the sperm, at least of the rat, hamster, and rabbit, un- dergo certain changes in the head, which in- clude loss of the "galea capitis" and partial dissolution of the acrosome. What other changes occur in spermatozoa, in vivo, during their transport through the genital tracts, and of what consequence such modifications are to either survival or fertilization can only be surmised. In some respects, the metabolic properties of epi- didymal and seminal sperm differ, as studied in vitro (see Metabolism). Pronounced changes, of course, follow activation at the time of ejaculation, changes associated with energy production and motility. Other bio- chemical activities are believed to occur, moreover, which may be regarded as part of the "resting" metabolism of sperm. This will be discussed in a later section. On the other hand, certain deleterious changes may also take place, particularly during sperm storage, to such an extent that large molecu- lar moieties, such as cytochrome c, are ap- parently lost from both bull and ram sper- matozoa (Mann, 1954). B. CYTOGENETIC DIFFERENCES IN SPERM As the result of meiosis and segregation, spermatozoa are haploid in chromosome number and bear one-half of the hereditary complement which is carried into the next generation at fertilization. The two main types of sperm, X- and Y-bearing (in mam- mals), are responsible for female and male offspring, respectively, on union with the X-chromosome egg. Attempts have indeed been made, with questionable success, to separate these two kinds of sperm both by electrophoretic (Schroder, 194:0a, b, 1941a, b, 1944; Gordon, 1957) and by countercur- rent centrifugal methods (Lindahl, 1956). Genetically distinct spermatozoa were long ago demonstrated by Landsteiner and Levine (1926), who showed that the A and B blood-group antigens occur in human sperm, without, however, making clear whether the specific phenotype of a given sperm is determined by its haploid set of genes or by the diploid set of the spermato- cyte from which it is derived. GuUbring (1957) has recently revived this issue and claims that the A and B antigens occur on separate sperm produced by a heterozygous AB blood-group male. Further evidence of gene-induced sperm heterogeneity is af- forded by the work of Beatty (1956), who studied the 3,4-dihydroxyphenylalanine (DOPA) reaction in sperm from pigmented and pale rabbits. A high correlation was found between the melanizing activity of the spermatozoa and the depth of coat color of the rabbits from which they came. It will be remembered that Snell (1944) found sig- nificant antigenic differences in the sperm of inbred strains of mice, those of strain C being readily distinguishable from those of strain C57 on a basis of their agglutination with specific antisera. Braden (1956, 1958a, 1959) has recently made an intensive study of sperm variation in pure strains of mice. Statistically significant differences in size and shape of the sperm head were demon- strated in the four inbred lines, CBA, C57BL, A, and RIII. IVIoreover, at fertiliza- tion, strain differences become apparent in the tendency for more than one sperm to penetrate the egg membranes, sperm of strain C57BL, for example, showing a sig- nificantly higher percentage (26 per cent) than those of other strains (12 to 14 per cent). Further, Braden (1958b) has found abnormalities in the segregation ratio of mice which tend to indicate that the actual allele present (e.g., at the T-locus) in the sperm determines certain of its properties. BIOLOGY OF SPERMATOZOA 711 including its relative fertilizing capacity. The precise nature of the impairment is un- known but seems to involve the ability of the sperm to traverse the uterotubal junc- tion (Braden and Gluecksohn-Waelsch, 1958). This is the same gene which Bryson (1944) found to affect both sperm morphol- ogy and motility in the heterozygous (^V^^) male. These results are yet fragmentary but are strongly indicative of the fact that spermatozoa reflect their haploid genotype and that when they bear an unfavorable al- lelic constitution they may display a de- creased fertilizing potential. The possibility exists that subviable mutants, recessive in the heterozygous condition, might have pro- found detrimental effects when segregated into particular gametes. C. REQUIREMENTS FOR LARGE SPERM NUMBERS Any suggestion at the present time to justify the large number, or ''excess," of sperm ordinarily involved in insemination is at best a hazardous supposition. The earlier speculations which presupposed a sperm ''swarm" to supply hyaluronidase for the dissipation of cumulus cells (McClean and Rowlands, 1942) are inconsonant with the facts, since only a very few sperm, on the order of 25 to 250, are to be found in the presence of fertilized eggs of rats, rabbits, and ferrets, for example, while the cumulus cells are still clustered about the eggs (Bra- den and Austin, 1953; Chang, 1959). That many more sperm are produced and in- seminated than are necessary for fertiliza- tion is not be to denied. A phenomenon im- plicating survival of the species can be expected to have built-in safety factors, and sperm production is no exception, particu- larly if the male is to be capable of frequent ejaculation. Very probably, the pattern for high gametic production was set long ago among animals which reproduced by means of external fertilization where sperm, egg, and larval loss are very high. In fact, ob- stacles to successful fertilization are present in mammals as well; definite blocks to sperm transport, for example, occur at the cervix, uterotubal junction, and tubal isth- mus in many animals. But it is to be em- phasized, in the light of evidence cited in the two preceding sections, that the waste of healthy spermatozoa may be less than previously conjectured. The exigencies of the complex series of cellular and functional changes which ensue during the passage of sperm through the genital tracts and the possibility of genetic variation with conse- quent differences in fertilizing capacity sug- gest that the number of physiologically ef- fective sperm in the ejaculate may be but a fraction of the total inseminated. III. Sperm Transport and Storage in the Male Tract Aside from the accessory reproductive glands that supply, in large measure, the constituents of the seminal plasma (see chapter by Price and Williams-Ashman), the male genital tract of vertebrates is es- sentially a collection and transport system, designed to convey the spermatozoa from the testis to the ejaculatory duct (Fig. 6.1). It does more than this, however, in that the gametes, on the one hand, are altered in their capacity for fertilization and, on the other hand, are stored, motion- less, often for long periods of time, prepara- tory to ejaculation. The intrinsic changes within the maturing sperm and the inter- relations between the gametes and the vari- ous segments of the male duct system are only just beginning to be appreciated. The cytologic integrity and the functional ac- tivity of the male reproductive ducts are directly influenced by the androgen output of the testicular interstitium and presuma- bly vary in their influence on the spermato- zoa within the tract. Spermiation, the re- lease and shedding of spermatozoa from the testes of amphibians, is, of course, hor- monally induced (Van Oordt, Van Oordt and Van Dongen, 1959; Witschi and Chang, 1959) ; the mechanism of release is dis- cussed elsewhere in this volume (chapter by Greep) . In the cock, which has no glands analogous to the seminal vesicles or pros- tate, "seminal" fluids are contributed by the seminiferous tubules and vasa efferentia (Lake, 1957). A. SPERM TRANSPORT It can be stated with reasonable assur- ance that sperm migration within the male 712 SPERM, OVA, AND PREGNANCY tract is, from the sperm's viewpoint, in the main a passive process (Simeone, 1933). The mechanism by which they are moved along the duct system, however, is not well understood; the mechanics may well vary in different segments. Certain workers have emphasized the currents of fluid which could sweep the sperm out of the seminiferous tubules and into the efferent ducts and epi- didymis (see Young, 1933; Macmillan, 1953). Resorption of fluid by the efferent ducts (Young, 1933; Ladman and Young, 1958) or cpididymal epithelium (Mason and Shaver, 1952; Cleland, Jones and Reid, 1959) would complete the fluid circuit and simultaneously concentrate the sperm mass in the distal reaches of the duct system. Certain ligation experiments in which the male ducts were occluded at various levels tend to support this concept of transport by fluid currents, and circumstantial evidence is further afforded by the presence of motile cilia in the upper segment of the genital tract. On the other hand, other experiments which involved separation of the testis from the efferent ducts, thereby cutting off the supply of fluid, demonstrate unequivocally that the sperm, under these circumstances, are carried distally by some other means of tubal transport (Young, 1933; Macmillan and Harrison, 1955). More recently acquired evidence indicates that muscular activity may play the pre- dominant role in sperm transport through the male ducts. Roosen-Runge (1951) has observed movement in the seminiferous tu- bules of the dog and rat, both in the intact testis and in vitro in physiologic saline at 36° C. The undulating motion was attrib- uted, by Roosen-Runge, to the contraction and relaxation of the Sertoli cells within the tubules. A more plausible explanation may rest in Clermont's recent (1958) elec- tron micrographic demonstration of fibrous elements which lie in the wall of the seminif- erous tubule of the rat and seem to re- semble smooth muscle cells. The ductuli efferentes of the adult rat can be cultured successfully in roller-tube tis- sue-culture preparations (Battaglia, 1958). Tubules maintained as long as 12 days show spontaneous movement, presumably due to muscular contractions. This activity could provide a mechanism whereby spermatozoa are carried along these ducts, in vivo. Migration of spermatozoa through the epididymis proper is mainly, although per- haps not exclusively, brought about by spontaneous peristaltic and segmental movements of the duct. Such activity was first clearly shown in the guinea pig by Simeone (1933) and in the rat by Muratori (1953) and has been confirmed and recorded cinematographically by Risley (1958, 1960). Rhythmic contractions sweep along the adult tubule at regular intervals of 7 or 8 seconds. After gonadectomy, contractions continue in the mature duct for two more weeks. Hypophysectomy results in the loss of activity within 10 days in the head, and within 13 days in the body and tail of the epididymis. In tissue-culture preparations, the spontaneous movement of the epididy- mis also continues for some time (12 days), the activity being the same whether the ducts are excised from normal or from gon- adectomized rats (Battaglia, 1958). It is of some historic interest to note that Moore and Quick, as early as 1924, suggested a neuromuscular mechanism for epididymal sperm transport as a result of their studies on vasectomized rabbits; at the same time they refuted the then hotly contested claims of Steinach and others that vasectomy re- ults in seminiferous atrophy and interstitial hypertrophy. Complete occlusion of the rat vasa eff'erentia, on the other hand, is claimed to lead invariably to si^crmatogenic destruc- tion (Harrison, 1953). Transport through the epididymis re- quires 2 to 4 days in the fowl, 4 to 7 days in the rabbit, 9 to 14 in the ram, 14 to 18 in the guinea pig, 8 in the mouse, about 15 in the rat, and 19 to 23 days in man (Toothill and Young, 1931; Munro, 1938; Edwards, 1939; Brown, 1943; MacMillan and Harrison, 1955; Asdell, 1946; Dawson, 1958; Oakberg and DiMinno, 1960). The guinea pig determinations of Toothill and Young (1931 ) made use of the migration of India ink particles, injected into the head of the epididymis, and not of sperm trans- port per se. The apparently rapid rate of migration of sperm in the fowl may be at- tributed to the relatively short length of the epididymis (Munro, 1938). Isolation of the BIOLOGY OF SPERMATOZOA 713 testis from the epididymis of the guinea pig increases transport time by 1 to 2 weeks, possibly as a result of interruption of flow of fluid through the excurrent ducts, or perhaps as a consequence of operational disturbances which involve changes in the local vascularization and nerve supply. The vas deferens serves mainly for the accumulation and storage of sperm, but what sperm migration does occur seems pri- marily dependent upon the muscular ac- tivity of the duct. The vasa of the rat and dog are normally quiescent in sexually in- active males, but are capable under experi- mental conditions, in vitro, of a high degree of muscular activity (Martins and do Valle, 1938; Valle and Porto, 1947). Belonosch- kin (1942) claimed that peristaltic activity of the vas deferens aids in sperm transport in man. B. SPERM SURVIVAL Spermatozoa may reside in the genital tract for considerable periods of time before being discharged at ejaculation. They gen- erally lose their capacity for fertilization before their capacity for motility during storage in the ducts. Survival times vary from several weeks to many months in dif- ferent species. Bats normally store sperma- tozoa over the winter months, and this may be typical of certain other hibernating mammals as well. Knaus (1933) claimed that epididymal spermatozoa remain viable and fertile for a year in vasectomized rab- l)its, but the process of sperm renewal was not eliminated in his experiments. Mouse spermatozoa in the excurrent ducts main- tain their capacity for fertilization for 10 to 14 days after spermatogenesis has been inhibited by x-ray irradiation (Snell, 1933). Rat epididymal spermatozoa, in animals with ligated vasa, remain capable of mo- tility for about 6 weeks, but lose their abil- ity to fertilize eggs within 3 weeks (White, 1933b); castration further reduces sperm survival to approximately 2 weeks (Moore, 1928). Likewise, in the guinea pig, after ligation of the efferent ducts, epididymal spermatozoa retain their capacity for mo- tility some 60 days and for fertility 20 to 35 days; castration reduces motility to about 3 weeks (Moore, 1928; Young, 1929b). Translocation of the epididymis to the ab- dominal cavity further limits sperm sur- vival to about 2 weeks. When the rabbit epididymis is anchored in the abdomen, sperm motility and fertility are reduced from about 60 and 38 days in the controls to 14 and 8 days, respectively. Demonstra- tions such as these seem to indicate that body temperature may have a pronounced effect even on relatively mature spermato- zoa (Knaus, 1958) ; however, the transloca- tion procedure may primarily affect the epi- didymis, which in turn alters the longevity of the spermatozoa. In at least one type of natural experiment we have evidence that excessive body temperature is seasonally avoided by the gametes. In certain passerine birds during the active breeding season a transient thermal adaptation provides lower temperatures for the storage of morphologi- cally mature spermatozoa (Wolfson, 1954). The sperm-engorged, distal ends of the vasa deferentia here increase prominently in size and become tortuously coiled, so as to re- sult in a cloacal, scrotum-like swelling, the internal temperature of which is about 4°C. less than body temperature. When the testes have been separated from the epididymides and time allowed for recovery, the potential sperm capacity of the duct system can be determined. Young (1929b) found that guinea pigs, prepared in this manner, can copulate successfully as many as 20 times over a 2-month period. The relative storage facilities of the major segments of the ducts can be determined by actual sperm count. Chang (1945) diligently counted the sperm in the vasa and epi- didymides of several ram genital tracts and found the greatest accumulation in the tail of the epididymis (Table 13.1). By frequent ejaculation of the ram, approximately twice a day, he further was able to estimate the average rate of sperm production to be about 4.4 X 10^ cells per day. In the bull the rate is less, about 2.0 X 10^ sperm daily (Boyd and VanDemark, 1957) ; most of the epididymal sperm storage here is also in the tail (45 per cent) compared with that in the head (36 per cent) (Bialy and Smith, 1958). 714 SPERM, OVA, AND PREGNANCY TABLE 13.1 Distribution of spermatozoa in male genital tract of ram (From M. C. Chang, J. Agric. Sc, 35, 243-246, 1945.) Segment Sperm Count (X 109) Percentage of Total p]pididymis i. Caput Corpus 17.3 8.4 104.3 1.5 0.3 13.1 6.4 79.1 Efferent duct Vas deferens Ampulla .... 1.1 0.2 C. THE FUNCTIONAL MICROANATOMY OF THE EPIDIDYMIS The epididymis has received considerable attention from microscopists bent on the elucidation of the role this part of the duct system plays in the reproductive physiology of the male. A number of recent papers have contributed to our understanding of the segmental organization of the epididymis, the cytochemistry of the mucosa, and the response of the duct to steroid influences. For many histochemical details, and histori- cal surveys of much of the earlier literature, the following papers should be consulted: Reid and Cleland (1957), Cavazos (1958), Maneely (1958, 1959), and Reid (1958, 1959) concerning the rat; Ladman and Young (1958) on the guinea pig; Nicander (1957) for the rabbit; and Nicander (1958) concerning the stallion, ram, and bull. Al- though exquisite in detail and extensive in scope, these papers, with few exceptions, have added little to the earlier contributions concerning the function of the epididymis vis-a-vis the physiology of the spermatozoa within the lumen (c/. Young, 1933; Mason and Shaver, 1952). With the cytochemical background now available, however, and the current interest in epididymal physiol- ogy, the expectations to be derived from a more functional approach should now be fulfilled. Emphasis has again been placed on the epididymal mucosa, and particularly on the vacuolar and endoplasmic reticular system as a site for the reabsorption of fluid (Ni- cander, 1957; Reid and Cleland, 1957; Lad- man and Young, 1958) , in contrast to its function as a secretory organ (Hammar, 1897; Henry, 1900; Benoit, 1926; Maneely, 1954; Goglia and Magh, 1957). The old question as to the cause of increasing sperm density has apparently been resolved re- cently by ligation experiments in the rat (Cleland, Jones and Reid, 1959) ; a spe- cialized region of the epididymis absorbs fluid from the lumen at the point where sperm concentration suddenly increases. Virtually nothing is known about the trans- port of substances, other than water and possibly inorganic ions, across the mucosal boundary, despite the elaborate cytochemi- cal reports, which include data for acid and alkaline phosphatase activitv (Bern, 1949a, b, 1951; Wislocki, 1949; Maneely, 1955, 1958; Montagna, 1955; Allen and Slater, 1957, 1958; Cavazos, 1958; Allen and Hunter, 1960), metachromatic substances (Cavazos, 1958), glycogen (Leblond, 1950; Montagna, 1955; Nicander, 1957, 1958; Ca- vazos, 1958; Maneely, 1958), lipids (Chris- tie, 1955; ]\Iontagna, 1955; Cavazos and Melampy, 1956; Nicander, 1957, 1958), gly- coprotein (Cavazos, 1958) , and nucleic acids (Nicander, 1957, 1958; Cavazos, 1958). It would be of interest to know how these cyto- chemical characteristics vary, if indeed they do, with sexual activity, on the one hand, and, on the other hand, with certain func- tional processes, such as the reabsorption of fluid from the duct, the possible transfer of tagged molecules across the limiting membrane, the elaboration and secretion of, for example, glycerylphosphorylcholine present in the epididymis (Dawson and Rowlands, 1959) , and the uptake of large molecular moieties into the mucosa from the lumen, as demonstrated with trypan blue, pyrrhol blue, fuchsin, and India ink parti- cles (von Mollendorf, 1920; Young, 1933; Mason and Shaver, 1952; Shaver, 1954). Nicander's studies have the added merit that cytochemical demonstrations are cor- related with regional differentiation of the epididymis; the duct is divided into 6 to 8 cytologically distinct segments. Such divi- sion includes the efferent ducts as part of the epididymis, whether they appear to be nested within a depression of the testis, as in the guinea pig, or quite external to it as in the stallion, ram, bull, and rabbit. All BIOLOGY OF SPERMATOZOA 715 told, the epididymis is an imposing duct, a single continuous tube about 10 feet long in the guinea pig and up to 280 feet in the stallion (Ghetie, 1939; Maneely, 1959). An impressive series of contributions per- taining to the regional differentiation and histology of the rat epididymis has been published by Reid (1958, 1959) and Reid and Cleland (1957), of the University of Sydney. They divide the rat epididymis into six discrete zones, plus the rete and efferent ducts, on the basis of cell type. The efferent ducts and zones 1 to 4 constitute the head, part of zone 4, the isthmus, and zones 5 and 6, the tail of the epididymis (Fig. 13.1). The relative lengths and di- ameters of the successive zones and the cellular types are represented in Figure 13.2. Six major cell types are discernible: principal, basal, ciliated, apical, halo, and clear cells (Fig. 13.3). Ciliated cells are confined to the efferent ducts — "the most beautiful ciliated cells of the vertebrate body" 1 8 1 E 11 1^ s M 1 E 3 2 §■ 4 1 1 s 1 2 ill tN JJ •J aT s 111 4) ^ 1 0. S c £ 1 E 3 8 -c T3 !C c "^ 5 C m H s 2 1 £ pii •^ t3 1 1 e ^ £ nil i ^ t3 1 3 3 S^ § S - P 3 -t; i a .C J= JJ "s S ° « .^ H .- 1 J-- — 3 H 5 3 ^ a . S 3 - --S S § =i 5 -5 -§ -0 .s -o 1 W u .2 11 II g a e 2 IS s 1 iPiJ i 1 s 1 i« 5 8 3 ' ^ 1 c -I •i ° 1 1 ^ H -< S < & M „ s s? Q BIOLOGY OF SPERMATOZOA 719 ^ s s >> 1 ■§ ^ 3J -g ■o CM (M CM « m CO M u u .2 •2 O a o •ii i ^ so 5 < ■Ao^Si ft o ^ -3 ?^2 C — i 3 differeni ■om vas d it 39 days toplasm wi rows of n * m oj SJ2 ftj2 c3 J2 & e m .- S ^ -c: -^ li^i :- H ft ■W -3 _ o Becomes ated fi ferens i pale cy several clei 1 aS^ 111 II 11-1 llll < H 1 < S 1 w-i 1 eg O 3 S > o 111 5:1 ^ '£• J '1 'S 00 1 00 i 3 •"■ ■ m , >. « 4 4 -g T3 •O 3 t~- ^ 0= CO 11 * M lO tn lO a o a .2 h •- « i2 > OS 03 (fl 03 •o a, ^ •fl T3 T! > 2 CO CO v3 ^ iO •o U5 S ■s| o o o •s '•9 ? a; Oi 10 » <: Q < < < li (5S » c m oo m 00 II >> >> >> >> •§ 4 •g 4 s?a. CM CM CM CO eo CO CO ^ll ^ 1 a .2 00 - :s < < < < •< s CO ^ CO s OJ '^ 720 SPERM, OVA, AND PREGNANCY TABLE 13.3 Mineral concentrations in male reproductive fluids (From R. G. Cragle, G. W. Salisbury and J. H. Muntz, J. Dairy Sc, 41, 1273-1277, 1958.) Testicular Ampullar Fluid Seminal Ve- Seminal Ele- Fluid sicular Fluid Plasma ment (average (average of (average of (average of of 12 samples) 3 samples) 10 samples) 10 samples) mg. per mg. per mg. per mg. per 100 ml. 100 ml. 100 ml. 100 ml. B 0.80 0.59 0.73 1.48 P 229.00 328.00 10.00 55.00 Mg 14.90 8.50 17.50 11.60 Ca 4.60 32.00 70.00 51.00 Fe 2.59 1.08 0.38 0.35 Cu 1.36 2.10 0.95 1.36 Na 178.00 137.00 251.00 273.00 plete analyses of inorganic ions in the fluids of the male tract are those by Cragle, Salisbury and Muntz (1958) for the testicu- lar and ampullar fluids of the bull. The values are compared with those of seminal plasma and seminal vesicular fluid in Table 13.3. One type of epididymal reaction which may be of considerable importance, al- though the mechanism of the process is little understood, is that concerning ionic ex- change, alluded to above. Salisbury and Cragle (1956) showed that shifts in the sodium-potassium ratio occur in the luminal contents of the goat and bull when sampled at different levels of the tract. The com- bined "semen" (sperm and fluid) tends to show a relative increase in sodium ion and an increase in K+ + Na+ when compari- sons are made of tubal contents from suc- cessively lower regions of the tract. Freez- ing-point determinations indicated that the fluid is initially hypertonic (— 0.600°C.), with respect to blood, and decreases in tonicity with passage through the tube. De- terminations of epididymal plasma and seminal plasma of ejaculated bull semen tended to confirm these results with respect to increase in Na+ and the combined K+ -l- Na+ values (S0renson and Andersen, 1956). In a general way, the capacities for mo- tility and fertility seem to be acquired about the same time, but in neither case is this brought about by a sudden change. The capacity for fertilization increases as the gametes are taken from more distal regions of the tract. In the fowl, for example, in- semination with sperm from the testis, epi- didymis, and vas deferens, respectively, gave 1.6, 18.8, and 65.3 per cent fertile eggs (Munro, 1938). Similarly, in the guinea pig, sperm removed from the proximal and distal portions of the epididymis and used in arti- ficial insemination resulted in 33.4 and 68.0 per cent pregnancies (Young, 1931). After ligation of the vasa deferentia and aging of the sperm, the percentage of fertility from proximal and distal sperm shifted to 44.2 and 32.5 per cent, respectively, for 20-day postligation sperm, and to 49.0 and 25.0 per cent for 30-day stored sperm. It seems clear that, with storage, the maturation of the sperm is followed by a process of senescence. This was further suggested by Young's ex- periments, since the percentage of aborted and resorbed fetuses increased apparently when fertilization was accomplished by aged spermatozoa. Whether or not the relative fertility rates of spermatozoa from different levels of the male genital tract can be explained entirely on the assumption that motility and fer- tilizing capacity go hand in hand remains to be seen, since other aspects of sperm behav- ior also change with transit through the ducts. Young (1929c) pointed out, for ex- ample, that the heat resistance (to 46°C.) of guinea pig, rat, and ram sperm decreases as they migrate through the tract, and Las- ley and Bogart ( 1944) showed that the re- sistance of boar sperm to "cold shock" is likewise reduced. E. THE FATE OF NONUTILIZED SPERM IN THE MALE In the absence of ejaculation, the question arises as to the fate of the millions of gam- etes which are continuously generated dur- ing spermatogenesis. It hacl been previously assumed that sperm elimination is by "in- sensible ejaculation"; sperm have been de- tected in the urinary outfiow (Wilhelm and Seligmann, 1937). It was shown, however, by Young and Simeone (1930; Simeone and Young, 1931), and since confirmed by others, that the sperm of the guinea pig, for example, undergo degeneration and dissolu- tion within the epididymis. The disposal of the degradation products of the sperm, on the other hand, is not clear from these ex- periments. They could very possibly be BIOLOCiY OF SPERMATOZOA 721 voided tliroii^li the vas deferens or he ab- .sorl)ed by the duct iniicosa and i)hago- cyto.scd, as suggested l)y Mason and Sliaver (1952) and Montagna (1955). The possi- bihty of absorption of si)enn and of sperm products by the epithelium poses a signifi- cant problem relating to self -immunization whicli is discussed in a later section. F. ACQUISITION BY SPERM OF THE CAPACITY FOR MOTILITY By the time spermatozoa are primed for union with the eggs they must be sufficiently activated to undergo independent move- ment, since motility, with rare exceptions, is a prerequisite for fertilization. Sperm ac- tivation is delayed in many species until the gametes are in intimate association. Among l)oth invertebrate and vertebrate animals, instances are known in which sperm are shed in a nonmotile condition and are activated only when passively brought into association with homologous eggs as noted above. Frequently the gam- etes are transferred in large bundles or packets, enclosed in spermatophores, and l)ecome motile only after the casings are rui)tured when in contact with the female I Drew, 1919). Generally, however, the gam- etes are stimulated when shed externally or ejaculated into the female genital tract. This event corresponds to a spectacular mo- ment in the metabolic life of the cell when tlie exergonic processes are shifted into high gear by the abrupt supply of oxygen, sub- strate, or cofactors, in mammals copiously provided by the secretions of the accessory reproductive glands. Before ejaculation, and for much of the time during their storage in the ducts, sperm are quite capable of motility but, so far as can be determined, remain, in vivo, in a (|uiescent state (Simeone, 1933). The repro- ductive advantage of this is obvious since, before activation, sperm survival is esti- mated in terms of months; after activity has been acquired, survival is a matter of days or hours (sec Table 13.8). The blocks both to the excessive utilization of energy and to motility, in vivo, are regarded as largely of a pliysical nature — the relative or absolute absence of oxygen which otherwise would encourage aerobic respiration, and the lack of glycolytic substrate, such as glucose or fi'uctose, which when present fosters an- aerobic processes (Mann, 1954; Walton, 1956). Infrequent reports of transient mo- tility by sperm immediately after removal from the genital tract, thereby implying that the cells are motile in vivo (White, Lar- sen and Wales, 1959), must be confirmed and may be attributable to the admission of oxygen during the sam]>ling procedure. Ear- lier supi^ositions that sperm immobilization within the ducts is due to high CO2 concen- tration or low pH level (Redenz, 1926) have been contraindicated (Bishop and Mathews, 1952a). Other physiologic factors, involving both intrinsic features of the gam- etes and exchange reactions between them and the luminal fluids, may play a role, but if so, their nature and action are unrecog- nized. The capacity for motility on a general scale is first attained by sperm during transit through the epididymis (Redenz, 1926). Cells removed from the tail of the duct, of the bull for example, immediately become highly active when suspended in physiologic saline and given access to oxy- gen; under anaerobic conditions, glucose, fructose, or mannose initiates vigorous flag- ellation. Sperm removed from the head or the isthmus of the epididymis, on the other hand, rarely become motile and at best show only a slow nonprogressive type of undulation. Other mammals present a simi- lar picture, the precise epidiclymal region where motile capacity is attained varying among species. Some degree of flagellation, albeit of a leisurely, low-frequency type, can be ob- served in sperm recovered from the testes of various mammalian species (Tournade, 1913; Young, 1929a; Bishop, 1958d). These gametes are incapable of activation to full motility by the addition of oxygen, glyco- lytic substrate, divalent cations (Mg+ + , Ca+ + ), or ATP (Bishop, unpublished data). Austin and Sapsford (1952) have ob- served that the axial filament of the living rat spermatid undergoes mo^-ement even be- fore the flagellum begins to push out from the margin of the roughly sjiherical cell. In lower forms as well, particularly among insects, sperm motility can be seen during the period when the gametes are still at- tached to their nurse cells within the gonad 722 SPERM, OVA, AND PREGNANCY (Anderson, 1950). In conclusion, then, it would seem that spermatozoa must develop much of the machinery for movement while undergoing spermiogenesis in the tes- tis, that subtle changes occur while they are in the mammalian epididymis such that the full capacity for motility is here ac- quired, and finally that this ability for flagellation is realized normally only on activation at the time of ejaculation. Although the problem has been recognized for many years, only recently has serious attention been paid to the nature of the pos- sible changes in spermatozoa that are re- sponsible for the acquisition of the capacity for motility within the epididymis. In set- ting forth a hypothesis to account for this phenomenon, Salisbury (1956) has focused needed attention on the problem. His sug- gestion follows from determinations of cat- ion concentration and freezing point de- pression values of fluids of the genital tract of the ram and the bull, noted above (Salis- bury and Cragle, 1956). The supposition is that a decrease in K+/Na+ and an abso- lute increase in K+ -t- Na+ concentration, nevertheless accompanied by a total reduc- tion in tonicity of the fluids, bring about permeability changes such that the sperm become hydrated and, as a result, capable of full metabolic activity (Salisbury, 1956). An ingenious theory, it is, nevertheless, not easily reconciled with the generally ac- cepted demonstration that sperm lose water rather than gain it as they mature (Lindahl and Kihlstrom, 1952; IVIann, 1954). Until more precise information is available con- cerning such details as the sodium and po- tassium concentrations of the sperm vis-a- vis those of the fluid of the ducts, the actual water- and cation-permeability of sperm at various stages, and the effect of shifts in the sodium-potassium ratio on motility of epi- didymal sperm, in vitro, this problem cannot be fully resolved. IV. Insemination At the time of mammalian sperm transfer, many millions of vigorously motile sperma- tozoa are introduced into the female genital tract. Mixed with the fluid component of the semen only at the moment of ejaculation, the sperm normally are activated by their sudden access to both oxvgen and the hex- ose energy substrate of the plasma. The source and composition of the seminal fluid have been reviewed elsewhere (Mann and Lutwak-Mann, 1951 ; Mann, 1954) and are further discussed in the chapter by Price and Williams-Ashman. Only certain charac- teristics of semen, relevant to sperm trans- port and welfare, need be noted here. What is the normal function of seminal plasma, and to what extent is it dispensable? The fluid component contributed by the ac- cessory glands can conceivably serve several functions which include its role as (1) a vehicle for sperm transport, (2) a medium containing essential inorganic ions and of adequate buffering capacity, (3) a satisfac- tory osmotic milieu, and (4) a source of energy substrate. Seminal plasma, by virtue of its very complex composition, also per- forms other duties. It supplies, for exam- ple, the enzyme and substrate responsible for vaginal-plug formation; it contains cer- tain substances, unique to the reproductive fluids, such as antagglutin which ostensi- bly prevents undue sperm agglutination; it provides such ingredients as ascorbic acid, ergothioneine, and possibly glutathione, which may play a role in the adjustment of the oxidation-reduction potential. On the other hand, some components of seminal plasma may indeed be by-products with no obvious beneficial role and even, perhaps, with harmful effects on the gametes; both alcohol and sulfonamides, for example, are excreted into the plasma (Farrell, 1938; Osenkoop and MacLeod, 1947). Since the vital process of sperm activation is accomplished by their admixture with plasma, and since a fluid vehicle is essential for sperm transport, it goes without saying that seminal plasma normally is necessary for the reproductive process. To suggest that artificial insemination with epididymal sperm suspended in saline, successful as it is, proves the dispensability of seminal plasma, is to ignore the normal biologic accomplish- ments of natural insemination. Neverthe- less, it is a fact that artificial insemination has proved highly successful in the repro- duction of many types of animals. More- over, the collection and analysis of the ejac- ulate, coupled with artificial insemination by natural or modified semen, constitute the basis for much of our knowledge concerning BIOLOGY OF SPERMATOZOA 723 tlie entire field of i'e|)r()(liu'tive physiology and animal breeding. A. EJACULATION The ejaculatory response in many mam- mals occurs seasonally, corresponding to periodic activity of the testes and accessory glands, and is dependent on a variety of neurohumoral factors. In some animals, in- cluding man, potency continues throughout the period of reproductive maturity. Vol- ume of semen and s})erm concentration, be- ing contingent on both secretory activity of the accessory glands and spermatogenic ac- tivity of the gonads, vary with successive collections. Repetitive ejaculation can "ex- haust" the sperm supply (Table 13.4), and >uch procedures have been used, albeit with- out great practical success, to test the sper- matogenic productivity of man and of vari- ous domestic animals. The potential for ejaculation of many animals is quite striking. Carpenter (1942) observed that free-ranging macaque mon- keys are capable of ejaculating 4 times a day for 3 or 4 days, whereas the so-called "black ape" (a baboon, Cijnopithecus) , studied by Bingham (1928), ejaculated 3 times in 20 minutes. Domestic cats have been known to inseminate 10 females within 1 hour, and rabbits, 38 to 40 does in 8 hours (Ford and Beach, 1951). White rats can ejaculate 4 times in 15 minutes and as many as 10 times during a 3-hour period. Chang and SheafTer (1957) reported that a golden hamster copulated 50 times in an hour, with ejaculation occurring during most of the mounts. ]\IcKenzie and Berliner (1937) col- lected 20 ejaculates in 1 day from a ram, the 19th sample of 0.66 ml. containing over 1 l>illion sperm, compared with the first ejaculate of 0.7 ml. which contained 3.5 liillion cells. There is relatively little correlation be- tween the number of intromittent thrusts and the number of actual ejaculations, or between the duration of copulation and the volume of seminal discharge. In rodents, for example, intromission may occur as many as 80 to 100 times before ejaculation, or in- semination may occur on the first intromis- sion. Copulation in the macaque may in- volve several dozen mountings and well over a hundi-ed pelvic thrusts before ejaculation TABLE 13.4 Chanyes in volume and sperm (lensitt/ of hull ejaculates collected frequently throughout a one-hour period (From T. Mann, Advances EnzvmoL, 9, 329-390, 1949.) Number of Ejaculate Collection Time Volume Number of sperm, mil. per ml. semen min. ml. 1 4.2 1664 2 10 3.9 680 3 18 3.7 254 4 28 3.7 648 5 38 3.4 135 6 45 3.5 342 7 55 2.7 390 8 63 2.9 98 occurs. The prolonged copulation of the fer- ret and sable, as long as 3 hours, represents, not excessive ejaculation or insemination, but rather a functional adaptation to de- layed ovulation (Ford and Beach, 1951). In the dog, however, the mounting time is roughly proportional to the duration of ejaculation, averaging in several breeds about 6V2 minutes (Perez Garcia, 1957). An important accomplishment of genital activ- ity, at least in the rat, is the stimulation of sufficient corpus luteal function to support pregnancy (Ball, 1934). Cerebral and constitutional influences in- cite and modify the physiologic processes of both erection and ejaculation (Rommer, 1952) under the direct innervation by lum- l)ar centers operating through muscular and vascular mechanisms. Spinal section in man does not necessarily prevent seminal emis- sion (Ford and Beach, 1951). The relevant neural pathways in man have been sum- marized by Whitelaw and Smith wick (1951) from their observations on partially sympa- thectomized patients (Fig. 13.4, a and b) ; sympathetic fibers and the second, thii'd, and fourth sacral parasympathetic outflows are involved. The abolition of ejaculation through bilateral presacral sym})athectomy, without loss of erection, has been demon- strated in dogs (Van Duzen, Slaughter and White, 1947) and rodents (Bacq, 1931). In man and other mammals (rat, cat, and dog) , the cerebral cortex inlays an important role in male sexual activity (Ford and Beach, 1951), but an e\-aluati()n of the co-ordina- ^4. Erection — normal Sensory (stimulation of glans) i via pudendal nerve i Lumbar center Psychic (higher centers of cerebral cortex) i diencephalon i cord Inhibition of vasoconstrictor fibers (sym- pathetic) dorsal and lumbar Parasympathetic sacral (S2 — S4) i Vesical plexus i Vascular supply of penis i Dilatation of vessels i Engorgement of sinuses i Passive compression of veins and retarda- tion of venous outflow B. Ejaculation — normal Sensory stimuli from glans I via pudendal nerve Psychic stimuli from higher centers i diencephalon i cord i Summation of sensory and psychic stimuli producing so-called orgasm i Lumbar center Sympathetic motor i Smooth-muscle contraction of prostate, seminal vesicles and vas deferens. Closure of internal sphincter i Emission Parasj-mpathetic motor i Contraction of striated muscle, ischiocaver- nosus, bulbocavernosus and contractor- urethrae muscles i Ejaculation Fig. 13.4. The probable neural pathways involved in (A) erection and (B) ejaculation in man, based on observations after partial sympathectomy. (From G. P. Whitelaw and R. H. Smithwick, New England J. Med., 245, 121-130, 1951.) 724 BIOLOGY OF SPERMATOZOA 725 150 140 130 120 < 110 cr h- en < 100 LxJ X 90 80 70 - n nP / ■ ^ r- •\J IIU ! t ^ INTROMISSION ORGASM 1 1 1 1 1 1 1 \ 1 1 10 20 30 60 70 80 90 40 50 MINUTES Fig. 13.5. Heart rate of man during coitus recorded by cardiotachometer. (From E. P. Boas and E. F. Goldschmidt, The Heart Rate, Charles C Thomas, 1932.) 100 tion between cerebral and spinal centers warrants considerable further study. The subject is further discussed in the chapter by ]\Ioney. Ejaculation in men and dogs is accompanied by pronounced cardiovascular intensification (Pussep, 1921; Boas and (ioldschmidt, 1932; Bartlett, 1956) affecting both blood pressure and pulse rate (Figs. 13.5, 13.6). Androgen administration in- creases libido and copulatory arousal (see cha])ter by Young ) , but the manner in which this is related to the preceding neurogenic factors is not well understood (Cheng and Casida. 1949). B. COLLECTION OF SEMINAL COMPONENTS Various methods of seminal collection may be employed for cither whole or frac- tional analyses. Normal ejaculates are cxix'ctcd when induced l)y masturbation, electrical stinuilation, or discharge into an artificial vagina. Sperm samples without seminal plasma are readily obtainable from the epididymis and vas deferens of the ex- cised tracts of many animals (e.g., the guinea pig, rat, boar, bull, and stallion) by backflushing the vas and cutting the epi- didymis. Relatively uncontaminated pros- tatic fluid is procurable, e.g., from men and dogs, and vesicular fluid from men, by man- ual massage of the appropriate glandular regions. Incomjilete ejaculates are produced after extirpation of such organs as the semi- nal vesicles and Cowper's glands; the pros- tatic isolation operation, perfected on dogs l)y Huggins, Masina, Eichelberger and Wharton (1939) and illustrated in Chapter 6. Figure 6.2, permits the collection of large amounts of uncontaminated prostatic fluid. C. SEMINAL VOLUME AND SUCCESSIVE FRACTIONS Scniiiial xolunie bears some relation to animal sizt'. but the individual contril)utions 726 SPERM, OVA, AXD PREGNANCY 2Z0 210 en 200 X £ E I 190 LJ cr z> ^ 180 if) LlJ cr CL 170- 160 150 140 WITHDRAWAL 8 MINUTES 10 16 Fig. 13.6. Blood pressure of dog during coitus. (After L. M. Pussep, Der Bhitkreidauj im Gehirn beini Koitus, Dorpat, 1921.) TABLE 13.5 Volume and sperm density of mammalian ejaculates (From T. Mann, Advances Enzymol., 9, 329-390, 1949; S. A. Asdell, Patterns of Mammalian Re- production, Comstock Publishing Co., 1946.) Volume of Single Ejaculate Density of Sperm in Semen Species Most Range com- mon value Range Average ml. n,l. cells per til. cells per til. Man 2-6 3.5 50,000-150,000 100,000 Dog 2-15 6 70,000-900,000 200,000 Rabbit ... 0.4-6 1 100,000-2,000,000 700,000 Boar 150-500 250 25,000-300,000 100,000 Bull 2-10 4 300,000-2,000,000 1,000,000 Ram 0.7-2 1 2,000,000-5,000,000 3,000.000 Goat 0.1-1.25 0.6 3,000,000-4,000,000 3,500,000 Stallion.... 30-300 70 30,000-800,000 120,000 of the respective accessory glands determine the quantity of semen in the ejaculate. The volumes of seminal discharge from several mammals are presented in Table 13.5. The enormous volume of the boar ejaculate, as much as half a liter, may be of importance in "washing" the sperm through the uterus, inasmuch as in the sow the semen is de- posited directly into the cervix, and the ejaculate is so proportioned that the sper- matozoa are concentrated in the earlier frac- tions and are followed by a copious flood of relatively sperm-free fluid (McKenzie, Mil- ler and Bauguess, 1938) . When fractional collection is possible, the spermatozoa are found generally concentrated in the initial or middle portion of the ejaculate (ram, dog, boar, horse, and man). The ejaculate of the dog consists of a small, initial, clear, rela- tively sperm-free portion, followed by a milky fraction containing the bulk of the spermatozoa, and finally a slow but copious dribble largely derived from the prostate (Evans, 1933; Hartman, unpublished data). Collection by means of the electrically stim- ulated "split-ejaculation" technique has BIOLOGY OF SPERMATOZOA 727 (Icinonstrated in the bull an abundant sperm-free initial portion which apparently is derived from the urethral glands and pre- sumably serves to clear the urethral passage b(>fore the transport of spermatozoa (Lut- wak-jNIann and Rowson, 1953). In man, however, approximately three fourths of the sjierm are present in the first 40 per cent of the ejaculate (MacLeod and Hotchkiss, 1 942 ) . Qualitative contributions to the total ejaculate by the several accessory glands liave been carefully studied and are dis- cussed elsewhere (see chapter by Price and Williams- Ashman ) . D. EFFECTIVE SPERM CONCENTRATION It is not a simple matter to determine what might be the minimal effective sperm count necessary to insure fertilization. The earlier standards of what constitutes a sub- fertile human seminal density have under- gone considerable re-evaluation. The once- acceptable value of minimal concentration, 80 to 100 million cells per ml. of semen, has now been reduced to one half or less. On the other hand, the spotty records of pregnan- cies in women whose husbands' sperm counts consistently average 1,000,000 per ml., or less, may be viewed with some skepticism CMichelson, 1951; Sandler, 1952). The ex- tensive studies of ]\IacLeod and Gold (1951) on human subjects of proved fertility, com- pared with men of infertile marriages, indi- cate a significant break between the two groups in the neighborhood of 20,000,000 cells per ml. of semen. Current trends in the evaluation of semen tend to minimize sperm density, as such, and to regard this prop- erty only with reference to other criteria, in- cluding volume, total sperm number, mor- phology, and, of course, motility. A reasonable gauge of minimal effective si)erm count necessary to insure fertilization lias been provided by dilution tests and arti- ficial insemination of domestic and labora- tory animals. In cattle, the normal ejacu- late, which contains some 4 billion sperm, can be reduced 500 to 1000 times without sacrificing high productivity (Salisbury and Bratton, 1948; Braden and Austin, 1953). Ral)l)it fertilization is unimpaired when the normal inseminate is decreased 500-fold (Cheng and Casida, 1948; Chang, 1951a; Chang, 1959; Braden and Austin, 1953). That mere number of sperm is not the only factor was clarified by Chang (1946a, b) who showed that the concentration and na- ture of the diluent are also important. The percentage of fertile eggs recovered from does inseminated with a suboptimal num- ber of sperm (ca. 40,000) decreases as a function of the volume of saline diluent (from 0.1 to 1.0 ml.). On the other hand, if rabbit seminal plasma is substituted for sa- line as the diluent, fertilizing capacity is enhanced (Chang, 1947b, 1949). The nature and the effect of the sperm diluent are fur- ther discussed below ; it is sufficient to point out here that many factors may determine the absolute number of sperm required for a high rate of fertilization. E. SITE OF INSEMINATION The location of the deposition of semen during ejaculation differs in various animals and may account, in part, for the variations recorded for time of transport through the female genital tract. Intravaginal insemina- tion predominates in the rabbit, dog, ewe, cow, and man, whereas intrauterine depo- sition occurs in the mouse, rat, sow, mare, and probably the hamster (Braden and Aus- tin, 1953; du Mesnil du Buisson and Dau- zier, 1955; Chang and Sheaffer, 1957). Experimental insemination has been at- tempted by a number of routes. Administra- tion of sperm into the ovarian bursa of re- ceptive mice proved highly satisfactory (Runner, 1947). Intraperitoneal insemina- tion has been accomplished in fowl (Van Drimmelen, 1945), guinea pigs (Rowlands, 1957), rabbits (Hadek, 1958b), and, with bare success, in the cow (Skjerven, 1955; McDonald and Sampson, 1957). In an ex- tensive animal breeding investigation, Salis- bury and VanDemark (1951) showed that artificial insemination was equally effective in cattle, as judged from the nonreturn rate, when semen was deposited in the vagina, the body of the uterus, or the uterine horns. F. .\RTIFICIAL INSEMIN.\TION Little more than a brief account of this special and applied subject seems appropri- ate at the moment. Excellent surveys of the development, techniques, and accomplish- ments of artificial insemination have ap- peared from time to time, two of the more 728 SPERM, OVA, AND PREGNANCY general being those of Anderson ( 1944) and Emmens and Blackshaw (1956). Legal and ethical aspects of artificial insemination in man have been dwelt upon extensively in the semiclinical literature (Haman, 1947; Nicolle, 1949; Guttmacher, Haman and MacLeod, 1950; Ellis, 1952; Pope Pius XII, 1957). According to various historic accounts, the Arabians have for centuries practiced, if not thoroughly understood, the art of arti- ficial insemination in the breeding of their horses.^ In more recent times Spallanzani developed a method for the artificial in- semination of amphibia and, in 1782, first successfully inseminated a dog. Shortly thereafter, the first successful insemination of a woman was recorded (Home, 1799). Now it is a common ])ractice, the world over, for the selective breeding of various species of mammals (Walton, 1958). The techniques have also been applied, both for academic and for practical aims, to other types of animals, including fowl (Quinn and Burrows, 1936; Van Drinnnolen, 1945), vi- viparous fish (Clark, 1950), and insects (Laidlaw and Eckert, 1950; Lee, 1950). Constant efforts are being made to im- prove the dilution and storage media of sperm for routine use in artificial insemina- tion (see Salisbury, 1957). At present, 5- or 6-day survival of bull semen, diluted with egg yolk-sodium citrate and stored in the presence of antibiotics at 2 to 3°C., is about all that can be expected. Most types of se- men lose their fertilizing capacity much sooner than this. It is a curious fact that fowl sperm, which survive so well (2 or more ^ Walter Heape ( 1898) recounted an interesting tale which probably has some basis in fact : "It is taken from a book written in the year 700 of the Hejira, and therein is described how an Arab of Darfour, the owner of a valuable mare on 'heat,' armed with a handful of cotton wool which had been saturated with the discharge from the vagina of his mare, approached by stealth a valuable stal- lion belonging to a member of a neighbouring hos- tile tribe, a stallion whose services for his favourite mare the owner was desperately anxious to obtain ; and having sufficiently excited the animal with the scent of the material he had brought, he obtained spermatic fluid from him on the same handful of cotton, and hastening back to his mare, which he had been obliged to leave some distance away, pushed the whole into her \agina, and obtained by that means a foal." weeks) in the female genital tract, cannot be iireserved in vitro more than a few hours without decline in fertilizing capacity (Gar- ren and Shaffner, 1952; Carter, McCartney, Chamberlin and Wyne, 1957) . The most significant advance — certainly the most striking — in the field of sperm preservation during the past decade is the remarkable success in maintaining cells in a viable condition at extremely low tempera- ture. The very early history began with Mantegazza's (1866) and Davenport's ( 1897) successful demonstrations that deep- frozen ( — 17°C.) human sperm could regain motility. Despite other attempts to improve the degree of recovery by the addition of various substrates and by control of tem- perature changes, a marked measure of suc- cess was to await the discovery of Polge, Smith and Parkes (1949), who showed that eciuilibration of the semen with glycerol be- fore freezing greatly enhances sperm recov- ery and motility after warming to room temperature. This work, on rooster and hu- man spermatozoa, catalyzed many investi- gations of the problem M'ith the result that today there are few common mammals whose sperm have not been vitrified, stored at —79° or — 196°C., and warmed up for observation of motility or used in breeding experiments (Emmens and Blackshaw, 1956; Polge, 1957; VanDemark, Miller, Kinney, Rodriguez and Friedman, 1957; Martin and Emmens, 1958). The presence of glycerol is essential, in concentrations be- tween 10 and 15 per cent, for bull sperm, to 20 per cent for those of fowl (Martin and Emmens, 1958). Bull spermatozoa have been stored successfully in this fashion for periods up to 6 years (Walton, 1958). Artificial insemination with previously deep-frozen, thawed sperm has resulted in conception and viable young in a number of animals. The degree of fertility varies, be- ing low in the rabbit and as high as in nor- mal matings in the bull (Emmens and Blackshaw, 1956). Pregnancies have been reported for several women inseminated with spermatozoa treated in this manner (Bunge and Sherman, 1954). The advantages of the perfection of the low-tempcrature method for the preserva- tion of animal sperm are obvious. In the case of bull semen, for example, the procedure BIOLOGY OF SPERMATOZOA 729 permits long-term storage and, in the long i-un. greater use of the sperm. An additional advantage is that the storage intervals al- low for i)rogeny testing, a procedure which takes time and is of considerable importance in identifying the breeding value. As applied to man, on the other hand, the method would seem to have only limited usefulness in ex- (■e|)tional instances. One might suppose, for example, that successive ejaculates of an ()lig()s|)erniic individual could be stored and pooled in this fasliion and give, upon insemi- nation, a sufficiently high sperm count to insure fertilization. The advantage of trans- portability of frozen semen, i)ractical in ani- mal husl)andry, would not be expected to play a significant role in matters concerning human fertility. 'The changes which may occur in cells during storage at such low temperatures, or during the freezing or thawing process, can only be surmised. Based on the resumption of motility at room temperature and ferti- lizing cajjacity, the alterations in bull sper- matozoa must be minor. In other kinds of spermatozoa, those of the rabbit for exam- ple, metabolic and permeability changes may l)e more pronounced. Subtle changes, sucli as might be induced in the cytogenetic api)aratus, are unknown; there is the ques- tion of whether they have been sought. The mechanism of the protective action of glycerol in maintaining the spermatozoa during the relatively slow freezing process and while in storage is obscure. The effect is probably not merely one of the prevention I if ice crystal formation, but rather one which involves the stability of the internal ionic concentration of the cell. One can sup- pose that without glycerol, the withdraw^al of fi-ee water would result in severe changes possibly involving an increase in ionic strength, alteration in jiH, the production of toxic concentrations of such substances as urea and dissolved gases, and an actual liliysical I'eorganization of intracellular components (Lovelock, 1957). One sugges- tion is that the elements sensitive to deep fieezing are lii)oprotein complexes which, in the ])resence of glycerol, aic pic\-ented fi'oni • lenaturation (Lovelock, 1907). Although deep freezing and cold storage of sperm are currently receiving the great- est attention, othei- methods of controlling metabolism and motility are being consid- ered and may ultimately prove useful in the preservation of sperm for artificial insemi- nation. Such metabolic blocking agents as tetrazolium compounds (Bishop and Math- ews, 1952b) and carbon dioxide (Salisbury and VanDemark, 1957; VanDemark and Sharma, 1957; du Mesnil du Buisson and Dauzier, 1958) can reversibly inhibit the processes involved in the utilization of sub- strate and the expenditure of energy. An- other api)roach has recently been suggested by the work of Petersen and Nordlund ( 1958) whose })reliminary experiments in- dicate that bull sperm can be subjected to 150 atmospheres of pressure, in nitrogen, and sur\-i\-e such treatment for two weeks, after which motility is regained. Whether such a procedure destroys fertilizing capac- ity has not yet been ascertained. V. Sperm Transport and Survival in the Female Tract The vigorous motility of seminal sper- matozoa has long been a source of fascina- tion and naturally gave strong support to early suppositions that migration in the fe- male tract is due to the activity of the cells themselves. This is now known not to be generally true, and only in certain limited segments does active sperm motility seem of possible importance in transport from the vagina to the site of fertilization in the ovi- duct. Suggestions have been made, in fact, that sperm motility may be unnecessary even for egg penetration (Allen and Grigg, 1957) , but such has never been demonstrated in studies of fertilization of either inverte- brate or vertebrate gametes. The over-all transport system for mam- malian spermatozoa is principally provided by muscular contractions of the walls of the tract, with a questionable role played by ciliary activity of the mucosa; under some circumstances, however, active flagellation of the gametes themseh^es is important (cf. Hartman. 1939). A. DUR.\TION OF TRANSPORT The most striking evidence that sperm mi- gration in the female tract cannot be attrib- uted solely to sperm motility is afforded by the results of studies of the rate of transport and the time required to pass from the point 730 SPERM, OVA, AND PREGNANCY of insemination to the site of fertilization or to intermediate levels of the reproductive system. Thus, for example, the mean velocity of bull sperm is on the order of 100 /x per sec. (Moeller and VanDemark, 1955; Gray, 1958 ) , and if a straight path were followed, it would require about IV2 hours for the gametes to cover the entire length of the tract; actually the time required after nat- ural mating is less than 2V2 minutes (Van- Demark and Moeller, 1951). Rapid sperm migration through the uterus was first demonstrated by Hartman and Ball (1930) in the rat; within 2 minutes after copulation myriads of sperm had entered the tubes (Table 13.6 ) . A subsequent investi- gation showed that a few sperm were present at the periovarial sac within IVk minutes after copulation (Warren, 1938). Blandau and Money (1944) later indicated that at copulation, rat sperm are catapulted through the cervix into the uterine cornua, and within 15 minutes have entered the Falloi^an tubes in considerable numbers. By clamping the middle of the tubes at various times after copulation, the distribution of sperm could be determined. After 15 minutes, sperm were found in 42 per cent of the uterine (lower) segments of the oviducts examined and 21 per cent of the ovarian (upper) segments; after 30 minutes, 85 per cent and 62 per cent, respectively; after 45 minutes, 90 per cent and 96 per cent; and at 60 minutes, both the uterine and ovarian portions of oviducts of all animals studied contained sperm. After insemination of the mouse, sperm reach the tubal infundibulum, the site of fertilization, within 15 minutes (Lewis and Wright, 1935). In the bitch, 20 minutes or TABLE 13.6 Time of passage of rat spermatozoa into female genital tract (From C. G. Hartman and J. Ball, Proc. Soc. Exper. Biol. & Med., 28, 312-314, 1930.) Animal Killed after Ejaculation Uterus Clamped near Apex after Ejac- ulation Sperm Located 1 2 3 4 1 min. 1 min. 30 sec. "Immediately" 2 min. 100 sec. 54 sec. 54 sec. Apex of uterine cornua Apex of cornua Lower part of cornua Vagina less are required (Evans, 1933; Whitney, 1937), and in the hamster about 30 minutes (Chang and Sheaffer, 1957). Rubenstein, Strauss, Lazarus and Hankin (1951) claimed that human sperm deposited at the cervix just before hysterectomy, can be recovered from the Fallopian tube 30 minutes later ; the nature of the operation, however, might se- riously affect the rate of transport. Other re- ports of sperm-transport time in women range up to 3 hours (Chang and Pincus, 19511. As part of a series of marvelously planned and executed experiments on cattle, Van- Demark and co-workers have shown that sperm migration requires only 2 to 4 minutes whether the heifers are mated or artificially inseminated. Indeed, even dead sperm, after artificial insemination, were transported to the uj^pcr reaches of the oviduct within 4.3 minutes (VanDemark and Moeller, 1951). Sperm-migration times reported for the ewe have varied considerably, ranging from sev- eral hours (Green and Winters, 1935) down to 6 to 16 minutes (Starke, 1949; Schott and Phillips, 1941). This variation is to be ac- counted for less by changes dependent on the estrous cycle (Dauzier and Wintenberger, 1952) than by improvements in technique. The rabbit, in many ways a domestic anomaly in reproductive matters, appar- ently requii-es several hours for transport of a significant number of sperm, although the "vanguard" may reach the ampulla within an hour after insemination (Chang, 1952; Adams, 1956). Heape's demonstration in 1905 of approximately 4 hours for migration seems to have stood the test of time. On the basis of recovery of spermatozoa from sepa- rate segments of the genital tract, Parker (1931) andBraden (1953) found that 2.5 to 3 hours are required for transport. Confirma- tion is afforded by experiments involving ligation of the tubes at various intervals after copulation (Adams, 1956; Gr,'enwald, 1956) ; whereas some eggs are fertilized when the tubal blocks are made prior to 2.5 hours, ligations made 3 to 5 hours after copulation do not prevent a high percentage of fertility. Whether this order of transport time is an adaptation to induced ovulation or is other- wise unique to the rabbit is not known ; com- parable experimentation on the cat and fer- BIOLOGY OF SPERMATOZOA 731 ret, for example, which also normally ovulate only when stimulated by copulation, might be instructive. The time for sperm migration in fowl is of the same order of magnitude as that in most mammals (Mimura, 1941). Fowl sperm, labeled with inorganic P-^-, were recovered from the infundibulum within an hour after insemination; the number found depended on the site of administration, i.e., intravagi- nal or intrauterine (Allen and Grigg, 1957). Killed sperm also reached the infundibulum when placed in the uterus, but not when in- troduced intravaginally. The study of seminal components, other than sperm, indicates that tubal transport must involve a muscular mechanism. In both the sow and the mare, certain natural semi- nal constituents, e.g., fructose, citric acid, and crgothioneine, are found in the uterine horns within an hour after mating (Mann, Polge and Rowson, 1955). Gunn and Gould ( 1958 ) produced a Zn*'^-labeled component of prostatic fluid in rats which served as a marker for tubal transport. In animals killed at intervals between 0.5 and 1.5 hours after mating, a significant quantity of the isotope had reached the uterotubal junction by 1 hour, and radioactive labeling was found throughout tlie oviduct at 1.5 hours. B. MECHANISM OF TRANSPORT IN THE UTERUS AND OVIDUCT The muscular contractility of the genital tract has been implicated in the process of sperm migration since the earliest studies of mating behavior and insemination (see Austin and Bishop, 1957). The normal ac- tivity of the uterus and Fallopian tube is well known (Westman, 1926; Parker, 1931; Reynolds, 1931, 1949). The contractions of the tract are not, however, peristaltic waves which might favor rapid, directed sperm transport, but rather segmentation waves which encourage dispersal from the source. Indeed, what peristalsis can be observed in the estrous oviduct (e.g., the rabbit) is di- rected from the fimbriated toward the uter- ine end (Reynolds, 1949j. Both mechanical and psychic factors in- fluence the contractility of the genital tract and api^ear to augment sperm migration. In the ral)bit (Heape, 1898; Krehbiel and Car- stens, 1939), and probably in many other animals, stimulation of the external geni- talia increases uterine activity. The mating response also enhances uterine action in the mare (Millar, 1952) and cow (VanDemark and Hays, 1952). According to VanDemark and Hays (1952) , the mere sight of the bull is sufficient to induce strong uterine contrac- tions in the estrous and postestrous heifer (Fig. 13.7). The activity of the Fallopian tube of the rabbit also appears to be stimu- lated by the presence of a suitable buck (Westman, 1926). In the oviducts of rabbits, Parker (1931) emphasized both the segmentation contrac- tions and the local ab- and adovarian ciliary currents in accounting for dispersal of sperm, once they pass the uterotubal junction. In a recent series of interesting experiments, how- ever. Black and Asdell (1958) tended to minimize ciliary activity, which is generally directed toward the uterus, and to attribute sperm distribution in the rabbit oviduct to the segmentation process brought about by the circular musculature of the tube. Tubal MINUTES Fig. 13.7. Uterine responses in an estrous cow stimulated by various mating activities: A, bull brought within sight of cow ; B, bull allowed to nuzzle vulva ; C, bull mounts but does not copulate; D, bull copulates; E, bull ejaculates. (From N. L. VanDemark and R. L. Havs, Am. J. Physiol., 170, 518-521, 1952.) 732 SPERM, OVA, AND PREGNANCY secretions, pronounced at the time of ovula- tion (Bishop, 1956a), serve as vehicle of transport for the sperm. The copious uterine fluid secreted in the rat during tlie proestrum performs the same role (Warren, 1938). It is probable that ciliary activity plays a greater role in some animals than in others in distributing sperm throughout the female tract. Thus, Parker ( 1931 ) stressed the im- portance of adovarian ciliary currents in the oviducts of the turtle, pigeon, and chicken. With respect to a6ovarian currents, more- over, it should be pointed out that these, too, could serve a function by orienting the sperm toward the infundibulum ; whereas unneces- sary emphasis should not be placed on this as a transport mechanism, considerable evi- dence exists to show that sperm orient against a current and, when free-swimming, make considerable progress upstream (Adol- phi, 1906a, b; Yamane and Ito, 1932; von Khreninger-Guggenberger, 1933; Brown. 1944; Sturgis, 1947). The activity of the several segments of the female genital tract varies with phases of the ovarian cycle and, as a consequence, may alter the rate of sperm migration (see Austin and Bishop, 1957). The active motil- ity of both the Fallopian tube and uterus, characteristic of estrus, is depressed by pro- gestational conditions, although little change is found immediately after ovulation (Rey- nolds, 1949; Borell, Nilsson and Westman, 1957; Black and Asdell, 1958). Cyclic changes in sperm-transport time through the uterus and oviducts have been noted in the cow (Warbritton, McKenzie, Berliner and Andrews, 1937) and sow (du Mesnil du Buis- son and Dauzier, 1955 ) . Recent work of Noyes, Adams and Walton (1959) suggests that estrogen enhances fertilization of rab- bit ova transplanted into castrates by in- creasing the efficacy of sperm transport, i.e., by reducing the obstacles to sperm migra- tion present in nonestrous does (Noyes, 1959a). The most spectacular development involv- ing endocrine control of sperm transport during the past decade has been the demon- stration that oxytocin, as an important me- diator of uterine activity, is essential, in some cases at least, for the rapid migration of sperm from the cervix to the site of fer- tilization (VanDemark and Moeller, 1951; VanDemark and Hays, 1952; Hays and VanDemark, 1951, 1953a). Excised and per- fused cow uteri function as a transport sys- tem so long as oxytocin is present in the per- fusate. Motile sperm, artificially inseminated into the cervix, are carried to the ovarian end of the oviduct in as few as 2.5 minutes. Even nonmotile sperm are transported throughout the tract within 5 minutes. In the absence of oxytocin, however, sperm mi- gration does not occur; in fact, the cells do not even enter the fundus. Oxytocin is also apparently released during natural and ar- tifical insemination of the cow (Hays and VanDemark, 1951, 1953b). and its admin- istration, during mating, augments uter- ine contractility (Hays and VanDemark, 1953a ) . Oxytocin may have a general role in the uterine responses to mating and rapid transport of spermatozoa through the geni- tal tracts of some other animals as well (Harris, 1951; Cross, 1958), although it is to be noted that coitus is claimed to abolish temporarily uterine contractions in women (Bickers and Main, 1941). C. CRITICAL REGIONS OF SPERM TRANSPORT The unrestricted passage of sperm, which is apparently characteristic of the heifer, is not, however, exhibited by all mammals. The cervix, the uterotubal junction, and, to a lesser degree, the isthmus of the Fallopian tube can each constitute an obstacle to free sperm transport. In these regions, active sperm motility may then assume some sig- nificance as a means of migration. In the rabbit only 1 sperm in about 50,000 reaches the site of fertilization; in the ewe and rat, the proportion is even smaller (Braden, 1953). According to Braden, of the total number of sperm deposited in the rabbit vagina during a normal insemination (about 60 X 10" cells), the proportions transmitted are roughly as follows: approximately 1 out of 40 traverses the cervix ; of these, one-third reach the uterotubal junction; 1 out of 160 passes the uterotubal junction and enters the Fallopian tube; and of these, one-fourth ultimately reach the ampulla. The distribu- tion of spermatozoa throughout the rabbit genital tract at various times after copula- tion is presented in Figure 13.8. BIOLOGY OF SPERMATOZOA 733 10 14 18 22 HOURS AFTER MATING Fig 13 8 Changes in sperm number in various sections of the genital tract of rabbit after copulation. (From A. W. H. Braden, Australian J. Biol. Sc, 6, 693-705, 1953.) 734 SPERM, OVA, AND PREGNANCY 1. The Cervix This portal connecting the vagina and the uterus is generally regarded as constitut- ing a partial block to sperm transport in cer- tain animals in which ejaculation occurs in the vaginal vault, e.g., the rabbit, ewe, and man (Warbritton, McKenzie, Berliner and Andrews, 1937; Chang, 1951b; Braden, 1953; Noyes, Adams and Walton, 1958). Sperm migration through the rabbit cervix is a gradual process (Florey and Walton, 1932; Braden, 1953) and, although the mechanism certainly is not definitely known, is possibly to be attributed to active flagellation of the sperm themselves, with little or no help from the cervical duct (Noyes, Adams and Wal- ton, 1958; cf. Hartman, 1957). Dead cells, according to Noyes and colleagues, fail to negotiate the cervical passage, as do radi- opacjue media. It should be noted that the latter finding is inexplicably at variance with a similar experiment of Krehbiel and Car- stens (1939), who found that radiopaque medium does pass the rabbit cervix in sig- nificant amounts. Noyes, Adams and Wal- ton (1959) also indicated that estrogen treatment facilitates cervical transport of spermatozoa, that is, decreases the resistance to migration shown by untreated animals, in this case castrated does. Whether the effect is actually on the cervical musculature, the secretion of cervical mucus, uterine motility, or some other system is not clear from these experiments. The cervix should not be regarded as al- ways constituting an obstacle to sperm trans- port. In at least two species with intravagi- nal insemination, namely, the heifer and dog, sperm transport is extremely rapid. The cervices in these animals, therefore, rather than retarding progress, must aid consider- ably in the migration of spermatozoa. A frequently suggested theory to account for the passage of spermatozoa into the uterus envisages "insuck" of the semen through the cervix. Indeed, a transient nega- tive uterine pressure of about 0.7 lb. per square inch has been demonstrated during coitus in the mare (Millar, 1952). However, the significance of such determinations in this animal is obscure since ejaculation nor- mally occurs directly into the uterus (Braden and Austin, 1953) . Nevertheless, in consider- ation of the concept relevant to women, it is reasonable to assume that the uterus can aspirate sperm and mucus into the uterine cavity by virtue of the elasticity of that organ following contraction (Belonoschkin, 1949, 1957) . This subject is more extensively reviewed by Hartman (1957). Much attention has been focused on the questions of the nature of cervical mucus, its cyclic changes, and its penetrability by sper- matozoa in vitro (Shettles, 1949). The im- portance of cervical mucus is obvious if sperm reside in the cervix for considerable periods of time, as in women, or if the sperm have to negotiate the canal by their own motile faculties; it is of much less signifi- cance when the sperm are shot through the cervical canal at ejaculation, as in the sow, or are carried through rapidly by muscular contractions, as in the heifer. The secretory activity of the cervix re- sponds to variations in the ovarian cycle, and the physicochemical composition of the mucus changes accordingly. The response of the cervix to cyclic changes was first clearly stated by Allen (1922) for the mouse. Much of our current understanding stems from the important monograph of Sjovall (1938) concerning investigations of human and guinea pig cervices. There now exists ade- quate evidence that changes in human cer- vical mucus correlate well with ovulatory and with endometrial, vaginal, and other indications of estrogenic activity (Sjovall, 1938; Vicrgiver and Pommerenke, 1946; Shettles, 1949; Bergman, 1950; Cohen, Stein, and Kaye, 1952; Odeblad, 1959). Cervical mucus in women is most copiously secreted during the estrogenic phase; its dry weight at this time is minimal (Bergman, 1950), tonicity is low (Bergman and Lund, 1950 », and pH, as generally determined, is elevated. Estrogenic mucus is also claimed to be richer in glucose and polysaccharide, but these components may be derived less from the cervical secretion than from the uterine glands higher in the tract (Bergman and Werner, 1950). Lipid is present in lower amounts at ovulation. Benas (1958) found changes, as determined by paper electro- phoresis, in the extractable protein, with a predominance of albumin in pre-ovulatory mucus and a prevalence of /?- and y-globulins BIOLOGY OF SPERMATOZOA 735 in iiiucus collected after ovulation. No cy- clical changes, however, were found by Bergman and Werner (1950) in carbohy- drate hydrolysates of cervical mucus, which, when tested chromatographically, showed the presence of galactose, mannose, fucose, and hexosamine. A recent investigation of cervical mucus from the cow (Gibbons, 1959a) has demon- strated the presence of glucose, glycogen, protein, alkaline phosphatase, lysozyme, an- tagglutin, and common inorganic ions. Iso- lation and relative purification of mucoid, prepared from bovine mucin, show that it changes in physical consistency with phases of the cycle; molecular configuration, as de- termined by sedimentation, viscosity, and flow-birefringence measurements, is altered and is probably due to changes in state of liydration (Gibbons and Glover, 19591. Chemically, bovine mucoid consists of about 75 per cent carbohydrate and 25 per cent amino acid residues and resembles human blood-group substances (Glover, 1959b). The presence of glucose and hydrolyzable l)olysaccharide in cervical mucus suggests the availability of metabolic substrate for the spermatozoa, but the utilization of these energy sources can only be conjectured. Moricard, Gothie and Belaisch (1957) have indicated that inorganic S^^ is apparently taken up by human sperm from cervical mucus, but the significance of this uptake cannot at present be evaluated. Many investigators have attempted to correlate cyclical changes in cervical mucus with capacity for sperm progression, in vitro (Fig. 13.9). Maximal penetration by human sperm, observed in capillary tubes, occurs in estrogenic cervical secretion when the mucus is most copious and least viscous (Lamar, Shettles and Delfs, 1940; Gutt- macher and Shettles, 1940; Shettles, 1940; Pommerenke, 1946; Leeb and Ploberger, 1959). Very little or no penetration is ob- served in pre-ovulatory or postovulatory mucus. Just before menstruation penetra- l)ility sharply increases, a change probably correlated with the premenstrual rise in circulating estrogen. During pregnancy, cer- vical mucus is only slightly penetrable, although the endocervical glands are hyper- active at this time (Guttmacher and Shet- tles, 1940; Atkinson, Shettles and Engle, 1948). Postmenopausal mucus is relatively impenetrable by spermatozoa, but after ade- quate estrogenic administration, a mucus is secreted which is characteristic of that of the ovulatory phase. Ovariectomized women or- dinarily produce a scant, viscous mucus which is increased upon estrogen administra- tion (Moricard, 1936; Abarbanel, 1946, 1948; Pommerenke and Viergiver, 1946). It has been claimed (Gary, 1943), although not confirmed, that mucous secretion in women is enhanced by orgasm and that this facili- tates sperm penetration. These studies on sperm, in vitro, have in a general way largely confirmed the earlier work of Sjovall ( 1938 ) , whose investigations of sperm penetration through the guinea pig cervix were mainly confined to observations in vivo. The penetration of sperm through the cervical mucus, in vitro, however, is at best only an approximation to the normal process of insemination and cervical trans- port, and the meaning of these carefully compiled results is not easy to assess. Their full significance must await further correla- tion between sperm migration in vitro and transit in situ. Certain evidence, indeed, tends to suggest that the condition of the cervical mucus in women may be of rela- tively little importance in sperm transport. In the series of 51 women studied by Ruben- stein, Strauss, Lazarus and Hankin (1951), spermatozoa were found to have passed rap- idly through the cervix at all stages of the cycle. No particulars were given concerning the condition of the cervical mucus, the pre- surgical coital history of the patients, or the possible effect of the operation (hysterec- tomy) on sperm transport. Their re])ort, however, seems to conflict with many of the above-cited observations in vitro which in- dicate that sperm migration is limited to the ovulatory phase of the cycle. 2. The Uterotubal Junction The speed of sperm transport through the upper genital tract is in general so rapid that in only two species, the rat and rabbit, is the junction between the uterus and the oviduct stressed in current literature as be- ing an obstacle to sperm migration (Braden, 1953). Yet, for almost half a century, de- 736 SPERM, OVA, AND PREGNANCY mm/mm. 2.0 1.5 1.0 0.5 mg. 500 400 300 200 7 14 12 10 8 37.1 37.0 36.9 36.8 36.7 SPERM PENETRATION DRY CONTENT OF MUCUS BASAL TEMPERATURE 98.8 98.6 98.4 98.2 98.0 12 16 DAY OF CYCLE 20 24 28 Fig. 13.9. Variation in human sperm penetration in vitro through cervical mucus during a single cycle (from J. K. Lamar, in Problems oj Human Fertility, George Banta Publishing Company, 1943), correlated with cyclical changes in the mucus and in body temperature based on 35 cycles (from P. Bergman, Acta obst. et gjmec. scandinav., Suppl. 4, 29, 1-139, 1950). scriptive and experimental investigations have pointed out the complexity of the junc- tion and the high pressures often required to force an opening through the lumen in this region. Rubin's initial paper (1920) indi- cated that gas pressures of 40 to 100 mm. Hg could be considered a normal range for hu- man tubal insufflation, the uterotubal junc- tion being the major source of resistance. In the cat, fluid pressures of 250 to 300 mm. Hg are incapable of "forcing" the opening when injections are made through the uterus (Lee, 1925a; Anderson, 1928). On the other hand, tubo-uterine injections of fluid, that is, those from tube to uterus, recjuire very little pres- sure to force the opening. Other species be- have differently. With relatively little pres- sure, between 25 and 40 mm. Hg, fluid can BIOLOGY OF SPERMATOZOA 737 l)e forced through the junction from the uterine to the ovarian side in both the cow and ewe (Anderson, 1928). The resistance to flow, in the cow at least, is greatest during estrus (Anderson, 1927; Whitelaw, 1933). During this early and important period of investigation, the structural aspects of the uterotubal junction of a wide variety of mammals were described, particularly the villi and folds which ajipear to guard the opening of the Fallopian tubes (Lee, 1925b; Anderson, 1928). Anderson's paper should be consulted for details of the comparative structure of the junction in 25 species of mammals and for her particularly thorough discussion of this region in the sow. A general conclusion which arises from these considerations of the uterotubal junc- tion is that the structure is sufficiently com- plex (Fig. 13.10) to render spurious many attempts to correlate forced-fluid determina- tions with sperm transport. It seems likely that in a case like the cat, for example, the fluid pressure applied would occlude the uterotubal orifices with villi or folds, and that the greater the pressure, the tighter the seal; under normal conditions the junction would remain more or less patent, at least between muscular contractions, and allow for sperm transport. That migration through the uterotubal junction in the rat, under some circum- stances, is probably accomplished by the gametes themselves was indicated by the ingenious investigation of Leonard and Perl- man ( 1949). They injected live spermatozoa of one or more species, as well as dead sperm and India ink particles, into the rat uterus. Spermatozoa of the rat, mouse, guinea pig, and bull were injected singly, and combina- tions of rat-guinea pig, rat-mouse, and rat- bull sperm were introduced together. Dis- tribution throughout the reproductive tract was determined 1 to 14 hours later. Under these conditions icf. Table 13.6) motile rat si)ermatozoa freely penetrated the utero- tubal junction in both estrous and diestrous animals, but dead spermatozoa and inert particles did not; foreign spermatozoa passed through only very rarely. A similar experiment on the rabbit, which also shows evidence of uterotubal blockade, should pro^•e rewarding. Fig. 13.10. Uterotubal junction of the rabbit. (From D. H. Anderson, Am. J. Anat., 42, 255-305, 1928.) 3. The Isthmus The lower segment of the oviduct consti- tutes a partial obstacle to sperm migration in both the rat and rabbit (Chang, 1951b; Braden, 1953). In the latter, transport of both sperm and eggs is slowed by a decrease in muscular activity, in contrast to the move- ments characteristic of the upper segment of the duct (Black and Asdell, 1958) . The small diameter of the lumen of the isthmus, along with its kinks and extensive mucosal fold- ing, may also retard sperm transport. In a recent extensive study to ascertain the source of the fluctuations in gas pres- sures during tubal insufflation of the rabbit, Stavorski and Hartman (1958) demon- strated that the isthmus is more important than the actual uterotubal union in the de- gree of resistance offered to applied pressure. Sphincters were observed at both the utero- tubal and tubo-ampullar junctions, but the elbow-like kinks in the isthmus were found to be the major source of resistance. The pressures necessary to force an opening were of the same order of magnitude whether a uterotubal or a tubo-uterine approach was employed. A suddenly applied high jiressure 738 SPERM, OVA, AND PREGNANCY was found to meet with great resistance ; the more slowly the pressure was built up, the lower the peak pressure required to open the isthmian and uterotubal constrictions. The reciuired pressures generally were higher in those animals receiving estrogen. D. NUMBER OF SPERM AT THE SITE OF FERTILIZATION In the few species subjected to careful in- vestigation, the number of spermatozoa re- covered from the ampulla, or what is re- garded as the site of fertilization, at the approximate time of fertilization, is sur- prisingly low. A summary of available evi- dence is included in Table 13.7. Whereas these data represent, in some instances, only single determinations and, in others, mean values within a very wide range, they show quite clearly that only a minute fraction of the inseminate is present in the vicinity of the ova when fertilization occurs. In some of these studies (Moricard and Bossu, 1951; Blandau and Odor, 1949), search failed to reveal many more sperm than the number of eggs undergoing fertilization. The presence of so few sperm at this critical point is evi- dence enough against the once-popular view that a sperm "swarm" is essential for ferti- lization — either to denude the ova of their TABLE 13.7 Number of spermatozoa found at the site of fertilization in several mammals TABLE 13.8 Sperm survival times in, the female tract Species Mean No. Sperm Tube Post- coital Time Reference hr. Rat 43 ? Austin, 1948 12 12 Blandau and Odor, 1949 30 24 45 12 Braden and Austin, 1954; Moricard and Bossu, 1951 Mouse 17 10-15 Braden and Austin, 1954 Rabbit 500 ? Chang, 1951a 38 4 Braden, 1953 250 10 Ferret 200 6 Hammond and Walton, 1934 500 24 Sheep 184* 24-48 Braden and Austin, 1954 673 1 24-48 Maximal Maximal Animal Duration Duration Reference Fertility Motility hr. hr. Rabbit 30-32 _ Hammond and Asdell, 1926 Mouse. 6 13 Merton, 1939b Guinea Yochem, 1929; Soderwall an.l pig 21-22 41 Young, 1940 Rat 14 17 Soderwall and Blandau, 1941 Ferret 36-48 — Hammond and Walton, 1934 Sheep 30-48 48 Green, 1947; Dauzier and Win- tenberger, 1952 Cow . 28-50 — Laing, 1945; Vandeplassehe and Paredis, 1948 Horse. 144 144 Day, 1942; Burkhardt, 1949 Man 28-48 48-60 Farris, 1950; Rubenstein et al., 1951; Home and Audet, 1958 Bat 135 days 159 days Wimsatt, 1944 * Ovarian third of oviduct. t Entire ampulla. cumulous auras by hyaluronidase or to sup- ply some ingredient for sperm penetration. Conversely, Braden and Austin (1954) have suggested that an accomplishment of the fil- tering out of the overwhelming majority of sperm during transport is to so limit the number of male gametes present that mul- tiple sperm penetration of the ova is reduced, thereby preventing polyspermy and anoma- lous development. E. DUR.\TIO\ OF FERTILIZING CAPACITY The retention of fertilizing capacity by mammalian spermatozoa is relatively lim- ited (Table 13.8). As in the male tract, the capacity for fertilization is lost more promptly than is their ability to move. In the female guinea pig, for example, motility of sperm continues for as long as 40 hours after mating, whereas fertilizing capacity is lost about 22 hours after copulation (Yo- chem, 1929; Soderw^all and Young, 1940); in the mouse these periods are approximately I3Y2 and 6 hours, respectively (Merton, 19391)). In the consideration of sperm sur- vival in parts of the tract other than the fer- tilization site, sperm motility is the most convenient, although not necessarily the only, criterion of longevity. The values presented in Table 13.8 are the most accurate available, but the degree of precision with which such data can be ob- BIOLOGY OF SPERMATOZOA 739 tained varies considerably among species, dependent as they are upon the estimates of the time of fertilization. Reliable figures may l)e expected in such forms as the guinea pig, which is known to ovulate some 10 hours after the onset of heat, or the rabbit which ovulates about 10 hours after copulation. But in women the exact time of ovulation cannot be determined with sufficient accu- racy to permit a precise statement as to the duration of fertilizing capacity of the sper- matozoa. The relatively long survival time reported for the mare may reflect a kind of thermal adaptation of the spermatozoa, be- cause in the stallion the testicles are carried in shallow scrotal sacs, the temperature of which is probably close to that of the body. Hil)ernating mammals which copulate in the autumn often show excessively long pe- I'iods of sperm survival in the female (Hart- man, 1933). In bats of the genera Myotis and Eptcsicus, the spermatozoa inseminated in the fall are capable of motility and of fer- tilization at the time of ovulation in the spring (Wimsatt, 1942, 1944), even though subseciuent copulations may occur in nature during the spring mating season (Pearson, Koford and Pearson, 1952). Long-range sperm survival is, of course, well known in various poikilothermic animals, including arthropods and lower chordates (see Hart- man, 1939). Custodians of reptiles, particu- larly, have recorded interesting breeding data relevant to the longevity of sperm in the female. Fertile eggs have been laid by the diamond-back terrapin and various snakes, 4 to 5 years after isolation; due to the unlikelihood of delayed development, this indicates sperm survival for periods of several years (Barney, 1922; Haines, 1940; Carson, 1945). Some attention has been directed toward the possible deleterious effect of the aging of sperm in the female tract ; although still capable of fertilization, they might give rise to abnormal or nonviable embryos (Austin and Bishop, 1957). This change with senes- cence has been well established in fowl (Crew, 1926; Nalbandov and Card, 1943; Van Drimmelen and Oettle, 1949; Dhar- marajan, 1950), and might be expected to occur in mammals; the evidence, however, does not support it. Young's early data ( 1931 ) indicated that guinea pig sperm, aged in the male tract, could lead to an increase in the percentage of abnormal embryos ; but no such "overaging" effect was demonstrated in sperm maintained in the female tract (Soderwall and Young, 1940; Soderwall and Blandau, 1941). Somewhat more recently, another type of sperm behavior was discovered which in- volves the capacity for fertilization (Austin, 1951; Chang, 1951b). This concerns not the maximal limit of survival, but rather the ini- tial attainment of full fertilizing compe- tency, a continuation, in a sense, of the proc- ess of sperm maturation long since begun in the male genital tract. This phenomenon of "capacitation," demonstrated thus far only in rats and rabbits, requires 2 to 6 hours of conditioning of the male gametes and prob- ably involves both physiologic and struc- tural alterations in the cells which enable them to penetrate the zonae pellucidae of the eggs (Austin, 1952; Austin and Braden, 1954; Chang, 1955, 1959). Capacitation is assumed to occur normally in the female genital tract. Under experimental conditions, the injection of rat sperm into the periovar- ian sac (Austin), or the introduction of rab- bit sperm into the Falloi)ian tube (Chang), accomplishes fertilization only after a de- lay of several hours, unless the sperm have been previously capacitated in another suit- able reproductive environment. Such a mi- lieu for rabbit sperm is afforded by the re- productive ducts of female rabbits under a variety of hormonal conditions, and by the reproductive tracts of both immature ani- mals and castrates, with or without the addi- tion of gonadotrophin or estrogen (Chang, 1958). The uteri of pseudopregnant rabbits, however, and those treated with progester- one, were found unsuitable for sperm capaci- tation. Some doubt has been cast upon the s]iecificity of the factors which bring about sperm conditioning by the demonstration, in the rabbit, that not only does capacitation occur in the uterus and Fallopian tube, but also in such unusual environments as the isolated bladder and colon of either male or female animals and in the anterior chamber of the eye as well (Noyes, Walton and Adams, 1958a, b; Noyes, 1959b). As to the nature of the changes induced in 740 SPERM, OVA, AND PREGNANCY the spermatozoa during capacitation, ear- lier suppositions leaned toward the view that something is lost or gained by the gametes which results in enzyme activation recjuired for fertilization (Austin and Bishop, 1957). It has since been suggested that the change, in rat sperm at least, involves processes lead- ing to the disintegration or loss of the acro- some from the sperm head, thereby exposing structures responsible for egg penetration (Austin and Bishop, 1958a, b). The revers- ible counteraction of capacitation by rabbit seminal plasma, demonstrated by Chang (1957), casts some doubt, however, on the likelihood of pronounced structural changes occurring during this phase of si:)erm matu- ration. Until the physiologic changes respon- sible for the suggested morphologic altera- tions are clarified, the mechanism of capacitation will remain obscure. F. DUR.\TION OF SPERM MOTILITY THROUGHOUT THE TRACT The viability of spermatozoa in the am- pulla, assessed by motility, outlasts their fertilizing capacity (Table 13.8). Elsewhere in the tract, motility serves as a criterion for sperm longevity, and considerable variation in the ability of separate segments of the tract to support it has been demonstrated. Rat spermatozoa, for example, survive in the cornua about 12 hours, compared with 16 or 17 hours in the oviducts (White, 1933a) . Sperm motility in the human fundus appears to be less than that in the Fallopian tube (Farris, 1950; Rubenstein, Strauss, Lazarus and Hankin, 1951). In most mammals the alkaline cervical mucus sustains motility well, whereas the acidic vaginal depository is detrimental. Mo- tile spermatozoa have been reported in hu- man cervical mucus a week after coitus, al- though the average duration of motility here is closer to 2 days. The duration of motility in cervical mucus varies with the cycle, maximal motility coinciding with the time of ovulation (Beshlebnov, 1938; Cohen and Stein, 1951). Estrogen-induced hyper- secretion of mucus is claimed to increase sperm viability as well as penetrability. Longevity in the cervical mucus of the es- trous macaque is approximately 24 hours. The primate vagina is notably inhospita- ble to spermatozoa, presumably because of its high acidity. Motility is sustained in the human vagina rarely longer than 3 to 4 hours (Weisman, 1939), and the duration is believed to vary inversely wdth changes in vaginal acidity (pH 4 to 5). The human vaginal pH, curiously enough, has been claimed to reach a minimum at the time of ovulation, an overt sign, according to Schockaert, Delrue and Ferin (1939), of high estrogenic activity (Fig. 13.11 ). On the other hand, a sharp rise in vaginal pH of approximately 0.5 unit was claimed by Zuck and Duncan (1939) to be coincident with ovulation; this elevation is inconstant and, when it does occur, may be due to the pres- ence of alkaline cervical mucus. Normally, in the cow, the influx of mucus renders the vagina alkaline at estrus (Lardy, Pounden and Phillii)s, 1940). Under normal circum- stances, the inseminate is only briefly, if at all, exposed to the vaginal medium. When not ejaculated directly into the cervix or uterus, the semen may be conducted rap- idly toward the cervical canal by longi- tudinal contraction waves (Noyes, Adams and Walton, 1958). It is doubtful, therefore, whether the high hydrogen-ion concentra- tion, characteristic of the vagina, is of any great significance in the reproductive econ- omy of most mammals. G. SPERM VIABILITY IX RELATION TO TUBAL PHYSIOLOGY In view of the great wealth of information concerning uterine and tubal transport and sperm survival, on the one hand, and uterine function and hormonal responses, on the other, there has been an appalling lack of in- terest in the nature of the genital fluids and the immediate environment surrounding the spermatozoa during their sojourn within the female genital tract. The corresponding defi- ciency of our knowledge of the male genital tract was previously noted. Difficulties in technique exist, to be sure, but they are far from insurmountable, and rich rewards should result from exploration in this virgin, but obviously fertile, field. A review of the extensive literature on the cytochemistry of the endometrium and tubal epithelium and on the changes with varia- tions in the estrous cycle reveals consider- BIOLOGY OF SPERMATOZOA 741 14 7 DAYS BEFORE NEXT MENSES Fig. 13.11. Cyclical changes in midvagina 37 normally menstruating women. (After A, Obst. & Gynec, 47, 467-494, 1944.) I i)H. A\erage values of 632 determinations on E. Rakoff, L. G. Feo and L. Goldstein, Am. J. able secretory activity (Joel, 1940; Hadek, 19oo, IQoSa; Borell, Gustavson, Nilsson and Westman, 1959; Fredricsson, 1959a, b), but little correlation with the behavior of the gametes within the lumen. When the se- cretory history of specific substances has l)een followed, the interest has generally l)een in postfertilization stages, as, for ex- ample, the mucopolysaccharides released into the oviduct of the rabbit several days after ovulation (Greenwald, 1957; Zachariae, 1958). On the other hand, several studies of the genital fluids afford some data on pH, oxy- gen tension, potassium and sodium ratio, enzyme content, and possible metabolic sub- strates. Warbritton, McKenzie, Berliner and Andrews, (1937) reported that the pH levels of the Fallopian tube, uterine horns, cervix, and vagina of the ewe are, respectively, 6.4 to 7.3, 6.6 to 7.3, 6.1 to 7.5, and 6.5 to 7.8. The wide variations to be noted here are more striking than the actual determina- tions. More recently, Blandau, Jensen and Rumery (1958) recorded pH values for the fluid of rat periovarian sac, ampullae, and uteri as follows: 7.7. to 8.4, 7.3 to 8.5, and 7.4 to 8.3. There thus appeared little change throughout the tract, but all regions were alkaline with respect to the peritoneal fluid and blood. These wide variations and the pronounced alkalinity suggest that the loss of carbon dioxide from the fluids may have been responsible for the high pH values re- ported. The oxygen tension of rabbit genital fluids has been determined and found adequate to support aerobic respiration (Bishop, 1957). Uterine values, determined by equilibration, range from 25 to 45 mm. Hg (Campbell, 1932 ) . The oxygen tension of Fallopian tubal fluid, measured directly with an oxygen elec- trode, is approximately 40 mm. Hg (Bishop, 1956b). Birnberg and Gross (1958), how- ever, claimed that changes in the human Fallopian tube during the ovulatory phase render it anaerobic (determined enzymati- cally) ; if this finding is confirmed, it bears significantly on the anaerobic preferences of hiunan sperm as studied in vitro (see be- low ) . Ionic and organic components of the luminal fluids of the cow have been analyzed (Olds and VanDemark, 1957a. b, c; Van- Demark, 1958). The data for follicular, tubal, uterine, and vaginal fluids are pre- sented in Table 13.9. Reducing substances, possibly glucose, were found in uterine fluid but were not detected in oviductal fluid. Shih. Kennedy and Huggins (1940) iiave 742 SPERM, OVA, AND PREGNANCY TABLE 13.9 Composition of bovine genital fluids (From N. L. VanDemark, Internat. J. Fertil., 3, 220-230, 1958.) Dry matter (per cent) Ash (percentage of dry matter) Sodium (mg. per 100 ml.) Potassium (mg. per 100 ml.) Calcium (mg. per 100 ml.) Total N (gm. per 100 ml.) Reducing substance (as mg. glucose per 100 ml.) Source of Fluid > 2 3 6 2.4 10.0 15.9 41.1 10.3 7.1 170 220 208 . 166 183 223 11 15 12 0.17 1.09 — 9 50 7.5 9.3 304 36 12 0.96 contributed extensive data concerning the chemical composition of the uterine fluids of the rabbit, rat, and dog (Table 13.10). An interesting report on potassium and sodium concentrations of uterine fluid in the proestrous rat indicates that K is relatively high (37 niEci./l.) and remains constant after copulation with a vasectomized male; Na decreases, however, by about 11 per cent from the initial value of 115 niEq./l. (Howard and DeFeo, 1959). The shift may be due to the change from follicular to luteal phase, but because of the contribu- tions of the several accessory glands, the significance of the change is not clear. None- theless, the high initial K/Na ratio (0.32) suggests a marked K-tolerance on the part of the spemi and, further, a secretory action of the genital mucosa leading to the ac- cumulation of potassium within the lumen. The paucity of data concerning enzymatic activity by the uterine fluids was indicated by Reynolds (1949). Since that time little has been added, except for two suggestive papers dealing with amylase activity of the tube and its fluids. Human tubal cysts con- tain high concentrations of such an enzyme and have led to the supposition that intra- luminal glycogen — if any should exist — might be hydrolyzed to provide a substrate for sperm (Green, 1957). McGeachin, Har- gan, Potter and Daus (1958) confirmed the presence of amylase in the cysts and found high activities also in tubal epi- thelium of man, rabbit, cow, and sheep, but not of other species studied. In an elec- trophoretic study of the cornual fluids of the estrous rat, low concentrations of 4 ma- jor proteid components were found, which appeared to differ in their mobility charac- teristics from serum proteins (Junge and Blandau, 1958). It is clear that energy substrates and other biochemical components of seminal plasma are introduced into the tubes in animals in which intrauterine ejaculation occurs (Mann, Polge and Rowson, 1955). However, the significance of these constit- uents for tubal i)hysiology is highly doubt- ful after intravaginal insemination. Rela- tively little glycolytic substrate seems to be present in the fluids recovered from the tract. In the rabbit, for example, little or no hexose, and only traces of phospholipids, can be detected, either before or after cojmlation (Table 13.11) ; lactate is present in appreciable quantities and might con- ceivably serve as a metabolic substrate (Bishop, 1957; Mastroianni, Winternitz and Lowi, 1958). At the present time, it is not easy to ascertain which metabolic sub- strates and products are associated with the activities of the spermatozoa and which with the activities of the mucosal cells lin- ing the tract. More work is necessary to fill in the metabolic and physiologic details of the sketch just barely outlined. Abundant evidence indicates that the tubal contents are a product of active se- TABLE 13.10 Chemical composition of uterine fluids (From H. E. Shih, J. Kennedy and C. Huggins, Am. J. Physiol., 130, 287-291, 1940.) H2O PH CO2 Total N NPN Protein CI Na Ca K Glucose Inor- ganic P Rabbit 979 982 984 7.78 7.55 6.09 mmoles perl. 53.6 61.8 3.0 0.8 1.0 0.8 gm per 0.37 0.29 0.20 gm per 2.7 5.1 3.8 mmoles perl. 98 98 167 mmoles perl. 158 169 162 mmoles perl. 4.7 1.5 3.5 mmoles perl. 6.1 4.3 5.2 mg. perl. 0-160 0-150 0-80 mmoles perl. 0-0.20 Rat 0-0.03 BIOLOGY OF SPERMATOZOA 743 TABLE 13.11 Metabolic substrates in rabbit tubular fluid (From D. W. Bishop, Internat. J. Fertil., 2, 11-22, 1957.) Condition of Animal Substrate Glucose Fructose Lactate Phospho- lipid Estroiis Pregnant Castrate 7of:L 0-2 0-2 0-1 mg. per 100 ml. <1 <1 <1 mg. per 100 ml. 6.8 15.0 7.5 lo'otl 0-8 Trace cretion and not merely a transudate from the vascular system or overflow from the peritoneal cavity. The presence of secre- tory cells in the tubal epithelium is well known; they undergo morphologic and ap- parent physiologic alterations which paral- lel changes in ovarian activity (Hadek, 1955, 1958a; Borell, Nilsson, Wersall and Westman, 1956). In the rabbit the se- cretion is regarded as essential for normal development of the egg (Westman, Jorpes and Widstrom, 1931), and it may be neces- sary for the normal functioning of sper- matozoa and the process of fertilization as well (r/. Whitten, 1957). The secretory activity of the rabbit Fal- lopian tube has been investigated and the volume of flow and secretory pressure in singly and doubly ligated tubes determined (Bishop, 1956a). Mean tubal secretion rates in lightly anesthetized estrous, pregnant, and castrate rabbits were 0.79, 0.37, and 0.14 ml. per 24 hours, respectively. Secretory activity was maximal at the time when the spermatozoa are in the ampulla. Active se- cretion, as opposed to passive diffusion or transudation, was demonstrated by mano- metric determinations of the pressures de- veloped within a closed tubal system over a 36-hour period. Pressure maxima in es- trous, pregnant, and castrate rabbits aver- aged 46.0, 15.6, and 11.8 cm. HoO, respec- tively (Fig. 13.12). Both secretory volume and pressure decreased from the 11th to the 21st day of pregnancy. Further indication that tubal secretion is an active process is shown by its sensitivity to pilocarpine; a single injection of 1 mg. of pilocarpine hy- drochloride almost doubled the secretory pressure, to a value of 75 to 80 cm. HoO, in estrogen-dominated animals (Fig. 13.13). A program initiated by Clewe and Mast- roianni (1959, I960) permits the continuous cm. HgO Fig. 13.12. Secretion pressures in rabbit oviducts. A, estrous; B, 14-da.v pregnant; C, 51- day castrate, (from D. W. Bishop, Am. J. Physiol., 187, 347-352, 1956a.) 744 SPERM, OVA, AND PREGNANCY Fig. 13.13. Effect of pilocarpine on tubal secre- tory pres.sure: right and left oviducts recorded. A, pilocarpine-HCI (1 mg. in 1 ml. saline, I.M.) in- jected 51/2 hours after catheterization; B, control estrous records. (From D. W. Bishop, Am. J. Phys- iol., 187, 347-352, 1956a.) collection of oviduct secretion over a period of many weeks. Their values for secretion rate are somewhat higher than those noted above, for example, 1.29 ml. per 24 hours for the rabbit in estrus. Although the present state of knowdedge permits only a fragile evaluation of the sig- nificance of these secretory products on the activity and viability of the gametes and fertilized eggs, tubal secretion can hardly be denied. Further chemical and physical analysis of the components of the fluids might profitably be attempted, not only in the rabbit and cow, but in other mam- mals as well. In the final analysis, the survival and fertilizing capacity of the sperm are functions of the relation between the cell's intrinsic properties and the en- vironment in which it operates. H. THE FATE OF NONFERTILIZING SPERMATOZOA Relatively soon after insemination, ex- cess sperm have disappeared from the lumen of the genital tract. Within 20 to 24 hours in the mouse and rat, little indication of the sperm mass can be found (Blandau and Odor, 1949; Austin, 1957) . In the sow uterus, a few sperm are present about 50 hours after copulation, but none can be found 25 hours later (du Mesnil du Buisson and Dauzier, 1955). The general fate of the unsuccessful sperm, recently reviewed by Austin (1957), has long been held to be enzymatic dissolution and phagocytic en- gulfment in the lumen (Konigstein, 1908; Sol)otti, 1920). Except for a brief spate in the Russian literature (Kushner, 1954; Voj- tiskova, 1955), little credence has been given to the many claims of Kohll)rugge ( 1910, 1913) that sperm and sperm products are incorporated into the genital epithelium and have profound effects on the maternal physiology (see Hartman, 1939). Indeed, a subsequent paper by Vojti?kova (1956) and others by Posalaky and colleagues in Prague (1956, 1957a, b) have been quite explicit in stating that the earlier histologic demonstrations of sperm in the epithelial mucosa can be explained on the basis of tech- nical artifacts, principally incurred during the sectioning of tissues. Within the past year or two, however, a number of instances have come to light which make it amply clear that sperm do, under some circum- stances, enter or are conducted into the uterine and tubal mucosa. Sperm in, or in association with, leukocytes have been found in the uterine glands of the guinea pig and in the tubal mucosa of other species, including the rat, rabbit, hedgehog, mole, stoat, mouse, and bat (Austin, 1959, 1960; Austin and Bishop, 1959; Edwards and Sir- lin, 1959). How commonly this occurs and what its significance may be for the sub- sequent reproductive capacity of the female remain to be seen. The findings within seven groups of mammals indicate that the phe- nomenon may be widespread. The associa- tion of the spermatozoa with leukocytic in- filtration further suggests that the genital tract may, under some circumstances, be re- garded as a route of foreign cell invasion. A natural skepticism regarding the ability of spermatozoa to penetrate somatic tissues is somewhat lessened by the realization that the process is a normal feature of reproduc- tion in certain invertebrate animals. Manton (1938) cites records of this among rotifers, turbellarians, leeches, and the bedbug {Ci- mex) ; in Peripatopsis (Onychophora) , the sperm are described as invading the body BIOLOGY OF SPERMATOZOA 745 wall at the attachment site of the sper- matophore and then passing into vascular channels through which they actively mi- grate to the ovary where sperm penetration and fertilization occur. VI. Immunologic Problems Associated with Spermatozoa A. ANTIGENICITY OF SPERM The antigenic properties of spermatozoa have been recognized since the turn of the century through the pioneer studies of Landsteiner (1899), Metchnikoff (1899), and Metalnikoff (1900), who, almost simul- taneously, discovered that guinea pigs pro- duce antibodies against heterologous and homologous sperm. Landsteiner's work is of classic interest not only because it was barely the first, but also because he used an in vivo method to demonstrate an im- mune response against sperm. Bull sperma- tozoa, he found, remain active when in- jected into the peritoneal cavity of normal guinea pigs, whereas if the pigs have been l^reviously injected parenterally with bull sperm, the peritoneally administered sperm rapidly become immotile. These early dis- coveries were to be followed by a great wave of interest in sperm antigens, generally assayed by in vitro methods, and attempts to induce sterility in female animals by in- jection of suspensions of spermatozoa or testicular homogenate. After a lull in ac- tivity, interest was rekindled by the de- velopment of new immunologic procedures and concepts, and the awareness of the im- l^lication of immune processes to problems of fertility and fertility control (Katsh, 1959a; Tyler and Bishop, 1961). Specific antigens have been demonstrated in, or on, spermatozoa of many mammals, including the rabbit, rat, mouse, guinea pig, dog, ram, bull, and man. The methods used for their determination have generally in- volved the classical serologic procedures — agglutination, immobilization, precipitin, and complement fixation — and the more recently introduced Oudin and Ouchterlony agar gel-diffusion techniques. The results, in general, indicate a relatively high degree of species-specificity, but some cross-re- activity does occur (Mudd and Mudd, 1929; Henle, 1938; Smith, 1949a). Tissue-spec- ificity is also incomplete. The AB-blood group antigens, for example, as pointed out previously, are present in human sperm (Landsteiner and Levine, 1926; Gullbring, 1957), and a comparable similarity of sperm-erythrocyte agglutinins has been claimed in cattle (Docton, Ferguson, Lazear and Ely, 1952). Common antigenicity be- tween brain and testicular tissue has been demonstrated (Lewis, 1934; Freund, Lipton and Thompson, 1953; Katsh and Bishop, 1958) and may relate to the mature germ cells themselves. As routinely determined by means of ag- glutination or immobilization of fresh sperm in the presence of antisperm serum, the antigenicity of the gametes is customarily attributed to surface moieties and exposed reactive groups. Smith (1949b), however, called attention to the reactivity of the more deeply situated antigenic substances in her study of heterologous reactions among rodent sperm. In part, of course, the mask- ing and unmasking of combining groups are a function of the technical procedures to which the cells are exposed and arc features wliich have to be circumvented or recog- nized in investigations of this kind. The surface properties of, and "leakage" from, spermatozoa are known to change with storage, dilution, washing, and centrifuga- tion, which, when severe enough (Mann, 1954), can be expected to alter the apparent natural antigenicity of the cells (Smith, 1949b; cf. Pernot, 1956). The number of antigenic sul)stancL's on the sperm surface is a moot ])oint and may prove merely a matter of definition, if not of semantics, depending on the techniques involved (see Table 3 in Tyler and Bishop, 1961). Henle, Henle and Chambers (1938) localized three distinct antigens in bull sperm by preparing, in rabbits, agglu- tinating and complement-fixing antibodies against the head and tail fractions. One antigen was found to be head-specific, an- other tail-specific, and the third common to both head and tail of the intact sperm. On the other hand, when the agar-diffusion method was applied to the study of sperm antigenicity, many reactive substances ap- peared which seemed to be surface antigens. 746 SPERM, OVA, AND PREGNANCY In one series of experiments, 7 precipitin bands were observed with washed human sperm tested against rabbit antihuman sperm sermii (Rao and Sadri, 1959). This investigation further indicated that 4 of the sperm antigens were common to seminal plasma, but were not present merely as contaminants. A parallel investigation, em- ploying essentially identical procedures, led to the conclusion that all of the human sperm antigens are also present in plasma and the two materials cannot, immunologi- cally, be distinguished (Weil, Kotsevalov and Wilson, 1956). A similar conclusion grew out of a study of rabbit semen, and the suggestion was made that "the effec- tive antigens found in seminal plasma and spermatozoa of semen appear to originate in the seminal vesicle" (Weil and Finkler, 1958). Because practically all large molecu- lar moieties and cells are potentially anti- genic, and because spermatozoa may safely be assumed to arise in the testis, this state- ment obviously oversimplifies the facts. The point is brought up merely to emphasize the caution that should be exercised in the use of and interpretations derived from vari- ous techniques. There are sperm antigenic differences in strains of animals and in in- dividuals within strains, as Snell ( 1944 ) and Landsteiner and Levine (1926) have long since pointed out. More, rather than less, immunologic differentiation will proba- bly be forthcoming in the future. Indeed, Weil (1960) has recently found that the antigenic properties differ in epididymal and seminal sperm of the rabbit; the sperma- tozoa apparently take up and bind antigenic material from the seminal plasma during ejaculation. B. SPERM-INDUCED IMMUNE RESPONSES IN THE MALE Antibodies against spermatozoa, both foreign and those of the same individual, are produced with facility by members of both sexes. Why an animal should so react against autologous antigen, i.e., a male against its own sperm, is not clear. Ac- cording to the concepts put forward by Burnet and Tenner (1949), Billingham, Brent and Medawar (1955, 1956), and others, an organism undergoes a state of "recognition" of its own native substances during the tolerant period before antibodies are produced. Thereafter, it does not con- sider these, or other substances initially introduced during the tolerant period, as foreign. The formation by an adult animal of antibodies against injected autologous spermatozoa, moreover, is generally attrib- uted to the fact that sperm are not normally produced until late in development; thus they have not had a chance to be "recog- nized" as native and are treated as foreign material when injected. The further sup- position must be made that spermatozoa in the testis are somehow normally insulated from the rest of the body, at least from the antibody-forming sites, and therefore fail to evoke antibody production and an im- mune response. Such speculations are tenta- tive and must await further understanding of the general nature of antigenic stimula- tion, antibody production, and antigen-an- tibody complex formation, subjects which are currently undergoing rapid growth and ]icrplexing change (Talmage, 1957, 1959; Lederberg, 1959). Because antibody production is evoked by autologous sj^erm, the question arises whether auto-immunization occurs and, fur- ther, whether it is of any biologic signifi- cance. Sperm agglutination occurs in other- wise normal ejaculates of rabbit, bull, and man, and the seminal plasma can be shown to contain agglutinating antibodies (Wilson, 1954; see Tyler and Bishop, 1961). The pos- sibility exists that an antigenic stimulus for antibody formation may arise following sperm absorption or penetration into the epididymal mucosa during a period of in- flammation, a process often associated with intense leukocytic infiltration (Mason and Shaver, 1952; Montagna, 1955; King, 1955). In man, such a reaction is claimed to be common after mild epididymal infection; it causes no impairment of testicular func- tion, but produces a tissue response char- acterized by granulomatous lesions (Stein- berg and Straus, 1946; Cronqvist, 1949; King, 1955). Similar lesions have been de- scribed in cases of granulomatous orchitis, in which spermatozoa were present in macro- phage cells and in the lymphatic system (Friedman and Garske, 1949). Cruickshank BIOLOGY OF SPERMATOZOA 747 and Stuart-Smith (1959) have recently de- scribed circulating antisperm antibodies in men who had previously suffered orchitis. It seems not unlikely, therefore, that certain cases of auto-agglutination of ejaculated sperm may have arisen from some kind of autosensitization and passage of the anti- bodies into the seminal plasma. If auto-im- munization does occur by such means, it is assumed that tubal inflammation or infec- tion must be present to effect the immune reaction; otherwise the condition should be much more common since resorption of non- ejaculated sperm from the epididymis seems to be a normal process (see above). The seminal and follicular component, antag- glutin, discovered by Lindahl and Kihl- strom (1954), which tends to prevent ab- normal clumping of sperm, does not coun- teract agglutination by prepared antiserum (Lindahl, 1960) ; it seems rather to operate through another, nonserologic type of mech- anism. Bocci and Notarbartolo (1956) suggested that immunologic factors might contribute to a state of sterility on a basis of their finding of positive antisemen skin reactions in some men suspected of infertility. Rumke (1954) and Riimke and Hellinga ( 1959) made extensive studies of sperm ag- glutinins in the sera of sterile men. In a series of over 2000 cases, they found a considerably higher incidence of sperm-ag- glutinating antibodies in the sera of child- less men (4.1 per cent) than in those of normal fertile controls (1.0 per cent). Among a small group of 21 relatively aspermic patients, all of whose sera had sperm agglutinins, 16 showed occlusions or obstructions of the male tract. In the light of these demonstrations, the suggestion may be ventured that auto-immunization occurs in the male, that the mechanism may result from spermatozoal reactions involving the tubal epithelium, and that the antibodies produced may impair fertility. To what ex- tent, if any, variations in androgen levels modify the epididymal reactivity in this regard is completely unknown. An unusual syndrome, aspermatogenesis, can be readily induced in the guinea pig by injecting homologous spermatozoa or ho- mogenized testis combined with adjuvant (Freund, Lipton and Thompson, 1953; Freund, Thompson and Lipton, 1955;'.'Katsh and Bishop, 1958; Tyler and Bishop, 1961). The immune response is due to a delayed sensitization and is apparently not associ- ated with the high levels of circulating anti- sperm antibodies which can be detected by such methods as sperm agglutination, immobilization, and complement-fixation (Freund, 1957; Katsh and Bishop, 1958). The testicular lesion, as observed 1 to 2 months after injection, is characterized by loss of germinal epithelium and decrease in gonadal weight and volume (Fig. 13.14). The Sertoli elements are affected very little, if at all. The interstitial tissue remains functional, as judged by the normal size and activity of the accessory glands. Since the induction of aspermatogenesis by the in- jection of spermatozoa has been established only in the guinea pig and rat, the implica- tions for reproductive physiology may be limited; its occurrence and the possible mechanism, however, are of substantial im- portance to the general areas of delayed sensitization and the immune response (Katsh, 1958, 1959c; Voisin, Toullet and Mauer, 1958). In contrast to these investigations of ac- tive immunization with sperm, the intro- duction of antisperm serum into male ani- mals has been shown to affect fertility in a limited number of instances. Mice and rabbits both show reproductive impairment after injection of homologous antibody serum (de Leslie, 1901; Guyer, 1922). In rats, a considerable weight loss (24 per cent) of the testes is accompanied by sloughing of germinal epithelium after injection of rat sperm antiserum produced in the rabbit (Segal, 1961). The testicular reaction ap- pears to be a specific response against the homologous sperm. C. SPERM-INDUCED IMMUNE RESPONSES IN THE FEMALE The memorable statement of Charles Darwin (1871) that ''the diminution of fer- tility may be explained in some cases by the profligacy of the women" may be taken to imply a sensitization against the male reproductive products, although another not unlikely explanation may involve the im- 748 SPERM, OVA, AND PREGNANCY It:^^*: b: ?K ^>- ■•4 ^ Vi^:^ * C D i'-.*.-^' %iM v><.;; '.•/^•'«;f...- ■ ^ ' ^-^ v^ .^;-:r..;v. r-^'^:--. .; -I ^ •- - •,. \- 1°""- •"'7 ■». . ;.'■• - „.. V.vr-' ."-K'.-'^^'- .; ^/t- /.' ^^- >/:•■■ *i« ^rvfe. . Fig. 13.14. Aspermatogenesis induced in the guinea pig by injection of testicular ho- mogenate and adjuvant. A, normal adult guinea pig testis used as donor, approx. 65 X, B, same, approx. 260 X ; C, testis of semicastrate 2 months after injection of autologous testicu- lar homogenate, 65 X ; £>, same, 260 X, note normal interstitial tissue; E, testis of guinea p-g injected at 1 week and sacrificed at 5 months of age, 65 X ,: F, same, 260 X. (From D. W. Bishop, unpublished photographs.) BIOLOGY OF SPERMATOZOA 749 paired health of the subjects. Three decades after Darwin, the immunization of labora- tory animals against spermatozoa suggested a mechanism by which sensitization could come about. During the following half cen- tury the pros and cons of this issue were to rage. The parenteral introduction of either homologous or heterologous spermatozoa was early claimed by many workers to in- duce some degree of female sterility in a wide variety of animals (see Parkes, 1944; Tyler and Bishop, 1961). Some experiments seemed so successful that a patent was once granted for an antisterility preparation based on this procedure (Baskin, 1937). Such experiments, however, are beset with difficulties of control and natural biologic variation. It is not surprising, therefore, that more recent investigations have tended to discredit the earlier reports of sterility induced by sperm injection, and to provide adequate explanation for many of the ap- l)arent positive results (Eastman, Gutt- macher and Stewart, 1939; Hartman, 1939; Henle and Henle, 1940; Lamoreux, 1940). The ancient role of spermotoxins in inducing female sterility seemed thus to be laid at rest. The issue was again raised with the ad- \ent of adjuvants which have the ability of potentiating the effect of an antigenic stimulus. Quite recently, evidence has ac- crued indicating that reproductive capacity may indeed be impaired in female rabbits and guinea pigs when they are injected with sjierm or testis homogenate combined with adjuvant (Katsh and Bishoj), 1958; Isojima, (h-aham and Graham, 1959; Katsh, 1959b). In treated guinea pigs, the fertility (number bearing litters) was reduced to 24 per cent compared with 84 per cent for the controls. The rate of fetal death and resorption was high, but there seems to have been little effect on ovulation or fertilization. High titers of circulating antisperm antibodies were present, but their connection with the decreases in fertility is not clear. One reasonable explanation for the occurrence of these induced effects on reproductive ca- pacity was suggested by Katsh (1957), who attributed the fetal loss to a possible ana- phylactoid response of the uterus to foreign antigen. Other plausible mechanisms may involve the gametes or develoi)ing embryos directly; circulating antibodies can pass into the uterine and tubal fluids and might impair development (McCartney, 1923). These recent results, then, not only give some credence to the early claims for in- duced sterility, but also raise the question as to the possibility of naturally acquired sensitization in breeding females. Little di- rect evidence can be cited in support of such a hypothesis since only fragmentary im- munologic studies have been made which indi'-.'ate sensitization or antisperm antibody titers in the sera of animals not previously inoculated (see Tyler and Bishop, 1961). However, in a series of over 200 women, Ardelt (1933) found a positive correlation between frequency of coitus and comple- ment-fixation titer against human sperma- tozoa. Studies of this sort on various species should ]irove rewarding. Whereas the evidence concerning the de- gree of sensitization of the female is scant, the means by which antigenic stimulation might occur seems adequate. The penetra- tion of the tubal epithelium by sperm has been noted ; under some circumstances, this phenomenon may be relatively common, as when mild infections or lesions occur within the tubal mucosa. Another possible site of antigenic stimulation, particularly in ani- mals like the rabbit, is the peritoneum, for not only do sperm pass through the tract and enter the body cavity (Hartman, 1939; Home and Audet, 1958) , but the peritoneum is an adequate site for antibody formation. Furthermore, repeated deposition of sper- matozoa into the rabbit vagina results in high titers of circulating antisperm anti- bodies (Pommerenke, 1928). A comparable situation has also been demonstrated in heifers in which genetically tagged erythro- cytes, rather than sperm, were introduced into the intra-uterine cavity, with the re- sult that specific antibodies subsequently^ appeared in the blood (Kiddy, Stone, Tyler and Casida, 1959). These results have been interpreted as demonstrating the passage of antigen into the circulation where access is gained to the sites of antibody formation ; it is to be noted, however, that the tissues of the reproductive tract itself do on oc- casion produce antibodies (Kerr and Rob- ertson, 1953). It is worth pointing out that, in other experiments, antibodies, rather than 750 SPERM, OVA, AND PREGNANCY antigens, seem to be transported across the genital epithelium, or to migrate by way of the peritoneal cavity. Parsons and Hyde (1940), for example, fomid circulating anti- bodies after introducing antisperm serum into the vaginas of rabbits, and McCartney (1923) claimed that circulating antibodies, actively produced in rats against sperm, could be detected in the uterine and vagmal fluids. Antibodies are known, of course, to pass into the uterine lumen of rabbits dur- ing pregnancy (Brambell, Hemmings and Henderson, 1951). Very little has been attempted in altering the fertility of female animals by means of passive immunization with spermatozoa, perhaps because the outstanding investiga- tion of Henle, Henle, Church and Foster (1940) was so conclusive. Repeated injec- tion of mice with antisperm serum, pro- duced in rabbits, failed to modify reproduc- tive capacity in any significant way. The treatment of fresh sperm with spe- cific antisperm serum has profound effects on the gametes, the basis, in fact, of the sperm-agglutination and sperm-immobiliza- tion test methods. The treatment generally renders sperm, both invertebrate and verte- brate, incapable of fertilizing eggs (God- lewski, 1926; Tyler, 1948; Kiddy, Stone and Casida, 1959). A significant contribution, moreover, has been the recent demonstra- tion that if the exposure to antiserum is carefully controlled, surprising and subtle effects may occur when these sperm are used for artificial insemination. Rabbit sperm, treated for 15 minutes with high concentra- tions of bovine antirabbit antiserum be- fore insemination, were incapable of effect- ing fertilization, as judged from the recovery of unfertilized ova. However, a 15-minute exposure of sperm to the same, but diluted, immune serum permitted fertilization, but resulted in a high percentage of embryonic deaths (Kiddy, Stone and Casida, 1959). No such fetal wastage occurred when rabbit sperm were similarly exposed to normal bovine serum. The antisera employed in these experiments were prepared against whole semen, rather than against washed sperm, but any additional antigenic com- ponents in plasma would not be expected to have altered the results. Various inter- pretations can be placed on these findings, including the possibility that the fertilizing sperm might have carried antibodies into the egg which impaired development, or, an alternative possibility, that the antibodies had a mutagenic action on the spermatozoa leading to abnormal development after fer- tilization (Kiddy, Stone and Casida, 1959). There seemed to be no injurious effect on the sperm that resulted in delayed fertiliza- tion; thus the effects cannot be attributed to aging of the ova. An immunologic mechanism has been im- plicated by Gershowitz, Behrman and Neel (1958) to account for the variations from the expected ratio of offspring of couples with incompatible ABO-blood groups. These investigators found hemagglutinins in the cervical mucus of 17 out of 77 cases so dis- tributed that they might be regarded as con- stituting a preconceptive selection mecha- nism by blood group antibodies of the uterine secretions acting on the sperm. In conclusion, a brief survey of the im- munologic literature relating to fertility indicates that spermatozoa may be deeply involved in both experimentally and natu- rally induced modifications in reproductive performance and capacity. Other immune- like interrcactions between specific sub- stances, fertilizin and antifertilizin, ex- tracted from invertebrate eggs and sperm, also have been demonstrated; the possible role of these reactions in the fertilization process is discussed in the following chapter. VII. Morphology and Composition of Spermatozoa A. STRUCTURAL FEATURES As one of the first objects to be viewed microscopically (van Leeuwenhoek, 1678) , the spermatozoon has had a long morpho- logic history,- and still enjoys great popu- larity, particularly among cytochemists and electron microscopists. No exhaustive item- ^Reimer Kohnz (1958) calls attention to a re- cent "find" in the library of the Cologne Cathedral which, if genuine, would shed revolutionary light on the history of microscopic science. A manu- script, purported to have been illuminated by monks of the Reichenau Monastery ca. 1000 A.D., is interpreted as showing an egg with eight sperma- tozoa attached ! BIOLOGY OF SPERMATOZOA 751 ization of sperm morphology is intended here, and even less is necessary by virtue of many extensive surveys which, over the years, have reviewed and collated the litera- ture of the times, in the light of contem- porary interests and in relation to other areas of biologic progress (Retzius, 1909; Wilson, 1925; Bradfield, 1955; Hughes, 1955, 1956; Franzen, 1956; Nath, 1956; Colwin and Colwin, 1957; Bishop and Aus- tin, 1957; Anberg, 1957; Fawcett, 1958; Schultz-Larsen, 1958; Bishop, 1961). The two historical surveys of Hughes (1955, 1956) are of particular interest to anyone mindful of the past. Wilson (1925), among others, drew at- tention to the great variation in animal sperm, including the existence of nonflagel- lated and nonmotile gametes among certain invertebrate groups. More recently, Franzen (1956), in an admirable survey of many kinds of invertebrate spermatozoa, has em- phasized what he believes is a significant correlation between sperm morphology and physiologic demands of the particular type of reproductive process concerned. Con- siderable attention has been paid to sperm size, from the small, microscopic sea-ui'chin gamete, some 40 /j. long, to the relatively gigantic sperm of the hemipteran insect, Notonecta glauca, which is reputed to be about 12 mm. in length (Pantel and de Sinety, 1906; Gray, 1955). The claim was once current that, because of the difference in chromosome number, a sperm population displays a bimodal size-distribution curve, but careful biometric studies by van Duijn ( 1958) and others have shown this to be untenable. More recently, differences in size and shape of sperm have been demonstrated in different inbred strains of mice ; the char- acteristics seem to be genetically determined and, when intermingled, lead to extreme variation in hybrid crosses (Braden, 1959). Gravimetric, interferometric, and refrac- tometric methods have been applied to the study of sperm in an analysis of their physical properties. By such procedures, one can determine that bull sperm have a rela- tive density of 1.280 (Lindahl and Kihl- strom, 1952), a dry mass averaging 7.1 X 10~^ mg. (Leuchtenberger, Murmanis, Mur- manis, Ito and Weir, 1956), and a total weight of about 2.86 X 10~^ mg. (see Bishop, 1961). Human sperm contain at least 45 per cent "solid material" in the head, and possibly 50 per cent "solids" in the tail, as assessed by the method of im- mersion refractometry (Barer, Ross and Tkaczyk, 1953; Barer, 1956). Cytochemical procedures, frequently com- bined with extraction procedures, have proved useful in the investigation of sperm composition, particularly in tracing the differentiation of cellular elements, such as the Golgi apparatus and Nebenkern, through spermiogenesis, and in identifying the chemical nature of various structures in the mature gamete. By means of PAS-posi- tive tests for 1 ,2-glycol groups, for example, the acrosome was found to consist of poly- saccharide associated with some protein- aceous material, complexed possibly as mucopolysaccharide (Schrader and Leu- chtenberger, 1951 ; Leblond and Clermont, 1952; Clermont and Leblond, 1955). Fur- ther, on extraction and hydrolysis, this material from guinea pig sperm proved to contain galactose, mannose, fucose, and hexosamine (Clermont, Glegg and Leblond, 1955). These are precisely the same com- l)onents found by Bergman and Werner (1950) in carbohydrate hydrolysates of hu- man cervical mucus (see above). The electron micrographic studies of sper- matozoa, of which there have been a great number, are well summarized by Anberg's fine treatise (1957) on human sperm and Fawcett's eloquent review (1958) of mam- malian sperm in general (Fig. 13.15). Faw- cett makes the historic point that in some instances the electron micrograph has con- firmed details wdiich theoretically should be invisible with the light microscope, but were seen and described, nevertheless, by an earlier generation of able microscopists — the enumeration, for example, of the 11 tail filaments of the fowl sperm by Ballo- witz in 1888. But many other features have been discovered by electi'on microscopy. The postnuclear cap and cytoplasmic sheath, previously described as parts of the human sperm' head, apparently do not exist (Faw- cett, 1958). The acrosome system of the human sperm is less discrete than that ob- served in other types of gametes. The nu- 752 SPERM, OVA, AND PREGNANCY ■B Grnrsuie i E ^ I'K., 1:M.'i KliriMiii 1. 1,-1 I, M~ of mammalian >i>riiii. A.V> >tagcs m tlic lorniation of the Lead cap of the humau sperm. B, late spermatids of the eat (i) and guinea pig {2) in roughly longitudinal section; note approximation of axial filament to centriole. C, principal piece of guinea pig sperm tail ; note that each peripheral doublet appears as one tubular and one solid element: 7 outermost fibers are present at this level (see Fig. 13.175). D, terminal region of human sperm tail; the doublets appear as hollow cylinders. E, midpiece of human sperm ; the electron-dense outermost array of filaments is surrounded by many mitochondrial bodies. {A and B from D. W. Fawcett, Internat. Rev. Cytol., 7, 195-234. 1958; C, courtesy of D. W. Fawcett; D and E from A. Anberg, Acta ob.st. et gvnec. scandinav., Sup])l. 2, 36, 1-133, 1957.) BIOLOGY OF SPERMATOZOA 753 cleus, instead of occupying only the pos- terior portion, seems rather to extend the entire length of the head (cf. Bishop and Austin, 19571. During differentiation, the nuclear cln-omatin condenses into a homo- geneous, electron-dense mass, but Yasu- zumi, Fujinmra, Tanaka, Ishida and Ma- suda (19561 demonstrated in enzymatically treated bull sperm helical strands which may correspond to distinct chromosomes. During spermiogenesis in the guinea pig the four spermatids resulting from meiosis re- main attached by intercellular bridges until late in the development of the gametes (Fawcett, 1959). Such connections may al- low for significant interchange of materials and for mutual interaction among the mem- bers of the tetrad. Electron microscopy has confirmed the traditional view that there are two cen- trioles present in the neck region of the sperm which are directly or indirectly as- sociated with the axillary bundle extending into the flagellum (Fawcett, 1958). The homology of the centriolar body with the basal granule (blepharoplast) is assumed. The spiral body, typical of the middle piece of the sperm, is made up principally of the mitochondrial elements, arranged spi- rally but not in a continuous helix. The dis- tribution of the mitochondria, constituting in large measure the "power plant" of the cell by reason of their oxidative and phos- phorylative activities, is in close association with the flagellar apparatus, particularly the fibrillar elements of the tail. The mito- chondrial system is derived from or related to the Nebenkern, a prominent cell inclusion in spermatids of lower forms. In some in- sect sperm, in the absence of a true mid- piece, the mitochondria extend far down into the flagellum (Rothschild, 1955). What had been considered the helical covering of the sperm tail might better be regarded as a "fibrous sheath" since the structure is nei- ther continuous nor constituted of uniform successive gyres (Fawcett, 1958). The outer membrane, probably the true physiologic surface of the cell, is a continuous envelope and is apparently derived from the sper- matid cell membrane. Emanating from electron micrographic in- vestigations, a universal fibrillar pattern in flagella and cilia is generally acknowledgeu. ]\lodifications exist but incontestable evi- dence indicates that the basic arrangement, as seen in transverse sections, is the now familiar 2 X 9 -t- 2 array. Surrounding 2 central filaments is a ring of 9 double fibrils (Figs. 13.16, 13.17^1, B), all of which seem to extend, uninterrupted, from proximal to distal tip of the flagellum. On extensive, but nevertheless largely circumstantial evidence, the outer filaments are generally regarded as the motile organelles. Inoue (1959), how- ever, in summarizing the evidence pertinent to ciliary movement, suggests that the outer fibrils may actually be conductile elements, whereas the two central filaments take a more active part in motility. Certain other features of the sperm tail, including the Fig. 13.16. Electron micrographs of fowl sperm flagella. Of the 11 major filaments, two (M fibrils) are differentiated from the remainder and consti- tute the central pair. Sperm were exposed to dis- tilled water, fixed in formalin, and shadow-cast with platinum. (From G. W. Grigg and A. J. Hodge, Australian J. Scient. Res., ser. B, 2, 271-286, 1949.) 754 SPERM, OVA, AND PREGNANCY chemical nature of these longitudinal fila- ments, their proximal association with yet another array of 9 peripheral fibers, their relation to the matrix of the flagellum, and their relation to one another, are described elsewhere in considerable detail (Bishop, 1961 ) . As a general conclusion, the three main divisions of sperm into head, middle piece, and tail correspond roughly to their genetic, metabolic, and motile functions. B. BIOCHEMICAL FEATURES The availability and homogeneity of sper- matozoa have long appealed to the biochem- ist in choosing a cell tyjie for study. Both chemical and histochemical methods have been employed in investigations of the com- position of sperm, and recent developments in quantitative cytochemistry show good agreement in the results obtained by the two general procedures. Complete analyses of the chemical components of several types of spermatozoa are now available and include the full range of substances from ions to en- zymes, many of which have been roughly localized within the major regions of the cells. For more extensive treatment concern- FiG. 13.17^. Highly diagrammatic representation of transverse sections of sperm flagellum and cil- ium ; 2 central and 9 double peripheral fibrils typi- cal of all such motile organelles. Mitochondria (oblique hatching) present in midpiece. An addi- tional array of 9 outermost filaments (solid) in the midpiece of mammalian sperm extends into the proximal portion of the flagellum. The fibrous sheath of the tail is frequently ribbed as indicated. Fig. 13.17fi. Diagram of rat sperm tail at various levels from midpiece (A) to tip (G). Note bilateral symmetry of fibrillar arrangement and termination of outer longitudinal fibers at different levels of flagellum. (Courtesy of D. W. Fawcett.) ing the functional composition of sperm, the reader is referred to several reviews (Marza, 1930; van Duijn, 1954; :Mann, 1954; Bishop, 1961 ) ; only selected features of the volumi- nous literature will be noted here. Just short of a century ago, Miescher, and later Kossel, and their co-workers took up the study of the basic proteins — protamines and histones — of fish sperm nuclei, easily procurable by plasmolysis of the cytoplasm and collection of the heads by centrifuga- tion. Progress was rapid and by the 1920's more was known, it was claimed, about the chemistry of the spermatozoon than about any other cell (Marshall, 1922). These early studies have expanded into investigations of the basic proteins as conjugates with desoxy- ribonucleic acid (DNA), and particular at- tention has been directed toward the sig- nificant and systematic changes from the histone- to the protamine-type protein dur- ing sperm differentiation (Miescher, 1897; Kossel, 1928; Mirsky and Pollister, 1942; BIOLOGY OF SPERMATOZOA 755 Pollister and Mirsky, 1946; Stedman and Stednian, 1951 ; Felix, Fischer, Krekels and Mohr, 1951 ; Bernstein and Alazia, 1953a, b; Alfert, 1956; Vendrely, Knobloch and Ven- drely, 1957; Ando and Hashimoto, 1958; Felix, 1958). Histone is regarded as typical of somatic chromosomes, whereas prota- mines characterize the nuclei of mature sperm (Daly, Mirsky and Ris, 1951). The two types of basic proteins differ in their solubility and physical properties and in their chemical composition as well; prota- mines are found to have fewer amino acids when compared to histones from the same animal (Daly, Mirsky and Ris, 1951 ) . Both are very rich in arginine. This polyamino acid is reported to constitute some 70 per cent of the protamine, "gallin," of fowl sperm (Fischer and Kreuzer, 1953), and about 50 per cent and 30 per cent, respectively, of the solid matter of bovine and human sperm nuclei ( Leuchtenberger and Leuchtenberger, 1958). Total amino acid composition and other chemical characteristics of sperm nu- clcoprotein have been reported on numerous occasions (see Sarkar, Luecke and Duncan, 1957; Daly, Mirsky and Ris, 1951; Porter, Shankman and Melampy, 1951 ; Dallam and Thomas, 1953) . Sperm DNA has been isolated from a va- riety of species and its nucleotide composi- tion determined (Chargaff, Zamenhof and Green, 1950; Chargaff, 1951; Chargaff, Lip- shitz. Green and Hodes, 1951 ; Elmes, Smith and White, 1952). According to Elmes, Smith, and White, the purine and pyrimidine bases of human sperm — guanine, adenine, cytosine, and thymine — are present in the molar ratio of 0.92:1.23:0.84:1.01, which is consistent with the "thymus-type" composi- tion of nucleic acid. The absolute amount of DNA ])er sperm nucleus is measurable, both by direct chemical analysis and by ultra- violet microspectrophotometry (Vendrely and Vendrely, 1948, 1949, 1953; Mirsky and Ris, 1949, 1951 ; Leuchtenberger, Leuchten- berger, Vendrely and Vendrely, 1952; Walker, 1956; Knobloch, Vendrely and Ven- drely, 1957; Leuchtenberger and Leuchten- berger, 1958). Bull sperm contain approxi- mately 3.3 X 10-^ mg. of DNA per nucleus. Of particular significance was the Vendrelys' (1948) demonstration that the sperm nu- cleus contains half as much DNA as does the diploid nucleus of the corresponding so- matic cell, thereby giving strong support to the theory that DNA is identical with the substance responsible for hereditary transmission. In a recent study of the sperm of bull and man, the Leuchtenbergers (1958) indicated that, whereas the amount of DNA is constant in gametes from fertile individuals, there is a tendency for DNA deficiency in the sperm from infertile in- dividuals (see also Weir and Leuchten- berger, 1957). This finding is surely of great significance but its cause and meaning are at present obscure. The amount of ribonucleic acid (RNA) in sperm nuclei is small, but sufficiently large to be detected. Leuchtenberger, Leuchten- berger, Vendrely and Vendrely (1952) gave a value for bull sperm of about 0.1 X 10~® mg. of RNA per nucleus. C. THE LOCALIZATION OF ENZYMES The mammalian spermatozoon has a full spectrum of enzymes which enables it to carry on the usual glycolytic and oxidative processes associated with the production of energy (Mann, 1954). In addition, there are relatively specific enzyme systems asso- ciated with movement, others related to fertilization, and still others (e.g., amino acid oxidase) possibly concerned with modi- fication of the substrate wdth which the sperm come in contact. Some of these en- zymes have been tentatively localized in specific regions of the sperm, thereby shed- ding some light on the intracellular activi- ties of the gametes and their constituent structures. Since both mechanically separated and naturally ejaculated sperm tails, free from the heads, are capable of motility, oxidation, and glycolysis, it is obvious that the key enzyme systems concerned with these proc- esses are relatively self-contained within the flagcllum (Engelmann, 1898; Cody, 1925; Mann, 1958a). As used here, the term fla- gellum includes the mitochondria-containing middle piece, for without it the tail frag- ment rapidly loses its capacity for metab- olism and motility (Bishop, 1961). The en- zymes wdiich have, by direct or indirect means, been identified in the ram sperm fia- 756 SPERM, OVA, AND PREGNANCY gellum and are known to be involved in the Embden-Meyerhof glycolytic process, in- clude hexokinase, phosphohexoisomerase, phosphohexokinase, aldolase, enolase, and lactic dehydrogenase (Mann, 1949, 1954). Cytochrome oxidase, determined both man- ometrically (Zittle and Zitin, 1942) and spectrophotometrically (Nelson, 1955a), is present in the tail fraction of bull sperm, and the complete cytochrome system can be demonstrated in flagellar preparations which include the midpieces as well (Mann, 1954). From what is known about mitochondrial activity in general, one assumes that most, if not all, of the enzyme systems associated with respiration, oxidative phosphorylation, and electron transport through the cyto- chrome system are concentrated in the sperm midpiece. Succinic dehydrogenase can be demonstrated in flagellar fractions both by biochemical and cytochemical methods (Mann, 1954; Nelson, 1955a; Kothare and De Souza, 1957). Nelson (1959) has further been able to show in frozen-dried sections of the rat sperm flagellum what seems to be succinic dehydrogenase activity in the out- ermost longitudinal fibers of the tail. The sperm flagellum, at least in man and bull, when tested cytochemically, gives posi- tive reactions for acid phosphatase, and the bull sperm tail shows alkaline phos])hatase activity as well (Wislocki, 1950; ]\lelampy, Cavazos and Porter, 1952). Both types of phosphatase have been cytochemically lo- calized in the midpiece of the rat sperm (Friedlaender and Fraser, 1952; Melampy, Cavazos and Porter, 1952). The precise functions, however, of these enzymes in the sperm are not clear. One or more adenosinetriphosphatases (ATPases) have been extracted from or demonstrated in the flagella of inverte- brate and mammalian spermatozoa (Felix, Fischer, Krekels and Mohr, 1951; Nelson, 1954, 1955b; Engelhardt and Burnasheva, 1957; Burnasheva, 1958; Hoffmann-Berling, 1955; Bishop and Hoffmann-Berling, 1959). In frozen-dried sections of rat sperm fla- gella, ATPase has presumably been visual- ized in association with the outermost array of fibrils (Nelson, 1958a). In the head of the mammalian sperm, only acid and alkaline phosphatases have been reported and these determinations were achieved by cytochemical localization (Wis- locki, 1949, 1950; Melampy, Cavazos and Porter, 1952; Friedlaender and Fraser, 1952). Thus far, no enzymes have been identified in the mammalian sperm head which com- pare with the invertebrate sperm lysins, be- lieved to play some role in egg penetration (Tyler, 1948). Hyaluronidase, which effec- tively disperses the cumulus cell mass around mammalian ova, is present on the sperm but has not been localized in any one region. Buruiana (1956) found that hyalu- ronidase activity is common to mammalian sperm, whereas trypsin activity is charac- teristic of bird sperm; of the species studied, only the rabbit sperm showed both types of enzymatic activity. Amylase has been dem- onstrated in bull sperm, but because of the violence of the extraction procedure, little is known as to its site of action (Lundblad and Hultin, 1952). Other enzymatic activities have been found in intact sperm or cell homogenates, such as aconitase in bull (Lardy and Phillips, 1945; Humphrey and Mann, 1948), cholinesterases in boar and guinea pig (Sekine, 1951; Sekine, Kondo and Saito, 1954; Grieten, 1956), and gly- cosidases and sorbitol dehydrogenase in ram (Conchie and Mann, 1957; King and Mann, 1958). Sorbitol deiiydrogenase may serve to convert the seminal plasma constituent, sorbitol, to fructose, a normal metabolic substrate for spermatozoa. D. THE SPERM SURFACE As far as can be determined from electron micrographs, the sperm cell membrane is identical with, or at least derived from, the spermatid membrane. In the mature ram sperm, as in many invertebrate sperm, the membrane was claimed to swell osmotically in response to hypotonic changes in the medium (Green, 1940). This is not true of bull sperm (Rothschild, 1959) ; in fact most mammalian sperm are resistant or indiffer- ent to osmotic changes (Emmens, 1948; Pursley and Herman, 1950; Blackshavv, 1953a, b; M. W. H. Bishop, 1955). This feature is in contrast to the selective per- meability with respect to many organic molecules, both charged and uncharged BIOLOGY OF SPERMATOZOA 757 (Mann, 1954). Rothschild (1959) investi- gated the anaerobic heat production in buff- ered suspensions of bull semen under vari- ous anisotonic conditions and found that an initial shock reaction, marked by reduced heat production and metabolic activity, was followed by gradual recovery or adap- tation which in some cases was complete. Such adaptation seems particularly charac- teristic of bull sperm but the nature of the osmotic regulation is not entirely clear. Under severely unfavorable conditions the ])ermeability of ram and bull sperm is so altered as to permit the apparent leakage of large molecules such as cytochrome c (Mann, 1951a, 1954). Pronounced changes in permeal)ility accompany the phenomenon known as "cold-shock" (Mann and Lutwak- Mann, 1955). Chemical analyses of ram and bull sperm by Green (1940), Zittle and O'Dell (1941), and others indicate that the surface mem- brane contains lipid, probably bound as phosjiholipoprotein; the lipid-free mem- l)rane is high in nitrogen and cystine and bears a superficial resemblance to keratin (Mann, 1954). The toughness and the elas- tic properties of human sperm actually have been ciualitatively determined by dexterous microdissection technique (Moench, 1929). The sperm surface at physiologic ionic strength and pH bears a negative charge which has been claimed to be higher on the tail than on the head (Joel, Katchalsky, Kedem and Sternberg, 1951 ) . The gametes thus tend to migrate electrophoretically to- ward the anode. According to Machowka and Schegaloff (1935), this movement is counteracted, at certain field strengths, by a galvanotropic tendency to swim actively toward the cathode. The negative charge on the sperm surface may be attributable to phosphate, carboxyl, and/or sulfate groups attached to organic components of the membrane. Several attempts have been made to uti- lize the electrophoretic properties of sperm in order to separate X- and Y-bearing gametes. Schroder (1940a, b, 1941a, b, 1944) , in an interesting and apparently careful series of investigations, claimed to have ac- complished this with rabbit sperm ; the two types of gametes thus separated, when arti- ficially inseminated into does, gave pre- dominantly (78 to 80 per cent) male or fe- male offspring. More recent work by Gordon (1957) suggests concurrence in these find- ings, but both the technicjue employed and the conclusions derived indicate the need for further confirmation. If such electrophoretic separation of the two cytogenetically dis- tinct types of sperm is possible, it would be of interest to ascertain the reason for the behavior, whether, for example, the male- and female-producing gametes carry dif- ferent ^-i:)otentials or otherwise vary in sur- face composition. Schroder's studies did indeed indicate that the electrophoretic re- sponse might be attributable to differences in the comjionents of the lipoprotein sheaths of the two tyi)es of spermatozoa. VIII. Sperm Metabolism A. SOURCES OF ENERGY In biochemical investigations of sperma- tozoa the focus of attention has been on the metabolic processes associated with the pro- duction of chemical energy required for motility. Although the sperm of relatively few species have been extensively explored, a fairly consistent pattern of metabolic ac- tivities has been established. Mammalian sperm, in general, display extensive gly- colytic activity under both aerobic and an- aerobic conditions, and carry on oxidative respiration when conditions are appropriate (Mann, 1954). Invertebrate spermatozoa, on the other hand, rely almost entirely on oxidative processes and show little, if any, glycolysis (Rothschild, 1951a). Regardless, however, of the nature of the substrate and the pattern of metabolism, the importance of the chemical conversions lies in the coupling of these exergonic reactions with the synthesis of ATP as a utilizable source of chemical energy for the performance of work (Lardy, Hansen and Phillips, 1945; Lehninger, 1955, 1959). In active sperma- tozoa much of this energy source is consumed by the processes underlying motility ; an un- known fraction may be utilized in other ac- tivities, including possible synthetic proc- esses, conduction, and membrane transport. In mammalian spermatozoa, anaerobic glycolysis supplies sufficient ATP energy to 758 SPERM. OVA, AND PREGNANCY support motility for long periods of time; however, respiratory processes coupled with oxidative phosphorylation are far more ef- ficient and can be assumed to furnish sperm, as other tissues, 8 to 10 times as much ATP for the same amount of initial sub- strate degraded (for general discussion, see Lehninger, 1955; Slater, 1958). Sperm mo- tility is sustained so long as a minimal concentration of intracellular ATP persists ; with the exhaustion of ATP, motility ceases (Engelhardt, 1945; Lardy, Hansen, and Phillips, 1945). In 1945, Lardy and Phillips suggested the presence of ATP in bull sperm and Mann ( 1945) succeeded in isolating from ram sperm the nucleotide, as the bar- ium salt, and characterizing it as ATP. Soon thereafter it was shown to be func- tionally identical with ATP isolated from muscle (Ivanov, Kassavina and Fomenko, 1946). ATP has since been extracted from sperm of the sea urchin. Echinus esculentus (Rothschild and Mann, 1950). A consider- able body of evidence has suggested that phosphagen is present in mammalian sperm which might serve as a phosphorus donor for the reconstitution of ATP from adeno- sine diphosphate (ADP) (see Bishop, 1961, for review) ; recently, however. White and Griffiths (1958) re-examined the problem and failed to find any significant amount of creatine phosphate or the enzyme which might take part in transphosphorylation in the sperm of the ram, rabl)it, or bull. B. INVERTEBRATE SPERM METABOLISM The processes underlying motility and survival of invertebrate spermatozoa are oxygen-dependent and involve the utiliza- tion of endogenous reserves (Rothschild, 1951a). In sea urchin sperm, on which such investigations have almost exclusively cen- tered, the oxidative substrate seems to be phospholipid, mainly situated in the mid- piece (Rothschild and Cleland, 1952) . About 20 per cent of the intracellular phospho- lipid of the sperm of Echinus esculentus is depleted during incubation over a 7-hour period at 20°C. According to Rothschild, sea-urchin spermatozoa do not utilize glyco- lytic substrates (glucose or fructose), and there is scant evidence of a "sparing" of en- dogenous substrate by exogenous hexose. Among certain other forms which, like the sea urchin, reproduce by external fertiliza- tion, the spermatozoa rely principally, if not entirely, on oxidative mechanisms. This is true, for example, of the starfish, Asterias (Barron, 1932) as well as the frog, Rana (Bernstein, 1954). On the other hand, some invertebrate sperm are less restricted in their metabolic capacity. The sperm of the oyster, Saxostrea, for example, normally de- pend on respiratory processes, but if these are inhibited by an oxidative inhibitor such as cyanide, and suitable substrate is present, glycolysis can occur (Humphrey, 1950). Barron (1932) indicated that sperm of vari- ous marine animals differ significantly in their tolerance for anaerobic conditions, as determined by the saf ranin test for oxygen ; sperm of Arbacia, Asterias, and Nereis re- tain their motility and fertilizing capacity when exposed to anaerobiosis for 1, 2, and 5 hours, respectively. The importance of oxidative phosi)horyla- tion, in contrast to oxygen consumption per se, to sperm motility has been clearly dem- onstrated in the sperm of the clam, Spisula (Gonse, 1959). Dinitrophenol, an uncou- pling agent, inhibits sperm motility while increasing Oo uptake several fold. Amytal, on the other hand, at a concentration which severely depresses respiration, only slightly impairs motility. Determinations of respiratory cjuotients (R.Q.) of invertebrate spermatozoa yield values approximating 1.0 (Barron and Goldinger, 1941; Hayashi, 1946; Barron, Seegmiller, Mendes and Narahara, 1948; Spikes, 1949; Humphrey, 1950). Such data suggest carbohydrate rather than lipid or phospholipid as substrate. Rothschild (1951a), however, has emphasized the tech- nical difficulties besetting such determina- tions and the errors which may arise; in his view, loss of bicarbonate from the sea- water diluent gives erroneously high R.Q. values. Yet, in support of the possible utili- zation of glucose or fructose by sea urchin sperm { Arbacia and Psaynmechinus) stands Wicklund's demonstration that exogenous hexose significantly prolongs motility and fertilizing capacity of sperm (in Runnstrom, 1949 ) , a point also suggested by the work of Spikes (1949). BIOLOGY OF SPERMATOZOA 759 It seems that, although there may exist some variation in the ability of invertebrate sperm to withstand anaerobiosis or to utilize glycolytic substrates to a limited extent, these cells generally are dependent on re- spiratory i)rocesses for the major produc- tion of chemical energy. Since the conditions of external fertilization deny them ready access to glycolytic substrates in the en- vironmental milieu, the sperm have failed to develop, or have secondarily lost, their glycolytic capacity, so characteristic of mammalian and avian spermatozoa. It is unlikely, although, of course, possible, that failure to utilize hexoses rests on the im- permeability of the sperm to these sub- strates. C. MAMMALIAN SPERM METABOLISM As is the case with invertebrate sperma- tozoa, most of wdiat is known about the biochemical characteristics of mammalian sperm has been acquired from studies, in vitro. To the extent that experimental con- ditions may duplicate those within the genital tract, the behavior of sperm, in vivo, can only be surmised. Considerable varia- tion is seemingly inherent in the metabolic characteristics of sperm of different species and in the gametes removed from different levels of the tract (see Dott, 1959). There is little doubt that such variation exists, but the causes may not be so distinctive as is generally claimed. Discounting differences in sperm behavior attributable to variations in handling and experimental procedure, it seems likely, without implying fundamental differences in metabolic patterns, that sperm, like most other types of cells, possess a lability of subcellular activity which en- ables them to regulate to external and in- trinsic factors. The variations in sperm be- havior, which at times seem so unique, are not likely to conflict with the conservative concept of the ''biochemical unity of living matter" (Fruton and Simmonds, 1959) . The principal metabolic characteristics of mammalian spermatozoa have been ex- tensively reviewed by Mann (1949, 1954) ; elsewhere special attention has been paid to human sperm (MacLeod, 1943b; Ivanov, 1945; Westgren, 1946; Lundquist, 1949). It is now well established that both glyco- lytic and oxidative processes provide energy for mammalian sperm and either one or both types of metabolic pattern can serve the sperm after insemination into the fe- male genital tract. Motility of ram and bull sperm, in vitro, is enhanced by the presence of both hexose and oxygen to- gether (Walton and Dott, 1956). Whereas fructose is the common natural substrate at ejaculation (see chapter by Price and Williams-Ashman), most mammalian sperm also utilize glucose and mannose with equal or greater facility (Mann, 1954). The j)rin- cipal steps in the degradation of sperm hexose to lactic acid occur by the well known Embden-Meyerhof scheme involving ATP as phosphate donor and diphospho- pyridine nucleotide (DPN) as hydrogen carrier (electron transport system) ; this has been demonstrated in both ram and bull sperm, mainly by the identification of in- dividual enzyme systems and glycolytic in- termediates (Mann, 1954). The several components of the cytochrome-cytochrome oxidase electron transport system have been established by manometric and spectropho- tometric methods in a variety of sperma- tozoa, including those of man (MacLeod, 1943a; Mann, 1951a). Less direct, but nevertheless adequate, evidence further in- dicates that the Krebs tricarboxylic acid cycle is involved in the oxidative processes (Mann, 1954; White, 1958). Indeed, there is no evidence to suggest that the over-all metabolic systems of sperm, at least of the ram and bull, are significantly different from those of muscle or of most other mam- malian tissues. The rates of glycolysis and oxidation vary, but the mechanisms are basically the same. Moreover, it is probable that under many conditions, in vivo, there is considerable interaction between the gly- colytic and oxidative processes (for general discussion, see Packer, 1959; Packer and Gatt, 1959). Both types of metabolic path- ways, glycolytic and oxidative, are com- plete within the sperm flagellum. This is clear from the fact that in both the guinea pig (Cody, 1925) and bull (Mann, 1958) cases have been reported in which the fia- gella are naturally separated from the heads at the time of ejaculation; such flagella are actively motile and show high rates of lac- 760 SPERM, OVA, AND PREGNANCY tate production and oxygen consumption. Both turkey and cock sperm also utilize glycolytic and oxidative substrates, al- though at lower rates than those generally found in mammalian spermatozoa (Win- berg, 1939; Pace, Moravec and Mussehl, 1952; Bade, Weigers and Nelson, 1956; Lorenz, 1958). The range of substrates metabolized by mammalian sperm is extensive and includes carbohydrates, lipids, and amino acids. Of the three readily glycolyzable hexoses — glucose, fructose, and mannose- — glucose is preferentially utilized by sj^erm of the bull, ram, and man (Mann, 1951b; van Tien- hoven, Salisbury, VanDemark and Hansen, 1952; Flipse, 1958; Freund and MacLeod, 1958). Hexosc degradation is such that one mole of glucose gives rise to two moles of lactate (Flipse and Almquist, 1955; Mac- Leod and Freund, 1958). Lactic acid tends to accumulate, since the rate of glycolysis, in bull sperm for example, exceeds the rate of pyruvate oxidation ( Melrose and Terner, 1951). Evidence bearing on the possibility of direct oxidation of glucose by way of the hexose monophosjihate shunt is fragmentary and thus far negative (Wu, McKenzie, Fang and Butts, 1959). Glycolysis can, of course, occur under l)oth aerobic and anaerobic con- ditions. The addition of exogenous hexose to a respiring system of sperm tends to "spare" the respiratory substrate (Lardy and Phillips, 1941; O'Dell, Almquist and Flipse, 1959); this partial inhibition of oxidation by glycolysis is a manifestation of the well known Crabtree effect ( Crabtree, 1929; Terner, 1959) and can be interpreted as a form of metabolic regulation. Since the initial demonstration (Lardy and Phillips, 1941) that endogenous phos- pholipid seems to constitute the natural respiratory substrate of bull spermatozoa, many oxidizable substances have been shown to increase oxygen uptake or to sup- port sperm motility (Mann, 1954; White, 1958). Considerable species variation occurs in the apparent facility with which such substances are oxidized, but some of this variation depends less on utilization than on the extent to which the substances pene- trate specific kinds of sperm. Succinate and malate, for example, can increase the respi- ration and motility of washed ram sperm, but are without effect on bull sperm under similar conditions, presumably because of their failure to penetrate the cells (Lardy and Phillips, 1945; Lardy, Winchester and Phillips, 1945). Changes in cell permeability induced by rough handling, severe centrifu- gation, storage, or specific chemical treat- ment, such as exposure to surface-active detergents, can alter the rate and degree of substrate penetration and thereby produce profound changes in respiratory activity (Koefoed-Johnsen and Mann, 1954). Among the oxidative substrates which in- crease respiration of mammalian sj)erm may be included the end products of an- aerobic glycolysis — pyruvate and lactate — as well as acetate, butyrate, propionate, citrate, and oxaloacetate (Lardy and Phil- lips, 1944; Mann, 1954). Glycerol is oxi- dized to lactic acid by ram and bull sperma- tozoa (Mann and White, 1957; White, 1957), probably by entering the Embden- Meyerhof jjathway as glycerol phosphate at the triose phosphate level. In experiments involving C"-tagged glycerol, it has been claimed that bull sperm can complete the oxidation to C^'*02 under anaerobic condi- tions (O'Dell, Flipse and Almquist, 1956), a point which requires confirmation. The glycerol moiety of the seminal constituent, glycerylphosphorylcholine, apparently is not made available to the sperm for respira- tory activity (Mann and White, 1957). In the early work on phospholipid oxi- dation, it was concluded that endogenous reserves are readily utilized and that egg phospholipid can serve as an exogenous source of energy (Lardy and Phillips, 1941). This finding is supported by the study of Crawford, Flipse and Almquist (1956) who determined the uptake by bull spermatozoa of P'^--labeled egg phospho- lipid. Bomstein and Steberl (1957), on the other hand, found a negligible decrease in intracellular phospholipid and an inappreci- able utilization of exogenous lecithin during incubation of well washed preparations of bull sperm. Recent re-analysis of the nature of the lipids in ram sperm indicates that 55 to 60 per cent is in the form of choline-based acetal phospholipid or plasmalogen (Lo- vern, Olley, Hartree and ]\Iann, 1957). BIOLOGY OF SPERMATOZOA 761 Although this material can he oxidized hy sperm with an R.Q. of about 0.71, there is no detectable change in lij^d phosphorus (Har- tree and Mann, 1959) ; rather, it is the fatty acid residue which is oxidized. Whatever the precise composition and nature of the intracellular oxidizable reserves, the supply nuist be fairly copious and the utilization efficient; some 20 years ago Moore and Mayer (1941) showed that ram sperm can remain motile in neutralized seminal j^lasma for 20 hours or more after the sugar, and presumably other exogenous stores, are ex- iiausted (see Lardy, Winchester and Phil- lips, 1945). The details of lipid oxidation in spermatozoa have not been elaborated, but it is assumed that, as in other tissues, the fatty acid residues react with acetyl-Co- enzyme A and enter the tricarboxylic acid cycle to be ultimately oxidized to carbon di- oxide and water. Earlier work had established that the addition of amino acids, particularly gly- cine, to suspensions of fowl or bull sperm increases many fold the duration of motility and in fowl sperm stimulates oxygen con- sumption as well (Lorenz and Tyler, 1951; Tyler and Tanabe, 1952). No utilization of the amino acids was detectable and the phe- nomenon was interpreted on a basis of the chelation of heavy metal ions, such as occurs, for example, with ethylenediamine- tetraacetate (Versene) (Tyler and Roths- child, 1951). More recent experiments in- volving the use of C^'*-labeled glycine have shown that this amino acid is actually taken up and metabolized by sperm of the bull, without, however, increasing oxygen con- sumption (Flipse, 1956; Flipse and Alm- ([uist, 1956; Flipse and Benson, 1957). (ilucose depressed but did not eliminate, the utilization of glycine; on the other hand, the addition of glycine had little or no effect on the utilization of glucose (Flipse, 1958). The principal pathway of glycine catabolism in sperm seems to involve gly- oxylate, formate, and carbon dioxide. This is similar to the scheme of glycine oxidation in rat liver and kidney (Nakada and Wein- house, 1953). Certain other amino acids, namely phenylalanine, tryptophan, and ty- i-osine, also are metabolized by sperm of the bull and ram by a process of oxidative deamination catalyzed by the enzyme, l- amino acid oxidase (Tosic, 1947, 1951). Hydrogen peroxide is produced in this re- action and is toxic unless eliminated by catalase (Tosic and Walton, 1950). Thus it is clear that certain amino acids are oxi- dized by sperm, but the significance of these reactions to the total energy-producing metabolic processes of the cells cannot be regarded as great. D. EPIDmVMAL SPERM AND METABOLIC REGULATION Striking differences have been claimed for the metabolic behavior, in vitro, of bull sjx'rm, from different segments of the epi- didymis, suggestive of metabolic regula- tion in relation to sperm maturation in the male genital tract (Henle and Zittle, 1942). These differences are manifested by lower rates of endogenous respiration and aerobic glycolysis, and a higher rate of anaerobic glycolysis, by epididymal sperm as com- pared with the rates shown by washed sperm of semen (Lardy, Hansen and Phillips, 1945; Lardy, 1952). Inasmuch as the motil- ity of the spermatozoa from both sources is essentially similar, such metabolic be- havior indicates a higher biochemical effi- ciency of the epididymal sperm. One can indeed demonstrate an inhibition of gly- colysis by oxygen (the Pasteur effect) in epididymal sperm which is less readily dis- played by washed seminal sperm. In a search for the cause of these differ- ences. Lardy found evidence for a so-called metabolic regulator which is present in a bound or inactive form in epididymal sperm and which is released or becomes active at the time of ejaculation (Lardy, Ghosh and Plaut, 1949). The action of the regulator was thus considered to increase respiration and aerobic glycolysis to levels character- istic of semen. This regulating activity was tentatively identified with a sulfur-contain- ing component extractable from semen and from testicular tissue; its action w^as found to be similar to that of cysteine and reduced glutathione (Lardy and Ghosh, 1952; Mann, 1954). Relatively little work has since been done to identify further the metabolic reg- ulator or to demonstrate a similar agent in other species of sperm. -62 SPERM, OVA, AND PREGNANCY Further study would be desirable to dem- onstrate whether bull semen contains a spe- cific metabolic substance which might ac- count for these effects, or whether, on the other hand, the changes noted are part of a more generally applicable type of cell regulation. For example, both the low rate of endogenous respiration and the Pasteur effect, characteristic of epididymal sperm, indicate an efficient phosphorylating sys- tem; the metabolism of seminal sperm, on the other hand, suggests that uncoupling of respiration and phosphorylation may have occurred {cf. Bomstein and Steberl, 1959). The similarity of action of the sperm metabolic regulator and dinitrophenol, a known uncoupling agent, further supports this interpretation (Johnson and Lardy, 1950; Lardy, 1953). Lehninger (1955) has stressed the relationship between uncou- pling and the inhibition of the Pasteur effect in other (mitochondrial) metabolic systems. A general type of metabolic regulation such as this, rather than a system unique to one type of cell, might account for some of the apparent discrepancies reported by different investigators in their studies of mammalian gametes (Melrose and Terner, 1951). White (1960), for example, working with ram sperm has failed to confirm the work of Lardy and associates; he found no significant difference in oxygen uptake or in fructolysis whether the sperm were from the epididymis or from the ejaculate. At first glance this seems to represent a marked metabolic difference in the sperm of closely related animals; considering, however, the delicate balance of cell regulation at the metabolic level (for general discussion, see Krebs, 1957; Packer and Gatt, 1959) the variation is not necessarily profound. The striking differences shown by Dott (1959) in the epididymal sperm of 5 species of do- mestic mammals may also represent subtle effects of metabolic control rather than overt manifestations of fundamentally dif- ferent systems of cell metabolism. He found that the epididymal sperm of the bull, ram, and rabbit are activated, in vitro, either by oxygen or by fructose under anaerobic con- ditions; boar sperm, however, apparently require oxygen, and stallion sperm require fructose, to initiate motilitv. Once stimu- lated, boar sperm glycolyze hexose freely, indicating perhaps that the action of the oxygen is to metabolize an intracellular in- hibitor of some process necessary for motil- ity (Dott, 1959). The response of stallion sperm suggests that the main source of energy is derived from aerobic glycolysis and, further, that endogenous oxidative reserves are scant or that the respiratory processes are inhibited at some critical point. The oxygen uptake of seminal sperm from the stallion is generally low. In cer- tain features this situation in stallion sperm corresponds to the metal)olic behavior of human seminal sperm. E. HUMAN SPERM METABOLISM Princij^ally through the investigations of MacLeod (1941-1946), the metabolic ac- tivities of sperm in the human ejaculate are generally considered to present a rather unique picture. Human sperm show a high rate of anaerobic glycolysis which is only slightly depressed by oxygen; the rate of oxygen consumption in the presence of glucose is extremely low — according to Terner (1960j, about one tenth that of bull sperm. When hexose is replaced by any one of a number of amino or fatty acids, sperm motility is gradually lost; it is not known, however, to what extent these exogenous substances do or do not penetrate the cell. Of the nonglycolyzable substrates em- ployed, only succinate stimulated oxygen consumption, and this reaction was accom- panied, when glucose was present, by a 40 per cent reduction in lactate production (MacLeod, 1946). MacLeod further claimed that oxygen, even at low tensions, is detri- mental to human sperm suspended in Ringer glucose; after several hours at 38°C., aerobic motility is seriously impaired. Motility is also suppressed by glycolytic inhibitors (iodoacetate and fluoride), but is claimed to be unaffected by respiratory poisons (cyanide, azide, and carbon monoxide). These considerations led MacLeod to the conclusion that human sperm rely entirely on the energy of glycolysis for motility and are unable to utilize effectively oxidizable substrate, despite the fact that the cells contain the main components of the cyto- clirome system and tricarboxylic acid cycle. BIOLOGY OF SPERMATOZOA 76a The I'c'latively high glycolytic activity of Imiuaii sperm was compared by MacLeod 1 1942) to the metabolism of certain types of tumor cells (see Warburg, 1956a, b). The apparent toxicity of oxygen on human sperm in vitro was attributed to the production of hydrogen peroxide, inas- much as the effect could be eliminated by the addition of catalase. The enhanced res- piration induced by succinate, noted above, was also found to be accompanied by an increase in HoOo formation. As a possible mechanism of peroxide formation, the auto- oxidation of a flavo-like compound was suggested (MacLeod, 1943b, 1946). These investigations on human sperm cmpiiasize the preferential utilization of glycolytic substrates, under the conditions of the experiments. The relative failure, however, of respiratory substrates to sup- port motility might well bear further scru- tiny. This is particularly true in light of the rapid oxidation of succinate, as shown by MacLeod, and the recent report of Ter- ner ( 1960) that saline suspensions of human sperm oxidize both pyruvate and acetate, as shown by C^^Oo production from pyru- ger, 1959; Hacker, 1959). In human sperm, however, the rates of oxidative respiration and phosphorylation are low and appear to be initially metabolically suppressed. Thus the oxidative inhibition is not induced by high glycolytic activity itself (Crabtree effect), but rather, glycolysis is favored by the previous suppression of respiration. The inhibition of oxidation, in turn, can be attributed, if MacLeod is correct, to the production of toxic amounts of hydrogen peroxide, and this seems to be the relatively unique feature of human sperm metabolism. A plausible explanation for both the source of the peroxide and the failure of respiration and oxidative phosphorylation can be formulated following the suggestion by MacLeod (1942). Thus, it is characteristic of flavoi)rotein (FAD) that, as a "pace- maker" in the oxidative chain (Krebs, 1957), it can either transfer hydrogen atoms from reduced diphosphopyridine nucleotide (DPNH + ) to the cytochrome system or, by auto-oxidation, noncatalytically com- bine with molecular oxygen to form hydro- gen peroxide (Fruton and Simmonds, 1958) (see schema). vate-2-C^'* and acetate-1-C^^. Dinitrophenol stimulated C^'*02 production from both glu- cose-C^-* and pyruvate-2-C^*. The peculiar metabolic behavior of hu- man sperm may be partially clarified l)y reference to the principles of intracellular regulation and alternative metabolic path- ways, characteristic of other cellular and subcellular systems. Of the two main types of energy-producing pathways, which in a sense are normally in competition (Krebs, 1957; Racker and Gatt, 1959 (, the process of glycolytic phosphorylation in human s])erm dominates oxidative phosphorylation. This imbalance could be brought about by the unequal distribution of such rate-limit- ing substances as ADP or inorganic phos- phorus (for general discussion, see Lehnin- Unlike most respiring cells which follow the first of these alternative pathways, hu- man sperm seem to be shunted off into the nonphosphorylative peroxide-producing route. Succinate is known to bypass DPN and to donate hydrogen directly to FAD (Krebs, 1957). As previously mentioned, in human sperm succinate causes increases in both oxygen uptake and peroxide formation and a decrease in lactic acid accumulation (MacLeod, 1946). But whether this repre- sents a shift from glycolytic to oxidative pathways or merely an inhibition of gly- colysis, possibly by the poisoning of sulf- hydryl-containing enzymes by excessive amounts of i)eroxide (MacLeod, 1951), is not known. Speculative as these interpretations con- 764 SPERM, OVA, AND PREGNANCY cerning human sperm may be, they have some merit. New avenues of investigation are opened by a broader approach. More- over, some advantage is gained by attempts to relate certain aspects of the apparently exotic behavior of human sperm to the meta- bolic patterns and principles common to other mammalian tissues. Many issues are yet to be resolved, including the question of the utilization of oxidative substrates vis-d- vis their jiermeability, and the recently announced difference in sensitivity of hu- man sperm to endogenous versus exogenous hydrogen jieroxide (Wales, White, and Lamond, 1959 ». Tests might be applied to determine whether peroxide is produced in accordance with the scheme noted above or whether it may arise from endogenous ni- trogenous sources, comparable to its forma- tion from exogenous aromatic amino acids, as previously noted (Tosic, 1947; VanDe- mark, Salisbury and Bratton, 1949; Tosic and W^alton, 19501. F. METABOLIC-THERMODVX.\MIC INTERRELATIONS Underlying much of the above discussion are many quantitative data pertaining to the metabolic and thermodynamic proper- ties of sperm. Rates of oxygen consumption TABLE 13.12 Vital statistics of hull spermatozoa (Data obtained in buffered saline, 37°C.; calcu- lations based on free-energy change of hydrolysis of —8 kcal. per mole of adenosine triphosphate.) Anaerobic fructolysis (Mann, 1954) 1.7 mg./lO' sperm/hr. Energy liberated 6.27 X 10-6 erg/sperm /sec. Energy trapped as ATP 1.76 X 10-6 erg/sperm/sec. Endogenous oxygen uptake (Lardy, 1953) 200 fi\./W sperm/hr. 1000 meal./ 10' sperm/hr. 1.5 X IQ-s erg/sperm/sec. Anaerobic heat production (Clarke and Rothschild, 1957) 220 nical./109 sperm/hr. 2.55 X 10-6 erg/sperm/sec. ATP phosphorus liberated aerobically* (Nelson, 1954, 1958b) 21 Mgm. P/mg. sperm N/min. 2.5 X 10-'9m ATP/sperm /sec. 2 X 10-12 meal. /sperm /sec. 8.4 X 10-8 erg/sperm/sec. Energy required for motility (Nelson, 1958b) 3.15 X 10-8 erg/speim/sec. (Rothschild, 1959) 2.11 X 10-' erg/sperm/sec. * Based on fragmented cells and expressed as net result of balance between hydrolysis and syn- thesis of ATP. and of fructolysis have been determined for sperm of a wide variety of species (Mann, 1954). Values have also been obtained for heat production (Bertaud and Probine, 1956; Clarke and Rothschild, 1957; Roths- child, 1959), ATP hydrolysis, and the en- ergy requirements for flagellar movement. Some of these properties for one species are tentatively summarized in a table of vital statistics for bull spermatozoa (Table 13.12). Expressed on a per sperm basis, the energy, in ergs, calculated for substrate utilization and heat production indicate a wide thermodynamic safety factor in the balance sheet between energy generated and that required. In Rothschild's exacting study (1959) in which he has demonstrated the changes in sperm heat production with variations in environmental factors, including pH, tonic- ity, and centrifugation, attention is drawn to the narrow margin between the free- energy change of anaeorbic glycolysis which is associated with ATP synthesis and the energy expenditure involved in flagellation. The data suggest that in bull sperm under anaerobic conditions the rate of ATP syn- thesis does not keep pace with that of ATP hydrolysis. Although adequate data are available for the ATP-splitting activity of sperm frag- ments and siierni extracts (see Nelson, 1954; Burnasheva, 1958), the rate of ATP hy- drolysis in whole sperm is difficult to assess, inasmuch as the value of inorganic phos- phate liberated is the net result of hydrol- ysis over synthesis or the phosphorylation of ADP. This is clearly indicated in Table 13.12, in which the energy from ATP-split- ting is seen to be insufficient for the energy requirements of movement. This procedural quandary was noted by Lardy, Hansen and Phillips (1945) who demonstrated in aero- bic suspensions of bull sperm an increase in nucleotide-phosphate release in the presence of cyanide, an inhibitor of phosphorylation processes. G. BIOSYNTHETIC ACTIVITY Although spermatozoa are generally re- garded as fully differentiated by the time they reach the epididymis, some questions have arisen with respect to their biosyn- BIOLOGY OF SPERMATOZOA 765 thetic ability even after ejaculation. Such metabolic cofactors as ATP, for example, are most certainly synthesized (at least from ADP), at the expense of organic sub- strates, throughout the motile life span. More complex substances may also be synthesized. Hakim (1959) has reported that polynucleotide phosphorylases can be extracted from human sperm which, when incubated with nucleotide phosphates, cause the formation of dinucleotides, as determined chromatographically. Thus, for example, a mixture of ADP and guanosine diphosphate (GDP), in the presence of suitable enzyme, forms some ADP-GDP. In another type of study employing intact bull sperm, Bishop and Lovelock indicated that C^*-labeled acetate is incorporated into fatty acid (see Austin and Bishop, 1957). The possibility of jirotein synthesis by sperm was suggested by Bhargava (1957), who reported the incorporation of labeled amino acids into the protein fraction of bull spermatozoa as assayed by radioactivity counting. These conclusions have since been contradicted by Martin and Brachet (1959) who suggest, on a basis of autoradio- graphic data, that the uptake and synthesis can be attributed to cellular components other than to the sperm in the sample. This finding falls more nearly in line with the general conclusion that RNA, essential for protein synthesis, is absent from mature sperm or is present in only very small amounts (Brachet, 1933; Friedlaender and Frasei-, 1952; Leuchtenberger, Leuchten- berger, Vendrely and Vendrely, 1952; Mauritzen, Roy and Stedman, 1952). In this connection it is of interest to recall the observations of Wu, McKenzie, Fang and Butts (1959) on the contrasting metabolic capacities of testicular and seminal bull sperm. Relatively clean preparations of spermatozoa expressed from incised testis, but not sperm from the ejaculate, can oxi- dize glucose by way of the hexose mono- phosphate shunt, thereby supplying a source of ribosc which is available for RNA in the cai'lici' stages of sperm differentiation. IX. Sperm Flagellation The characteristics and mechanics of s]ierm movement are discussed in con- siderable detail in several recent reviews dealing with both invertebrate and verte- brate material (Gray, 1953, 1955, 1958; Gray and Hancock, 1955; Bishop, 1961). Sperm motility, closely related to muscular contraction, on the one hand, and to general flagellar and ciliary activity, on the other, represents an important physiologic process with implications l)eyond the specific be- havior of the gametes. For the present con- text, however, only certain more general aspects of the problem are pertinent. By the tiirn of the century the signifi- cance of the flagellum for sperm motility was well established (see Wilson, 1925). As early as 1898, Engelmann had succeeded in cutting off the tails of frog spermatozoa to find that the flagella continued to move if the separations were made close to the heads. Ciaccio (1899) and particularly Koltzoff (1903) discussed the elementary mechanisms of flagellation and went so far as to compare the process with contraction of muscle. In 1911, Heidenhain postulated that the chemical energy rec}uired for mo- tility must be distributed throughout the flagellum, a concept generally conceded to- day (Gray, 1958). Ballowitz (1888, 1908) emphasized the significance of the longi- tudinal fibrils of the axial bundle for mo- tility. In the history of sperm biology these two decades, immediately before and after 1900, constitute the "Age of Flagellation." A. WAVE PATTERNS Largely through the efforts of Sir James Gray (1953-1958) many details of the proc- ess of flagellation have been recorded, most attention having been focused on the sperm of the sea urchin and bull. Although there exists much natural variation among species in the overt characteristics of the phenome- non, basically the same fundamental mech- anism is involved. Propagated waves originate at the base of the flagellum and progress distally toward the tip. The major bending-couple is two-dimensional, but as it sweeps distally it is accompanied by, or is converted into, a three-dimensional wave which gives the sperm a helical spin about the axis of forward progression (Gray, 1955, 1958). In squid sperm under experimental conditions the two components of move- ment, lateral vibration and rotation, can SPERM, OVA, AND PREGNANCY be separated and analyzed individually (Bishop, 1958f). Wave co-ordination in- volves not only the initiation of the beat, which may be a function of the basal gran- ule, but also the propagation of the conduc- tion wave along the flagellum. The velocity of wave propagation has been calculated for bull sperm to be 600 to 700 /x per sec. (Bishop, 1961). The frequency of beat, stroboscopically determined, is on the order of 20 per sec. for the bull and 15 per sec. for man (Ritchie, 1950; Rothschild, 1953; Rikmenspoel, 1957; Zorgniotti, Hotchkiss and Wall, 1958). Wave amplitude in bull sperm is 8 to 10 fi, about 20 times the diameter of the tail. These values are at best only first approxi- mations, because wave characteristics change not only with progression along the length of the flagellum, but also with en- vironmental conditions such as temperature and viscosity of the medium. B. SPERM VELOCITY Many attempts have been made to de- termine the speed of sperm travel (see Bishop, 1961). As a general rule, the methods used give data for translatory rather than absolute velocities (Table 13.13). Speeds up to 350 /* per sec. have been recorded for bull sperm. Rikmenspoel (1957) has presented an extensive correla- tion of the variations in bull sperm velocity with changes in frequency and amplitude of wave formation and with alterations in viscosity and temperature of the environ- ment. The effect of current flow on stallion sperm velocity was demonstrated by Yam- TABLE 13.13 Translatory velocities of mammalian spermatozoa, in vitro (Buffered saline or saline-plasma, 37°C.) Species Average Velocity Reference H per sec. Man 23 Adolphi, 1905 14 Botella Llusia et al., 1957 Horse 87 Yamane and Ito, 1932 Ram 80 Phillips and Andrews, 1937 Bull 123 Rothschild, 1953 114 Moeller and VanDemark, 1955 105 Rikmenspoel, 1957 94 Gray, 1958 ane and Ito ( 1932 ) . They found that sperm orient themselves by rheotaxis, or are oriented physically, against a current, and that up to a limit, as the opposing flow is increased, the speed of movement also in- creases. When the opposing current flow was varied from to 20 ju. per sec, sperm velocity increased from 87 to 107 yu, per sec. Under the conditions of the experiment, the results might be attributable merely to the direction given the sperm, thereby reducing the randomness of movement. Nevertheless, these findings may have some bearing on the problem of active sperm transport in vivo, where ciliary or other currents play a role. From a comparison of the data on sperm velocities (Table 13.13) and those previously cited on sperm transport, the conclusion is inescapable that in most mam- mals, migration is not dependent on active swinnning movements alone. C. HYDRODYNAMICS Initiated by the theoretical speculations and mathematical derivations of Sir Geof- frey Taylor (1952), a considerable body of information has accrued which permits an evaluation of the mechanics and forces in- volved in si)erm movement (Gray and Han- cock, 1953; Hancock, 1953; Rothschild, 1953; Machin, 1958; Xelson, 1958b; Carl- son, 1959). From these considerations it is clear that a spiral or three-dimensional pattern of flagellation is more eflficient than a two-dimensional wave motion; Taylor calculates that the resulting sperm velocity in the former case may be up to twice as great, depending on the configuration of the sperm cell, for a given amount of energy ex- pended. Employing these mathematical der- ivations and experimental data for wave characteristics such as frequency and ampli- tude, Gray and Hancock (1953) found good agreement in calculated and observed values for the velocity of sea-urchin sperm of about 190 fx per sec. The power output required to effect this activity has also been calculated. For sea urchin sperm, Carlson (1959) obtained a value of about 3 X 10~" erg per sec. per sperm. Compa- rable figures for bull sperm have been esti- mated as ranging from 2xl0~^to3x 10~^ erg })er sec. per sperm, depending on BIOLOGY OF SPERMATOZOA 767 certain theoretical assumptions underlying the analysis (Rothschild, 1953, 1959; Nel- son, 1958b). At the present time, such information may seem limited in its application to problems of sperm physiology in relation to the reproductive process as a whole. From a broad point of view, however, it obviously affords a biophysical measure of what the sperm can accomplish, and constitutes a link between the metabolic energy produced, on the one hand, and the work performed during activity, on the other (Table 13.12). D. MECHANISM OF MOTILITY Speculation concerning the physical basis for activity of cilia and, by implication, flagella has a long tradition (Grant, 1833; Ankermann, 1857; Schafer, 1904). Of the various theories proposed, the only one to persist is that which conceives of the flagel- lum as a diminutive contractile system (Ciaccio, 1899; Koltzoff, 1903; Ballowitz, 1908; Heidenhain, 1911). Other types of biochemical systems can be imagined to ac- count for sperm movement, but the evi- dence, particularly of the past few years, favors the concept of a contractile protein mechanism, generally associated with the fibrillar system of the tail (see Bishop, 1961). Brief mention has been made of certain salient features of the motility process. It is clear that ATP is essential for sperm activity, as it is for many other physiologic processes reciuiring energy. A constant suji- ply of ATP is maintained by the glycolytic and/or oxidative processes of metabolism (Engelhardt, 1958 j. Certain experiments have indicated that extractable ATP is not significantly depleted during sperm activity (Hultin, 1958), thus further supporting the view that resynthesis of the nucleotide ac- companies its dephosphorylation. The pres- ence and general localization of ATPase in the flagellum have been noted; by its spe- cific action on ATP as substrate, chemical energy associated with ''high-energy" phos- phate bonds is liberated. The ATP-ATPase type of enzyme sys- tem is widely distributed throughout the animal and plant kingdoms; it has been extensively studied and closely identified Avith the contractile svstem of muscle. It was of major significance that the con- tractile protein itself, myosin, was found to possess the ATP-splitting activity which leads to contraction (Engelhardt and Lyu- bimova, 1939). ATPases thus represent the essential link between the biochemical and mechanical events (Engelhardt, 1958). Myosin alone is incapable of shortening, but when combined with actin, the complex un- dergoes contraction in the presence of ATP. This can be readily demonstrated in simpli- fied muscle systems such as glycerinatetl fiber models (Szent-Gyorgyi, 1949; Varga, 1950) or actomyosin thread preparations (Portzehl and Weber, 1950). As a result of their previous studies of the biochemistry of muscle, and the overt similarities of muscle contraction and sperm flagellation, Engelhardt and his as- sociates undertook a detailed study of mo- tility of bull sjierm. They extracted from sperm cell homogenates a partially purified l)rotein which showed ATPase activity and was tentatively called "spermosin" (Engel- hardt, 1946). Refinements in extraction and luirification procedures since that time have resulted in tlie jireparation of a product with many of the properties of myosin, isolated by similar techniques from muscle. Mean- while, work was being reported from several other laboratories confirming the occurrence of ATPase in sperm and sperm tail prepa- rations of a variety of species (Felix, Fischer, Krekels and Mohr, 1951 ; Nelson, 1954, 1955b; Utida, Maruyama and Nanao, 1956; Bishop, 1958a; Tibbs, 1959). Although not all of these preparations are unequivo- cally associated with contractile protein or contractile protein alone, the evidence seems clear that the sperm tail possesses high ATPase activity. More recent publications from Engel- hardt's institute indicate that material of a high degree of purity can be extracted from bull sperm tails which probably is the con- tractile protein, "spermosin," responsible for movement (Engelhardt and Burnasheva, 1957; Burnasheva, 1958; Engelhardt, 1958). Ai^iiroximately 80 per cent of the ATPase activity of the whole sperm is concentrated in the tail fraction, isolated by centrifuga- tion. Substrate specificity and cationic re- quirements of the enzyme have led to the conclusion that it is very similar to muscle •68 SPERM, OVA, AND PREGNANCY I On 9 ACTOMYOSIN ACTOSPERMOSIN O (D (D ® © (5) ©0 © F-Actin (g) Spermosin @ Spermosin + F-Actin (ratio I2:i) @ Spermosin + F-Actin + AT P ( 4 23 x 10"" M) © Myosin © Myosin + F-Actin (ratio 2:1) @ Myosin + F-Actin + AT P (4 23 x 10"" M) Fk;. 13.18. Complex-formation and viscosity change upon addition of adenosine triphosphate (ATP) in system composed of contractile protein extracted from bull sperm and actin from rabbit muscle. The response of muscle actomyosin is shown at the right for comparison. (From 8. A. Burnasheva, Biokhimiia, 23, 558-563. 1958.) myosin. Further similarity is indicated by the chiim that "spermosin" can combine with actin, extracted from muscle, to form an "actospermosin" complex (Burnasheva, 1958). This complex undergoes viscosity changes similar to those shown by actomyo- sin, upon the addition of ATP (Fig. 13.18). It is to be noted that, thus far, physical methods have not been applied to the study of the protein isolated from sperm by these investigators. Attempts to extract an actin- like protein from bull sperm have thus far proved unsuccessful. Whether the con- tractile system of sperm is eventually re- solved as a single component system, as suggested by Burnasheva, or a double com- ponent system as in muscle, remains for further investigation to demonstrate. Although these extraction experiments give strong evidence in favor of a myosin- like protein in sperm flagella, the picture is far from complete. Rather striking dif- ferences have been shown, for example, in the response of fish sperm ATPase to cation concentration when compared with the be- havior of muscle ATPase (Tibbs, 1959). Moreover, a comparison of structural details of the sperm flagellum before and after KCl-extraction procedures fails to indicate the source of the extractable protein; in- deed, very little change can be detected in electron micrographs of mammalian sperm subjected to such treatment (Bishop, 1961). The motile mechanism of spermatozoa has been investigated also by the prepara- tion and reactivation of cell models, com- parable to the glycerinated models of mus- cle. Hoffmann-Berling (1954, 1955, 1959) first accomplished this with sperm of the locust, Tachijcines; as in the case of muscle models, glycerol-extracted sperm were re- activated by treatment with ATP at suit- able concentration. This phenomenon has since been demonstrated with sjierm of the squid, Loligo, and of several species of mammals (Bishop, 1958b, e; Bishop and Hoffmann-Berling, 1959). The methods of extraction, ATP concentrations, ionic re- quirements, and response to sulfhydryl in- hibitors are roughly similar to those appli- cable to muscle models. The general nature of the response to ATP, however, is strik- ingly different in that the addition of the nucleotide initiates flagellation which may continue, in bull sperm for example, for as long as 2 hours (Bishop and Hoffmann- Berling, 1959). Apparently, contraction-re- laxation cycles are induced in the models which in frequency and amplitude are simi- lar to those of normal fresh sperm. How- ever, as a result of the complete loss of permeability and co-ordination properties of the flagellar models, wave propagation along the flagellum fails to occur and for- ward movement is insignificant. Among other interesting features of these virtually dead but ATP-reactivated sperm models is the fact that they can be reversibly im- mobilized by treatment with the Marsh- Bendall (relaxing) factor, prepared from rabbit muscle according to the method of Portzehl (Bishop, 1958c). Moreover, the models are capable of flagellation against a force inijiosed by increasing the viscosity BIOLOGY OF SPERMATOZOA 7(>9 of the surrounding medium (Bishop, 1958f ; Bishop and Hoffmann-Berling, 1959). Such biochemical approaches as these suggest that the molecular basis of sperm motility is very similar to that of the con- tractile protein system of muscle. The identification of this system in the sperm is less securely established, but it is assumed to be localized in the longitudinal fibrils of the flagellum. The universality of the 2 X 9 + 2 pattern of filaments seems to de- mand that considerable significance be at- tached to them. The filaments appear on chemical grounds to resemble a fibrous pro- tein which could be contractile in nature. Both solubility data (Schmitt, 1944; Brad- field, 1955) and the results of proteolytic digestion of sperm flagella (Hodge, 1949; Grigg and Hodge, 1949) support this view. The positive form birefringence of sperm tails further indicates an orderly arrange- ment of highly asymmetric structural units which may indeed be the components of the longitudinal fibrils themselves (Schmitt, 1944). X-ray diffraction measurements also suggest a high degree of organization with regular spacing of the structural elements (Lowman and Jensen, 1955). These reports on sperm flagella do not prove that the longitudinal fibrils are contractile protein, but they lend credence to that assumption. Excellent supporting evidence, moreover, is that obtained by Astbury and Weibull ( 1949) in their study of an entirely different type of flagellar system, the isolated flagella of bacteria. These investigators concluded that the x-ray diffraction pattern of flagellar preparations is characteristic of the k-m-e-f group of fibrous proteins and, further, that both the a- and ^-configurations can be demonstrated in unstretched and stretched fibrillar preparations. Astbury and Saha (1953) refer to these bacterial flagella as "monomolecular muscles." It is to be stressed that the longitudinal filaments of spermatozoa show no consistent cross-striation or periodicity which might be compared with that of the striated mus- cle fibril (Bishop, 1961). In human sperm prepared for electron micrography, Schultz- Larsen (1958) found an indication of peri- odicity with intervals of about 20 A, but this phenomenon is irregular and remains to l)e confirmed. Cross-striations at inter- vals of 500 to 700 A were found in Arbacia sperm by Harvey and Anderson (1943), but these have been interpreted as aggregation artifacts rather than as true components of structural periodicity. Whereas the physical basis for sperm mo- tility is thus fairly well established in a con- tractile protein system possibly associated with the flagellar filaments, no fully satis- factory theory of the operation of the mech- anism has been advanced. The suggestion of Bradfield (1955) that the cylindrically arranged i^eripheral fibrils fire off progres- sive contraction waves in successive order was put forth hypothetically to describe a plausible but untested description of flagel- lation. Afzelius (1959) proposes, on the basis of ultrastructural differences in mem- bers of the pairs of peripheral fibrils, that the mechanism may function along the lines of the interdigitating-fibril scheme de- scribed for striated muscle by Huxley and Hanson (1954, 1957). Other more conserva- tive speculations have been suggested (c/. Bishop, 1958f; Gray, 1958; Nelson, 1959), and final analysis of the precise nature of the contraction-relaxation waves and their synchronous operation in the sperm flagel- lum must await further experimental inno- vation and investigation. A striking gap currently persists between the ultrastruc- tural interpretations of spermatozoa and the molecular characteristics associated with motility. X. Fertilizing Capacity of Treated Spermatozoa A wide range of environmental factors has been employed in the study of mam- malian sperm responses, dating from the very earliest investigations of the gametes (van Leeuwenhoek, 1678). Three principal criteria have served as end points in the in- vestigations of sperm physiology — motility, metabolism, and fertilizing capacity. Inter- related and interdependent in vivo, any of these properties alone or in combination can be assessed following experimental manipu- lation of the sperm in vitro. The chemical factors known to modify motility and meta- bolic behavior of sperm may be arbitrar- ily grouped roughly as follows: electrolytes including the hydrogen ion, enzymatic in- hibitors, chelating compounds, and a variety 770 SPERM. OVA, AND PREGNANCY of uncoupling agents which include sulf- liyclryl-blocking agents, hormones {e.g., thy- roxine), antibiotics, and surface-active sub- stances (Mann, 1954, 1958bj. Other types of environmental factors which induce pro- found effects on sperm behavior involve di- lution of the cell suspension, temperature changes, ionizing radiation, and certain bio- logic fluids and cell extracts. The action of such agents on sperm mo- tility and metabolic activity, but not neces- sarily on fertilizing capacity, is reviewed in detail elsewhere (Hartman, 1939; Mann. 1954; Bishop, 1961) ; the effect on fertilizhig capacity per se will be briefly presented here. Alteration of the fertility rate by pre- treatment of spermatozoa is, of course, an established procedure. In an extreme sense, this is accomplished by the extension of the life span of sperm for purposes of artificial insemination (Anderson, 1945; Emmens and Blackshaw, 1956; Salisbury, 1957), or, con- versely, the curtailment of survival by spermicidal agents (Mann, 1958b; Jackson, 1959). A. DILUTION OF THE SPERM SUSPENSION Chang (1946a) drew attention to the di- lution effect on mammalian sperm by dem- onstrating that artificial insemination of rabbits was successful with a given number of sperm suspended in a small amount (0.1 ml.) of saline medium, whereas the same number of sperm in a larger volume (1.0 ml.) failed to bring about fertilization. Mann (1954) suggested that the dilution effect in mammalian sperm might in part be the same general type of response as that occurring in invertebrate sperm, in which the phenomenon has been extensively inves- tigated (Gray, 1928; Hayashi, 1945; Roths- child, 1948, 1956a, b; Rothschild and Tuft, 1950; Mohri, 1956a, b). The studies of mamn:ialian sperm by Emmens and Swyer (1948), Blackshaw (1953a), and White (1953) indicate that some essential sub- stance, or substances, is lost during dilution of the sperm suspension. Such loss can be partially counteracted by the addition to the diluent of K+ (Blackshaw, 1953b; White, 1953) or of seminal plasma or cer- tain large molecular compounds (Chang, 1959). The nature of the loss, the protective effect of colloidal substances, and the in- tracellular changes involved in the dilution eft'ect in mammalian sperm are still obscure. The alterations in the sperm are probably not mere physical changes but rather chemi- cal alterations wdiich involve the metabolic state. The dilution phenomenon in inverte- brate spermatozoa, for example, seems to in- volve an activation of the cytochrome sys- tem or other changes in respiratory pattern induced by such factors as pH or copper ions of sea water (Rothschild, 1950, 1956b). Rothschild (1959) has shown an increase in both the initial heat production and [pro- longed heat production of bull sperm diluted 1:3 with balanced saline solution, compared with the heat output of sperm in seminal plasma. B. TEMPERATURE EFFECTS Numerous studies have indicated a direct effect of temperature change on the overt behavior and survival of spermatozoa, but little attention has been directed toward the possible effect on fertilizing capacity of pre- treatment of the gametes. One earlier in- vestigation (Young, 1929c) indicated that exposure of guinea pig sperm in the epididy- mis to 45°C. for 30 minutes reduced the fer- tility rate, and treatment at 47°C. seriously impaired motility; nevertheless, those em- bryos which were produced by females in- seminated with sperm treated at 45 to 46°C. were apparently normal. Hagstrom and Hagstrom (1959) recently demonstrated that the fertilization rate of sea urchins is enhanced by exposure of sperm to either slight increases or decreases in temperature before union of the gametes. The pro- nounced temperature changes to which sperm are exposed during vitrification are of a far different order of magnitude and are surprisingly well tolerated when prop- erly controlled (see Artificial Insemination) . C. IONIZING RADIATION When very severe, irradiation can lead to impairment of motility and metabolism in animal spermatozoa; lower doses induce change in nuclear components with conse- ciuent abnormalities in development. Hert- wig (1911) first demonstrated the paradoxi- cal effect of fertilizing frog eggs with sperm BIOLOGY OF SPERMATOZOA 771 exposed to radium emanations. At lower levels of treatment, abnormal young were produced, increasing in percentage and se- verity with increase in dosage. At high levels of radiation, normal young developed. The latter effect was attributed to the parthcno- genetic development of eggs stimulated by sperm incapable of participating in fertili- zation. This was confirmed by Rugh (1939) who found an increase in embryonic al)nor- mality and death following fertilization by sperm x-irradiated with doses from 15 to 10,000 r; sperm treated with 50,000 r, how- ever, failed to enter the eggs and a high proportion (91 per cent) of the partheno- genetic young were viable. Since parthenogenesis is not readily in- duced in mammals, no such paradoxical effect is to be expected. Impairment of fer- tilization and induction of embryonic ab- normalities have, however, been caused by x-irradiation of sperm in vitro. Irradiation of rabbit and mouse sperm induced changes as manifested by embryonic abnormalities and chromosomal aberrations after fertili- zation of normal eggs (Amoroso and Parkes, 1947; Bruce and Austin, 1956). y-Radiation when administered at doses of 32,000 to 65,000 r from a radiocobalt source depressed the motility of rabbit sperm (Chang, Hunt and Romanoff, 1957). After treatment with these high exposures, the sperm that were able to reach the ova showed little if any impairment in fertilizing cai^acity. How- ever, even at a dosage of 800 r, blastocyst formation was retarded, and at 6500 r it was prevented altogether. Johansson (1946) had reported similar findings in fowl; high levels of x-irradiation (3000 to 12,000 r) reduced motility of sperm, whereas rela- tively low levels (600 to 1200 r) impaired development. The w^ork of Edwards (1954- 1957) on the mouse indicates that irradia- tion, either x-ray or ultraviolet (nonioniz- ing), while permitting fertilization, can render the male gamete incapable of taking part in development. Comparable radiomi- metic effects were obtained by treatment of mouse sperm with either trypaflavine or toluidine blue (Edwards, 1958). The effect of irradiation in mammalian sperm may be similar to that suggested for invertebrate sperm. At certain levels of x- irradiation the fertilizing capacity of sea urchin sperm is reduced, and the cause has been attributed to the formation in the medium of hydrogen peroxide, produced by splitting of water molecules and recombina- tion of free radicals (Evans, 1947). It has also been suggested that stable organic per- oxides, rather than hydrogen peroxide, are formed and that these are toxic to sperm, possibly acting by the oxidation of enzy- matic sulfhydryl groups (Barron, Nelson and Ardao, 1948; Barron and Dickman, 1949; Barron, Flood and Gasvoda, 1949). D. IONIC AXD OSMOTIC EFFECTS Despite a mass of data concerning the action of electrolytes and pH changes on sperm motility, and recently on sperm heat production (Rothschild, 1959), relatively little has been done to assess the fertilizing capacity of pretreated sperm. Although cer- tain ions in excess seem to have unusually detrimental effects on sperm survival, in vitro — for example, calcium, manganese, lithium, and chloride (Lardy and Phillips, 1943; MacLeod, Swan and Aitken, 1949) — of more surprising interest is the general resistance of sperm to nonbalanced saline media (see Bishop, 1961 ). Rabbit sperm, for instance, can tolerate 2.0 per cent NaCl for many hours if brought gradually into the hypertonic medium (Anderson, 1945), and bull sperm retain motility for several hours in isotonic KCl. Determinations of the de- gree to which fertilizing capacity is affected by such treatment might yield very signifi- cant results. Chang and Thorsteinsson (19581)) have made an important beginning with this aim in view. They found that the fertilizing capacity of rabbit sperm is unimpaired by exposure for brief periods (10 to 20 min- utes) before insemination in Krebs-Ringer solutions of one-half or twice isotonic con- centration. Of jiarticular interest was the finding that motility, but not fertility, was dein-essed by treatment with the hypertonic medium ; one can assume that some re- covery occui'red in the female tract. Beyond the limits of this range of tonicity, fertiliz- ing capacity was reduced, as judged by ob- servation of recovered tubal eggs; yet even witli solutions 0.1 oi' 4 times isotonic 772 SPERM. OVA, AND PREGNANCY strength (which completely inhibited mo- tility, in vitro) fertilization occurred, al- though at a low rate. Chang and Thorsteins- son also studied the tolerance of sperm to osmotic variation in relation to simulta- neous changes in pH. In isotonic Krebs- Ringer medium, rabbit sperm withstood short exposure to acid or alkaline condi- tions over a pH range of about 5.6 to 10.0, based on observations of motility and con- ception rate. Under hypo- or hypertonic conditions, however, the upper limit of the pH range tolerated was significantly de- pressed. This work emphasizes once again the unusual resistance or adaptation of the mammalian germ cell to changes in ionic environment. E. EFFECTS OF BIOLOGIC FLUIDS Some effects of certain biologic fluids with which sperm come in contact have been discussed in previous sections. It is clear, for example, that seminal fructose serves the gametes as glycolytic substrate at the time of ejaculation; uterine fluid, or certain of its components, aids in the capacitation phenomenon of sperm during transport through the genital tract. In studies of the effect on fertilizing capacity, sperm have been treated, in vitro, with seminal plasma, urine, normal blood serum, and antisperm serum, as well as with isolated products of the female tract itself. The beneficial effect of seminal plasma as sperm diluent, for example, was demon- strated in the rabbit by Chang (1947b). Tests on 33 rabbits showed the advantage (percentage of fertilized eggs I of homolo- gous plasma over saline when the does were inseminated with a minimal number of sperm. It was subsequently indicated that heterologous plasma from human semen was equally effective when used as a diluent for rabbit sperm (Chang, 1949). Bull seminal plasma, however, was injurious to rabbit sperm and caused a significant reduction in fertilizing capacity. It is not clear whether the favorable action of plasma, when it occurs, is due to a specific factor or set of factors, or whether it is caused by a non- specific action such as chelation by the amino acid or polypeptide components pres- ent. The role of chelating substances in extending the motility, metabolism, and fertilizing capacity of sperm has been dem- onstrated in several invertebrate and verte- brate species (Lorenz and Tyler, 1951; Tyler and Rothschild, 1951; Tyler and Tanabe, 1952; Tyler, 1953; Rothschild and Tyler, 1954). Such an effect was indeed sug- gested by the work of Chang, since prepara- tions of dead heterologous sperm were as effective as seminal plasma in augmenting fertility in the rabbit (Chang, 1949). These findings may have some bearing on those cases in which, it has been claimed, resus- pension of human sperm in foreign plasma improves motility and fertilizing capacity (see Rozin, 1958). A possible detrimental effect of seminal plasma on the fertilizing capacity of sperm was indicated by the demonstration of Chang (1957) that plasma destroys or counteracts the capacitation re- sponse of sperm within the rabbit genital tract (see above). Although it is generally believed that urine is harmful to spermatozoa, Chang and Thorsteinsson (1958a) have shown that rabbit sperm tolerate exposure to 50 per cent urine for 10 to 15 minutes with no disturbance in conception rate. A urine con- centration of 75 per cent can seriously im- pair sperm motility, in vitro, but even this treatment does not prevent fertilization when these same sperm are artificially in- seminated into receptive does. As has long been known, normal blood serum sometimes agglutinates spermatozoa, usually in a head-to-head type of aggrega- tion. This is regarded as a nonspecific ag- glutination response, and the serum factor which brings it about can be destroyed or effectively reduced by heating to approxi- mately 60°C. In an investigation of the effects of sera on homologous and heterolo- gous sperm, Chang (1947a) demonstrated a complement-like agglutinating component which generally was toxic to the sperm of both its own and of other species; the one exception was the factor in human serum which was ineffective on human sperm. The substance in rabbit serum was found chemi- cally unstable, thermolabile, and nondialyz- able. Such an agent was detectable in the sera of man, bull, rabbit, guinea pig, and rat; very little is known, however, concern- BIOLOGY OF SPERMATOZOA 773 ing its origin or possible role, if any, during normal reproductive processes. The effects of antiserum on the gametes have been discussed in a previous section. It will be recalled that the tyi)ical cell- specific agglutination and inniiobilization responses can render the sperm incapable of fertilization. Further, the experiments of Kiddy, Stone and Casida (1959) suggest a differential effect of treatment with high and low concentrations of antisperm serum. The former impairs fertilizing capacity; the latter results in abnormal development after fertilization (see section on Lnmunologic Problems) . The impendency of fertilization provokes consideration of several types of sperm re- sponses which can profitably be introduced here and further elaborated on in the chap- ter by Blandau. These responses involve interrreactions of the sperm and egg, or egg exudates, and concern such processes as chemotaxis, sperm activation, sperm agglu- tination, and the acrosome reaction. With respect to the occurrence of chemo- taxis, that is, the directed movement toward the egg in response to a chemical gradient from the egg, the evidence relating to ani- mal gametes is essentially negative (Roths- child, 1956b). Many earlier claims for sperm chemotaxis among lower animals (see Hcilbrunn, 1943), and certain recent reports on mammalian species (Hiibner, 1955; Schuster, 1955; Schwartz, Brooks and Zins- ser, 1958), can be readily ascribed to "trap- action," that is, the accumulation of sperm in the vicinity of the egg or egg substance, and not to a nonrandom movement of the k % - '^ ?^B '♦ W^ Fig. 13.19. Fertilizin reaction in nianinial. Agglutination of rabbit .sperm in immediate vicinity of egg collected from rabbit oviduct. Sperm agglutinates form predominantly in head-to-head patterns. (From D. W. Bishop and A. Tvler, J. Exper. Zool., 132, 575-601, 1956.) Fig. 13.20. Electron micrographs of sea iirchiu spermatozoa (llonicentrotus pulclicrriniu.s) : A, control, formalin-fixed in sea water; B, formalin-fixed two seconds after addition of egg water showing breakdown in acrosomal region and extrusion of protoplasmic mass; C, for- malin-fixed 20 seconds after addition of egg water; D, formalin-fixed three minutes after addition of egg water. The agglutination which results from the addition of egg water is re- versed at 2.5 minutes. (Photographs courtesy of J. C. Dan.) 774 BIOLOGY OF SPERMATOZOA spenn toward it. The iihenomenon of chemo- taxis has, however, been established as oc- curring in some primitive ph^nts, such as certain ferns, mosses, and brown algae, and the various attempts to determine the na- ture of the chemical stimulus and the mech- anism of the response have been attended by some success (Pfeffer, 1884; Shibata, 1911; Cook, Elvidge and Heilbron, 1948; Cook and Elvidge, 1951; Rothschild, 1951b, 1956b; Wilkie, 1954; Brokaw, 1957, 1958a, b). Activation of sperm by homologous eggs and egg exudates has been described in some invertebrate species, and the stimulating ac- tivity has been attributed to the fertilizin (gvnogamone I) present in the egg jelly coat (Lillie, 1919; Tyler, 1948; Rothschild, 1956b). The source of the activator and the specificity of the reaction are, however, somewhat controversial. The increase in motility, when observed, may or may not be accompanied by a substantial enhancement in respiratory activity (Rothschild, 1956b). The species-specific agglutination of in- vertebrate spermatozoa by fertilizin of ho- mologous eggs constituted the keystone of the fertilizin theory advanced by Lillie (1919) to account for the specificity and "cell recognition" inherent in the process of fertilization. The nature of the serologic- like gametic substances — egg fertilizin and sperm antifertilizin — and the role these sub- stances may play in the fertilization process have been extensively studied by Tyler and coworkers (1948-1959). Sperm aggluti- nation by egg exudates has been demon- strated in many species of animals in both the invertebrate and lower vertebrate groups (see Tyler, 1948). The phenomenon is also exhibited by mammalian gametes (Fig. 13.19), among which some degree of species specificity is displayed (Bishop and Tyler, 1956 ) . A current view of the possible signifi- cance of these gametic substances to fertili- zation may be found in the recent review by Tyler (1959). Spermatozoa not only seem to interreact serologically with egg exudates resulting in agglutination and/or loss of fertilizing ca- pacity; they also can be stimulated under some circumstances to undergo morphologic change, most spectacularly characterized by the acrosome reaction (Dan, 1952, 1956; Colwin and Colwin, 1955, 1957). The forci- ble release of material from the sperm head (Fig. 13.20), apparently induced by the presence of egg fertilizin, involves the pro- trusion of a filamentous projection which seems to play a vital, if, as yet obscure, role in the initial stage of the fertilization proc- ess. XI. Conclusion The notation of a conclusion to the Biol- ogy of Spermatozoa seems singularly in- appropriate. Both the intensity and the ex- panse of current research indicate that one is merely taking stock of accumulating data and transient concepts — that in the future lies the answer to most of the ciuestions raised in the pages above. On the one hand, the properties of spermatozoa can be ex- pected to become increasingly clear by our delving more deeply into the nature and ac- tivity of the cell, a fruitful approach in its own right and beneficial to the more practi- cal concerns of fertility, sterility, and ani- mal breeding. On the other hand, the recog- nition of the general characteristics of sperm behavior, movement, metabolism, and sur- vival seems likely to shed brighter light on comparable processes and systems in other cells and tissues, including the nature of cell regulation and adaptation, energy utiliza- tion, aging, and movement inherent in ciliary activity, flagellation, and muscular contraction. If much of the foregoing seems more frag- mentary than complete, more provocative and speculative than dogmatic or resolute, this survey may then serve some purpose. The accomplishments have been many, but even more fascinating developments lie ahead. XII. References Abarbaxfx, a. R. 1946. 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Recovery of attached embryos. . . . 799 B. Egg Culture and Preservation in Vitro 799 C. Intraspecific Egg Transfer 800 D. The Production of Eggs by Superovu- lation " 801 III. Biology of the Mammalian Egg 802 A. Oogenesis 802 B. Growth, Composition, and Size of the Mammalian Egg 807 C. Egg Membranes 811 1. The zona pellucida 811 2. The mucous or "albuminous" layer 815 D. The First Maturation Division ...... 81G E. The Ovulated Egg 817 F. Respiratorv Activity of Mammalian Eggs. . .". : 818 G. Transport of Tubal Ova 819 IV. Fertilization and Implantation 827 A. The Cumulus Oophorus and Sperm Penetration 828 B. The Zona Pellucida and Sperm Pene- tration 832 C. Sperm-Egg Interacting Substances.. 834 D. Sperm Penetration of the Vitelline Membrane 834 E. Fertilization in Vitro 835 F. Fate of the Unfertilized Egg 83(i G. Formation of the Second Polar Body, 837 H. Pronuclei Formation, Syngamy, and First Segmentation Division 838 I. Fate of the Cytoplasmic Components of the Fertilizing Sperm Flagellum. 841 J. Supernumerary Spermatozoa and Polyspermy in Mammalian Ova. . . 844 K. Stages of Development and Location of Eggs 845 L. The Age of the Elgg at the Time of Fertilization 848 M. Implantation 850 N. Spacing and Orientation of Ova in Utero 852 O. Blastocyst Expansion 858 P. Embryo-endomet rial Relationships . . 860 V. References 8(i5 797 I. Introduction In recent years there has been much more intense research activity on the morphology, physiology, and biochemistry of sperma- tozoa and semen of mammals than on their eggs and the fluids forming their environ- ment. The significant increase in the in- vestigations of the male gametes is due largely to stimuli resulting from the neces- sity of perfecting techniques of artificial insemination in domestic animals and of elucidating the problems of infertility and contraception in man. A distinct advantage with respect to investigations of the male is the ready availability and large number of gametes which can be obtained from a single subject. In contrast, the mammalian egg is available in restricted numbers and then only at very specific times in the re- productive cycle. Furthermore, there are very real difficulties in maintaining mam- malian eggs in a normal physiologic state after they have been removed from their usual environment. Even though there have been notable ad- vances in the investigations of the compli- cated physiologic and biochemical mecha- nisms which exist in the development, storage, transport, and syngamy of the gametes since Dr. Carl G. Hartman's erudite discussions of the subject in 1932 and 1939, our understanding of the fundamental prob- lems involved in maintaining the continuous stream of life from generation to generation is still in its infancy. As we proceed 20 years later, it will be clear that the older methods of classical histology have not yet outlived their usefulness. But it will also be ap- parent that many of the advances which have been made, particularly in the in- vestigation of mammalian materials, can 798 SPERM, OVA, AND PREGNANCY be attributed largely to the use of new and improved techniques for the collection and study of living gametes and embryos. For this reason, the subject to which this chap- ter is devoted will be introduced with an enumeration and description of some of the methods which have contributed so much to the work of the last two decades. Most im- portant of these are the methods which have been developed for recovering eggs and em- bryos from the oviducts and uterus, and they, therefore, will be described as a pre- liminary to the discussion which follows. Methods A. METHODS FOR RECOVERING MAMMALIAN EGGS AND EMBRYOS 1. Collecting Ova jrom the Oviducts In animals such as the guinea pig, rat, mouse, and hamster, in which the oviducts are highly coiled, several procedures may be followed for obtaining the tubal eggs. The coils of oviduct can be trimmed from the mesosalpinx with iridectomy scissors. By stroking the length of the tube with a fine, curved, blunt probe, the entire contents can be expressed and the ova separated from the debris. Another method is that of placing the oviducts in a balanced salt solution and Fig. 14.1. Apparatus for washing ova from the oviducts of mammals. mincing them into small pieces with a pair of fine, pointed scissors, and then searching for the ova. Both of the above methods are wasteful of time and material, because the ova may be damaged and the full number frequently is not recovered. The best method for obtaining ova from the coiled oviducts of the rat, mouse, hamster, and guinea pig is to insert a fine pipette filled with a suitable solution into the lumen of the fimbriated end. The pipette is held in place with fine watchmaker's for- ceps. Gentle pressure is exerted on the fluid in the pipette by a simple arrangement whereby air pressure can be controlled in the manner illustrated in Figure 14.1. If the oviducts are removed and cut just above the uterotubal junction, ova may be seen to escape slowly from the cut end. By control- ling the pressure, all of the ova can be kept within a circumscribed area and any other contents of the oviduct, such as spermato- zoa, can be accurately counted or evaluated (Rowlands, 1942; Simpson and Williams, 1948; Blandau and Odor, 1952; Noyes and Dickmann, 1960; Dickmann and Noyes, 1960). 2. Oollecting Free Ova jrom the Uterus Flushing of free ova from the uterus has been performed in the monkey (Hartman, 1944) and cow (Rowson and Dowling, 1949; Dracy and Petersen, 1951). In the monkey the uterine lumen may be entered with a hypodermic needle inserted into the uterus through the abdominal wall. The contents of the uterus are then flushed through a funnel, the stem of which has been inserted into the cervical lumen. Several segmenting eggs were obtained by this procedure. The disadvantages of this method are two : first, a large quantity of fluid must be examined, and, second, the presence of cellular debris in the washings makes it difficult to locate the single egg. In rodents the cornua may be removed from the body and separated into their right and left halves. Each cornu is then flushed with physiologic saline by inserting a fine hypodermic needle into the oviductal end. During the flushing, the cornu should be gently stretched so as to release ova that may be trapped within the endometrial folds. BIOLOGY OF EGGS AND IMPLANTATION 799 In the cow relatively large quantities of l)hysiologic solutions are used to flush out tlie cornu on the side on which the corpus luteum has been detected by rectal palpa- tion (Rowson and Bowling, 1949). The recovered fluid is poured into a series of French separatory funnels and allowed to stand for 20 minutes. Ordinarily, this inter- val is long enough for the ovum to gravitate to the bottom. A few milliliters of fluid are removed from each funnel and the egg searched for. By this method, Dracy and Petersen reported the recovery of 10 fer- tilized ova from a single cow which had been superovulated. 3. Recovery of Attached Embryos The techniques devised by Dr. Chester Heuser, thus far unsurpassed in the degree of their perfection, provide the safest method of obtaining blastocysts or early implanting embryos. Uteri of man or other primates which have been removed by hys- terectomy are completely immersed in Locke's solution. The uterus is cut coronally into dorsal and ventral halves. The sur- face of the mucosa can then be examined under a binocular dissecting microscope in order to locate the site of the implanting embryo (Heuser and Streeter, 1941 ; Hertig and Rock, 1951 ) . A somewhat similar procedure can be fol- lowed in observing and recovering implant- ing embryos of the guinea pig, rat, and rabbit. The cornu is cut longitudinally along the mesometrial border with iridectomy scissors and the entire cornu laid open as a book. The mucosa of the antimesometrial area is examined under a binocular dissect- ing microscope in order to find the implant- ing embryos and, when they are found, fixatives can be added directly and only a small segment of the uterus removed for sectioning (Blandau, 1949b; Boving, per- sonal communication). B. EGG CULTURE AND PRESERVATION IN VITRO Studies of the effects of various environ- mental conditions on mammalian eggs and zygotes are of more than academic interest. The possibility of applying such knowledge to artificial insemination and intergeneric and reciprocal transplantation of eggs is of economic importance, especially in animal husbandry. Consequently, for years special attention has been given to the problem of finding satisfactory media for the successful culture and transplantation of eggs. Gates and Runner (1952) compared Or- tho-bovine semen-diluter containing egg yolk with regular Locke's solution as a me- dium for transplanting mouse ova and con- cluded that the semen diluter was the more satisfactory medium. Many other media have proved successful. These include, to list only a few, Ringer-Locke solution with an equal volume of homologous blood serum (Pincus, 1936), Krebs' solution (Black, Otto and Casida, 1951), phosphate-buffered Ringer-Dale solution mixed with an equal volume of homologous plasma (Chang, 1952b), and Krebs-Ringer bicarbonate con- taining 1 mg. per ml. glucose and 1 mg. per ml. crystalline bovine plasma albumin (Ar- mour) (McLaren and Biggers, 1958). Rabbit eggs have been used most often as test objects in the evaluation of media. The eggs of this animal are particularly hardy during manipulation and storage in vitro, a condition which may be related to the presence of the mucous coat. Aqueous humor from sheep's eyes has been used successfully for the transfer of eggs from sheep to sheep (Warwick and Berry, 1949) . Willett, Buckner and Larson (1953) ob- tained pregnancies in cows from eggs sus- pended in homologous blood serum during transfer. Except when the rabbit was used, at- tempts at growing fertilized eggs in vitro in the same media used for their transfer have not been successful. The pioneering work on the cultivation of mammalian eggs under conditions of tissue culture must be attributed to Brachet (1913), Long (1912), Lewis and Gregory (1929), Pincus (1930), and Nicholas and Hall (1942). Lewis and Gregory recorded their notable success in culturing fertilized rabbit ova in homologous blood scrum in vitro by means of cine- microphotography. Fertilized rabbit ova will cleave regularly in vitro up to and be- yond the initial stages of blastocyst expan- sion (Pincus and Werthessen, 1938). Lewis and Hartman (1933) succeeded in culturing the fertilized eggs of Macacus rhesus for a 800 SPERM, OVA, AND PREGNANCY number of divisions. Eggs of guinea pigs, cultured in vitro, rarely divide beyond the first few blastomeres (Squier, 1932). Guinea pig blastocysts, however, grow quite well in a culture medium consisting of equal parts Locke's solution (pH 7.5), serum from guinea pigs pregnant from 20 to 24 days, and embryo extract prepared from 19- to 20- day-old guinea pig embryos (Blandau and Rumery, 1957). As yet, no success has been obtained with the very early fertilized eggs of the hamster and rat (Wrba, 1956) . Hammond (1949.) cultured fertilized mouse ova in dilute suspensions of whole hen's egg in saline to which had been added Ca, K, Mg, and glucose. No 2-cell ova de- veloped beyond the 4-cell stage; 8-cell ova ordinarily developed into blastocysts. Whit- ten (1956) found that 8-cell mouse eggs de- veloped into blastulae in an egg white-sa- line mixture or in Krebs-Ringer bicarbonate solution to which 0.003 m glycine had been added. There seems to be some physiologic difference between the 2- and 8-celled ova in this animal because the 2-celled mouse eggs are refractory to in vitro cultivation unless calcium lactate replaces the calcium chloride in the culture medium (Whitten, 1957). Considerable success has attended the in vitro culture of embryos which are beyond the blastocyst stage at the time of transfer to tissue culture (Brachet, 1913; Wadding- ton and Waterman, 1933; Jolly and Lieure, 1938; Nicholas, 1947; Moog and Lutwak- Mann, 1958). Nicholas (1933) obtained bet- ter growth in vitro when the embryos were cultured in a circulating medium. Several investigators have studied the ef- fects of cooling mammalian eggs in vitro. Chang (1948a, b) found that rapid lowering of the temperature of 2-celled rabbit ova that had been suspended in a mixture of equal parts of buffered Ringer's solution and rabbit serum was harmful to subsequent development. However, the important fac- tor was not the rate of cooling but whether the process was continued until +10°C. was reached. Apparently, that is the optimal temperature for the storage of fertilized rabbit eggs. At this temperature eggs can be kept in vitro up to 168 hours without loss of viability. At +22°C. to +24°C. ova lived for only 24 to 48 hours. Attempts to main- tain glycerol-treated rabbit ova at temper- atures ranging from -79° to -190°C. have so far been unsuccessful (Smith, 1953). C. INTRASPECIFIC EGG TRANSFER The technique for the transfer of unfer- tilized and fertilized eggs between the mem- bers of the same species was first described by Heape (1890). He used this method in rabbits to demonstrate that the genetical characteristics of mammals are fixed at the time of fertilization and are not influenced by the intra-uterine environment of the foster mother. Biedl, Peters and Hofstatter (1922) and Pincus (1930) used Heape's technique during investigations on fertility and demonstrated that it is possible to transplant fertilized rabbit eggs to pseudo- pregnant does. In animal husbandry artificial insemina- tion has been an important method for the widespread distribution of desirable genes by way of the spermatozoa. Similar geneti- cal improvement through the egg has been greatly limited in domestic farm animals by the small number of offspring. A single cow, for example, will produce 1 calf per year and seldom more than 5 in a lifetime. If transplantation of eggs could be per- fected, the number of genetical experiments could be increased at least 2-fold. That the prospect is favorable, is indicated by the fact that transfers which have resulted in pregnancies have been reported for mice (Bittner and Little, 1937; Fekete and Little, 1942; Fekete, 1947; Runner, 1951; Gates and Runner, 1952; Runner and Palm, 1953; McLaren and Michie, 1956; Tarkowski, 1959; McLaren and Riggers, 1958); rats (Nicholas, 1933; Noyes, 1952); rabbits (Heape, 1890; Bicdl, Peters and Hofstatter, 1922; Pincus, 1936, 1939; Chang, 1947, 1948a, b, 1949a, 1952b; Chang, Hunt and Romanoff, 1958; Venge, 1953; Avis and Sawin, 1951; Black, Otto and Casida, 1951; Adams, 1953); sheep and goats (Warwick and Berry, 1949; Averill and Rowson, 1958) ; swine (Kvasnickii, 1951) ; and cows (Wil- lett, Buckner and Larson, 1953). The majority of successful egg transfers have been accomplished by exposing the oviducts and cornua surgically and placing the eggs within them (Fig. 14.2). Introduc- ing fertilized eggs into the cornua by way of BIOLOGY OF EGGS AND IMPLANTATION 801 the vagina and cervix has usually failed to result in pregnancy (Dowling, 1949; Um- baugh, 1949; Rowson, 1951). Two excep- tions have so far been reported. Kvasnickii (1951) obtained one pregnancy in the sow from eggs placed in the uterus per vaginam and Beatty (1951) obtained 5 young from 55 mice morulae and blastulae introduced into the cornua by the same approach. Since the normal development of ova in artificial pregnancy is wholly dependent upon the environment into which they have been placed, day-old rabbit ova would develop into normal young only when transferred to oviducts of animals in which ovulation had been induced at approximately the same time. Similarly, blastocysts would develop into young only when transplanted into 2- day or 5-day cornua (Chang, 1950c). Again in transferring fertilized tubal ova to the cornua of rats, Nicholas (1933) reported that when the host animal ovulated later than the donors, implantations were greatly reduced as compared to those instances in which the cycles were more closely synchro- nized. Dickmann and Noyes (1960) trans- ferred ova that were one day younger than the cornua to host females and found that they developed at a normal rate until the fifth day, when they degenerated and failed to implant. On the other hand, ova that were one day older than the host's cornua delayed their development until the endometrium had "caught up" and was ready for im- plantation. This implies that there is a very critical egg-uterine interrelationship that is established on the fifth day of pregnancy in the rat. Transplantation of rat ova beneath the kidney capsule (Nicholas, 1942) and of mouse ova into the abdominal cavity and anterior chamber of the eye (Fawcett, Wis- locki and Waldo, 1947; Runner, 1947) have resulted in only partial embryonic develop- ment. D. THE PRODUCTION OF EGGS BY SUPEROVULATION Many studies have been directed to meth- ods for superovulating various animals, then fertilizing the eggs in vivo, recovering and transferring them to recipient females (Clewe, Yamate and Noyes, 1958; Noyes, 1952; and Chang, 1955a). Sucli possibilities have been realized es- Fk;. 14.2. Result of autotransfer of a 4-cell goat egg, B. Tlio mother was operated upon on the sec- ond day after breeding, the oviduct was removed and the 4-cell egg (A) was washed out. The egg was then injected into the opposite horn of its mother (Warwick and Berry, 1949). pecially by Chang (1948a), who obtained 53 2-celled rabbit ova from a single doe. These ova were transplanted to 4 other fe- males and yielded 45 normal young. Using somewhat similar techniques of superovula- tion and in vivo fertilization in rabbits, Avis and Sawin (1951) obtained 81 per cent suc- cessful impregnations and Dowling (1949) 78 per cent pregnancies. Subsequently, Marden and Chang (1952) performed the novel experiment of shipping superovulated, fertilized rabbit ova by way of aerial transport from Shrewsbury, Massa- chusetts, to Cambridge, England, for suc- cessful transplantation into recipient does. While in transport, the eggs were stored in a flask containing whole rabbit serum kept at temperatures from 12 to 16°C. In domestic animals, the economic importance of such transfer of eggs from genetically superior animals is receiving considerable attention (see Proceedings of the First National Egg Transfer Breeding Conference, 1951). Un- 802 SPERM, OVA, AND PREGNANCY fortunately, superovulation in cattle which has been achieved by the administration of gonadotrophic hormones (Casida, Meyer, McShan and Wesnicky, 1943; Umbaugh, 1949; Hammond, 1950a, b) has met with little success as a means of inducing preg- nancy (Willett, Black, Casida, Stone and Buckner, 1951 ». III. Biology of the Mammalian Egg A. OOGENESIS The literature is now revealing a more clear cut opinion as to whether or not the primordial germ cells from the yolk sac of the embryo are set aside at the beginning of ontogenesis, or whether they arise de novo from the somatic cells of the gonadal peri- toneum in the embryo and particularly the sexually mature female. Knowledge in this field has been significantly advanced by em- ploying the techniques of experimental em- bryology, organ and tissue culture, histo- chemistry, x-rays, ultraviolet irradiation, genetics and statistics. The Gomori alkaline phosphatase procedure lias been used by a number of investigators to distinguish selec- tively the primordial germ cells in the hu- man (McKay, Hertig, Adams and Danzigcr, 1953), the mouse (Chiquoinc, 1954; Mintz, 1959), and the rat (McAlpine, 19551. Using the same technique, Bennett (1956) reported the absence of germ cells in strains of mice known to be sterile. It has been suggested that the high alkaline phosphatase activity in the germ cells may be related to their ac- tive movement through tissues. This specu- lation has merit when it is noted that alka- line phosphatase activity is greatly reduced in amblystoma, in which the germ cells do not actively migrate, and in the chick where these cells are apparently transported by way of the blood stream (Chiquoine and Rothenberg, 1957, Simon, 1957a, b). It should be noted that the primordial germ cells may be identified by other techniques. For example, in the rat and man the use of the periodic acid-Schiff (PAS) reaction and a hematoxylin counter stain gives such excellent cytologic differentiation of the germ cells that they can be counted and their migratory course followed (Roosen- Runge, personal communication). It is beyond the scope of our discussion to present the details of the controversy of germ cell origin, migration, localization, and proliferation. Excellent reviews of the better- known theories are contained in the papers and monographs of Heys (1931), Cheng (1932), Swezy (1933), Pincus (1936 », Bounoure (1939), Everett (1945), Nieuw- koop (1949), Zuckerman (1951), Brambell (1956), and Nieuwkoop and Suminski ( 1959) . Evidence for the extragonadal origin of the primordial germ cells has been signifi- cantly enhanced by the more recent investi- gations in amphibia, birds, and various mammals such as the armadillo, mouse, rat, cat, rabbit, and man. In an excellent paper dealing with the migration of the germ cells in the human, Witschi (1948) points out that in embryos of less than 16 somites all of the primitive germinal elements are located in the endoderm of the yolk sac splanchno- l)leure near the site of evagination of the allantois (Fig. 14.3). From this location the individual germ cells appear to migrate to the genital folds by various routes. Witschi concludes from studies of sectioned human embryos that the migration of the germ cells is accomplished by active autonomous movements and cites evidence of proteolysis of the cells and tissues in the immediate vicinity of the forward moving cells. He suggests that the specific orientation of the cell is directed by some chemical substance released by the peritoneum of the gonadal regions. A very important contribution to the solution of the problem of seeding the primi- tive gonads by germ cells from extragonadal origin is described in the contributions of IVIintz (1957, 1959) and Mintz and Russell (1957). These authors noted that the gonads of mice of the WW, WW'' and WW^ geno- tyi^es are almost devoid of germ cells at birth. The application of the alkaline phos- phatase technique revealed that the cells are present in their usual numbers in the yolk sac splanchnopleure by the 8th day of development. The mutant genes appar- ently do not impair the initial formation of the primordial germ cells. By the 9th day of development, however, many of the germ cells had already degenerated at their site of origin. Some of them escape destruction and migrate toward the genital ridge. The migratory cells fail to divide so that the BIOLOGY OF EGGS AND IMPLANTATION Fig. 14.3. Drawings of graphic reconstructions of a 16- and 32-somite human embryo. A. The bhick dots within the circle represent the location of the germ cells in the yolk sac and ventral wall of the hind-gut in the 16-somite embryo. B. Position of indi\-idual germ cells (black dots) in the 32-somite embryo. Larger dots indicate an endodermal position. Few germ cells remain in the ventral mesenchyme. (After E. Witschi, Contr. Embryol., Carnegie Inst. Wasliington, 32, 67-80, 1948.) 804 SPERM, OVA, AND PREGNANCY total number reaching the gonads is small. These findings were in strong contrast to the behavior of germ cells of the normal mouse. By use of a genetical marker, further experimental proof of extragonadal origin of germ cells was obtained. From theoretic expectations, experimental matings using heterozygotes should yield 25 per cent de- fective offspring. The actual frequency of embryos with gonads containing few germ cells was 28 to 29 per cent. The observations of Mintz and Russell give significant veri- fication of the initial extragonadal origin of primordial germ cells in the mouse. Their work demonstrates further that mice of different strains lose oocytes at different rates depending on their genetical charac- teristics. In some of the mutant mice, there is a complete absence of ovocytes in the ovaries of the adults. Russell and Fekete (1958) have shown that when chimeric organ cul- tures were made in vitro, combining one- half of a fetal ovary from the mutant strain with one-half of an ovary from a normal animal, no germ cell differentiation occurred despite active proliferation of the germinal epithelium. The sterility pattern described for the female has been observed also in the male mouse. Primordial germ cells are very poorly represented in the testes of WW, WW" and W^'W"' embryos and newborn. The mature males of these strains are in- variably sterile. Veneroni and Bianchi (1957) reported some success in treating such sterile males with follicle stimulating hormone and testosterone propionate. They conclude that the problem of sterility is re- lated not only to the reduction in the num- ber of primordial germ cells but also to an endocrinologic deficiency. Willier (1950) studied the developmental history of the primordial germ cells in the chick by preparing chorio-allantoic grafts of the blastoderm at certain critical stages, namely, (1) at the time the germ cells were still near the site of their origin, (2) during their migration, and (3) when they had arrived in the prospective gonadal areas. He found that under these experimental condi- tions the ovarian cortex never forms; he attributed this deficiency, at least in part, to a failure of the development of a mecha- nism in the graft for transporting the pri- mordial germ cells to the areas of the devel- oping gonad. Swift (1914), Dantschakoff, Dantschakoff and Bereskina (1931 ) , Willier (1950), and Weiss and Andres (1952), sug- gested that the primary germ cells are car- ried to the primitive sex glands of the chick embryo by way of the blood stream. Thus the cells are originally distributed at ran- dom, but they accumulate and persist only in the gonadal primordium. Recently, Simon (1957a, b) confirmed the vascular transport of the germ cells in the chick by the application of several ingenious experimental embryologic techniques of transplantation and parabiosis. In the de- veloping chick of less than 10 somites the primitive germ cells are localized in the germinal crescent zone in the anterior part of the yolk sac. The caudal part of the em- bryo containing the future genital ridge was severed and moved some distance from the original embryo. Vascularity of both parts was interfered with as little as possible. Stained sections of embryos examined on the 4th day of development revealed that the gonads had been populated by germ cells which could have reached them only by way of the vascular stream. In other experiments the caudal areas of 10 somite embryos, where gonads were not seeded by germ cells, were transplanted to the area vasculosa of other 10 somite embryos. The developing gonads in the transplants were colonized by germ cells. In still another experiment chick embryos were placed in parabiosis. In one of the transplanted embryos the anterior crescent containing the primordial germ cells was cut away. In cases of successful parabiosis the gonads of both embryos were seeded by germ cells. Even though it is recognized that in many mammals and the chick the germ cells of the primitive sex glands are derived from migratory primordial germ elements, a more difficult problem remains of a possible second source of germ cells arising from so- matic cells in the gonad of embryos, fetuses, and mature animals. It has been proposed that the original germ cells degenerate after having reached the gonads and having ef- fected their inductive roles, and that new- cells arise secondarily by proliferation of BIOLOGY OF EGGS AND IMPLANTATION 805 cells in the germinal epithelium (Allen, 1911; Firket, 1914; Kingery, 1917). On the otlier hand, Essenberg (1923), Butcher (1927), Brambell (1927, 1928), and Swezy and Evans (1930) postulated a dual origin for the germ cells, i.e., they may arise both from the primordial germ cells, and directly from somatic cells. The ingrowth of new cells from the ger- minal epithelium, resulting in the produc- tion of new oocytes, was thought to have been demonstrated for both the eutherian mammals (Pincus, 1936; Duke, 1941; Slater and Dornfeld, 1945), and birds (Bullough and Oibbs, 1941). However, various opin- ions flourished as to whether these oocytes were produced continuously throughout the reproductive life of the female (Robinson, 1918; Papanicolaou, 1924; Hargitt, 1930j, or whether they arose from a cyclically stimulated germinal epithelium. On the basis of Allen's (1923) investigations on the mouse, and Evans' and Swezy 's (1931) work on a variety of mammalian species, it was widely accepted that a large number of oocytes make their appearance from the germinal epithelium about the time of es- trus. According to these investigations the oocytic population reaches its peak during the period of heat and ovulation. On the other hand. Green and Zuckerman (1951a, b, 1954) analyzed the difference in the number of oocytes during the menstrual cycle in 12 pairs of ovaries of Maccica mulatta by both quantitative and statistical methods. Their results did not support the accepted view that the total number of oocytes in the ovaries of the monkey varies during the cycle and reaches a maximum near the time of ovulation. They concluded that there is no significant difference be- tween the average total number of oocytes present at the beginning, middle, and end of the cycle. From the results of the ex- periments of Papanicolaou (1924), Moore and Wang (1947), Mandl and Zuckerman (1951), Mandl and Shelton (1959), Enders (1960), and others, one would assume that the germinal epithelium is not essential for oogenesis in the adult mammal. If oogenesis is to continue after puberty in the absence of a germinal epithelium, are there al- ternative sources for the new oocytes? It has been proposed that either the concentration of primordial germinal cells in the region of the hilum of the ovary, redescribed by Vin- cent and Dornfeld (1948), may be a source, or that specialized cells, histologically in- distinguishable from other stromal cells, may be transformed into germ cells. In support of the latter, Dawson (1951) sug- gested that in polyovular follicles in which there is a great disproportion in the size of the ova, the accessory egg may have arisen by delayed oocytic differentiation of a cell temporarily incorporated in the follicular epithelium. Of the numerous experimental approaches to the problem of the origin of the germ cells in the sexually mature animal, the action of various hormones on the ger- minal epithelium has received particular attention. Bullough (1946) claimed that at the time of ovulation the estrogen-rich fol- licular fluid which bathes the ovary induces mitotic activity of the germinal epithelium. Stein and Allen (1942) demonstrated a stimulating effect of estrogen on the pro- liferation of the germinal epithelium of the mouse when this hormone was injected di- rectly into the periovarial sac. On the other hand, thyroxine similarly applied retarded mitoses of the germinal epithelium (Stein, Quimby and Moeller, 1947). More recently Simpson and van Wagenen (1953) reported an enhancement of all the processes con- cerned with the development of oocytes and follicles in prepubertal monkeys (Macaca mulatta) that had been injected subcu- taneously with either highly purified follicle- stimulating hormone (FSH) extracted from the sheep pituitary or extracts from ho- mologous pituitaries ( also see van Wagenen and Simpson, 1957, and Simpson and Van Wagenen, 1958). The germinal epithelium was stimulated to such an extent that there was an active ingrowth of germinal cords which closely simulated the development of Pfliiger's tubes. Small oocytes appeared to be developing within the germinal cords and there were evidences which one could in- terpret as reactivated oogenesis. An attempt was made to carefully quantify the response of the ovaries by counting the number of oogonia and growing follicles. In general the follicular counts remained unchanged, but primary follicles with a single granulosa cell laver were fewer in the stimulated 806 SPERM, OVA, AND PREGNANCY ovaries than in the controls, indicating that more of them had been started on the course of fm^ther development. From the evi- dence presented in the monkey and from a variety of other observations one must con- clude that, once reproductive life has begun, there is no neonatal growth of germinal epi- thelium. One of the major difficulties is the prob- lem of distinguishing germinal epithelial cells from adjacent oogonia. A similar diffi- culty is encountered when attempts are made to remove only the germinal epithelial cells by surgical or chemical means (]Moore and Wang, 1947; Mandl and Zuckerman, 1951). This problem is further emphasized by Everett (1945) when he states, "It seems probable that the cells of the epithelium, which form functional sex elements, are not and never were a part of the mesothelial covering, but are cells which were segre- gated early and are merely stored in the epi- thelium." From some of the earlier work, it was felt that much would be gained if some tech- nique were devised whereby individual cells could be marked and their subsequent fate determined. Latta and Pederson (1944) initiated such experimentation when they injected India ink into the periovarian space and examined the ovaries at varying in- tervals thereafter. Ova and follicular cells with carbon particle inclusions were seen in various stages of growth and maturation and these observations were interpreted as demonstrations of the origin of ova and follicular cells from "vitally stained" ger- minal epithelium. It is suggested, however, in light of recent evidence that many cells are capable of moving such particles across the cells and transferring them to others (Odor, 1956; Hampton, 1958), that the va- lidity of using colloidal particles for labeling epithelial cells should be re-evaluated. Theoretically, the study of tissue culture preparations of fetal and adult ovaries by phase contrast and time-lapse cinematog- raphy might be a better approach to the problem of the neoformation of oocytes in mammals and a few experiments of this type have been performed. Long (1940) re- ported oocytes developing from newborn and adult mice ovaries growing in vitro. These findings were not confirmed by simi- lar studies of Ingram (1956) in which he found no signs of oogenesis in tissue culture preparations of either mouse or rat ovaries. Gaillard (1950) suggested that the germinal epithelium was essential for survival of ex- plants of human embryonic ovaries in that explants without germinal epithelium in- variably died. On the other hand, Martino- vitch (1939) cultured fetal mouse ovaries for as long as 3V2 months. Although the ovarian epithelium disappeared after one week in vitro, the ovocytes continued to grow. The covering epithelium of the ovary is capable of proliferation, and mitotic figures are frequently demonstrable. As the size of the ovary changes during the normal cycle or upon stimulation with exogenous hor- mones, the covering epithelium must keep pace with the changing surface contour. As mentioned above, the primordial germ cells in the embryo are strongly phosphatase- positive. Careful evaluation of the cells arising from the germinal epithelium have so far shown negative enzymatic reactions. Furthermore it is a consistent finding that when mice are x-rayed in late fetal life or at birth with sufficient dosages to eliminate the ovogonia, no new ovocytes form from the cells of the germinal epithelium (Brambell, Parkes and Fielding, 1927; Mintz, 1958) . It is an obvious conclusion that any attempt to ascertain the origin of germ cells cannot be considered adequate without thor- oughly investigating the entire germ-cell cycle from tlie very earliest stages to the formation of the definitive sex elements in the fetal and postnatal periods. This must include also the origin of the functional germinal cells in the sexually mature ani- mal. There is an urgent need for a compre- hensive comparative study of the cytology, distribution, and migration of these cells. Inasmuch as the germ cells often contain nuclear and cytoplasmic features which are highly characteristic, they offer unusual ad- vantages for various experimental analyses using some of the moi'e modern techniques of experimental embryology, tissue culture, and microscopy. Even though we have confined our re- marks here to the chick and mammal, we recognize the importance of the considerable body of descriptive and experimental in- BIOLOGY OF EGGS AND IMPLANTATION 807 formation that has been recorded for the amphibia and invertebrates (Tyler, 1955). Heteroplastic transplantations and other experimental procedures which can be per- formed more easily in these animals may lead to explanations of the fundamental patterns of germ cell-inducing influences by the surrounding cells and to other problems bearing on the question of the origin of second generation germ cells in the genital ridge. B. GROWTH, COMPOSITION, AND SIZE OF THE MAMMALIAN EGG The rate of growth of the oocyte in re- lation to the stage of development of the ovarian follicle has been investigated in a numl)er of placental mammals (Brambell, 1928, mouse; Parkes, 1931, rat, ferret, rab- l)it, pig; Zuckerman and Parkes, 1932, ba- boon; Green and Zuckerman, 1951a, 1954, Macaca mulatta and man). The available information indicates that size relationship of ovum and follicle has the same c^uantita- tive aspect in all animals studied. It is in- teresting that the regression line relating to the size of egg and follicle is steep in the first phase and almost horizontal in the second (Fig. 14.4). It is generally believed that the ovum attains its mature size about the time antrum formation begins in the follicle. Further, it is also believed that follicular response to pituitary hormones is confined primarily to those follicles in which the ova have attained their full dimensions (Pincus, 1936). It is well known that not all ova grow to mature size. Factors de- termining which of the ovarian eggs are destined to begin their growth or to com- plete their growth during a reproductive cycle are unknown and present very chal- lenging problems. Growth of the follicle be- yond the antrum stage may be quite in- dependent of the presence of an ovum. This has been demonstrated in a variety of ways, but particularly by the observation that in senile rats large anovular follicles are of common occurrence (Hargitt, 1930). The converse has been reported; ova may grow to full size within the stroma of an ovary without being invested by follicular cells. Of particular interest, also, are the ques- tions raised by Gaillard (1950) and Dawson (1951) of the histogenetic relationship be- tween the oocyte and follicular cells and the oocytic potentiality of the follicular cells themselves. In tissue culture explants from human fetal ovarian cortex, Gaillard de- scribed the development of cord-like groups of cells from the germinal epithelium. A second group of cord-like outgrowths de- veloped from the follicular cells of the pri- mordial follicles in which the oocytes had degenerated. New oocytes developed within these follicular cords and the surrounding cuboidal epithelial cells arranged themselves in a single layer to form the corona radiata. The observations of Gaillard emphasize the potential histogenetic interrelationships be- tween the egg and the first layer of follicular cells. The possible inductive relationships of the ovarian egg and the various components J I I L J I 10 20 30 40 50 60 70 80 90 gg 600 8001000 2000 3000 4001 Diameter of foMicle (/i) Fig. 14.4. Regression lines relating size of ovum and follicle in human ovaries (Green and Zuckerman, 1951b). SPERM, OVA, AND PREGNANCY of the follicle need to be clarified and offer excellent opportunities for more detailed investigation. Studies of the various microscopically visible components of the ooplasm of mam- malian eggs have not advanced as rap- idly and significantly as have studies deal- ing with similar elements in the eggs of the lower vertebrates and invertebrates (Claude, 1941; Holtfreter, 1946a, b; Schra- der and Leuchtenberger, 1952; Rebhun, 1956; Yamada, Muta, Motomura and Koga, 1957; Nath, 1960). Relatively little information is availal)lo on the historv, biocliemical significance, and function of the cytoplasmic inclusions dur- ing the period of growth, maturation, or fertilization of the mammalian oocyte. In the dog, cat, and rabbit Golgi material of the young oocyte is first localized in the region of the nucleus, but it is later dis- tributed throughout the ooplasm and finally aggregates near the cell periphery. The sub- microscopic details of these shifts in the organelles of the oocyte have now been described for the rat and mouse. In oocytes with a single layer of granulosa cells the large Golgi complex lies at one pole of the nucleus (Fig. 14.5). This position of the Golgi complex is characteristic of primary I 1 OOCYTE NUCLEUS r V . WITOCHONDWA-g MULTIVESICULAR BODIES P7-V // .COMPLEX ..>■■; y |ERGAST0 PLASM ^^<^;^'&V' .' ^IZ: _, -GRAWIIQSA C^^^y Fk;. 14.,5. Electron micrograpli of a portion of a imilammar or prniiary follirle obtained fronn a rat 2 days postpartum. The large mitochondria have much matrix and few cristae. The large Golgi complex is located at one pole of the nucleus. Note close apposition of granu- losa cell membranes to oolemma! membrane. (Courtesy of Dr. L. Odor.) BIOLOGY OF EGGS AND IMPLANTATION 809 ; «■,* GRANULOS^I^LL l'^^'' / / PROCESSES MICROVILLI ZONA PELtuClDA Fig. 14.6. An electron micrograph of a small segment of a multilaminar follicle from a 15- day-old rat. The peripheral location of the Golgi elements, its parallel stacked double mem- branes and associated vesicles are well shown. The relations between the microvilli and the granulosa cell profiles in contact with the oolemma may be observed. (Courtesy of Dr. L. Odor.) follicles before zona pellucida formation. Large mitochondria with relatively few cristae are present also and at this stage are rather evenly distributed throughout the egg. As the egg continues to develop the fol- licle becomes multilayered and the Golgi complex now appears as a number of smaller units with a complex of stacked, parallel, double membranes lying relatively near the surface of the egg (Fig. 14.6). The mito- chondria and other organelles also assume a more peripheral position. The behavior of the Golgi complex varies greatly from ani- mal to animal (Zlotnik, 1948), and there are diverse opinions concerning its role in yolk production. Some investigators sug- gest that the Golgi material is concerned with the production of protein yolk, where- as others, working on different animals, maintain that it is always associated with the fatty yolk (Gresson, 1948 ». During the early stages in the develop- ment of the follicle, the Golgi material in those cells arranged to form the corona radiata lies nearest the zona pellucida. Small granules from the vicinity of the Golgi ma- terial have been described, in fixed and 810 SPERM, OVA, AND PREGNANCY stained cells, as migrating toward the egg (Gresson, 1933; Moricard, 1933; Aykroyd, 1938; Beams and King, 1938; Zlotnik, 1948) . How the yolk material is transferred from the cells of the corona radiata into the egg itself has not been miequivocably demon- strated. A reversal of the polarity of the Golgi complex in the follicular cells of the more mature follicles suggested to Henneguy (1926), Gresson (1933), and Aykroyd (1938.) that it may be responsible, at least in part, for the elaboration of the follicular fluid. The appearance and distribution of the mitochondria in the mammalian egg also vary greatly from animal to animal. Rod- like or granular mitochondria have been de- scribed as being concentrated around the Golgi material in the fixed and stained eggs of the dog (Zlotnik, 1948) and in the cortical zones of the eggs of the bat, cat, and dog (Van der Stricht, 1923). In the mature unfertilized eggs of the rabbit, mouse, and hamster the mitochondria are concentrated in the peripheral zones. At the time of fer- tilization they migrate to the region of the developing pronuclei and tend to aggregate around them (Lams, 1913; Gresson, 1940). Observations of the living eggs of the rat and guinea pig by time-lapse cinematog- raphy at the time of fertilization do not reveal a significant displacement of the cy- toplasmic inclusions such as have been de- scribed in fixed and stained preparations. The ultracentrifuge has been used in an investigation of the cytoplasmic components of the eggs of the mouse and human (Gres- son, 1940; Aykroyd, 1941). In the human ovarian egg coagulated cytoplasm occupies more than one-half of the cell, whereas the nucleus, mitochondria, and Golgi material are confined in the remaining half. During ultracentrifugation the mouse egg is strati- fied into four distinct layers: (1) a cen- tripetal layer, which stains very lightly and which may contain a few small Golgi ag- gregations, (2j a thin layer of yolk, (3) a relatively wide band containing the major portion of the Golgi material and the nu- cleus, and (4) a wider band containing prin- cipally the mitochondria (Gresson, 1940). The distribution of nucleic acids in the developing and the mature rat and rabbit egg has been studied histochemically by Vincent and Dornfeld (1948),Dalcq (1956), Dalcq and Jones-Seaton (1949), Austin (1952b), Van de Kerckhove (1959); and Sirlin and Edwards (1959). As the oocyte grows, the desoxyribonucleic acid content of the nucleus is reduced and a perinuclear band of ribonucleic acid makes its appear- ance in the cytoplasm. Vincent and Dorn- feld attributed the organization of the pri- mary follicle to the evocating action of the ribonucleic acid elaborated by the oocyte. Alicrophotometric determinations of desoxy- ribonucleic acid (DNx\) have been reported on Feulgen-stained nuclei of mouse oocytes and of cleaving eggs (Alfert, 1950). The data indicate that the amount of DNA {present in a primary oocyte nucleus is con- stant, but that as the nucleus grows the DNA is progressively diluted. On the other hand, just before the first cleavage in fer- tilized eggs the amount of DNA in the pronuclei is doubled. The nuclei of each of the succeeding cleavage stages contain twice the amount of DNA present in the early pronuclei. In addition, studies were carried out on the protein concentration in oocytes and cleavage nuclei using the Millon re- action. The ripe egg contains a reserve of proteins which is divided among the cells and nuclei of the cleavage stages. Attention should be directed to the raj)- idly expanding literature dealing with the cytology and biochemistry of the eggs of amphibia and the chick. Clues for experi- mental methodology on the eggs of mam- mals may be found within these rejiorts (Bieber, Spence and Hitchings, 1957; Flick- inger and Schjeide, 1957; Rosenbaum, 1957, 1958; Wischnitzer, 1957, 1958; Bellairs, 1958; Tandler, 1958; also see Tyler, 1955, and Brown and Ris, 1959). The use of compounds labeled with radio- isotopes is an important tool for the study of the transport and utilization of various substances by eggs (^Moricard and Gothie. 1955, 1957, Lin, 1956; Friz, 1959). Most of the tracer experiments have been done in the chick and amphibia in which it is clear that such egg storage materials as lecithin, cepha- lin, and vitellin are formed in organs outside the ovary and transported by way of the plasma to the egg. Greenwald and Everett (1959) injected pregnant mice with S^*" me- thionine and subsequently studied the eggs BIOLOGY OF EGGS AND IMPLANTATION 811 by radioautographic techniques. Ovarian ova and the blastocysts recovered from the cornua showed active protein synthesis. Sim- ilar synthesis was noted in the early fertili- zation stages. However, eggs in the 2-cell through the morula stages contained no demonstrable S^^ methionine. From these observations one would conclude that there is a basic difference in the metabolism of tubal and cornual ova, and again raises the question of the importance of the en- vironmental fluids in providing materials necessary for the growth and development of the eggs. Earlier investigators directed attention to the fact that in many mammalian eggs the deutoplasm is arranged in such a way as to exhibit an obvious polarity. Such polarity was described particularly for the eggs of the guinea pig by Lams (1913) and is con- spicuous in a newly ovulated egg found in section by Myers, Young and Dempsey (1936). Such a polarity has been observed also in eggs of the cat (Van der Stricht, 1911), bat (Van Beneden, 1911), dog (Van der Stricht, 1923), and ferret (Hamilton, 1934). Attention has recently been redirected to the fact that the mammalian egg may have a specific cytologic organization which is important in establishing its symmetry and polarity. This pattern of symmetry is based on the crescentic distribution of a primary basophilia and the localization of the mito- chondria. The significance of the cytoplas- mic organization in relation to the morpho- genetic pattern in the mammalian egg must await the elaboration of new techniques of experimental embryology which can be ap- plied to mammalian material ( Jones-Seaton, 1949; Dalcq, 1951, 1955; Austin and Bishop, 1959). There are striking species differences in the amount and distribution of yolk material within the cytoplasm of living mammalian eggs. In the eggs of the horse, cow, dog, and mink the cytoplasm is so filled with fatty and highly refractile droplets that the vitel- lus under phase microscopy appears as a dark mass obscuring the nucleus (Squier, 1932; Enders, 1938; Hamilton and Day, 1945; Hamilton and Laing, 1946). In living eggs of the monkey, rat, mouse, rabbit, ham- ster, and goat the yolk granules are finely divided and vmiformly distributed; thus the various nuclear changes occurring during meiosis and fertilization are more readily visible (Long, 1912; Lewis and Gregory, 1929; Lewis and Hartman, 1941; Amoroso, Griffiths and Hamilton, 1942; Samuel and Hamilton, 1942; Austin and Smiles, 1948; Blandau and Odor, 1952). The ooplasm of human and guinea pig eggs is of interme- diate density when compared to the two groujis mentioned above (Squier, 1932; Hamilton, 1944). The mature mammalian egg is a cell of extraordinary size, and even the smallest (field vole, 60 //,) is large when compared with any of the somatic cells within its en- vironment. It is remarkable that through- out the eutheria there should be so little re- lationship between the size of the adult animal and the volume of the egg (Hartman, 1929). Data on the apparent sizes of the vitelli of living eggs of various animals are summarized in Table 14.1. The need for more accurate measurements on the diam- eters and volumes of the living eggs of mam- mals still exists. C. EGG MEMBRANES 1. The Zona Pellucida The zona pellucida is usually classified as a secondary egg membrane. It is believed to be a product of the primary layer of follicu- lar cells which surround the oocytes in the ovary (Corner, 1928a). Under the light mi- croscope the fresh zona pellucida appears as a more or less homogeneous membrane with a somewhat irregular surface, the amount of irregularity depending upon the species. As mentioned earlier the immature mammalian oocyte is surrounded by a single layer of cuboidal "follicle cells" whose plasma mem- branes are in intimate contact with the vitelline membrane. This relationship is par- tially altered in the growing egg by the gradual deposition of a mucopolysaccharide membrane which when fully formed consti- tutes the zona pellucida. At first the zona pellucida appears in irregular patches and in the form of an homogeneous secretion (Fig. 14.7). Slender microvilli which extend from the surface of the vitelline membrane are embedded in the zona. Short, blunt cel- lular processes also arise from the granulosa 812 SPERM, OVA, AND PREGNANCY TABLE 14.1 Estimates of the diameter of the viteUus of various mammalian ova (Modified from C. G. Hartman, Quart. Rev. Biol., 4, 373-388, 1929) Animal Most Probable Size of Egg M Monotremata Platypus Echidna 2.5 mm. 3.0 mm. Marsupialia Dasyurus 240 Didelphys 140-1 GO Edentata Armadillo 80 Cetacea Whale 140 Insectivora Mole (Talpa) Hedgehog (Erinaceus) Rodentia 125 100 Mouse 75-87.8 Rat 70-75 Guinea pig 75-85 Hamster 72.2 Field vole 60 Lagomorpha Hal)hit 120-130 ("arnivora Mink 107 Dog Cat 135-145 120-130 Ferret 153 Ungulata Cow 138-143 Horse 105-141 Sheep 147 Goat 145 Pig 120-140 Chiroptera Bat 95 105 Lemurs Tarsius 90 Primates Gibbon 110-120 M. mulatta 125-143 Gorilla 130-140 Man 130-140 cell surfaces facing the zona and as the cells recede clue to the thickening of the zona they maintain contact with the vitelline mem- Ijrane (Fig. 14.6). Several investigators have called attention to an agranular layer of cytoplasm of the granulosa cells in contact with the developing zona (Trujillo-Cenoz and Sotelo, 1959; Odor, 1960). This layer may indicate the elaboration of secretory material for the building of the zona pellu- cida. The agranular layer is certainly sug- gestive but not conclusive evidence for the follicular cell origin of the zona, for a simi- lar layer of dense substance has been de- scribed just below the oolemmal membrane (Fig. 14.8). Some interpret the granular layer below the plasma membrane of the egg as indicative of the transfer of ma- terial of large molecular weight from the granulosa cells into the egg. As the zona pellucida increases in thick- ness the number of microvilli also greatly increase and extend into the zona for ap- i:)roximately one-third of its width (Figs. 14.6 and 14.8). In eggs with fully developed zonae pellucidae, membrane profiles of the granulosa cell processes traversing this membrane have been observed in intimate contact with the oolemma (Fig. 14.8) (Ya- mada, Muta, Motomura and Koga, 1957; Odor, 1959; Sotelo and Porter, 1959; Ander- son and Beams, 1960). If the living tubal ova of mammals are ex- amined with the phase microscope, the pro- toplasmic extensions of the corona radiata cells also may be seen penetrating the zona pellucida in an obliciue or irregular direction. These canaliculi are the radial striations of the zona pellucida described by Heape (1886) andNagel (1888). It is well known that after ovulation and sperm penetration the egg shrinks and the ])eri vitelline space makes its appearance. At this time the surface of the vitellus appears quite smooth with the microvilli no longer demonstrable. As mentioned above, the jn'otoplasmic ex- tensions of the corona radiata are in inti- mate contact with the surface of the egg membrane. A number of investigators have described the passage of Golgi material from the follicle cells into the eggs in fixed prep- arations in fishes, reptiles, bii'ds, the sciuirrel, rabbit, and rat (Brambell, 1925; Bhatta- charya. Das and Dutta, 1929; Bhatta- charya, 1931). Zlotnik (1948) described the migration of small .sudanophilic granules from the vicinity of the follicular Golgi ma- terial into the oocytes of the dog, cat, and the rabbit. There is great need for clarifica- tion of the role of the cells of the corona radiata in the transport of various materials into the ooplasm and in the formation of yolk in the mammalian egg (Gatenby and BIOLOGY OF EGGS AND IMPLANTATION 813 GRANULOSA CELL rl m- 'H 1^: JiirMitOCHO I M.^% ■"' yiLLi Fic. 11.7. I'uiiKju ui uiiiLiiuiiiai- luilirli Hum :ai b-Jay-old rat. Tlic zona prlluri.la '././',' is just forming, and is deposited in irregular patches. The Golgi complex, not shown in this micrograph has begun to break up into smaller units. The mitochondria still have a random distribution. (Courtesy of Dr. L. Odor.) Woodger, 1920; Kirkman and Severinghaiis, 1938 ». Also awaiting clarification is the problem as to whether the retraction of the corona radiata cell processes alters the morphology and/or physical characteristics of the zona IK'llucida. The zona apparently is able to function as a differential membrane. It has been observed in the rat that accessory sper- matozoa within the perivitelline space re- main intact even until the time of implanta- tion whereas those suspended in the fluids of the oviduct and not incorporated in j^hago- cytic cells undergo complete disintegration 814 SPERM, OVA, AND PREGNANCY CQ^ PL EX wpMiT0CH0NDRj4 / GRANULOSA ^ CELL \ PROCESSES Z.R MlTdCHONDRIA NUCLEUS GRANULOSA CELL I'H,. 14.8. Small M-cUuii noiii all egg williin a laultilainiiiai l\)lln h in ui,i. w .< .-iu,.ll ,iii,.iiin was present. Continuity and extent of ovular microvilli are well shown. Note dense substance just inside the oolemma. (Courtesy of Dr. L. Odor.) within 12 to 24 hours after insemination. Furthermore, if the zona pellucida of a rat ovum is removed mechanically, the ooplasm then lying free in Ringer-Locke's solution will undergo visible plasmolysis within a few minutes. The physical properties of the zona pel- lucida vary according to the animal species and the experimental conditions under which the membrane is examined. Ordinarily the zona pellucida of a newly ovulated ovum is glassy, resilient, and tough. It is moderately elastic and may be considerably indented with fine needles without rupturing. Chemi- cally the zona is composed chiefly of neutral or weakly acidic mucoproteins (Leach, 1947; Wislocki, Bunting and Dcmpsey, 1947; Bar- ter, 1948; Leblond, 1950; Konecny, 1959; Da Silva Sasso, 1959). It is exceedingly sensitive to changes in hydrogen ion concen- tration: for example, the rat zona pellucida softens in buffers more acid than pH 5 and passes into solution in pH 4.5, but the rab- bit zona rec}uires buffers of pH 3 or lower to accomplish the same effect (Hall, 1935; Braden, 1952). The dissolution of the zona may also be effected by hydrogen peroxide and certain other oxidizing and reducing agents. Fur- thermore, the zona pellucida in fresh rat eggs may be dissolved readily by trypsin, chymotrypsin, and mold protease (Braden, 1952). In the rabbit the zona is removed by trypsin but is not affected by chymotrypsin or mold protease (Braden, 1952) . These data indicate that in both rat and rabbit ova the zona contains protein, but that the type of protein is not the same in the two species (Chang and Hunt, 1956). In rat eggs which are undergoing cleavage and which are ex- amined immediately after being flushed from the oviduct the external surface of the zona is suflEiciently smooth so that the eggs may roll down the incline of a concave BIOLOGY OF EGGS AND IMPLANTATION 815 dish containing them. But after a short in- terval in the new environment, the zonae may become sticky and chng to the glass surface of the dish or to the pipettes and needles used in transporting them. Nonmo- tile spermatozoa caught within or on the zona pellucida have been pictured many times in the eggs of the human (Shettles, 1953), the rhesus monkey (Lewis and Hart- man, 1941), the guinea pig (Squier, 1932), and the rabbit (Pincus, 1930). The same phenomenon has been observed only on rare occasions in rat eggs, again emphasizing dif- ferences in the physical characteristics of the zona from animal to animal. There is very little information as to the permeability of the various membranes en- closing the mammalian egg. Recently the eggs of the rabbit, rat, and hamster were ex- posed to dyes such as toluidine blue and alcian blue and to a 1 per cent solution of heparin and digitonin in order to test the selectivity of the membranes (Austin and Lovelock, 1958) . It was found that the zonae pellucidae of all three animals were perme- able to the dyes and digitonin but not to heparin. There is too little known of the changes which occur in the zona pellucida and other egg membranes under varying environmen- tal conditions to draw conclusions as to the nature of its selectivity. Techniques whereby invertebrate egg membranes are impaled with microelectrodes have yielded new in- formation as to membrane potentials and resistance at varying stages of fertilization (Tyler, Monroy, Kao and Grundfest, 1956). Similar investigations on mammalian eggs would be valuable in solving the problems of selectivity of the egg membranes and in evaluating the response of eggs to various environmental fluids. The question should also be raised as to whether or not the zona pellucida and/or the mucin coating may present barriers to the diffusion of gases and thus constitute a lim- iting factor to the rate of development. Fridhandler, Hafez and Pincus (1957) found no differences in the 0^ uptake when com- paring normal rabbit eggs and eggs in which the mucin coat and zona pellucida had been punctured. Other properties of the zona pel- lucida will be considered later when the problem of the means by which spermatozoa penetrate it is discussed. 2. The Mucous or "Albuminous'' Layer Unlike the zona pellucida, which is formed in the ovary, the "albumin" or mucous layer is deposited on the zona by secretions of the glandular cells in the oviducts or uterus and is therefore classified as a tertiary mem- brane. In the monotremes (Hill, 1933) and many marsupials (Hartman, 1916; McCrady, 1938) an abundant albuminous coat is de- posited on the zona pellucida as the egg moves through the oviduct. A similar de- posit, but composed principally of muco- polysaccharides has been described for the eggs of various animals forming the order Lagomorpha (Cruikshank, 1797; Gregory, 1930; Pincus, 1936). A thinner but chenii- cally identical coat has been described in the ova of the horse and dog (Lenhossek, 1911; Hamilton and Day, 1945). It is only in the rabbit that the mucous coat has been charged with limiting the period during which the ovum can be penetrated by sper- matozoa. A very thin layer of mucus has been observed on rabbit eggs removed from 5 to 8 hours after ovulation (Pincus, 1930; Braden, 1952). Furthermore, it has been shown that the rabbit egg must be pene- trated by a spermatozoon before the 6th hour after ovulation if normal development is to ensue (Hammond, 1934). That the mucous membrane inhibits sperm penetra- tion is confirmed by the fact that unferti- lized rabbit ova may be stored in vitro for 48 to 72 hours without, in many instances, losing their fertilizing capacity after being transferred into the oviducts of properly timed recipients (Chang, 1953). It has been clearly demonstrated that the mucin is stored in the secretory cells of the oviduct and that estrogens are necessary for the synthesis of the mucin granules (Greenwald, 1958a). Discharge of the mucin granules is apparently controlled by progesterone. The thickness of the mucin coat on rabbit eggs, or glass beads placed in the oviduct, can be either significantly increased by injecting progesterone in properly conditioned fe- males, or greatly reduced by injecting estro- gens immediately after ovulation. 816 SPERM, OVA, AND PREGNANCY Apparently the ovum plays only a passive role in the process of mucin deposition. The remarkably even distribution of mucin on living eggs or glass beads implies that the oviduct has a specific pattern of muscular contraction so as to rotate the eggs as they move forward. If the mucous coat is vitally stained with toluidine blue, one observes a concentric stratification which may indicate an apposi- tional growth as the egg proceeds through the oviduct. Chemically the mucous coat is composed chiefly of strongly acid mucopoly- saccharides. It is readily dissolved by tryp- sin, chymotrypsin, and pepsin. It is not af- fected by hydrochloric acid solutions as strong as 0.1 M but it may be slowly removed by solutions more alkaline than pH 9. A pe- culiar and important proi)erty of the al- buminous coat is that at pH 9 or 10 it be- comes exceedingly sticky. As will be noted later, this may be of importance for the ad- herence of the egg to uterine tissue at the time of implantation. The possible role of the mucous coat in the development of the egg was not realized until the investigations of Boving (1952c) in which certain details of rabbit blastocyst implantation were ob- served directly. A plastic chamber was de- veloped for examining the interior of the pregnant rabbit uterus. It was noted that the mucous coat participates actively in the initial adhesive attachment of the blastocyst to the uterus. Such localized attachment precedes by several days the cellular ad- hesion and invasion of the uterus by the blastocyst. Boving observed further that the adhesion to the uterus is localized in the abembiyonic hemisphere of the blastocyst, probably because it is in this region that an alkaline reaction, produced by secretions of the embryo, enhances the stickiness of the mucous coat. The polar localization of the adhesive attachment of the mucous coat not only provides a mechanism for the initial blastocyst attachment, but also is impor- tant in establishing the orientation of the blastocyst within the uterus (see section on "Spacing and orientation of ova in utero"). Boving (1954) observed that still another membrane is deposited on the rabbit egg by secretions of the uterus. The membrane forms a sticky covering that stains meta- chromatically in toluidine blue and func- tions as an adhesive attachment during po- sitioning and orientation of the blastocyst in utero. He proposed that the noncellular, adhesive layer be called the "gloiolemma." D. THE FIRST MATURATION DIVISION Meiotic division is not a phenomenon which is confined entirely to the ova in the preovulatory follicles. It may be encoun- tered in egg cells in the latter part of em- bryonic development, in immature follicles undergoing atresia, and in ovaries stimu- lated excessively by the animal's own pi- tuitary hormones, or by pituitary hormone preparations which have been injected (Evans and Swezy, 1931; Guthrie and Jef- fers, 1938; Dempsey, 1939; Witschi, 1948). Fairly complete descriptions of the vari- ous stages in the formation of the first po- lar body and second maturation spindles are available for a number of mammals (Hartman and Corner, 1941, the macaque; Hoadlcy and Simons, 1928, Hamilton, 1944, and Rock and Hertig, 1944, the human; Kirkham and Burr, 1913, Blandau, 1945, Odor, 1955, the rat; Long and Mark, 1911, the mouse; Moore, 1908, the guinea pig; Langley, 1911, the cat; Van Beneden, 1875, Pincus and Enzmann, 1935, the rabbit; Rob- inson, 1918, the ferret). Specific data on the temporal relationship between ovulation and the first maturation division are available primarily for the rab- bit (Pincus and Enzmann, 1935-1937), guinea pig (Myers, Young and Demi-)sey, 1936), cat (Dawson and Friedgood, 1940), rat (Odor, 1955), and mouse (Edwards and Gates, 1959). Tlie rabl)it is an animal particularly suited for studies of maturation phenomena because it ovulates regularly between 9 and 10 hours after copulation. The first evidence of change in the nucleus of a ripe ovum may be seen 2 hours after copulation. At this time the nuclear membrane is intact but tetrad formation is in evidence. Four hours after copulation the nuclear membrane has disappeared and the first polar spindle, with tetrads located on the metaphase plate, oc- cupies a paratangential position near the periphery of the ooplasm. Abstriction of the first polar body is completed about 8 hours BIOLOGY OF EGGS AND IMPLANTATION 817 after copulation. Shortly thereafter, the sec- ond metaphase spindle is formed and re- mains in position just below the surface of the primary egg membrane. It remains in this condition until the fertilizing sperma- tozoon penetrates the egg. Similar observations on successive phases of the first maturation division have now l)een completed for the rat (Odor, 1955). In over 1500 living and fixed eggs examined at specific times before and after the onset of heat it was observed that by the onset of heat, the germinal vesicle has lost its mem- iM'ane in most animals, and has been trans- formed into a a dense chromatic mass which then quickly moves towards the periphery of the ooplasm. Between the 3rd and 4th hours the chromosomes have arranged them- selves in the metaphase plate. Abstriction of the first polar body is usually completed between the 6th and 7th hour, and position- ing of the second metaphase spindle by the 8th hour. It is interesting that, even though there was considerable variation in the stages of maturation found in animals killed at the same time after the onset of heat, 83 per cent of all the ova were in the same stage of maturation or in a very closely related phase. In all mammals studied, except the dog and fox (Van der Stricht, 1923; Pearson and Enders, 1943), the first maturation division is completed within the ovarian follicle sev- eral hours before it ruptures. There is evidence that a specific correla- tion exists between the gonadotrophins and the maturation phenomena within the oo- cytes (Bellerby, 1929; Friedman, 1929; Friedgood and Pincus, 1935). Apparently the threshold of response of oocytes for mat- uration is lower than is the threshold for ovulation (Hinsey and Markee, 1933). Mor- icard and Gothie (1953) injected small quantities of chorionic gonadotrophin di- rectly into the ovarian follicles of unmated ral)bits and observed the formation of the first metaphase spindles and the abstriction of the first polar bodies. This was inter- preted as showing the direct effect of pitui- tary hormones in inducing meiosis. On the basis of a study on oocytes recovered from ral)bit ovaries Chang (1955b) concluded that once the oocytes have attained the ve- sicular stage maturation can be readily in- duced by a variety of experimental proce- dures the most effective of which is the subnormal temperature treatment of unfer- tilized ova. According to his investigations first polar body formation is not immedi- ately dependent on gonadotrophic stimula- tion. A number of investigators who have ex- amined mammalian ova have commented on the rapid disappearance of the first polar body. Sobotta and Burckhard (1910) saw the first polar body in only 2 of 100 recently ovulated mouse ova. The infrequent pres- ence of the first polar body in postovulatory ova in which the second maturation spindle was completed suggested that possibly the first polar body was not always formed (Sobotta, 1895) . Yet from a variety of stud- ies on meiosis in fixed and living eggs, it may be concluded that the abstriction of the first polar body invariably occurs. In addition it may not disappear as rapidly as some of the older investigators believed. The first and second polar bodies are visible in a 4-celled guinea ])ig embryo photographed by Squier (1932). There has been considerable speculation as to the method whereby the first polar body disappears. Kirkham ( 1907) suggested that the first polar body in the mouse either was forced through the zona pellucida or escaped from the perivitelline space by its own ameboid movement. Simi- lar theories have been held by Moricard and Gothie ( 1953) for the rat, in which they maintain that the polar body passes directly through the zona pellucida. From the obser- vations of Lams and Doorme (1908) in the mouse. Mainland (1930) in the ferret, and Odor (1955) in the rat, it is almost certain that the first polar body undergoes rapid fragmentation and cytolysis within the peri- vitelline space so that only some finely granular material remains. Ameboid move- ment of the first body has never been docu- mented in the thousands of living mammal- ian eggs examined. E. THE OVULATED EGG The appearance of tubal ova from a single animal varies considerably depending on the lapse of time between ovulation and ex- amination and the environmental fluids in 818 SPERM, OVA, AND PREGNANCY which they are kept. When the eggs are shed from the follicles they are ordinarily sur- rounded by a variable number of layers of granulosa cells and a matrix of more or less viscid follicular fluid. The vitellus does not completely fill the zona pellucida, and the first polar body, if it has not already disinte- grated, may be pressed between the zona and the ooplasmic membrane. An exception to this may be found in the Canidae in which formation of the first polar body is apparently delayed for some time after ovu- lation. The length of time that the coronal cells persist varies greatly in the eggs of dif- ferent species. A well developed corona radiata is regu- larly found in newly ovulated ova of the mouse (Lewis and Wright, 1935), the ham- ster (Ward, 1946), the rat (Gilchrist and Pincus, 1932), the rabbit (Gregory, 1930), the cat (Hill and Tribe, 1924), the dog (Evans and Cole, 1931) , the monkey (Lewis and Hartman, 1941), and man (Hamilton, 1944; Shettles, 1953). The rapid dispersal or even absence of the cells forming the corona radiata has been reported for the sheep (McKenzie and Terrill, 1937), the cow (Evans and Miller, 1935; Hamilton and Laing, 1946; Chang, 1949b), the pig (Corner and Amsbaugh, 1917; Heuser and Streeter, 1929), the horse (Hamilton and Day, 1945), and the deer (Bischoff, 1854), and would seem to be a characteristic of the newly ovulated guinea pig ovum (Myers, Young and Dempsey, 1936). The eggs of unmated females gradually lose their investment of granulosa cells as they pass through the oviducts. The cells be- come rounded and drop away from the cum- ulus, a process that occurs first in the more peripheral cells. The cells of the corona ra- diata which are adjacent to the zona pellu- cida are the last to fall away and when they are brushed from the surface of the zona in living eggs in vitro their long and irregularly shaped protoplasmic processes extending into the zonal canaliculi can be seen (Squier, 1932; Duryee, 1954; Shettles, 1958). The mechanism which effects the dis- persal and final dissolution of the cumulus oophorus and corona radiata in unmated fe- males is not known. It has been suggested that an enzyme, elaborated by the tubal mucosa, is responsible for the dispersal of the cells (Shettles, 1958). When an observer follows the cytologic changes in the cells forming the cumulus oophorus as ovulation approaches and notes their behavior in tissue culture preparations in vitro he is impressed with the suggestion that separation of the cells involves a grad- ual depolymerization of the intercellular cement substance and a change in the ac- tivity of the cell surface. If time-lapse photographs are made of the coronal cells surrounding ovulated eggs, a very active bubbling and "blister" forma- tion of the surface membranes is apparent. "Bubbling" activity of the cell surfaces is frecjuently seen in cells which are losing their vitality (Zollinger, 1948). These sur- face changes occur at the time when the cells are undergoing most active separation and accounts for the withdrawal of the cyto- plasmic processes from the zona pellucida. Further evidence that the behavior of the cell is related to loss of vitality is shown by their very poor growth in tissue culture. F. RESPIRATORY ACTIVITY OF MAMMALIAN EGGS There have been only limited investiga- tions on the energy-yielding mechanisms and energy-requiring processes of the de- veloping eggs of mammals (rat, Boell and Nicholas, 1948; rabbit, Smith and Kleiber, 1950, Fridhandler, Hafez and Pincus, 1957; and cow, Dragoiu, Benetato and Oprean, 1937). The fertilized ovum during its various stages of cleavage and differentiation is an ideal experimental object for such studies and has been used extensively in the inver- tebrates, amphibia, and birds where large numbers of eggs are readily available (Bo- ell, 1955). Refinements in the Cartesian diver technique have made possible the measurement of gas exchange of less than 1 mfxi.; thus the number of mammalian eggs required to obtain significant data need not be large (Sytina, 1956). Furthermore, the effectiveness of gonadotrophins in inducing ovulation in the sexually immature female rodents and their willingness to mate after such treatment provides a ready source of eggs independent of ovulation at specific phases of the sexual cycle. The type of information which can be ob- BIOLOGY OF EGGS AND IMPLANTATION 819 tained by measuring the Oo uptake of ferti- lized rabbit ova placed in the Cartesian diver and subjected to a variety of metabo- lites and inhibitors can be seen in Tables 14.2 and 14.3 (Fridhandler, Hafez and Pin- cus, 1957). In the rabbit egg, as in other cells, cyanide has a markedly inhibiting ef- fect on respiration. This inhibition is re- versible and presumably cyanide acts through the cytochrome oxidase system. Of significance is the finding that glucose is not an obligatory substrate for respiratory ac- tivity of the fertilized rabbit egg. If glucose is added to the medium con- taining 2- to 8-cell eggs, there is little ca- pacity to carry out glycolysis. However, such capacity develops during the late mor- ula and blastocyst stages. This change may indicate either an alteration in the mem- brane characteristics of the egg, or the de- velojmient of a new enzyme system as the egg develops. The electrical characteristics of eggs and their changes during activation and fertili- zation have been studied in frogs, echino- derms, and fish (Maeno, 1959; Ito and Maeno, 1960). The electrical properties and membrane characteristics of mammalian eggs are entirely unknown. The use of the ultramicro-electrode which has been so help- ful in nerve and muscle electrophysiology offers an unusual research tool for examin- ing the primary process of activation of G. TRANSPORT OF TUB.\L OVA The mammalian oviducts must perform a variety of functions in the transport and de- velopment of the gametes (also see "Sperm transport in the female genital tract") . They must provide some means for transporting the ovulated ova from the ovary or perio- varial space into the infundibulum. Se- cretions must be elaborated within the infundibulum in order to provide an en- vironment favorable for sperm penetration. In some animals, such as the rabbit, opos- sum, horse, and dog, specialized cells se- crete materials which form tertiary mem- branes for the eggs. Still other cells secrete nutritional and possibly other substances which may be essential for the normal growth and development of the fertilized eggs. Furthermore, the peristaltic and anti- peristaltic activities of the oviducts must be regulated in such a way that the ova are propelled forward at a definite rate and in proper rotational sequence so as to be evenly coated with the tertiary membranes. The oviducts are indeed highly specialized or- gans whose anatomic differences in the vari- ous regions have been described by many investigators but whose specific physiologic functions still present many unsolved prob- lems. As evidence accumulates, a happier mid- dle ground of opinion is forming as to the roles of the musculature and ciliary activity in the downward propulsion of the eggs and in the ascent of the spermatozoa. Compre- hensive summaries of observations and the- ories dealing with these particular problems may be found in the papers and monographs of Westman (1926), Parker (1931), Hart- man (1939), Alden (1942b), Kneer and Cless (1951). The more extensive investigations of the oviducts during the estrous cycle include: (1) The observations of Snyder (1923, 1924), Andersen (1927a, b), Anopolsky (1928), Westman (1932), and Stange (1952), on the lymphatics, the size of mus- cle fibers, and the cyclic changes in the epi- thelium of the Fallopian tubes of the rab- bit, sow, and man. (2) The alterations of rhythmic contractions in the oviducts of the rat (Alden, 1942b; Odor, 1948), the sow (Seckinger, 1923, 1924), the rabbit (West- man, 1926), the rhesus monkey (Seckinger and Corner, 1923; Westman, 1929), and man (Seckinger and Snyder, 1924, 1926; Westman, 1952). The specific method whereby the newly ovulated egg is moved from the site of rup- ture of the ovarian follicle to the infundibu- lum is poorly understood. There is consid- erable species variation in the relationship of the fimbriated end of the oviduct to the ovary proper. In the Muridae and Musteli- dae the ovaries are almost enclosed by the thin, membranous periovarial sac (Alden, 1942a; Wimsatt and Waldo, 1945). The medusa-like infundibulum is enclosed within the sac but occupies a relatively small area of the periovarial space. It is believed thai in those animals in which fluids accumulate within the ovarian bursa at the time of ovu- lation the ova are directed to the ostium by 820 SPERM, OVA, AND PREGNANCY TABLE 14.2 Effect of pre -incubation on O2 uptake of fertilized ova Incubating medium: Ca++-free Krebs-Ringer phosphate, pH 7.4. Gas phase: air. (After L. Frid- handler, E. S. E. Hafez, and G. Pincus, E.xper. Cell Res., 13, 132-139, 1957.) Developmental Stage Pre-incubation of Ova Metabolites Added to RP in Diver Average O2 Uptake Morphology Hr postcoitum r'c Time min. m fil . / oTu»! / hr . 23 23 23 120 120 120 None 0.1% ghicose 10~^ M pyruvate 0.45 0.49 0.47 2-4 cell 20-28 29 29 29 90 90 90 None 0.1% glucose 10"" M pyruvate 0.45 0.42 0.41 29 29 29 180 180 180 None 0.1%, glucose 10~" M pyruvate 0.39 0.59 0.53 Blastocyst 108 37 37 150 150 None 10^2 M pyruvate 1.84 2.42 TABLE 14.3 O2 uptake of fertilized ova in different media Gas phase: air. (After L. Fridhandler, E. S. E. Hafez, and G. Pincus, Exper. Cell Res., 13, 132-139, 1957.) Developmental Stage Medium in the Divers Average O2 Uptake Morphology Hr. Postcoitum Basic medium Added substances (m) mill./ ovum /hr. 2-8 cells 24-30 RPG None lO"'' M NaCN (appr.) 10^" M phlorizin 0.41 0.02 0.41 RPG None 2 X 10-3 M Na fluoride 0.56 0.47 Morulae 68 RP None 10-2 M malonate 10-2 M malonate plus 10-3 M fumarate 0.48 0.42 0.47 Blastocysts 78 RP None 10 2 M malonate 10-2 M malonate plus 10-3 M fumarate 1.71 1.69 1.74 Blastocysts 88 RP None 10-2 M malonate 10-2 M malonate plus 10-3 M fumarate 2.92 2.36 2.70 Blastocysts 115 RPG None 10-3 M NaCN (appr.) 78.00 0.00 BIOLOGY OF EGGS AND IMPLANTATION 821 movement of these fluids into the oviduct (Fischel, 1914). However, observations on normal fluid flow within the periovarial sac are very limited. It has been demonstrated that if dyes such as Janus green or particu- late material are introduced into the perio- varial space in the immediate vicinity of the ostium, the material quickly passes into the first loop of the oviduct (Alden, 1942b). Transport is effected primarily by the cili- ary activity of the fimbriated end of the ostium (Clewe and Mastroianni, 1958) . Fur- thermore, if newly ovulated eggs are placed on the surfaces of the fimbriae in the rat, mouse, or hamster the cilia will sweep them into the infundibulum within 8 seconds (Blandau, unpublished observations). How those ova located at some distance from the oviduct reach the fimbria has not been ob- served. Under normal physiologic conditions the ovary moves backwards and forwards within the periovarial sac. These movements are accentuated at the time of ovulation and are effected by the abundant smooth muscle in the mesovarium. Such activity keeps the fluids of the periovarial sac in motion. Those eggs ovulated at the opposite side of the ovary away from the infundibu- lum are passively moved into its vicinity where ciliary currents then aid in complet- ing transport. A potentially wide communication be- tween the ostium of the oviduct and the peritoneal cavity exists in a variety of ani- mals such as the guinea pig, rabbit, monkey, and man (Sobotta, 1917; Westman, 1952). The extent of the communication varies with the stage of the menstrual or estrous cycle. Ordinarily in a rabbit not in heat, the fiml)riae do not cover the ovary. As the time of ovulation approaches, there is a great in- crease in motility and turgidity of the fim- briae so that they almost enclose the ovary (Westman, 1926, 1952). Recently attempts have been made to observe the activities of the human fimbriae by means of abdominal l)eritoneoscopy or exploratory culdotomy. Elert (1947) has seen the elongated fimbria grasp the lower pole of an ovary for as long as 2 minutes. Doyle (.1951, 1954), how^ever, failed to observe either a sweeping or grasp- ing motion of the fimbriae before or during the rupture of the follicle. He suggests that in the human female the initial transport of the ovum is by a process in which it floats into the cul-de-sac and from there is si- phoned into the ampulla by simple peristal- tic contractions which originate at the re- gion of the fimbriae. Doyle's (1956) recent observations are more in line with those de- scribed by Elert above. It has been suggested that the activity of the abundant smooth musculature of the adnexa and the fimbriae produces a power- ful suction effect on the ovary, thus drawing the ovulated eggs into the tube (Sobotta, 1917; Westman, 1952). It is a fact, however, that no one has made measurements of this presumed negative pressure, nor, as pointed out earlier, has anyone observed a newly ovulated mammalian ovum transported from the surface of the ovary into the ovi- duct in animals in which the ovaries are not enclosed in periovarial sacs. During lapa- rotomy there are very real problems in maintaining the normal anatomic position and physiologic condition of the oviducts so that their actual function in vivo can be assessed accurately. In general the muscu- lar activity of the fimbriae has received more enthusiastic support than the cilia as being the agent for the transport of eggs from ovary to oviduct. However, in the few instances in which eggs were placed close to the fimbriae and egg transport observed di- rectly, the ciliary activity of the fimbriae appeared to be primarily responsible. The rate of the ciliary beat in the rabbit Fallopian tubes has been studied by Borell, Nilsson and W^estman (1957) ; during estrus the cilia beat at a rate of 1500 beats per minute. The rate increases about 20 per cent on the 2nd and 3rd day after copulation and at the time of implantation. By the 14th day of pregnancy the rate of beat had returned to normal. There was no significant differ- ence in the rate of beat in cilia removed from various segments of the oviduct. Many more direct and continuous observations on the intact oviducts of different animals are needed before definite conclusions may be reached as to the mechanics of egg trans- port from the ovary to the infundibulum. In the rat, mouse, and hamster, one of the 822 SPERM, OVA, AND PREGNANCY most striking changes in the oviduct is the dilation of the ampulla during the heat pe- riod (Sobotta, 1895; Alden, 1942b; Burdick, Whitney and Emerson, 1942). In the rat several of the loops of the ampulla begin to dilate between the 3rd and 4th hours after the onset of heat, maximal dilation being about the time of ovulation (Odor, 1948) . A constriction at the distal end of the dilated loop is frequently visible as a distinct blanched segment a few millimeters in length and in which the mucosal folds fit snugly against each other. This valve-like constriction is responsible for the retention of the oviducal fluids and eggs for at least 18 to 20 hours. Nothing is known of the nervous or hormonal mechanisms effecting the constriction, nor how spermatozoa cope with the stenosis as they proceed through the oviducts to reach the ampullae where sperm penetration occurs. The eggs of the mouse, rat, and hamster are fertilized in the dilated ampullae and I'emain there for ap- proximately 20 to 30 hours after ovulation (Burdick, Whitney and Emerson, 1942; Odor and Blandau, 1951 ; Strauss, 1956 1 . In the rabbit the freshly ovulated eggs pass through the upper half of the oviduct within 2 hours after ovulation and come to lie at the junction of the ampulla and isthmus. They remain here for the next day and a half (Greenwald, 1959). Normally sperm penetration into the eggs of mammals takes place in the ampullae. There are, however, several interesting ex- ceptions. In ferrets, tenrecs, and shrews spermatozoa somehow enter the ovarian fol- licles containing the ripe eggs and penetrate them before ovulation. Both ciliary activity and peristalsis are involved in moving the eggs into the dilated ampullae. Burdick, Whitney and Emerson (1942) showed that ciliary action in the second loop of the oviduct in the mouse is sufficiently strong to rotate a whole cluster of eggs. Vigorous, localized peristaltic waves, spaced 12 to 16 seconds apart, seemed to be more important than the cilia in moving the eggs towards the entrance of the isthmus. Almost identical observations have been reported for the transport of eggs in the ampulla of the rat (Alden, 1942b; Odor, 1948). As the time of ovulation approaches in the rat, the contractions of the dilated loops of the ampulla increase in amplitude more than in rate. The force of the aduterine con- traction waves, measured by the rate of movement of particulate matter in the lu- men, greatly exceeds that of the antiperi- staltic activity. The contraction waves do not extend beyond the constriction at the uterine end of the dilated ampulla. Clumps of ovulated eggs, stained lycopodium spores, or ascaris eggs were moved vigorously back- wards and forwards within the lumen of the tube and then forced gradually into the dis- tal, most dilated loop. This vigorous ac- tivity subsided rapidly after ovulation and would have been missed completely if con- tinuous observations had not been made. It would be important to determine more ac- curately the temporal relationship between ovulation, the dilation of the ampulla, and the changes in the pattern of muscular con- tractions of this area as compared with the remaining coils of the oviduct. The passage of ova through the isthmus and intramural regions proceeds at a re- markably constant rate in various animals. The principal forces invoked are muscular or ciliary or both. Whatever the mechanism for propulsion may be, it is not necessarily similar for all species nor for any particular segment of the oviduct within a single ani- mal (Sobotta, 1914; von ]\Iikulicz-Radecki, 1925; von ]\Iikulicz-Radecki and Nahm- macher, 1925, 1926; Kok, 1926; Alden, 1942b; Burdick, Whitney and Emerson, 1942; Odor, 1948). On the basis of their studies on the be- havior of the rabbit oviduct in vitro, Black and Asdell (1958) suggested that the move- ment of the luminal contents imparted by the circular muscles is ample to account for the transport of sperm and eggs through all of the oviduct except the isthmus. When the ova reach the isthmus they w^ait until suf- ficient fluid "surges down the tube to sweep them through the tubo-uterine junction" (Black and Asdell, 1959). When the in vivo movements of oviducts are studied by short interval time-lapse cinematography one is impressed wdth the variety of contraction patterns exhibited at different times in the cycle. These observa- tions re-emphasize the importance of ap- BIOLOGY OF EGGS AND IMPLANTATION 823 plying a host of techniques to chirify the physiology of the oviducts. The normal functional state of the ovi- ducts is dependent on the maintenance of a delicate balance between estrogen and pro- gesterone. In the mated mouse and rabbit, injections of estrogen result in tube-locking the ova for as long as 7 days after copula- tion, at which time the eggs degenerate (Burdick and Pincus, 1935; Burdick, Whit- ney and Pincus, 1937). By contrast, the in- jection of progesterone (Alden, 1942c) and induced superovulation (Wislocki and Sny- der, 1933) accelerate the passage of eggs. Fertilized ova introduced into the oviducts of pseudopregnant rabbits will continue to develop normally but they are not trans- ported into the uterus. Similarly the eggs of donor rabbits will not be transported if they are introduced into the oviducts of estrous females in which there is no luteal growth (Austin, 1949b). Alden (1942c) carefully removed the ovaries from the periovarial sacs in mated rats and observed the position and development of ova. Ovariectomy after ovulation did not prevent the normal de- velopment or transport of the eggs through the oviduct and, in fact, hastened their transport. Noyes, Adams and Walton (1959) ovariectomized rabbits and found that when freshly ovulated eggs from donor females were transplanted into the ampulla of the oviduct, the eggs were transported into the uterus in 14 hours. There is very little pertinent information concerning the role of the cilia in moving the ova through the isthmus and intramural portions of the oviduct. Because of the thickness of the muscular wall in these areas it is difficult to observe the activity of the cilia in living specimens even by trans- illumination (Alden, 1942b). Also the num- ber, size, and arrangement of the ciliated cells in the oviduct varies greatly from spe- cies to species. In addition, individual vari- ations within a given species have been de- scribed throughout the reproductive cycle (Sobotta, 1914; Novak and Everett, 1928; Hartman, 1939; Burdick, Whitney and Em- erson, 1942; Odor, 1948). The earlier observations of Parker (1928, 1931) on the ciliary currents in the opened oviduct of the turtle. Chryseunis picta, have recently been repeated and extended by Yamada (1952) to the tortoise, Clemmys japonicus, and the frog, Rana nigromacu- lata. Yamada described a reverse ciliary movement beating toward the ovarian end of the oviduct in both animals. The rate of the descending current was about two times faster than that of the ascending current. In the frog the activities of the cilia cause the eggs to rotate as they descend. This may be an important mechanism for coating the eggs evenly with egg jelly. Crowell (1932) also described a tract of cilia beating to- ward the infundibulum in the oviducts of several species of lizards. It is generally assumed that during the period in which eggs are being transported the oviducts of most mammals undergo a secretory phase, but it is not known what proportion of the fluid within the lumen is contributed by the secretions of the oviduct, the lining of the periovarial sac when present, the follicular fluid, and the peritoneal fluid. Even less is known concerning the chemistry of these fluids. The rabbit, hare, opossum, and pos- sibly the dog and horse present peculiar problems because of the specialized mucous secretions which coat the eggs and form the tertiary membranes. The cytology and secretory activity of the epithelial lining of the oviduct have been the subjects of many studies in mammals, but there is little unanimity of opinion re- garding (1) the changes in cellular mor- phology during the cycle, (2) the types of secretions elaborated, and (3) the cyclic variations of the particular secretory prod- ucts which have been identified. In the ovi- ducts of the pig and man both secretory and ciliated cells are present in the same pro- portions in all phases of the cycle. The height of the ciliated cells varies periodi- cally, reaching a maximum during the time the eggs are passing through the tubes (Snyder, 1923, 1924; Novak and Everett, 1928; Bracher, 1957). Allen (1922), among others, expressed the view that there are no ciliated cells in the isthmus of the oviduct of the mouse or rat. This interpretation must be modified at least for the rat, in view of the findings of Alden (1942b), Kel- log (1945), and Deane (1952) that both ciliated and secretory cells are present in 824 SPERM, OVA, AND PREGNANCY the isthmus of this animal. Alden (1942b) and Deane (1952) were unable to observe cyclic variations in the histologic or histo- chemical picture of the oviducts of the rat. In the mouse the primary cyclic alteration of the epithelium is restricted to a slight but significant variation in the height of the ciliated cells ('Espinasse, 1935). In the sheep the majority of the secretory cells are confined to the ampulla, few being found in the isthmus (Hadek, 1953). Hadek de- scribes a significant increase of secretory products in the lumen of the oviduct during estrus and early in the metestrum. Studies of electron micrographs of ultra- thin sections of oviducts of the mouse, man (Fawcett and Porter, 1954), rabbit (Borell, Nilsson, Wersall and Westman, 1956; Nils- son, 1957), and rat (Odor, 1953; Nilsson, 1957, 1959) have demonstrated the similar- ity of the ciliary apparatus of epithelial cells in the various species. Of special interest was the presence of tiny, filiform projections on certain of the cells interspersed among the ciliated cells (Fig. 14.9). Similar projections are also found on the luminal surface of what are probably the secretory cells. These processes do not have the longitudinal fibrils nor basal corpuscles that are essential com- ponents of cilia. A comparative study of the fine structure of the mammalian oviducts at carefully timed intervals and under dif- ferent hormonal influences may lead to im- portant observations of cyclic variations in both the ciliated and secretory cells (Borell, Nilsson and Westman, 1957). The histochemical characteristics of the epithelium of the oviduct have been studied particularly by Deane (1952) and Milio (1960) in the rat, Hadek (1955) in the sheep, Fredricsson (1959b) in the rabbit, Fawcett and Wislocki (1950) and Fredrics- son (1959a) in man. In the rat alkaline phosphatases occur on the ciliated borders of the cells of the isthmus, which suggests that this material has a role in the trans- fer of phosphorylated compounds. The rat differs from many other species in that gly- cogen could not be demonstrated in the epi- thelium of the oviduct at any time of the cycle. Quantities of esterase were present in the cells of all regions but only the cells of tr/s.;^y ,Mv, ^ .-V^ Fig. 14.9. Electron microgiaph of a thin section of the oviduct of the rat. Note nonciliated cell with microvilli wedged between ciliated cells. NN, nucleus of nonciliated cell ; NC, nu- cleus of ciliated cell; BB, basal bodies; C, cilia; MV, microvilli. (Courtesy of Dr. L. Odor.) BIOLOGY OF EGGS AND IMPLANTATION 825 the fimbriated end contained lipid droplets. It is interesting as noted earlier that in the rat no histochemical changes could be dem- onstrated during the various phases of the estrous cycle. In the sheep an acid muco- polysaccharide is secreted by the oviduct most profusely at the time of ovulation (Hadek, 1955). Amylase is present in the .secretions of the oviducts of man, cow, rab- bit, and sheep in concentrations above that found in homologous sera. The significance of the relatively high concentrations of this enzyme in relation to the reproductive proc- ess is not clear (McGeachin, Hargan, Potter and Daus, 1958). In man glycogen occurs not only in the ciliated cells but also in the nonciliated epi- thelia. Even though it is impossible to draw a firm conclusion regarding the correlation of glycogen in tubal epithelium with the menstrual cycle, it is generally believed that the maximal amount is present during the follicular phase (Fawcett and Wislocki, 1950). It is generally assumed that the luminal fluids of the oviducts and cornua undergo cyclic changes, not only in amounts se- creted, but also in their chemical composi- tion. Such assumptions are based on very tenuous evidence; actually these fluids have received very little attention primarily be- cause of the problems in obtaining ade- quate samples and in correlating the chemi- cal and physical characteristics of the tract fluids with the endocrinologic and histo- chemical activity of the cells forming the stroma. With the development of a method for the volumetric collection of tubal fluid O / / . J / L V / > '^ - 30 /lX i/» -^ ^ 20 J3 ^ - ' 3 y^ 1 ' ' ^ 10 '0--^"^ P M 12 16 Hours 20 A M 24 28 32 Fig. 14.10. Tubular secretion pressure of right and left oviducts of rabbit under Dial anes- thesia. Vertical bars indicate pulsations due to visceral movements at the time of reading. (After D. W. Bishop, Am. J. Physiol., 187, 347-352, 1956.) BIOLOGY OF EGGS AND IMPLANTATION 827 observed after ovariectomy. Injections of corjius luteum extract into the operated ani- mals prevented degeneration of the ova. Current investigations on fluids of the rabbit oviduct have shown that the secre- tions of the upper, fimbriated third are nec- essary for normal enlargement of the blasto- cyst (Bishop, unpublished data). The oviducts of pregnant females and castrates who have received progesterone secrete co- pious quantities of fluids. If these fluids are prevented from entering the uterus about the 5th day, by double ligation, the blasto- cysts remain small and do not reach their normal size by the 8th day or the time of implantation. If fertilized ova of the Muridae are pre- A-ented from entering the uterus, either by ligation of the oviduct or by the administra- tion of hormones which inhibit the normal jiropulsive mechanism of the tube, the eggs develop to the blastocyst stage before de- generation begins (Burdick, Whitney and Pincus, 1937; Burdick, Emerson and Whit- ney, 1940; Alden, 1942d). The occurrence of tubal pregnancies, especially in the hu- man female, indicates that under some cir- cumstances development may continue within the oviduct beyond the stage of nor- mal implantation. IV. Fertilization and Implantation Fertilization involves the penetration of a fully developed egg by a motile, mature spermatozoon, and the subsequent forma- tion, growth, and karyogamy of the sperm and egg nuclei. An integral part of this process is the physical act of penetration of the spermatozoon into the "karyocyto- plasm" which results in the "activation" of the egg. The classical experiments of Loeb (1913) in the invertebrates and Rugh (1939) in amphibia have shown that "ac- tivation" does not depend on a specific prop- erty of the spermatozoon, but may be ef- fected by chemical, mechanical, or physical stimuli (see also Wilson, 1925). Unfertilized mammalian eggs may likewise be activated by a variety of stimuli, but ordinarily do not proceed far in embryonic development (Pincus and Enzmann, 1936, Chang, 1954, 1957. in the rabbit; Thibault, 1949, Austin, 1951a. in the rabbit, rat, and sheep). Although Barry (1843) was the first in- vestigator to observe a spermatozoon within the mammalian egg, no detailed description of the process of fertilization appeared until Van Beneden published his observations on the rabbit in 1875. Since then, numerous in- vestigations on the cytology and physiology of fertilization in the mammal have formed a large volume of literature (Van der Stricht, 1910, the bat; Sobotta, 1895, Lams and Doorme, 1908, Gresson, 1948, the mouse; Rubaschkin, 1905, Lams, 1913, the guinea pig; Gregory, 1930, Pincus and Enz- mann, 1932, the rabbit; Tafani, 1889, So- botta and Burckhard, 1910, Kirkham and Burr, 1913, Huber, 1915, Kremer, 1924, Gil- christ and Pincus, 1932, MacDonald and Long, 1934, Austin, 1951a, b, Blandau and Odor, 1952, Austin and Bishop, 1957, the rat; Van der Stricht, 1910, Hill and Tribe, 1924, the cat; Mainland, 1930, the ferret; Van der Stricht, 1923, the dog; Pearson and Enders, 1943, the fox; Wright, 1948, the weasel; Hamilton and Laing, 1946, Piykia- nen, 1958, the cow; Amoroso, Griffiths and Hamilton, 1942, the goat; and others). The specific point of emphasis and the degree of completeness of these studies vary widely and in a number of instances only discontin- uous and isolated stages were observed and reported. Certain of the many changes occurring during the process of sperm penetration and fertilization can be studied best in fixed material properly sectioned and stained. Many features, however, can be observed most clearly only in the living egg. Obvi- ously one way of studying fertilization phe- nomena is to look at them. But microscopic observations on the living egg even with the newer phase-contrast objectives and other techniques have been disappointing to many because of the problems in establish- ing and maintaining an environment in which the processes can take place. There is such an array of observations of sperm pen- etration and fertilization in the inverte- brates that there has been a tendency to translate these observations directly to the mammalian egg. It is becoming increasingly clear that there is not necessarily a common denominator for these vital processes and that they vary widely. The interesting dif- ferences in the shape of the heads of sper- 828 SPERM, OVA, AND PREGNANCY Fig. 14.11. A living rat ovum with cumulus oophorus intact and a fertilizing spermatozoon in the ooplasm (A). B. Living rat ovum with cumu- lus intact and showing the earW development of the male and female pronuclei. X 450. matozoa from species to species alone may indicate the existence of a variety of mecha- nisms for penetrating the various barriers encountered before the vitellus can be en- tered. Quantitative data on the temporal rela- tionship between ovulation, penetration of sperm, and syngamy are lacking for most mammals. Before this information can be had for any animal, the time of ovulation must be easily and accurately determinable, the rate of ascent of spermatozoa to the site of fertilization must be known, and the rate of sperm passage through the cumulus oophorus, zona pellucida, and vitelline mem- brane must be established. Information of this sort is now available for several species, particularly that obtained by the use of phase-contrast microscopy and time-lapse cinemicrophotography in the study of living eggs. These methods have supplemented the earlier observations and made possible a more complete account of the process of fertilization (Austin and Smiles, 1948; Odor and Blandau, 1951; Austin, 1951b, 1952a). A. THE CUMULUS OOPHORUS AND SPERM PENETRATION The number of layers of cells and the compactness of the cumulus oophorus of newly ovulated eggs varies greatly in dif- ferent animals. Cumulus cells and the muco- polysaccharide matrix enclosing them have been reported as sparse or absent in the tubal eggs of the sheep (Assheton, 1898; McKenzie and Allen, 1933; Clark, 1934), the roe deer (Bischoff, 1854), the cow (Hartman, Lewis, Miller and Swett, 1931 1, the pig (Corner and Amsbaugh, 1917), the horse (Hamilton and Day, 1945), and the opossum (Hartman, 1928). In other species such as the rat, mouse, hamster, mink, rab- bit, monkey, and man (Boyd and Hamilton, 1952), many layers of granulosa cells form the cumulus oophorus. Furthermore, in cer- tain rodents the ovulated eggs clump to- gether within the dilated ampullae of the oviducts, greatly increasing the number of cell layers and viscous gels the spermatozoa must penetrate in order to reach the more centrally lying eggs. If attempts are made to remove the cells forming the cumulus of newly ovulated eggs by pulling them away with fine needles, the tenaciousness of this investment is impressive and one wonders how a spermatozoon ever reaches the vitel- lus (Fig. 14.11). In the preovulatory follicle the cells of tiie cumulus oophorus become loosened from the follicular wall and somewhat separated one from another. This is seen most spec- tacularly in the guinea pig and cat (Myers, Young, and Dempsey, 1936; Dawson and Friedgood, 1940). The ovum and enveloping cumulus cells have frequently been ob- served to lie free within the antrum before BIOLOGY OF EGGS AND IMPLANTATION 829 the follicle ruptures. Although only limited observations have been made, some reports indicate that the cumulus oophorus in the preovulatory follicles cannot be dispersed as readily by the methods that are effective in ovulated eggs (Farris, 1947; Shettles, 1953). It is important to determine what chemical or physical alterations occur in the inter- cellular cement substances of the cumulus during the time the follicle is ripening and to learn why this should differ in the cells surrounding the egg from other similar cells lining the walls of the follicle. The existence of a "cumulus-dispersing" factor in mammals was brought to light by the experiments of Gilchrist and Pincus (1932), Yamane (1935), Pincus (1936), and Pincus and Enzmann (1936). These in- vestigators demonstrated that either living sperm suspensions or sperm extracts of the rabbit, rat, and mouse rapidly disperse the cells of the cumulus oophorus of tubal ova. Yamane (1930) inferred that the presence of a proteolytic enzyme in the spermatozoa was responsible for both follicle-cell dis- persion and "activation" of the egg to pro- duce the second polar body. In a series of carefully controlled experi- ments Pincus (1936) showed that a heat- labile substance was present in sperm ex- tracts which caused follicle-cell dispersion, but that this substance would not effect second polar body formation. Pincus dem- onstrated further that the rate of cell dis- persion in vitro was roughly proportional to the number of spermatozoa in the sus- pension. It was discovered later that the "cumulus-cell-dispersing substance" was the enzyme hyaluronidase (Duran-Reyn- olds, 1929). The enzyme depolymerizes and liydrolyzes the hyaluronic acid cement sub- stance binding the granulosa cells together. This discovery at first seemed to provide a happy solution to the problem of how sper- matozoa penetrate the cumulus oophorus (McClean and Rowlands, 1942; Fekete and Duran-Reynolds, 1943; Leonard and Kurz- rok, 1945). Numerous observations cpickly demonstrated that the testes and spermato- zoa of mammals are the richest sources of animal hyaluronidase. The enzyme first ap- pears in the testes when spermatogenesis begins in the pubertal animal and before fully developed spermatozoa are present in the tubules (Riisfeldt, 1949). It became clear that there is a propor- tional relationship in vitro between sperm count and the hyaluronidase concentration ; further, that the enzyme is associated with the spermatozoa and not with the seminal plasma (Werthessen, Berman, Greenberg and Gargill, 1945; Kurzrok, Leonard and Conrad, 1946; Swyer, 1947a; Michelson, Haman and Koets, 1949). Hyaluronidase concentration per sperm is highest in the bull and rabbit, somewhat less in the boar and man, still lower in the dog, and very low in birds and reptiles (Swyer, 1947a, b; Mann, 1954). Observations on the in vitro dispersal of granulosa cells by hyaluroni- dase suggested that large numbers of sper- matozoa are necessary in the semen in order to provide a sufficient concentration of the enzyme. The in vitro observations of Pincus and Enzmann (1936) strengthened this assump- tion when they demonstrated that a mini- mum number of 20,000 spermatozoa per cubic millimeter of rabbit semen is neces- sary if the cumulus cells surrounding one ovulated egg are to be dispersed. Such ob- servations seemed to explain the necessity of the "sperm swarms" described in the oviducts of mated rabbits. The swarms created and maintained a sufficiently high concentration of the enzyme to permit the denudation of the eggs so that certain of the spermatozoa could approach and pene- trate the zona pellucida. Attempts were then made to increase the fertilizing capacity of a subnormal number of spermatozoa by adding hyaluronidase extracts to semen suspensions used for arti- ficial inseminations. In 1944, Rowlands pro- posed that such a procedure had increased the fertilizing capacity of rabbit spermato- zoa. This could not be confirmed by Chang (1950b) ; indeed, it was observed that semi- nal plasma in which the hyaluronidase had been inactivated by heat was as effective as untreated plasma. Kurzrok, Leonard and Conrad (1946) outlined a method for adding bull hyalurodinase to oligospermic speci- mens of human semen which was to be used for artificial insemination. This method was employed in the treatment of sterility and reported to have been notably successful. 830 SPERM, OVA, AND PREGNANCY Many further attempts to demonstrate the therapeutic value of hyaluronidase in mammalian infertility have met with fail- ure (see Siegler, 1947; Tafel, Titus and Wightman, 1948; Johnston and Mixner, 1950). The generally poor results obtained by the addition of hyaluronidase to semen introduced into the vagina or uterus by artificial insemination may be explained by the later experiments of Leonard, Perlman and Kurzrok (1947), which conclusively demonstrated that hyaluronidase inserted into the lower reproductive tract is not transported to the oviducts. The systematic studies of Austin (1949b) and Chang (1947, 1951a) revealed that in the rabbit only 100 to 1000 spermatozoa reach the site of fer- tilization. Even though in one experiment 600,000,000 spermatozoa were artificially introduced into the female reproductive tract, only approximately 2000 of them were found in the tubes. An even smaller number (10 to 50) have been shown to reach the ampulla of the rat oviduct at the time of sperm penetration (Blandau and Odor, 1949; Moricard and Bossu, 1951). It is probably correct to assume that any hyaluronidase which reaches the cumulus at the time of semination is transported by relatively few spermatozoa. Although the enzyme has not been localized in the sperm itself, it is assumed that it is an integral part of the cell and is liberated in a rela- tively localized region as the spermatozoon makes its way through the cement sub- stance. The spermatozoon is remarkably permeable in that such large molecules as cytochrome c or hyaluronidase can detach themselves from the sperm cell and pass into the extracellular en^'ironment by the so-called "leakage" phenomenon (Mann, 1954). In vitro tests have shown that the enzyme hyaluronidase diffuses into the suspending fluid at a definite rate depending on the type of medium and the temperature. New for- mation of the enzyme by spermatozoa does not seem to occur (Meyer and Rapport, 1952). The possibility exists that the en- zyme may be able to exert its action while still bound to the sperm cell. A recent development in the study of hyaluronidase action and its possible role in fertilization has been the attempt to utilize certain inhibitors of the enzyme as systemic contraceptives. Among the natu- rally occurring and extraneous inhibitors may be listed heavy metals, heparin, qui- nones, "rehibin" or trigentisic acid, and antihyaluronidase antibodies, as well as a nonspecific, electrophoretically identifiable serum factor (Leonard and Kurzrok, 1945; Beiler and Martin, 1947; Glick and Moore, 1948; Meyer and Rapport, 1952; Hahn and Frank, 1953; Parkes, 1953). Many of these substances are highly active inhibitors of hyaluronidase and may reduce or prevent fertilization when added to semen in vitro before artificial insemination. Attempts to inhibit fertilization by giving these sub- stances orally or by injection have not been repeatedly successful, but several deriva- tives of hyaluronic acid obtained by acety- lation or nitration and added to rabbit semen in vitro seemed to have inhibited dis- persion of follicle cells and to have im- paired fertility (Pincus, Pirie and Chang, 1948). It has now been demonstrated repeatedly that ova in the ampulla of the oviduct may have been penetrated by spermatozoa with- out evident dispersal of the granulosa cells (Lewis and Wright, 1935; Leonard, Perl- man and Kurzrok, 1947; Austin, 1948b; Bowman, 1951; Odor and Blandau, 1951, in the rat; Chang, 1950b, in the rabbit; Amo- roso, personal communication, in the cat). Again, dog spermatozoa do not contain hyaluronidase yet they are capable of pene- trating the many layers of granulosa cells comprising the cumulus. Inasmuch as a gen- eralized dispersal of the cells of the cumu- lus does not occur at the time of sperm penetration, the pendulum has swung to the present view that the individual spermato- zoon carries sufficient enzyme to make a path for itself through the cumulus layer and the gel matrix. If rat spermatozoa are added to slides containing cumulus masses from freshly ovulated eggs and their move- ment through the cumulus matrix observed with phase objectives, one is led to conclude that an intact cumulus is essential if sperm penetration is to be successful, i.e., the cumulus may act as a base against which the sperm flagellum can push as it moves BIOLOGY OF EGGS AND IMPLANTATION 831 forward towards the zona pelliicida. The spermatozoa may move through the cumu- lus with broad sweeps of their flagella and at a rate of forward i^rogression which makes it difficult to conceive of the de- l)olymerization of the matrix to form a tunnel for the sperm. It must be concluded therefore that the role of hyaluronidase in sperm penetration is unknown and that much more critical evaluation needs to be directed into this area. Even though the outer layers of the cu- mulus oophorus of ovulated eggs iti vitro may be removed readily by hyaluronidase, the corona radiata may not be dispersed with the same rapidity, especially in eggs treated immediately after ovulation. The basis for this difference lies in the fact that the cells forming the corona radiata send liolar, cytoplasmic extensions into the zona liellucida, thereby anchoring them firmly, although temporarily. In the newly ovulated eggs of the rat, hamster, and mouse the corona cells cannot be removed mechani- cally without breaking the zona pellucida. It is only after the eggs have been in con- tact with spermatozoa or have resided in the oviducts for a number of hours that the corona cells may be either brushed off the zona pellucida or drop away spontaneously. Swyer (1947b) and Chang (1951b) sug- gested that the coronal cells are removed mechanically by being more or less brushed off by the ciliary and muscular activity of the oviduct. This may be true for human and rabbit eggs, but in the rat, mouse, and hamster in which the eggs lie in the dilated ampulla, and thus at a distance from the wall of the oviduct, it would seem appropri- ate to assume that factors other than me- chanical are involved in dispersing the corona. If rat eggs are examined approxi- mately 24 hours after ovulation, one can observe that their zonae are completely free of the coronal cells, but that they may be still enclosed in an abundant viscous matrix. It appears that the corona cells gradually retract their cytoplasmic exten- sions from the zonal canaliculi. Interesting observations can be made by growing freshly ovulated eggs and their at- tached corona cells in tissue culture. Time- lapse cinematography reveals that the cells forming the cumulus and corona, although alive, have lost much of their vitality. The surfaces of the cells undergo peculiar bub- bling movements. This "bubbling" is simi- lar to that described in cells in the late stages of cell division or in cells which are about to die. Changes in the fluidity of the cell surface apparently account for the bubbling which continues for hours in fa- vorable preparations. This i)henomenon accounts for the retraction of the cell proc- esses from the zona and the gradual dis- persal of the cells. That the cumulus and coronal cells lose their vitality rather quickly after ovulation is shown further by their very poor growth in tissue culture compared with that of similar cells removed from young follicles. The rate of the dispersal of cumulus cells after ovulation varies in different animals. In mated ral)bits the eggs are completely denuded of cumulus and corona cells 4 to 6 hours after ovulation. After sterile mat- ings, however, the cumulus and corona are not dispersed until 7 to 8 hours after ovula- tion (Pincus, 1930; Chang, 1951b; Braden, 1952). In the rat there is relatively little change, either in the cumulus mass or in the corona cells for many hours after ovulation and fertilization (Blandau, 1952). Shettles (1953) suggested that in addition to hy- aluronidase there may be a tubal factor which is important in the removal of the cumulus oophorus in the human egg. He found that hyaluronidase had little effect in removing the cumulus cells in ovarian eggs, but, if bits of homologous tubal mucosa were added, the cumulus oophorus was dis- persed readily. In spite of the formidable barriers inter- posed by the cumulus and corona, they do not prevent the entrance of sperm into the egg ; in fact, as suggested earlier, their pres- ence seems to be important in some animals if penetration is to be effected (Fig. 14.11). Chang (1952a) demonstrated that, in the rabbit at least, there is a relationship be- tween the loss of the granulosa cells and fertilizability. He counted the spermato- zoa in eggs fixed at different intervals after ovulation and found that the greatest num- ber entered the eggs between the 2nd and 4th hours. Once the denudation of the eggs 832 SPERM, OVA, AND PREGNANCY is completed (approximately 6 hours after ovulation), penetration of spermatozoa no longer occurs, despite the presence of ade- quate numbers in the environs. It is im- portant to remember that the deposition of the mucous coat in the rabbit ovum may limit its fertilizable life (Pincus, 1930; Hammond, 1934). The actual time after ovulation that mucous deposition begins has been variously reported as 5, 6, 8, and 14 hours (Pincus, 1930; Hammond, 1934; Chang, 1951b; Braden, 1952). It remains to be determined whether failure of sperm penetration into the rabbit egg after 6 hours' sojourn in the ampulla is related to the loss of the cumulus, the deposition of the mucous coat, or to a specific change in the physical characteristics of the zona pellucida itself. B. THE ZONA PELLUCIDA AND SPERM PENETRATION The general appearance and pi'operties of the zona pellucida were described earlier. The manner whereby spermatozoa pene- trate the zona pellucida and the conditions influencing this process are poorly under- stood. Despite the numerous attempts to fertilize mammalian ova in vitro, only a few investigators have described isolated stages in the process of sperm penetration through the zona pellucida or into the vitel- lus. Shettles (1953) described in some detail the behavior of a human spermatozoon passing through the zona pellucida of an isolated follicular ovum. As the spermato- zoon became attached to the zona it ro- tated on its longitudinal axis. As the head was observed in focus in the equatorial plane, the rate of rotation decreased until, by the time the tip of the head was midway in the zona, the front and side views of the head could be seen to alternate. The progression of the head through the zona pellucida was intermittent until only the tail lay within it. The head and body then underwent several intermittent side-to-side, jerky movements and finally slipped into the peri vitelline space. It required 18 min- utes for a spermatozoon to traverse the zona pellucida. Duryee (1954) described the consistency of the zona pellucida of the human follicular egg as jelly-like, much less tough and resilient than the tubal egg. It would be interesting to know whether these differences in the physical properties of the zonae of ovarian and tubal eggs in the human affect the manner of spermatozoon penetration. On two occasions Pincus (1930) found rabbit ova with the heads of spermatozoa partially embedded within the zonae, and described the slow yet perceptible forward progress until the heads penetrated the vitelli. Pincus believed that the flagellae did not enter the ooplasm but were left be- hind in the zonae pellucidae. There is no sound evidence of a prede- termined pathway or "micropyle" in the zona pellucida of mammals. In the few instances where attention has been paid to this matter, spermatozoa seem to be able to penetrate the zona at any point on its sur- face. A small elliptical slit with the sperm tail partially projecting through it has been noted in the zona pellucida of living ferti- lized eggs of the rat, guinea pig, and Libyan jird (Austin, 1951b; Austin and Bishop, 1958). The slits in the zona are not seen in eggs which do not contain spermatozoa. It is usually possible to discern as many slits as there are sperm within the peri- vitelline space. The general appearance of the slit and the manner in which the per- foratorium of the sperm head attacks the zona pellucida in vitro creates the impres- sion that the zona may be fractured by the spermatozoon. Similar slits can be made by fracturing rat zonae with tungsten needles sharpened electrolytically to several micra in thickness. Recently Austin and Bishop (1958) have presented observations suggesting that the acrosome is lost as the sperm passes through the female reproductive tract and postulate that the perforatorium elaborates an en- zyme which depolymerizes the zona pel- lucida in a very restricted zone as the sperm moves through it. Discussions on the mechani.sms involved in sperm penetration of the zona have im- plicated a variety of conditions and sub- stances as being of importance in changing the physical characteristics of the zona in the localized area of contact. As mentioned earlier, the zona pellucida can be softened BIOLOGY OF EGGS AND IMPLANTATION 833 or disintegrated in rat and rabbit eggs by buffers with pH values from 3 to 5 (Hall, 1935; Harter, 1948; Braden, 1952). Various reducing agents such as glutathione and cysteine in Tyrode's solution cause rapid dissolution of the zona. Oxidizing agents such as the hydrogen peroxide which is pro- duced by sperm (Tosic and Walton, 1946) are particularly efficacious in removing this membrane. Several investigators favor the possibility that a specific mucolytic en- zyme, "zona lysin" (Austin and Bishop, 1958) may be secreted by the sperm as it makes contact with the zona pellucida (Le- blond, 1950; Austin, 1951b). It seems likely that the passage of the spermatozoon through the zona pellucida may occur in a variety of ways in different animals. Too few observations have been made to sig- nificantly implicate any of the physical, chemical, or mechanical mechanisms sug- gested for sperm penetration of the zona l)ellucida in the mammalian egg. It has been suggested that the physical jiroperties of the zona pellucida in the dog, hamster, and sheep are altered after the first sperm passes through it and enters the vitellus. It is postulated that a substance is secreted by the vitellus which "tans" the zona so that additional sperm cannot pene- trate it (Braden, Austin and David, 1954). Smithberg (1953) reported that the zonae l)ellucidae of the unfertilized mouse eggs are more readily removed by proteolytic enzymes than those of fertilized eggs. Chang and Hunt (1956) tested the effects of a variety of proteolytic enzymes on the zonae pellucidae of fertilized and unferti- lized eggs of rabbits, rats, and hamsters. Even though none of the fertilized hamster eggs contained more than one sperm, there was no evidence that the zonae pellucidae of the fertilized eggs were more resistant to digestion than those of unfertilized eggs. In contrast Austin (1956c) reported that the zonae pellucidae of fertilized hamster eggs were dissolved more quickly by trypsin than those of unfertilized eggs. Blockage of the zona pellucida in the rat and rabbit egg is not as definite, yet there are indica- tions that fertilized and unfertilized eggs react differently to proteolytic enzymes. In many animals the sequence of the re- productive processes are arranged in such a manner that spermatozoa must wait at the site of fertilization for several hours be- fore ovulation occurs and the eggs have ar- rived in the ampullae. If freshly ejaculated spermatozoa of rats or rabbits are trans- ferred directly to oviducts containing newly ovulated eggs, relatively few if any of the eggs will be fertilized. If, however, sperma- tozoa are introduced into the genital tract several hours l)efore the expected time of ovulation, they undergo some kind of change by which they gain the capacity to fertilize eggs on contact. Chang ( 1951 ) was the first to report this j^henomenon in the rabbit and termed it "development." In the same year, Austin (1951) working in Australia inde- pendently described the phenomenon and called it "capacitation." Chang (1959a) fur- ther api)i-oached this question by artificially inseminating rabbits that acted as "incuba- tor" hosts. He subsequently withdrew sperm samples at stated intervals and injected them into the oviducts of rabbits that had just ovulated. Chang concluded that 6 hours of such "host incubation" was neces- sary before rabbit sperm could fertilize the majority of ova ovulated. Similar observa- tions by Austin (1951), Noyes (1953), and Noyes, Walton and Adams (1958) on rats indicated that approximately 3 hours is the time required for capacitation in this ani- mal. There has been some success in the intrajieritoneal insemination of the rabbit doe 8 hours before ovulation with sperm which had been washed several times in a sodium citrate buffer solution (Hadek, 1958). Attempts to induce capacitation in vitro by exposing rabbit spermatozoa for varying lengths of time to a variety of physiologic solutions and solutions contain- ing endometrial tissue have been largely unsuccessful (Chang, 1955b). Partial ca- pacitation has been reported when rabbit spermatozoa are incubated in diverticula of the bladder and colon which had been created surgically (Noyes, Walton and Adams, 1958). Capacitation was also ef- fected when spermatozoa were stored in the seminal vesicles and anterior chamber of the eye. There is no evidence as yet which favors the need for capacitation in the mouse and guinea pig during normal mating. According 834 SPERM, OVA, AND PREGNANCY to Austin and Bishop (1958) there are changes in the optical properties of the acrosomes of rabbit, rat, and hamster sper- matozoa as they traverse the female repro- ductive tract. When a sperm reaches the egg in the ampulla, the acrosome is detached, exposing the perforatorium. Austin and Bishop propose that the acrosome is the carrier of the enzyme hyaluronidase which allows the sperm to depolymerize the hy- aluronic acid jelly of the cumulus oophorus. The exposed perforatorium, then, may be a carrier of a lysin which may alter the physi- cal characteristics of the zona pellucida so that the sperm may pass through it. There has been much speculation on the impor- tance of capacitation in fertilization, but there is little significant evidence to support the various theories proposed (Chang, 1955a, b, and 1959b; Strauss, 1956). C. SPERM-EGG INTERACTING SUBSTANCES The phenomenon of agglutination by "egg water" has been observed and described many times for the spermatozoa of echino- derms, annelids, molluscs, ascidians, cyclo- stomes, fish, and amphibia (Rothschild, 1956; Tyler, 1957) . The compound in the egg water responsible for tlie effect is derived from a jelly-like membrane which is secreted on the egg by the follicular cells. On ovula- tion the jelly gradually dissolves in sea water and composes the fertilizin first described by Lillie (1919). Experiments with inverte- brate eggs have demonstrated that fertilizin is responsible for the specific sperm-agglu- tinating power and for the initial specific adherence of the sperm to the egg. One of the interesting chapters in biology has been the attempt to characterize the biologic and chemical properties of these interacting substances. Whether sperm-egg interacting substances are present in the fluids forming the en- vironment of ovulated mammalian eggs has been very little investigated. Recently Bishop and Tyler (1956) and Thibault and Dauzier (1960) have reported the presence of fertilizin in the eggs of rabbits, mice, and cows. The reaction was found to be pri- marily species specific and its source is believed to be the zona pellucida. Much more experimental testing must be done to amplify knowledge in the field of interacting substances of mammalian eggs and spermatozoa. D. SPERM PENETRATION OF THE VITELLINE MEMBRANE The penetration of a spermatozoon into the ooplasm in vitro has been observed on so few occasions in mammals that it is not yet possible to give an accurate account of this phenomenon. Pincus (1930) records a slight bulging of the ooplasm in rabbit eggs at the point where the head of the sperm made con- tact with the vitelline membrane. Because of the opacity of the egg cytoplasm, no fur- ther i^rogress of the head could be observed. Studying rat, mice, and hamster eggs, Austin (195ibj and Austin and Braden (1956) de- scribed a more or less passive penetration of the ooplasm by the fertilizing spermato- zoon, as if the ooplasm "pulled" the entire sperm into its substance or "phagocytized" it. Austin (1951b) and Austin and Bishop (1957) ascribed some peculiar property to the head of the sperm which results in its being "absorbed" into the vitellus. The in- vestigations of Dan (1950) on the changes in the acrosome of the sea urchin at the time of sperm penetration of the egg have an in- teresting bearing on this problem. She be- lieved that as the spermatozoon swims ac- tively through the jelly layer of the egg, the acrosome responds to the chemical stimula- tion of the egg jelly by a localized break- down of its membrane. By the time the spermatozoon reaches the vitelline mem- brane a few seconds later, it carries at its tip a labile mass of lysin with which it effects penetration of the ooplasm. The observations of Austin are at variance with those made by others also in the rat and in which it appeared that ooplasmic pene- tration was accomplished primarily by the activities of the flagellum of the fertilizing sperm (Blandau and Odor, 1952). Although discontinuous, the forward progression of the spermatozoon into the ooplasm seemed to depend on a propulsive type of undulating movement of the tail which forced the head forward a distance of 10 to 20 /x at a time. While that portion of the flagellum within the ooplasm was retarded in its amplitude of motion by the viscosity of the egg cyto- BIOLOGY OF EGGS AND IMPLANTATION 835 plasm, that which was still in the pehvitel- line space lashed about vigorously. These observations are similar to those described by Shettles (1960) in the human. As men- tioned earlier, the technical problems in ob- serving in vitro fertilization will no doubt be solved when the molecular species of the fluids forming the normal egg environment is known. There is no specific information with re- spect to the nature of the vitelline membrane of the mammalian egg or to the changes it may undergo on sperm entry. It would be de- sirable to know whether the vitelline mem- brane undergoes modification after penetra- tion by the fertilizing spermatozoon. An interesting procedure for measuring the so- lidification of the egg membranes of salmo- nid eggs has been described recently by Zotin (1958). Even though there is no clear evi- dence of a comparable phenomenon in mam- malian eggs, some factor appears to control the number of spermatozoa which enter the vitellus. Cortical granules have been de- scribed in the unfertilized hamster egg which disappear on fertilization, but apparently they are not associated with the block of l)olyspermy (Austin, 1956a). Quantitative data are necessary to clarify the relationship between the number of spermatozoa which may enter the periovarial space, the rate of the "tanning" reaction of the zona, if such a ])henomenon exists, and the reaction of the perivitelline membrane which blocks the en- try of further spermatozoa. Shrinkage of the vitellus after sperm pen- etration has been described in the rabbit and rat (Gilchrist and Pincus, 1932; Pincus and Enzmann, 1932), but a comparable shrink- age can be noted in unfertilized ova recov- ered from the oviduct several hours after ovulation, and thus shrinkage per se cannot be used as a criterion for sperm penetration. The shrinkage of the vitellus is related in some way to changes in the vitelline mem- brane because the numerous microvilli pres- ent in the young ovarian egg have disap- peared and the total surface of the egg has been greatly reduced. E. FERTILIZ.\TIOX IN VITRO During the past century one of the most challenging and frustrating problems was the attempt to fertilize mammalian ova in vitro and to follow their cleavage. Even though several successes were recorded, it could not be maintained unequivocally until the recent work of Chang ( 1959a) that sperm penetration has been accomplished and that the divisions of the eggs noted were the re- sult of fertilization rather than of an "ac- tivation" of the egg instituted by some other factor in the environment, or just plain frag- mentation. Relatively little has been added to our un- derstanding of the mechanism of sperm pen- etration into the ooplasm since the extensive experiments of Long (1912) in which he at- tempted to fertilize rat and mice eggs in vitro. He described penetration of the fol- licle cells and observed the sinuous move- ments of the sperm as they advanced within the cunmlus. The role of the spermatozoa in the dispersal of the granulosa cells was noted and this was interpreted as being due to the lashing activities of the sperm fiagellum. Long also described the formation of the second polar body in eggs which had been placed in sperm suspensions. Polar body for- mation began within 2 hours and abstric- tion was completed within 4 hours of the time of immersion. Unfortunately, his de- scription leaves one uncertain as to whether penetration by the sperm was actually ob- served or merely confirmed by sectioned ma- terial. Some success with fertilization in vitro was also achieved by Pincus (1930, 1939), Pincus and Enzmann (1934, 1935), Venge (1953), and Thibault and Dauzier (1960) in their extensive experiments with both ovar- ian and tubal eggs of rabbits. These in- vestigators described the abstriction of the second polar body, the shrinkage of the vitellus, the penetration of the zona by spermatozoa partially embedded within it, and the presence of spermatozoa in the peri- vitelline space in fixed and stained prepara- tions. Transplantation of living eggs into the oviducts of pseudopregnant rabbits, follow- ing the addition of sperm to the eggs, re- sulted in the birth of live young possessing the genetic characteristics of coat color which had been used as markers. It is sug- gested in a later report (Chang and Pincus, 1951) that the results "may have been due 836 SPERM, OVA, AND PREGNANCY to adherent sperm effecting fertilization in the fallopian tubes." The mammalian egg may be "activated" to various degrees according to the balance of thermal, osmotic, and chemical factors in its environment. Thus eggs "activated" by being placed in a cold environment may form double nuclei which closely resemble normal pronuclei (Thibault, 1947a, b, 1948). The eggs of the opossum, rat, mouse, hamster, mink, and ferret also will show varying de- grees of "activation" and may be difficult to differentiate from normally cleaving ova (Smith, 1925; Chang, 1950a; Austin, 1951a, 1956c; Blandau, 1952). Attempts to fertilize the timed human ovarian ova recovered by Corner, Farris and Corner (1950), were un- successful. Rock and Menkin (1944) and Menkin and Rock (1948) also attempted to achieve fertilization of human ovarian eggs in vitro and reported several successes. The first egg recovered from a large follicle was cultured in the patient's serum for 27 hours. It was then placed in a washed suspension of sperm for 1 hour and observed continuously. Penetration of the ovum by sperm was not observed. When the same egg was inspected 40.5 hours later, it consisted of two blasto- meres each measuring 86 /a in diameter. A second egg treated in much the same manner also was found to contain two blastomeres 45 hours after exposure to spermatozoa. The stage of maturation of these ovarian eggs could not be determined and it is assumed that the meiotic divisions occurred in vitro. Since the fertilizable life of the human ovum is unknown, and there is no specific informa- tion on sperm penetration, the role of the flagellum in semination, pronuclei formation, karyogamy, and the rate of cleavage, it is clear that the true identification of a fer- tilized human ovum has