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^ 0 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 ^ £ 0 nil i ^ t3 1 3 3 S^ § S - P 3 -t; i a .C J= JJ "s S ° « 0 .^ H .- 1 J-- — 3 H 5 3 ^ a . S 0 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 0 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 0-0.03 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 0 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 0 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 0 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 not been achieved. In the rat, for example, one can find unfer- tilized cleaved ova which on first inspection closely resemble fertilized eggs even contain- ing modified nuclei or nuclear fragments. When examined in detail the fragmenting eggs do not contain the flagellae of spermato- zoa, a positive indication that penetration has not been accomplished (compare 2 and 3 in Figure 14.15). Various criteria have been accepted as an indication of fertilization in vitro such as polar body formation, shrinkage of the vitel- lus, presence of one or more pronuclei, and cleavage of the ooplasm. As emphasized ear- lier, all of these phenomena have been ob- served many times in eggs which have not been penetrated by a spermatozoon and which are in varying stages of degeneration and fragmentation. Too little is known con- cerning the processes of semination and fer- tilization in mammals, with the possible ex- ceptions of the rat and rabbit, to judge uneciuivocally whether normal sperm pene- tration and fertilization have been accom- plished in vitro. The freshly ovulated eggs of most mam- mals are notoriously sensitive to changes in environment and one is concerned lest the eggs cultured in vitro may simulate the events occurring in vivo without activation by a spermatozoon. If sperm penetration and the various fertilization i)henomena cannot be followed continuously by direct visuali- zation, it is generally agreed that, unless viable embryos are obtained by transplant- ing the supposedly fertilized eggs to recipient animals, the success of fertilization is not sufficiently proven. Recently Chang (1959a) was successful in fertilizing the rabbit egg in vitro and obtaining living young by trans- planting them to host animals. Thus for the first time a repcatable procedure for fertiliz- ing mammalian ova in vitro has been per- fected. Chang obtained unfertilized rabbit eggs by intravenous injection of sheep pi- tuitary extract into estrous rabbits. Sperm were obtained 12 hours after mating females with fertile bucks by washing the uterus with a Krebs-Ringer bicarbonate solution. Un- fertilized ova were obtained by flushing the oviducts of the animals which had received the gonadotrophins. Both sperm and eggs were placed in a small Carrel flask and kept at 38°C. Three to 4 hours later the ova were transferred to a second Carrel flask contain- ing rabbit serum and cultured for another 18 hours. At this time the eggs were recov- ered and examined, and those that appeared to be cleaving were transferred to recipient rabbits. Approximately 42 per cent of the transferred ova that appeared to be ferti- lized were delivered at term as viable young. F. FATE OF THE UNFERTILIZED EGG Evidence that ovulation without fertiliza- tion is followed by rapid degeneration and BIOLOGY OF EGGS AND IMPLANTATION 837 fragmentation of the vitellus has been ob- tained for many different species (Hartman, 1924, Smith, 1925, in the opossum; Sobotta, 1895, Kirkliam, 1907, Long, 1912, Charlton, 1917, in the mouse; Chang and Fernandez- Cano, 1958, in the hamster; Long and Evans, 1922, Mann, 1924, Blandau, 1943, 1952, in the rat; Squier, 1932, Blandau and Young, 1939, in the guinea pig; Chang, 1950a, in the ferret; Heape, 1905, Pincus, 1936, in the rabbit; Dziuk, 1960, in the gilt; Hartman, Lewis, Miller and Swett, 1931, in the cow; and Allen, Pratt, Newell and Bland, 1930, in man ) . With the possible exception of the rat, the jiroblem of the ultimate fate of the degener- ating ova has not been satisfactorily resolved for any mammal. It is generally accepted that as the unfertilized eggs undergo com- l)lete fragmentation and dissolution they are absorbed either in the oviducts or uterus (Corner, 1928a; Pincus, 1936). Charlton { 1917) suggested that final disintegration of unfertilized ova in the mouse is effected by means of phagocytic leukocytes. It is as- sumed further that the unfertilized ova dis- appear from the female reproductive tract before the succeeding ovulation. However, Hensen (1869) described the retention of approximately 100 rabbit ova in a blocked oviduct in which presumably the eggs had accumulated from a number of ovulations. The unfertilized ova in the rat do not un- dergo complete dissolution during the nor- mal 4- to 5-day estrous cycle. The vitellus fragments ordinarily into a number of units of varying sizes and the eggs, with their zonae intact, are eliminated near the end of the succeeding heat period by being washed out through the vagina (Blandau, 1943). Attention has been directed to the freciuent occurrence of abortive "cleavages" in the un- fertilized tubal eggs of the ferret and rat (Austin, 1949a; Chang, 1950a). This phe- nomenon is more common in the prepubertal rat treated with gonadotrophins than in the adult animal. In the "cleaved" unfertilized ova, the blastomeres and their nuclear con- figurations may appear identical with those of fertilized ova and can, indeed, be differ- entiated only by the absence of the flagellum of the fertilizing sperm. Most unfertilized ova. however, fragment into a number of units of unequal size, each containing one or more abortive nuclei. G. FORMATION OF THE SECOND POLAR BODY The penetration of the vitellus by a sper- matozoon is not the only stimulus which will induce the formation of the second polar body. Yamane (1930) observed that if rab- bit eggs are placed in solutions containing rat or horse spermatozoa, or immersed in pancreatic solutions, cytoplasmic masses similar to the second polar body will be ab- stricted. Similar "false polar bodies" or ex- trusions of clear, chromatin-free masses were produced when rabbit eggs were immersed in various concentrations of trypsin ( Pincus and Enzmann, 1936). Both the abstriction of the second polar body and shrinkage of the ooplasm may be induced in rabbit, rat, and mouse eggs by a variety of other non- specific stimuli such as ether, Nembutal, nitrous oxide anesthesia, and "cold shock" (Pincus and Enzmann, 1936; Thibault, 1949; Austin and Braden, 1954b; Braden and Aus- tin, 1954). By contrast, colchicine or "hot shock" inhibits the emission of the second polar body (Austin and Braden, 1954b). Austin (1951b) described the formation of the second polar body in rat eggs in which spermatozoa were in the perivitelline space but had not yet penetrated the vitelline membrane. It is uncertain whether "activa- tion" is caused by a substance released into the perivitelline space by the spermatozoa, or by the mechanical impact of the sper- matozoa on the vitelline membrane. There are relatively few data on the temporal re- lationship between penetration of the vitel- lus by the sperm and the abtrusion of the second })olar body. Pincus and Enzmann (1932) reported that in rabbit ova 45 min- utes or more elapse between the time the sperm enters the vitellus and the formation of the second polar body is completed. For- mation of the second polar body in vitro has also been observed in mouse eggs that had been penetrated by spermatozoa. The time required for the complete process was over 2 hours (Lewis and Wright, 1935). Long (1912) pointed out that second polar body formation in the rat began within 5 minutes to 2 or more hours after the spermatozoa were added to tlie eggs in in vitro prepara- 838 SPERM, OVA, AND PREGNANCY tions. Abstrictions of the polar bodies were completed 45 minutes later. The interesting observations of Austin (1951c) on the sequence of events during formation of the second polar body in the living rat ova deserve special mention. In the unfertilized egg the chromosomes are arranged on the metaphase plate with the spindle lying paratangentially to the sur- face, usually in close association with the abstricted first polar body. Within a few minutes after the sperm head has penetrated the vitellus, and before it shows any de- tectable change, the chromosomes on the second maturation spindle pass to anaphase. The telophase stage is reached about 75 min- utes after the initial penetration by the sperm. Then, there is a 20-minute period dur- ing which no further change is noted. Subse- quently, the spindle slowly moves away from the surface and begins to rotate in such a way that its final position is at right angles to its original location. Rotation is com- pleted in about 50 minutes. The spindle then elongates and becomes narrower, the process terminating in abstriction of a clear vesicle containing the clumped chromosomes. Since it was necessary to flatten the egg consider- ably in order to be able to observe the spin- dle under the phase microscope, complete abstriction of the polar body did not occur. Similar observations on the formation of second polar bodies in rat ova were re]iorted by Odor and Blandau (1951). Approxi- mately 2000 eggs were removed at varying intervals after ovulation and sperm penetra- tion. The eggs were examined either in the fresh condition or after histologic prepara- tion. In the majority of ova, the second polar body had been abstricted completely by the end of the 4th hour after semination. H. PRONUCLEI FORMATIOX, SYNGAMY, AND FIRST SEGMENTATION DIVISION As mentioned earlier, the general concept of the mechanism of fertilization in mam- mals has been based almost entirely on the examination of fixed and stained material. Even so, it is remarkable that a story of con- tinuing development should have evolved by the piecing together of evidence from killed eggs, the age of which could not be deter- mined within narrow limits. The more recent advances involving an evaluation of the temporal relationship between ovulation and the various phenomena of fertilization may be said to be due largely to the applica- tion of phase contrast microscopy to the studies of living rat ova (Austin and Smiles, 1948; Odor and Blandau, 1951; Austin, 1951a, b, 1952a; Blandau and Odor, 1952; Austin and Braden, 1954a, b). Employing this method, Austin and Smiles observed fertilized eggs that were obtained by inducing ovulation in immature rats by means of gonadotrophins and subsequently allowing the females to mate. The recovered zygotes were kept at body temperature and development was followed continuously with the phase microscope. The details of the fer- tilization process described by Odor and Blandau were the result of examining sev- eral thousand living and fixed fertilized eggs recovered from sexually mature females at specific time intervals after ovulation and fertilization. In the rat the complete process of fertiliza- tion, from the penetration of the ooplasm by sperm until the first segmentation division, requires approximately 24 hours. In general, the first 8 hours after sperm penetration is the period of the formation of the second polar body and the initial development of the male and female pronuclei (Fig. 14.12). Changes in the morphology of the living sperm head can be noted as early as 10 min- utes after penetration of the ooplasm and involve a loss of sharpness of outline and contrast, first in the posterior and caudal re- gions of the head. The decrease in contrast continues until finally the whole nuclear part is almost invisible in the living specimen, even under the phase-contrast objectives (Fig. 14.12, Jf). Concomitantly the head in- creases greatly in size and fluidity. During the initial period of swelling of the nuclear portion, the bifid perforatorium becomes de- tached (Fig. 14.12, 3). Approximately 2 hours after the sperm has entered, the j^ri- mary nucleoli make their appearance within the enlarged sperm nucleus. Time-lapse cine- microphotography has shown that the nu- cleoli enlarge by the fusion of minute nucleo- lar aggregations. The larger nucleoli then fuse one with another until only a single large nucleolus is present (Fig. 14.13, 1 and BIOLOGY OF EGGS AND IMPLANTATION 839 ^ -^ ■ 1, . 1 ^" 1 \p .ji* "*\ *:- . 3 A %,■ ♦■ • • 1 • • * % ^ i . .9 \\ ^ N SF 6 Fig. 11.12. \'nri()U.< ,-l;i-iv.s in the traii.-loiiuat ion ol I lie Ina.l nl iIm I. i i ili/iii- -|m i m leading to the formation of the male pronucleus. Note loss of contrast of the liead as it enlarges. The changes in the head from 1 through 6 require 2 to 3 hours. Observations were made on the living egg, in vitro, and examined with phase contrast objectives. P, perforatorium; N, sperm nucleus; SF, sperm flagellum (Austin, 1951c). 2). Throughout this period of transforma- tion, the fiagellum may remain attached to the head and may undergo a very fine, inter- mittent, vibratory motion, especially in the region of the middle piece. The formation of the definitive female pronucleus begins soon after the second polar body has been com- l)letely abstricted. The chromosomes re- maining within the ooplasm after extrusion of the polar body are clumped together in the form of a small, compact mass (Fig. 14.13). The first indication of transforma- tion of this chromosomal mass into the fe- male pronucleus is the appearance of several minute nucleoli within a homogeneous nu- cleoplasm. As the nucleoli increase in size 840 SPERM, OVA, AND PREGNANCY Fig. 14.13. Further transformation of the sperm head into the male pronucleus, 1 and 2. Note the large nucleolus, formed by fusion of smaller nucleoli. NC, nucleolus. This stage has been reached approximately 5 hours after that in part 1, Figure 14.12, 1 and 2 (Austin, C. R., 1952). Developing male ( i ) and female ( $ ) pronuclei, in situ, as observed in living rat eggs, 3, 4 and 5. The entire sperm flagellum enters the ooplasm at the time of sperm penetration, 3 (Odor and Blandau, 1951). and number, certain of them coalesce, even- tually producing one or two large nucleoli. As the pronucleus grows, the optical density of its nucleoplasm decreases to such an ex- tent that it becomes clear and translucent. Although there may be considerable varia- tion in the development of the pronuclei be- tween the 9th and 19th hours after sperm penetration, this is the time of active growth of the pronuclei and of increase in the num- BIOLOGY OF EGGS AND IMPLANTATION 841 her of their nucleoH (Fig. 14.13, 5). During the early hours of this period, the male pro- nucleus grows at a more rapid rate than that of the female, and this differential is main- tained even until karyogamy. At the stage of greatest development, the number of nu- cleoli in the male pronucleus may have in- creased to approximately 30 and that within the female nucleus to 10. Near the end of this interval, the pronuclei gradually ap- proach one another. For some time after ac- tual contact, the pronuclei retain their identity and the female pronucleus may con- siderably indent the larger male pronucleus (Fig. 14.14, 2). Approximately one-half hour before karyogamy begins, the nucleoli in both in'onuclei disappear from view and there is some shrinkage in the size of the pronuclei (Fig. 14.14, 3). Even after the complete disappearance of the nucleoli, the nuclear membranes may still be intact. Soon, however, they become irregular in outline and disappear. Shortly before the first seg- mentation division, an aggregation of the pi'ophase chromosomes may be observed. Within a brief period, the chromosomes are arranged on the metaphase plate. After an interval of 30 to 40 minutes, the chromo- somes begin to divide and pass through the anaphase and telophase stages (Fig. 14.14, 4 and 5). The first segmentation spindle is observed most commonly between the 21st and 23rd hours after the entrance of the sperm. Even though Austin ( 1951c) followed the formation of the segmentation spindle, cleavage of the rat zygote did not occur in vitro. It is often difficult to differentiate between the male and female pronuclei in sectioned material. Hence, their identification has not been clearly established for most mammals. The male pronucleus has been reported to be larger in the cat (Hill and Tribe, 1924) , vole ( Austin, 1957 1 , guinea pig ( Lams, 1913 ) , and rat (Odor and Blandau, 1951 ; Austin, 1951c; Austin and Braden, 1953) , and of approxi- mately equal size in the mouse, guinea pig (Lams and Doorme, 1908), bat (Van der Stricht, 1910), cat (Van der Stricht, 1911), and hamster (Boyd and Hamilton, 1952; Austin, 1956b). Edwards and Sirlin (1956a, b, 1959) have demonstrated that the male pronucleus within the fertilized mouse egg could be identified by injecting adult males with C^"*-labeIed adenine approximately 1 month before mating. The male pronuclei showed autoradiographs which could be related to the labeled sperm particularly in di- and tri- spermic eggs. Lin ( 1956) labeled unfertilized mouse eggs with DL-methionine while they were still within the follicles. Ovulation was induced by gonadotrophins and the unferti- lized eggs were transplanted to mated fe- males where they were fertilized and subse- quently delivered as normal young. The acridine orange-staining tcchni(iue has been applied recently to living rat eggs and the localization of the stain determined by fluorescence microscopy (Austin and Bishop, personal communication). The dis- tribution of DNA may be determined by this technique and the ]H-eliminary data give sup- port to the earlier rejjorts of Dalcq and Pas- tcels (1955) that duplication of DNA occurs within the jironuclei. Information regarding the temiwral re- lationship between the formation of the first segmentation spindle and karyogamy is also very meager. In the guinea pig (Rubaschkin, 1905; Lams, 1913), bat (Van der Stricht, 1910), and rat (Odor and Blandau, 1951), the pronuclei have not completely fused by the time the spindle is formed. Isolated phases of this stage have been described also for the mouse (Lams and Doorme, 1908), rabbit (Gregory, 1930), and goat (Amoroso, Griffiths and Hamilton, 1942). I. FATE OF THE CYTOPLASMIC COMPONENTS OF THE FERTILIZING SPERM FLAGELLUM Observations on the extent to which the flagellum of the fertilizing spermatozoon is carried into the ooplasm of the mammalian egg are contradictory and incomplete. The majority of the reports deal with sectioned material in which the identification of the whole flagellum may be very difficult. Yet, knowledge of the fate of the cytoplasmic components of the sperm is essential to an understanding of the role of the male gamete and must be pursued further. In the mammals, the entire tail has been reported to be lodged within the ooplasm in the bat (Van der Stricht, 1923 ) ; mouse (Van der Stricht, 1923; Gresson, 1948) ; guinea pig 842 SPERM, OVA, AND PREGNANCY Fig. 14.14. Migration of the male and female prouuclei towards the center of the egg, 1 and 2. The male pronucleus is frequently indented by the female pronucleus, 2. At 3, note the dis- appearance of the nucleoli in the pronuclei immediately before the appearance of recognizable chromosomes as seen in 4. Telophase stage during first segmentation division in 5. PB, polar body (Odor and Blandau, 1951). (Lams, 1913) ; rat (Gilchrist and Pincus, 1932; Austin, 1951b; Blandau and Odor, 1952) ; and ferret (Mainland, 1930). Pineus (1930) and Nihoul (1926) were not con- vinced that in rabbits the flagellum enters the ooplasm. In the vole the flagellum enters the vitellus in only 55 per cent of fertilized eggs (Austin, 1957). Except for the investigations by Gresson in the mouse, and Blandau and Odor in the rat, there are no detailed accounts of the fate of the flagellum after it enters the fer- BIOLOGY OF EGGS AND IMPLANTATION 843 tilized egg. In the mouse the mitochondria and Golgi material of the sperm become dis- persed throughout the egg cytoplasm and the axial filament of the flagellum disap- pears before the first cleavage. But in the rat the flagellum is of such length and rigidity that it assumes an eccentric position within the periphery of the cell. Probably this ex- plains why the male pronucleus ordinarily begins its development in the outer zones of the egg. Between the 15th and 19tli hour after penetration, the external sheaths of the middle- and main-pieces begin to lose their smooth contours and they gradu- ally disappear (Fig. 14.15). When this has been accomplished, the spiral mitochondrial sheath of the middle piece and the axial filament of the main piece can be clearly visualized. Immediately before the first cleavage, the continuous helical mitochon- drial thread begins to swell. During the 2-cell stage, the mitochondrial thread is Fig. 14.15. Chromosomes from the metaphase of the first segmentation division removed from a living, fertilized rat egg, 1. The sperm flagellum from the same egg lies just below the chromosomes. Note that the spiral mitochondrial sheath (SMP) is still present. At 2, two- cell rat egg with the remains of the sperm flagellum at arrow. At 3, unfertilized rat egg in which the fragments appear similar to the blastomeres of a normally fertilized egg but there is no sperm flagellum present (Blandau and Odor, 1952). 844 SPERM, OVA, AND PREGNANCY Fig. 14.16. Somewhat flattened, living rat ovum with 13 accessory spermatozoa in the perivitelline space and a single fertilizing spermatozoon in the ooplasm. X 450. broken vip into globules that are dispersed throughout the egg cytoplasm. The remains of the axial filament have been observed in the 2-cell stage of the bat (Van der Stricht, 1902), guinea pig (Lams, 1913), and vole (Austin, 1957) and as late as the blastocyst stage of the rat (Blandau and Odor, 1952). Van der Stricht (1902) and Lams (1913) believed that, in the 2-cell stage of the bat and guinea pig, the sperm tail is pres- ent in only one of the blastomeres. This was partially substantiated for the rat by Blandau and Odor, who noted that in 58 per cent of 329 2-celled ova a greater por- tion of the axial filament was located within one blastomere and that in 12 per cent it lay entirely within a single blastomere. In the remaining 30 per cent, the axial filament was equally divided between the two. The significance of the various posi- tions of the axial filament in the cleaving egg is not clear. It may represent merely the mechanical difficulty of moving an inert body. Of greater significance is the meaning of the cytoplasmic contribution of the sperm midpiece to the developing embryo in those animals in which its component parts are despersed within the vitellus. J. SUPERNUMERARY SPERMATOZOA AND POLYSPERMY IN MAMMALIAN OVA The terms "supernumerary sperm," "ac- cessory sperm," and "polyspermy" have been used to mean either the penetration of more than one spermatozoon into the ooplasm with the subseciuent development of multiple sperm nuclei, or the location of one or more spermatozoa in the perivitelline space. Inasmuch as polyspermy is used widely in the literature of invertebrates to designate the penetration of the ooplasm by multiple spermatozoa, it is suggested that this meaning should be retained for mammals and that the terms supernumerary or accessory spermatozoa should be utilized just to indicate the presence of nonfertiliz- ing spermatozoa in the perivitelline space. Intact spermatozoa have been observed many times within the perivitelline spaces of ova of various mammals (Sobotta and Burckhard, 1910, Gilchrist and Pincus, 1932, Odor and Blandau, 1949, Austin, 1951b, in the rat; Lams and Doorme, 1908, Lewis and Wright, 1935, in the mouse; Van der Stricht, 1910, in the bat; Hcnsen, 1876, Lams, 1913, in the guinea pig; Hill and Tribe, 1924, in the cat; Heajie, 1886, in the mole; Harvey, 1958, in the i)ika; Pincus and Enzmann, 1932, Chang, 1951c, in the rabbit; Hancock, 1959, in the pig). Quanti- tative data on the presence of supernumer- ary spermatozoa are available for the rat and several strains of mice. Austin (1953) and Odor and Blandau (1951) found that approximately 23 per cent of seminated rat ova contained supernumerary spermatozoa. The number of sperm per egg ranged from 1 to 23 (Fig. 14.16). After mating various strains of mice, Braden (1958a, b); and Piko, 1958) reported that the percentage of ova containing more than one sperm was more significantly related to the strain of the male than to the female. Matings with C57 males resulted in a consistently higher number of eggs with more than one sperm, irespective of the strain of the fe- males used. Apparently supernumerary spermatozoa have no effect on the rate of development of the ovum. In the rat, at least, the fluids of the perivitelline space offer an environ- ment which is considerably more favorable for these spermatozoa than that of the oviduct. Except for a separation of the head from the neck-piece, the accessory spermatozoa in the rat, at least, show no evidence of cytolysis in any of the de- BIOLOGY OF EGGS AND IMPLANTATION 845 velopmental stages including the late blast- ocyst. As mentioned earlier, spermatozoa from the same insemination that are lying free in the oviduct will have undergone extensive cytolysis in less than 24 hours. Finally, with the disappearance of the zona pellucida at the time of implantation, the accessory spermatozoa are cast forth into the uterine lumen. Austin (1957) suggests that the flagellum within the perivitelline space of the vole egg may undergo dissolu- tion in situ. The penetration of more than one sperm into the ooplasm is a common phenomenon in birds, rei:»tiles, urodeles, selachians, and insects (Fankhauser and Moore, 1941). Ordinarily, the additional sperm nuclei do not interfere with the development of the egg. Until recently, polyspermy in cutherian eggs was considered to be relatively rare (Austin and Braden, 1953). Nevertheless, trinucleate eggs have been described in the rat (Tafani, 1889; Kremer, 1924; Pesonen, 1949; Austin and Braden, 1953); cat (Van der Stricht, 1911; Hill and Tribe, 1924); ferret (Mainland, 1930) ; and rabbit (Amo- roso and Parkes, 1947; Austin and Braden, 1953). According to Austin and Braden, the in- cidence of polyspermy in the normally mated rat is approximately 1.2 per cent; in the rabbit 1.4 per cent. If mating is de- layed until after ovulation or if rats are subjected to hyperthermia, the figure rises to as much as 8.8 per cent. The incidence of polyspermy is no doubt influenced by a variety of conditions including hereditary variations within various strains (Odor and Blandau, 1956; Braden, 1958a, b). Austin and Braden (1953) concluded from their work that polyspermy in rats gives rise to triploidy in the embryo and that the poly- spermic male pronuclei and the female pro- nucleus contribute to the formation of the first cleavage spindle. To the present, the polyspermic rat embryos have been found to develop to at least the 8-cell stage without showing abnormality. Fischberg and Beatty (1952a, b) have observed a normal-appear- ing triploid mouse embryo at 9^2 days. It is not known wdiether the triploid embryos can survive to birth. More recently. Gates and Beatty (1954) have stated that delay of fertilization by 5^/2 hours or more in the mouse did not result in an increased number of triploid embryos. K. STAGES OF DEVELOPMENT AND LOCATION OF EGGS The zygotes of the eutherian mammals are remarkably similar in their appearance and rate of development through the vari- ous stages of cleavage and formation of the blastocyst. Cleavage consists of a succession of mitotic divisions of the zygote at specific time intervals after karyogamy. The par- titioning of the zygote occurs with little or no increase in the total amount of cyto- plasm. Salient features of the mechanism of cleavage in different vertebrate types have been reviewed bv Bovd and Hamilton (1952). Data on the rate of cleavage and trans- port of fertilized ova through the oviduct in different animals have accumulated much more slowly than one would expect from the availability of material. The most complete information has been obtained for some of the ungulates and laboratory rodents and is presented in tabular form (Table 14.5) from the summary of Hamilton and Laing (1946). The rate of cleavage is an inherent property of the zygote. Thus the cleavage rates of different species of amblystoma reared at the same temperature are signifi- cantly different. Similarly, in the rabbit the cleavage rate is consistently more rapid in strains of larger-sized animals than in the smaller-sized races (Castle and Gregory, 1929; Gregory and Castle, 1931). It is in- teresting that, although the zygotes of the larger-sized race divided more rapidly and contained more cells, embryonic differentia- tion occurred at the same rate in both races. Altering the environment of zygotes may also effect the rate of cleavage. Thus the early fertilized eggs of the rat, mouse, ham- ster, and guinea jiig cleave only irregularly if at all under tissue-culture conditions. If various thio-amino acids are added to the medium in which rabbit zygotes are being cultured, cell division will proceed normally and may even be accelerated (Pincus, 1937; Pincus and Werthessen, 1938; Miller and Reimann, 1940). 846 SPERM, OVA, AND PREGNANCY ^1 III I 00 t: ~— - c3 00 C^ 00 t^ 1 1 02 -^ r^ CO r-l -^ 00 o '^ 05 o O -^ ^ !C o ^ t^ rt t^ C3 ' lO ^H lO 00 »o i I I I A ~ ^ 3 o3 2 ^ J ^3: 1 3 I I I I -I- "H c^ ^ o 00 I I S8 lO ~ t^ cc ,^ iCi - o ^ 12 hours, experimental Blandau and Jordan (1941) Guinea pig >20 hours, experimental Blandau and Young (1939); Row lands (1957) Ferret >30 hours, experimental Hammond and Walton (1934) Rabbit 6 hours, experimental 8 hours, experimental Hammond (1934) Chang (1953) Braden (1952) Sheep 24 hours, estimation Green and Winters (1935) Cow 18-20 hours, experimental Barrett (1948) Mare Short Day (1940) Monkey 23 hours, estimation Lewis and Hartman (1941) Man 6-24 hours, estimation Hartman (1936) interference with either the normal proc- esses of producing or liberating evocators, or the capacity of the embryonic tissues to respond to induction. Of special interest is the finding that the older fertilized eggs frequently gave rise to teratomatous pro- liferations in the endoderm. When these tumor-like masses were transplanted to older larvae they grew rajjidly and metasta- sized. Needham (1950) proposed that, be- cause the primary evocator, the principal sex-hormone, and various carcinogens be- long to the steroid compounds, the effect of over-ripeness may be related to a disturb- ance of embryonic sterol metabolism. The fertilizable life of the mammalian ovum has been experimentally determined in only a few rodents, carnivores, and ungu- lates (Table 14.6). In the ferret, for exam- ple, Hammond and Walton (1934) found that the ovum remains capable of fertiliza- tion for not more than 30 hours after ovula- tion. In the rabbit, delay in fertilization results in lowered fertility and smaller size of litters (Hammond, 1934). In the hamster 50 per cent of ova are incapable of fertiliza- tion 4 to 5 hours after ovvdation (Chang and Fernandez-Cano, 19581. In rats the spermatozoa may penetrate eggs which have been aged 12 hours before fertilization or to a point of devitalization but not of death. In such eggs they may even undergo transformation into the male pronuclei and form segmentation spindles, but the female nucleus in the same egg either fails to develoj) or fragments into a number of nuclei of varying sizes. Even though 70 per cent of the greatly over-ripe rat eggs may be penetrated by spermatozoa, various abnormalities of development result which are not compatible with continued growtii and development. Thus, at the time of implantation only 4 per cent of the ex- perimental rats are impregnated. Further- more, the ova which do implant successfully are retarded in their development, and the «50 SPERM, OVA, AND PREGNANCY majority die before the fetal period is reached (Blandau, 1952; also see Braden, 1959). A strikingly similar picture is presented by delayed fertilization in the guinea pig. The fertilizable life of the egg in this species is approximately twice (20 hours) that of the rat (Blandau and Young, 1939; Row- lands, 1957). The first effects of over-ripe- ness are seen in embryos from females in- seminated approximately 8 hours after ovulation. No normal development followed inseminations more than 20 liours after ovu- lation. As far as could be determined, the principal effects of aging were either the early death of the zygote in the pre-im- plantation period or retardation in the rate of growth in embryos which were capable of implanting. A moderate delay in fertiliza- tion has been shown to lead to polyspermy particularly in rats and rabbits (Austin and Braden, 1953; Odor and Blandau, 1956). M. IMPLANTATION The blastula of the placental mammal is called the blastocyst. In the fully developed stage it is still enclosed in the zona pellu- cida and shows the inner cell-mass attached to the embryonic pole of the trophoblast. During the early period of its existence the blastocyst is spherical to somewhat oval in shai)e and except for size appears remark- ably similar from animal to animal (Fig. 14.i7). In most mammals, the blastocyst does not come into firm contact with the maternal .•^ ^. Fig. 14.17. The similarity of the free uterine bhistoc-y.^t.s of various mammals: 1, 5!/2-day human blastocyst (photograph courtesy, Hertig, A., and Rock, J.); 2, 6-day guinea pig blas- tocyst; 3, 9-day monkey blastocyst (Heuser and Streeter, 1941); and 4, 9-day sheep blasto- cyst (Boyd and Hamilton, 1952). BIOLOGY OF EGGS AND IMPLANTATION 851 endometrium for a number of days after reaching the uterus. In the mouse, mole, shrew, and guinea pig, the free uterine pe- riod is from 3 to SVk days; in the rabbit, 5 to 6 days; in the rhesus monkey and possi- bly the human, 4 to 6 days ; in the cat, 8 to 9 days; in the dog, 9 to 10 days; and in the ungulates probably somewhat longer. Under the conditions of "developmental diapause" or delayed implantation, the free uterine period of the blastocysts may be significantly prolonged. Delayed implanta- tion occurs naturally in a variety of species such as the pine marten, 6 months; Ameri- can badger, 2 months; European badger, 3 to 10 months; European roe deer, 4 months; armadillo, 14 weeks; fishers, 9 months; and bears, 6 months. Delayed implantation has also been recorded in the stoat, weasel, sa- ble, and fur seal. In the rat, mouse, and cer- tain insectivores, implantation may be de- layed several days to 2 wrecks if there is concurrent lactation (Lataste, 1887; Daniel, 1910; King, 1913; Hamlett, 1935; Brambell, 1937; AVeichert, 1940, 1942). In the mouse and rat the delay varies roughly with the number of young suckled, and this, in turn, prolongs the period of gestation. According to Lataste, the duration of gestation is nor- mal in mice suckling only 1 or 2 young but prolonged in those suckHng 3 or more. If certain hormonal conditions are satisfied, implantation will occur in normal females suckling large litters (Kirkham, 1916; Weichert, 1940, 1942, 1943; Krehbicl, 1941). Delay of implantation is very likely due to an inhibitory effect by some uterine or nu- tritional factor acting on the blastocysts (Whitten, 1958). Various experimental methods may successfully delay implanta- tion without destroying the ova. Ovariec- tomy the second day after mating in the rat, followed by subliminal doses of progester- one (0.5 mg. per day ) , will keep the eggs alive for 6 to 45 days, but the decidual cell response and implantation do not take place. If more progesterone than 0.5 mg. per day is injected into these animals, implanta- tion may occur. A combination of injections in which a small dose of estradiol benzoate is added to the subliminal dose of progester- one is very effective in consummating im- l^lantation. In contrast, if the ovaries are removed from pregnant rats on the fourth day when the blastocysts have reached the cornua, progesterone, even in dosages of 10 mg., cannot effect implantation. If estrogen and progesterone are injected simultane- ously, the blastocysts will resume their growth and will implant (Canivenc and Laffargue, 1957; Cochrane and Meyer, 1957; Mayer, 1959). The blastocysts of pregnant rats spayed the 4th day may remain alive for as long as or longer than 21 days. Rat blastocysts apparently do not reciuire adrenocortical hormones to remain viable. Mayer (1959) and his co-workers have demonstrated that the blastocysts in the cornua of rats which have been ovariectomized and adrenalecto- mized on the 4th day after mating can im- plant on the 10th day, provided estrogen and progesterone are both injected simul- taneously. The experiments of Cochrane and Meyer, and others that have been mentioned, sug- gest that the optimal conditions for embryo attachment and implantation depend on a delicately balanced, synergistic action of estrogen and progesterone on the endome- trium. But nothing is known as to what is happening within the egg during its dormant state and what factors control the dor- mancy, nor do we understand what changes occur within the uterine lumen which may eventually satisfy the conditions of the em- bryo to continue its growth, make attach- ment to the uterine epithelium, and implant. Our point of view will no doubt be broad- ened as experimental approaches to the problem are varied and more species are studied. Runner <1947), Fawcett (1950), and Kirby (1960) found tliat, irrespective of the state of the host's gonads, im- plantation occurred when mouse ova were transplanted either to the kidney cap- sule or to the anterior chamber of the eye. Whitten (1958) transplanted 8-celled mouse eggs to the surface of the kidneys of normal and hypophysectomized mice. Ten days later successful grafts were found in 10 of 15 normal and in 13 of 18 hypophy- sectomized animals. Successful implantation of mouse eggs onto the kidney apparently does not depend on the secretion of the pi- tuitary. Buchanan, Enders and Talmage (1956) 852 SPERM, OVA, AND PREGNANCY reported that implantation occurs in ovari- ectomized armadillos that are not receiving hormonal replacement. In the European badger ovulation occurs during delayed im- plantation. The new set of corpora lutea does not hasten implantation because delay in implantation may continue for 2 months after the last ovulation (Harrison and Neal, 1959). The phenomenon of delayed implantation offers an excellent experimental approach to the general problem of embryo-endometrial interrelationships and the specific factors that control embryo attachment and im- plantation. N. SPACING AND ORIENTATION OF OVA 7^- UTERO The specific sites of implantation in mam- mals having multiple young, as related both to the longitudinal axis and to the surface of the endometrium, are remarkably con- stant (Mossman, 1937). Even in animals having only a single young and a simplex uterus, such as man, monkey, sloths, and others, the location of the implantation site and the orientation of the blastocyst to the endometrium are quite definitely regulated (Mossman, 1937; Heuser and Streeter, 1941). Various explanations have been proposed to account for the intra-uterine spacing of blastocysts in polytocous mammals. Moss- man suggested that the implanting blasto- cyst may interact in some manner with the surrounding endometrium so as to create a local refractory zone in which no other em- bryos can implant. The results obtained by Fawcett, Wislocki and Waldo (1947) after transplanting several mouse ova into the same anterior chamber of the eye are of in- terest in this connection. They found that fertilized eggs continue to develop in close proximity to one another only until one of them begins to implant. Thereafter, the re- maining embryos degenerate. The onset of the degenerative changes in the surrounding blastocysts is coincident with the extravasa- tion of blood into the tissues in the immedi- ate vicinity of the attaching embryo. They suggest that possibly a cytolytic ferment of the trophoblast may cause edema or hemor- rhage into the maternal tissues which so al- ters the local environment that it is unten- able for the remaining blastocysts. According to Mossman's theory, the blastocyst that enters the uterine cavity first establishes a refractory zone near the uterotubal junction and begins the process of attachment. The remaining blastocysts establish similar zones in the fashion of a gradient toward the cervix until all become evenly spaced. It has been frequently ob- served in pregnant animals with bicornu- ate uteri that the embryos which are im- planted nearest the oviducts are slightly more advanced in development than those nearest the cervix. It has also been observed that the embryos which are implanted near- est the cervix show a higher incidence of resorption than those implanted at other sites. Recently McLaren and Michie (1959) have taken issue with Mossman's theory that implantation is serial and that refrac- tory zones are established. These investiga- tors induced ovulation and mating in mice by hormone treatment. At 18V^ days after mating, the cornua were divided into 6 equal segments and the embryos weighed. They found that the embryos in the middle of the cornua actually weighed less, on the aver- age, than those at either end. The embryo lying nearest the oviduct was usually sig- nificantly lighter than its neighbor. It may be questioned whether the differ- ences in weight of mice fetuses at ISV^ days post coitum have any relationship to differ- ences in size and differentiation of the em- bryos during the first 5 to 10 days of devel- opment or during the period of orientation in utero or of attachment and implantation. Investigators who have observed blasto- cysts and implanting embryos have fre- quently commented on the variations in the early stages of development in the same ani- mal and the variation from animal to ani- mal when they are killed at identical times after mating. The variations in the rate of differentiation are particularly striking if the development of the attachment cones of the guinea pig embryos are observed in tis- sue culture. The attachment cones of each of the 2 to 3 blastocysts recovered from the cornua of the same animal may be in a dif- ferent stage of development and may retain BIOLOGY OF EGGS AND IMPLANTATION 853 this difference throughout the period of cul- tivation. The successful transplantation of eggs from animal to animal in certain rodents is feasible and may be the means whereby an experimental approach to the problem of spacing can be made. One or more fertilized eggs could be transferred to the oviducts of properly timed hosts and their sites of at- tachment observed. One of the problems in evaluating implantation grossly in trans- plantation e.xperiments is the possibility of inert objects (lint, clumps of cells, etc.) affecting the decidual response and mimick- ing imiilantation. In normal, pregnant rats the embryos are more evenly spaced in cornu when the num- ber of young is 5 or more. If the number of implanting blastocysts is less than 4, there is a tendency to occupy chiefly the caudal halves of the horns (Frazer, 1955). Information is needed as to the manner in which eggs enter the cornua, i.e., whether they enter singly or as a group and what the relationship of the multiple eggs may be one to another during the several days that they lie free within the uterine lumen. It is ciuite clear that embryonic spacing in utero is more even than random. This raises the cjuestion as to what controls the size of the refractory area if the cornu is crowded by superovulation, transplantation of eggs, or more than normal numbers of eggs from compensatory hypertrophy in cases where one ovary has been removed. It has long been known that in bicornu- ate uteri blastocysts may pass from one cornu into the other through the body of the uterus (Boyd, Hamilton and Ham- mond, 1944; Boyd and Hamilton, 1952; and many others). Bischoft' (1845) interpreted transuterine migration as a method by which the distribution of embiyos could be equalized in cases where there is a disparity in the number of eggs ovulated from each ovary. The means by which this migration is accomplished has been the subject of speculation and some investigation. At present, there is no direct evidence that the unimplanted embryo has the power of independent movement. If this is true then the positioning of the blastocyst in utero and its orientation in relation to the endo- metrium must depend on chemical and/or physical forces. Markee and Hinsey (1933) suggested that alternate contractions of the cornu transport blastocysts from one to an- other. Krehbiel (1946) anastomosed the cornua of ovariectomized rats in a variety of ways and concluded that each uterine cornua retains its individuality in effecting: the distribution of embryos. The role of the myometrium in the distri- bution and spacing of the blastocysts in utero has received considerable attention. Corner (1923) and Wislocki and Gutt- macher (1924) found active myometrial contractions in the sow during the pre- implantation period. Even though the postovulatory contractions occurred with greater frequency, they were greatly dimin- ished in amplitude compared with those re- corded during the estrous phase. The motil- ity pattern of the myometrium changes gradually from day to day so that, by the time of implantation (12th or 13th day), the spontaneous contractions continue at a rate of 4 to 8 per minute, but their ampli- tude is so slight that the kymographic trac- ings are almost level. Similar observations were reported for the excised uterine horns of the rabbit (Knaus, 1927). Using a more refined technique and beginning their ob- servations immediately after the muscle strips were put into the bath, Csapo and Corner (1951) and Csapo (1955) showed that uterine muscle under the dominance of progesterone displays a high state of irrita- bility but poor conduction, and it develops spontaneously a state of "contracture" when it is first placed in the muscle bath. Spontaneous contractions begin after a short interval but they are of very low amplitude. The initial "contracture" is reversible and may be suspended by electrical stimulation or anoxia. Progesterone in some way alters the response of the myometrium to stimuli. The motility pattern of the myometrium under the dominance of progesterone is cer- tainly different from that when the animal is in estrus, but the nature of these differ- ences is still puzzling (Reynolds, 1949; Csapo and Goodall, 1954). A strip from an estrous uterus placed in the bath relaxes immediately. After a short interval, spon- taneous contractions begin and continue 854 SPERM, OVA, AND PREGNANCY with increasing amplitude. Thereafter, con- tractions occur at intervals of 1 to 2 minutes followed by prompt relaxation. In contrast, similar relaxation was not observed in uter- ine strips under the influence of progester- one. Instead they slowly shorten. Ivy, Hartman and Koff (1931j observed that muscular contraction waves in the monkey uterus originate from an area slightly ventral and cranial to the insertions of each of the oviducts and then proceed medially to meet in the midline. They con- cluded that in the monkey the area of the endometrium where implantation usually occurs is affected by contractions to a lesser extent than the remainder of the uterus. Nicholas (1936) interposed a section of duodenum into the rat's uterus and found embryos in the lower uterine segment. Lim and Chao (1927) reversed the middle por- tion of one or both cornua of the rabbit and reported that pregnancy was not prevented. Markee (1944) introduced sea urchin eggs, celloidin balls, and glass beads into the tubal ends of rabbit cornua and observed their distribution at varying intervals from estrus to 10 days after ovulation. He found that the sea urchin eggs were distributed most evenly in the uteri of cstrous rabbits, especially at the time of ovulation. Fairly good distribution was recorded at 5 days and poor distribution at 10 days after ovu- lation. As noted below, none of these inert objects or sea urchin eggs expand with time as do rabbit blastocysts before attachment. It is doubtful that the movements of these objects in utero could be considered as the normal state of affairs in the transport of blastocysts. In order to study this problem further, Markee observed uterine contrac- tions directly through a glass window which had been sewn into the abdominal wall. Three types of contractions were observed during estrus and for 5 days after ovulation : (1) local ring-type contractions persisting for approximately 10 seconds, (2) peristal- tic contractions proceeding throughout the length of the cornu, and (3) antiperistaltic waves of approximately the same intensity as the peristaltic contractions. After the 5th day, the peristaltic and antiperistaltic con- tractions decreased greatly in amplitude and in the length of their excursions. Recent studies on the mechanisms con- tributing to the distribution of the implant- ing rabbit blastocysts have directed at- tention to the possibility that both physical and chemical interactions between the blas- tocyst and uterus are important (Boving, 1952a, b, 1954, 1956, 1959). Boving has found that by 7 days post coitum, rabbit blastocysts have achieved an almost even distribution, not only with reference to the space between them, but also with respect to the entire length of the uter- ine cornu (Fig. 14.18). If the num- ber of blastocysts in utero varies, the spacing is nevertheless appropriate to their number. The cornua reacts to the presence of each blastocyst and po- sitions it in relation to all other blasto- cysts present until a remarkably even dis- tribution is achieved by the 7th day post coitum. There is evidence from the work on the rabbit at least that the movement and i)ositioning of blastocysts in utero coincide with their increase in size. Rabbit blastocysts of approximately 1-mm. size are propelled much more slowly than blasto- cysts or glass beads 3 to 6 mm. in diameter. Boving suggested that each blastocyst acts as a localized stimulus which initiates the propulsive muscular activity and that the size of the blastocysts determines the way in which the myometrium responds. Cessa- tion of positioning is coincident with a local loss of uterine tone and a ballooning out of the antimesometrial wall to form a "dome." The blastocysts of the leporid family of rodents, the carnivores, some insectivores, and bats undergo considerable expansion in the uterine cavity before and at the time of attachment. In these animals, then, the spacing of the blastocysts may be arranged according to Boving's theory that myogenic uterine contraction is the effector of both propulsion and spacing. As mentioned earlier, during the 6th and 7th days after copulation in rabbits, the expanded blastocysts occupy a distended, antimesometrial "dome" caused by a local decrease in uterine muscle tone. From in vivo observations of the pregnant uterus, Boving (1952b, c) observed that the blas- tocysts of the rabbit undergo a rotational BIOLOGY OF EGGS AND IMPLANTATION 855 8 + + + + + • • • • • • ^ ( • • • • • 1— • • • • • o o b • • • • 1— Q_ D • • • 00 >- Q 4 • • •• .^ ... t 1 1 1 1 1 0 20 TUBAL END 7 'a 40 60 80 OF UTERINE LENGTH 100 Fig. 14.18. Positions of rabbit blastocysts (dots) in utero (bars) from the 3rd to the 8th day post coitum. There is little change in position during days 3 and 4. Even distribution is achieved 6 to 7 days -post coitum. The crosses in the 8-day uterine horn represent the position of blastocyst models which had been in the uterus for 2 days (Boving, 1954). and to-and-fro motion approximately every 30 seconds. This seems to be effected by a change in the tone of the muscles forming the uterine dome. The rotational motion could provide an orientational mechanism, because eventually all surfaces of the blas- tocyst would come in contact with the dome. In the in vivo observations, it seemed that the blastocyst is "grasped" by the muscular action of the uterus, and by the 7th day post coitum is confined along the antimeso- metrial border. As implied earlier, the orientation of the blastocyst with reference to the uterus and mesometrium varies considerably in differ- ent species. It may be mesometrial as in the Pteropodidae and Tarsiidae, antimesome- trial as in most rodents and insectivores, or orthomesometrial as in the Centetes and Hemicentetes (Mossman, 1937). The orientation of the embryonic disk within the uterus is remarkably constant in closely related species but varies greatly in different orders. Thus the inner cell mass at the time of attachment may be di- rected toward the mesometrium in the rodents, toward the antimesometrial side in the vesperilionid bats and some insectivores, or toward the lateral side as in the golden mole. With the possible exception of the rabbit and guinea pig, the role of the blas- tocyst in determining the pole of attachment is unknown. Alden (1945) reversed the mesometrial- antimesometrial axis of the uterus of the rat by surgical means and demonstrated that, regardless of the position of the altered segment, the implanting embryos were cor- rectly oriented relative to the uterus. Ap- parently, gravity alone is not of great importance in determining the pole of at- tachment, at least not for the rat egg. Before the cells of the trophoblast can come into contact with the uterine epi- thelium, either the tough and resistant zona pellucida must be removed or the cells of the living trophoblast must penetrate the zona. A number of investigators have thought chiefly in terms of the removal of the mucous coat and zona pellucida by uterine factors. As we will see, others have been impressed by the possibility of par- ticipation by the trophoblast. In 1935 Hall presented evidence which seemed to support the former view. He found that in rats and mice the zonae pellucidae disappear rapidly when im- mersed in fluids of pH 3.7 or below. In less acid solutions (pH 4 to 5), they were affected much more slowly. Acidified Ring- 856 SPERM, OVA, AND PREGNANCY er's fluid at first caused a swelling of the zonae, and the ordinarily smooth outer contour became wavy and fringe-like. In measuring the hydrogen ion concentration of the fluids in the vicinity of deciduomata of the rat, values as low as pH 5.7 were recorded. Such values w^ere of sufficient acidity to effect the gradual softening of the zona pellucida. Pincus and Enzmann (1936) also measured the pH of uterine luminal fluids in pseudopregnant rats and at no time observed values below 6.7. From Hall's work it was concluded that "as the decidua develops around the implanting egg and as the metabolic activities of the divid- ing blastocyst increase, the fluid bathing the blastocyst may become sufficiently acid to be a factor in the removal of the oo- lemma." Fertilized mouse ova, transplanted to the anterior chamber of the eye, lost their zonae independently of a change in hydrogen ion concentration of the envi- ronmental fluids r()xiiiiaii'l\- oiic hour before at- tachment of the abembryonal pole to the endometrium. Note the increase in the number of the abembryonal pole cells and the cytoplasmic extensions of these cells through the zona pellucida. X900. 858 SPERM, OVA, AND PREGNANCY Fig. 14.21. Living guinea pig blastocyst removed appioximatel}' one-half hour before at- tachment to the endometrium. The blastocyst is slightly rotated to show the extensive pro- toplasmic projections at the abembryonal pole. X 900. scribed in fixed preparations of a number of genera of grovnid squirrels and chip- munks (Lee, 1903; Mossman, 1937). Al- though the extension of conclusions based on the study of one species to other species is precarious, it is possible that the same re- lationship of the attachment cone to the zona pellucida exists in other forms (Moss- man, 1937). Boving noted a change in the viscosity and adhesiveness of the rabbit egg investments at the time of implantation which he attributed to local alkalinity (pH 9) released from one or more regions of the abembryonic hemisphere. Following ad- hesion, the outer investments of the blasto- cyst in this area disintegrate. Inasmuch as remnants of the membranes are sometimes observed in the areas between the implant- ing blastocysts, their final removal ap- jiarently is similar to that described for the guinea pig. When the membranes have been shed the abembryonic trophoblast adheres to the uterine epithelium, particularly in areas where blood vessels are subjacent to the epithelium. The trophoblast pene- trates the epithelium by displacement, and the invasion of the stroma at first is not destructive. O. BLASTOCYST EXP.\XSION In the guinea pig, rat, mouse, and ham- ster, the diameter of the blastocyst at the time of attachment is approximately the same as that of the tubal ova. In these species implantations are more or less regu- larly spaced but not invariably so, because placental fusion occurs frequently. Thus there does not seem to be the same purpose- BIOLOGY OF EGGS AND IMPLANTATION 859 fill interplay between the embryos and cor- nua as described for the rabbit. The blas- tocysts of these rodents are definitely polarized in relation to the uterine epithe- lium at the time of attachment and in- vasion. Although it is universally stated that the blastocyst does not have the ca- l^acity for independent movement in utero, observations on the behavior of the guinea pig blastocyst in tissue culture and the cytologic descriptions of the attachment cones in the monkey, ground squirrels, and chipmunks suggest that the blastocyst plays an active role in its positioning at the time of attachment. This possibility would en- courage one to examine more carefully the living blastocysts of various animals at the time of attachment. From some of the earlier investigations, it would seem that the expansion of the rabbit blastocyst is dependent on physiologic factors external to the egg itself (Pincus and Werthessen, 1938). Thus, blastocyst expansion is inter- fered with if ovariectomy is performed or estrogen is injected 3 to 5 days after mat- ing. On the other hand, injections of pro- gesterone can reverse the effect of estrogen (Burdick and Pincus, 1935; Pincus, 1936; Pincus and Kirsch, 1936). Allen and Corner (1929) showed that if progesterone is in- jected into rabbits ovariectomized shortly after fertilization, the fertilized eggs will implant normally. If fertilized rabbit ova are grown in watch glass cultures, they will ■ cleave normally, but they herniate and col- lapse during the blastocyst stage (Lewis and Gregory, 1929) . If crystalline progester- one is added to these cultures, there is no increase in the rate of cleavage nor is herniation or collapse prevented (Pincus and Werthessen, 1937). The same investi- gators have shown that regular expansion of the blastocyst is obtained if the morulae or blastocysts are cultured in homologous serum and the medium is continually circu- lated. Recently, Bishop observed that ex- pansion is suppressed if the oviducts of rabbits are ligated soon after the blastocysts have entered the uterus. The implication is that some oviducal factor is necessary for expansion. The problem is complicated by the fact that the egg does not expand dur- ing its 3 day sojourn in the oviduct. From the observations recorded above, it seems that in order to stimulate normal growth and expansion of the blastocyst, in the leporid family of rodents at least, pro- gesterone must act in some way on oviducal and uterine metabolism since both parts of the genital tract are probably involved. The specific physicochemical processes in blastocyst expansion are not known. A plausible explanation is that the expansion may be due simply to the processes of osmosis, the changes in size being related to ionic variations of the fluid within the blastocyst cavity and the surrounding en- vironment. It is more likely, however, that comjMex processes of active transport are involved, and, if these are to be elucidated, help from the biochemist and physical chemist is essential. One of the difficulties confronting in- vestigators so trained is the small amount of material obtainable for study by the conventional chemical methods. This is par- ticularly true in such laboratory animals as the mouse, rat, hamster, guinea pig, and monkey, in which the blastocyst undergoes very little expansion before implantation and in which uterine secretions are present in very minute amounts. Nevertheless the recent approaches to the study of embryo attachment and implantation in the rabbit, particularly those by Boving (1954), Ben- nett (1956) and Lutwak-Mann (1959), offer a methodological approach that is essential if the dynamic aspects of nidation are to be understood. Lutwak-Mann espe- cially and her co-workers have been the most active in discerning the practical prob- lems in the handling of early embryologic material for biochemical study and in de- vising sound methodologic approaches. In 1938 Pincus and Werthessen described a crystalline deposit in the abembryonal membranes of certain blastocysts of rabbits removed on the 5th day after mating from females which had been ovariectomized 18 to 20 hours after copulation. Boving (1954) identified this crystalline material as cal- cium carbonate and noted that there is little or none present 3 to 4 days after mating, but that the deposit increases to a maximum at the 6th day post coitum. He suggested that the osmotic effect of the 860 SPERM, OVA, AND PREGNANCY blastocyst fluid is increased by the ioniza- tion of the inherent calcium carbonate re- serve. Deficient respiration of the free blas- tocyst may perhaps lead to the production of acids which react with the calcium car- bonate reserve. At the time of uterine at- tachment, there is improved gas exchange due to the embryo's close proximity to sub- epithelial blood vessels. Thus the bound alkali is liberated, the ionic concentration of the fluid is decreased, and blastocyst turgidity is lessened. In measuring the bicarbonate of the rab- bit blastocyst cavity fluid, Lutwak-Mann and Laser (1954) found a remarkably high content in 6- and 7-day-old embryos. There- after, the level of bicarbonate fell rapidly so that on the 8th day, when implantation is completed, the level was somewhat below that for maternal blood. The occurrence of high concentrations of bicarbonate in the unattached blastocysts led to assays of carbonic anhydrase activity in extracts of pregnant and nonpregnant rabbit uterine mucosa. It was found that carbonic an- hydrase activity was very low in the uteri from nonpregnant animals but very high in the uteri from pregnant individuals. The oviducts, endometrium, and placental tis- sues are the main loci of carbonic anhydrase activity in the female reproductive tract. There are, however, species differences in the extent and the time at w^hich the enzyme can be demonstrated. The endometria of pregnant or nonpregnant hamsters, rats, and guinea pigs do not contain measurable quantities of carbonic anhydrase. However, significant enzyme activity has been found in the maternal portions of the placenta of these animals (Lutwak-Mann, 1955). It has been clearly established for the rabbit that the enzyme is hormone-de- pendent. Progesterone and progesterone-like compounds greatly increase the amounts of the enzyme measured in the endometrium and this increase is proportional to the dosage of the hormone injected. There is no concomitant increase of carbonic an- hydrase in the blood (Lutwak-IVIann and Adams, 1957a, b). There is a 10- to 30-fold increase in the weight of the blastocyst between the 5th and 6th days. Dry weight measurements have shown that this increase is due pri- marily to water. The enzyme system re- sponsible for the active transport of water is as yet unknown, but is being actively sought. Concentrations of Na, K, and CI ions in the yolk sac fluid approach or, in the case of K, exceed that of the maternal serum. Glucose, on the other hand, is present in less than half the amount found in mater- nal blood on the 7th day and two-thirds the amount on the 8th day. Data are also available on total nitrogen, phosphorus, bi- carbonate, and various vitamins, particu- larly the components of the B complex, in the unimplanted blastocyst (Kodicek and Lutwak-Mann, 1957; Lutwak-Mann, 1959). Obviously the opportunities for uti- hzing isotopes for transfer studies in the fresh and implanting blastocysts are many indeed, and one may confidently expect a rapid unravelling of the manifold functional aspects of implantation if these techniques are employed by competent investigators. p. EMBRYO-ENDOMETRIAL RELATIONSHIPS The interrelationship between the blasto- cyst and the endometrium at the time of at- tachment and implantation is not only exceedingly comjilex but also highly vari- able in different species. Irrespective of the complexity of the attachment, each type has as its purpose the apposition or intimate fusion of the fetal mem- branes to the maternal endometrial epi- thelium or stroma so that adequate phys- iologic exchange can take place. Earlier studies on the experimental pro- duction of deciduomas by mechanical stim- ulation of the sensitized endometrium, and the dependence of implantation on the proper hormonal stimulation of the uterine mucosa, had the effect of swinging the pendulum of opinion toward the endometrium as being the most active agent in the process of nida- tion (Huber, 1915; Kirkham, 1916; Selye and McKeown, 1935; Krehbiel, 1937; Ross- man, 1940). More recently, however, the ob- servations ( 1 ) on the development of the attachment cone in some specific area of the trophoblastic wall just before attachment, (2) the changes in the viscosity and ad- hesiveness of the egg envelopes at the time of attachment, and on the develop- mental potentialities of ova transplanted to the anterior chamber of the eye and BIOLOGY OF EGGS AND IMPLANTATION 861 other sites have swung the pendulum back to the embryo and the role that it may l)lay in nidation (Asshcton, 1894; von Spec, 1901; Schoenfeld, 1903; Mossman, 1937; Fawcett, Wislocki and Waldo, 1947; Run- ner, 1947; Blandau, 1949a; and Boving, 1954, 1961 j. The extensive i)rolil'eralion and differen- tiation in the endometrium of certain ani- mals after ovulation undoubtedly arc im- l)ortant in the nourishment and maintenance of the ovum in utero and in providing a suit- able implantation site. The considerable growth and differentiation which the blasto- cysts of many animals undergo before they make contact with the uterine mucosa would indicate that more nutrients are required than are stored in the ooplasm of most mam- malian eggs. The widespread occurrence of glucose, glycogen, lipids, phosphatases, iron, calcium, and many other substances, includ- ing vitamins and enzymes, in the endome- trium may provide the necessary nourish- ment during the very early stages of implan- tation ( Wislocki and Dempsey, 1945) . Bloch (1939) described the secretion of an osmo- l)hilic substance by the uterine epithelium which is thought to be absorbed by the free mouse blastocyst. The work of Daron (1936), Markee (1940), Phelps (1946), Parry (1950), and Boving (1952a, 1961) has demonstrated that there is an increased blood supply immediately below the uterine epithelium at about the time of blastocyst attachment. The increased vascularity may not only provide nutrition to the uterine epi- thelium, but more importantly it provides blood vessels for specific physicochemical re- actions between the trophoblast and endo- metrium (Boving, 1959a) . A similar increase in the blood supply in the antimesometrial area has been observed in the guinea pig (Bacsich and Wyburn, 1940). This is the area in which implantation invariably oc- curs in this species, and the localized hyper- emia is considered to be a factor in tlie anti- mesometrial implantation. It is well established that the presence of an actively secreting corpus luteum is essen- tial if implantation is to be complete and successfully maintained. In rabbits proges- terone is necessary, not only for the nutri- tion of the free blastocyst in utero, but also for implantation (Fraenkel, 1903; Corner, 1928b; Corner and Allen, 1929; Hafez and Pincus, 1956a, b) . Histochemical and quan- titative tests have indicated that lipids are present in the endometrium in greater amounts during the luteal phase of the re- productive cycle than at any other time (Krehl)iel, 1937; van Dyke and Chen, 1940; Alden, 1947). It is clear that the presence of an embryo in the cornu exerts a significant effect on the secretion of luteotrophic hormone and on the functional life of the corpus luteum. How these effects are producecl remains a chal- lenging problem. We need to determine whether direct invasion of the endometrium is essential or whether mere expansion of the embryo can act as a trigger mechanism. Nalbandov and St. Clair (1958) have shown that if plastic beads of more than 2 mm. in diameter are inserted into the cornua on the 8th day of the estrous cycle in sheep, the cycle is significantly lengthened. Denerva- tion of the cornu containing the beads pre- vented this change in length. It has been found repeatedly that endo- metrial sensitivity to the formation of de- ciduomata is limited normally to the period of implantation and placentation (Loeb, 1908; Allen, 1931; Selye and McKeown, 1935; Krehbiel, 1937; Greenwald, 1958b). The traumatizing substances were physical, chemical, and electrical stimuli. From these studies, three facts were revealed: (1) The formation of the "maternal placenta" can be induced in the complete absence of the blastocyst (Krehbiel, 1937; Mossman, 1937; Dawson and Kosters, 1944). (2) Even though tissue destruction in the endome- trium can be brought about by specific and nonspecific stimuli and even though the end- result may appear similar, the mechanisms producing the changes do not necessarily stem from the same basic stimulus. (3) All of the stimuli used are presumed to have as the basis of their action some kind of tissue injury.^ Notwithstanding, the histologic transformations of the deciduomas corres- pond exactly to those occurring normally in ^ The passage of an electric current of sufficient magnitude through the endometrium to induce the decidual response gives no evidence of tissue dam- age that can be detected microscopically. This of course does not eliminate the possibility^ that cel- lular injury has not occurred. 862 SPERM, OVA, AND PREGNANCY early pregnancy. Krehbiel (1937) found, for example, in the experimentally induced de- ciduomas of the rat that glycogen and lipids appeared intracellularly in cells which cyto- logically seemed identical with those of the normal endometrium of pregnancy. It would be interesting to know whether the same intensity of artificial stimulus would induce the decidual response in the uteri of a variety of animals. In the rat, for example, the slightest pressure against the superficial uterine epithelium, at the proper time after ovulation, is sufficient to initiate the decidual response. Thus a bit of lint, small clumps of cells, and glass or paraffin beads the approximate size of eggs effect an endometrial response identical with the re- sponse to the normally implanting embryo (Blandau, 1949a). In this species the very earliest changes in the subepithelial stroma begin when the blastocyst is attached only very tenuously to the uterine epithelium (Fig. 14.22). From this response of the endometrium, perhaps localized pressure is sufficient to induce the decidual reac- tion. Equally impressive is the fact that the decidual response begins before there is any alteration in the superficial uterine epithe- -r^ H^l:y.' 'f/. •}^v::i/L. Fic. 11.22. I.MiigiiiKhii.il ^.riinn 1 hrough the anti- mesometiial wall of a pregnant rat killed on the 5th day. The loosely attached rat blastocyst has initiated the subepitheUal decidual response. There is no detectable alteration in the superficial epi- thelium. X 450. limn detectable by microscopic means. Thus, any stimulus from living eggs or inert ob- jects within the lumen is transmitted to the underlying stroma directly through the in- tact lining epithelium. Wimsatt (1944), in describing the earliest phases of implanta- tion in the bat, came to the conclusion that the changes in the epithelium of the pocket into which the blastocyst comes to rest is "an expression of a localized physiologic re- action of the uterus to some chemical stimu- lus of unknown nature liberated by the ovum, which may produce this effect by act- ing locally on the epithelium or by inducing a local relaxation in the uterine muscle." It is important to recall again that the de- struction and removal of the uterine epithe- lium by the trophoblastic cells of the rat blastocyst do not begin until the embryo lies deeply within the decidual crypt and a siz- able decidual response has been elicited (Al- den, 1948). Therefore, the initiation of the decidual reaction and the active invasion of the endometrium by the trophoblast are two distinctly different phenomena separated by a considerable interval of time. In the guinea pig, rabbit, monkey, man, and possibly other mammals, the normal decidual response is not elicited until the embryo has effected the removal of the sujierficial uterine epithe- lium. Recently, it has been shown that there is a definite species difference in the response of the endometrium to glass or paraffin beads inserted into the uterus of properly timed females (Blandau, 1949a). In the rat, the beads initiated the decidual response and were implanted in a manner similar to blas- tocysts. In the guinea pig, the beads did not effect the removal of the uterine epithelium, and only occasionally was a minimal decid- ual response induced. Thus it would appear for the guinea pig, at least, not only that the .stimulus must be a direct one to the under- lying stroma but that a certain amount of tissue injury or invasion is necessary before the decidual response can be initiated. As we suggested earlier, the initiation of the decidual reaction may be the result of a localized pressure exerted by the blastocyst, or of the action of some chemical substance secreted by the egg, which is transmitted to a properly sensitized subepithelial stroma. Recently, Shelesnyak (1952, 1954, 1959a, BIOLOGY OF EGGS AND IMPLANTATION 863 19591)1 undertook to investigate the na- ture of the non-specific stimulus required to initiate the deciduomas by determin- ing the effects of histamine and histamine antagonists on the endometrium. He the- orized that some degree of injury was a common factor to all methods of uterine stimulation, that a histamine or histamine-like substance was present at the site of injury, and further, that at the time of blastocyst attachment there is an "estrogen surge" which acts to release his- tamine from the endometrium and which in turn initiates the decidual cell response. Evi- dence for the role of histamine in deciduoma production also includes the depletion of the mast cell population of the endometrium just before attachment. On this basis, after in- stilling diphenhydramine hydrochloride or other antihistamines into one horn, both cornua of pseudopregnant rats were stimu- lated to induce deciduoma development. Definite inhibition of deciduoma was noted in the cornu receiving the antihistamine, particularly if the drug was instilled before the transformation of endometrial cells to decidual cells. Consistent with this finding are the indications from extensive tests that drugs having a specific histamine antago- nism are effective in suppressing the de- cidual cell reaction when introduced into the uterine lumen of rats and mice during pseu- dopregnancy. On the other hand, antihis- tamines injected subcutaneously in these animals ordinarily fail to prevent implanta- tion. Species differences must also be con- sidered. Boving (1959) was unable to find mast cells associated with rabbit tropho- blast invasion. The theory that some mechanism of his- tamine release is responsible for initiating the decidual cell reaction would logically imply that the blastocyst is an active his- tamine secretor or that it indirectly effects a rise of "free" histamine in the cornua, or interferes with its destruction. In all of the work that has been reported in the attempt to establish histamine as the primary evo- cator in the decidual cell response and im- plantation, the blastocyst has been ignored. There has been no attempt to examine the living blastocyst itself and to determine the effects of the various drugs used on it. Con- sequently, the conclusions drawn as to the failure of implantation are equivocal because the condition of the implanting agent in the experiment has not been evaluated. Also rel- evant is the fact mentioned earlier that the decidual response in the rat and mouse is evoked, not only by living embryos, but also by many inert objects inducing the response without evidence of epithelial destruction. The mechanism of histamine release under these conditions must be based on some un- known factor. The appearance of implantation cones, just before and durine attachment of the blastocyst to the endometrium in guinea pigs, rabbits, squirrels, chipmunks, and prob- ably primates, raises the question as to whether the embryo may not initially send protoplasmic extensions between the epi- thelial cells lining the lumen and thus se- crete some substance which not only ini- tiates the decidual response, but also effects the removal of underlying endometrial tis- sue (compare Figs. 14.23, 14.24 and 14.25) (Mossman, 1937; Wislocki and Streeter, 1938; Boving, 1954, 1959a j. It is interesting that during this initial invasion in the guinea pig, rabbit, and man, there is a negligible amount of endometrial necrosis. In the description of the implantation stages of the macaque, Wislocki and Streeter also emphasized that during the earliest INNER CELL MASS 1 |i|" 'WIF ATTACHING ,^ -^^^ mm TROPHOBLAST-^^ - •' . ^ « Fig. 14 23. Section of a ciuili.'a pig lila.-lo.-yst showing the \eiy earliest stage in the attachment of the abembiyonal pole cells to the maternal endometrium. X 500. 864 SPERM, OVA, AND PREGNANCY '^^^^^^^0-^^^^^-' INNER CEUL MASS X ^ # ^- - - ^5#^- ^ '.>4-%^- i lis ©• Fic. 14.24. The (•.-Illy M.-itic of attacliiucnt of the O-day maca.iuc M im.hxm The .Miihryonic pole is directed towards the uterine epithelium (Wislocki and Streeter, 1938). :^^M- Fig. 14.25. A section through an implanting rabbit blastocyst showing an unusually narrow trophoblast invasion of the uterine epithelium. There is no evidence of epithelial debris within the trophoblast cells. A group of clumped uterine epithelial nuclei surrounded by pale cytoplasm lies to the left of the invading foot (Boving, 1959a). Fixation: Sousa, Azan stain. X600. BIOLOGY OF EGGS AND IMPLANTATION 86;: l)hases of embryonic attachment to the uter- ine epithelium, the subepithelial mucosa shows no reaction whatever. When joined, these observations remind us that as yet there is no conclusive evidence that the im- planting embryo secretes cytolytic enzymes but may secrete other substances. The most imaginative experimental ap- proach to the problems of embryo spacing, attachment, and implantation is the work of Boving (1959a, b and c, 1961) on the rabbit. He has clearly shown that, in this animal, invasion is promoted by a chemical substance elaborated witliin the blastocyst and transferred to the maternal circulation. The invasion-promoting substance has been characterized as being in the form of bi- carbonate which induces a localized high 1)H. Circulating progesterone increases the level of endometrial carbonic anhydrase and accelerates the removal of bicarbonate from the embryo by catalyzing the forma- tion of carbonic acid. The carbonic acid is converted to carbon dioxide which is re- moved by the maternal circulation. The local pH rises and the various blastocysts' membranes become very sticky, particu- larly at the site of attachment. The physico- chemical interrelationship of the tropho- blast and endometrial epithelium effects a dissociation of the epithelium, thus opening a path for the trophoblast. At this writing, the precise roles of the egg and endometrium during implantaton are unknown and remain a challenging problem. The numerous modifications of the implan- tation processes in the different mammalian families create difficulties of interpretation in what is already an unusually complex problem. As more detailed descriptions of the embryo-endometrial relationships ap- pear, it seems clear that neither the ovum nor the endometrium is primarily responsi- ble for implantation, but that both play mu- tual and overlapping roles. One of the greatest gaps in our knowledge of implanta- tion for any animal is a detailed description of the process itself and the precise timing of the events in this phenomenon. The various experimental approaches to the physiologic and biochemical mechanisms of implanta- tion have quickened our interest and broad- ened our view of the complex metabolic processes required if implantation is to be successful, but our efforts to interpret cor- rectly the data from biochemical, physio- logic, and pharmacologic investigations will be limited until more accurate information has been obtained bearing on the morpho- logic features of the process itself. V. References Adams, C. E. 1953. Some aspects of ovulation, reco\'eiy and transplantation of ov^a in the im- matuie rabbit. In Mammalian Germ Cells, pp. 198-216. Boston: Little, Brown and Company. Alden, R. H. 1942a. The periovarial sac in the albino rat. Anat. Rec, 83, 421-434. Alden, R. H. 1942b. 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Uber den Mechanismus der Aufnahme der Eier der Saugetiere in den Eileiter und des Transportes durch diesen in den Uterus. Anat. Hefte, 54, 359-446. SoBOTTA, J. 1924. Beitriige zur lc .f^c^.^ 5 ''^ ' - .s^.*, r ■ • ^- ^'i^.v */ ^•^'- -- ^ '-A •■ jfe^^ _ ■. .- /-.-* ... '^^ >^t. • //^ " ^^*-.r ^ ?■ %.K-.-^:^% '-/ £;7u^ -"^fe^..^. ,/ Plate : 15. IV 894 SPERM, OVA, AND PREGNANCY itself in the puerperium from the basal zone and the deep residual portion of the stratum spongiosum. The trophoblast forms the parenchyma of the placenta and the major element of the placental barrier. It mediates the metabolic exchange between mother and fetus. It pro- vides for the nutrition of the embryo, at first, by the local destruction and absorp- tion of the uterine decidua, and later, by transmission of metabolites through the syncytial trophoblast from the maternal to the fetal blood streams. It serves also as an avenue for the excretion of various fetal waste products. The human trophoblast is also an important endocrine organ which produces steroid hormones, chorionic gon- adotrophin and other hormones. B. CHORIONIC VILLI 1. Trophoblasts: the Langhans Cells These conspicuous cells which possess large nuclei constitute a germinal bed in which mitoses are frequently seen and from which in the early part of gestation the syn- cytium is evidently derived. The Langhans cells gradually diminish in number, but some of them survive until the end of ges- tation (Wislocki and Bennett, 1943; Wis- locki and Dempsey, 19551. Electron micro- graphs show that the Langhans cells are closely apposed on their outer surfaces to the syncytium and on their basal surfaces to the basement membrane of the stroma of the chorionic villi. The cytoplasm of the Langhans cells is characteristically chromophobic (Wislocki, Dempsey and Fawcett, 1948 j, exhibiting only faint cytoplasmic basophilia (Fig. 15.10), little affinity for acid dyes (Fig. 15.9), and no metachromasia. This lack of basophilia correlates with their meager en- doplasmic reticulum, i.e., ergastoplasm (Wislocki and Dempsey, 1955) . In the first 4 to 6 weeks of gestation their cytoplasm contains a considerable amount of glycogen, stainable by the PAS method. This glycogen subsequently disappears. Except for slight staining adjacent to the nuclear membrane (Fig. 15.54) , the cytoplasm is negative with the PAS reagents following the removal of glycogen. There are no lipid droplets. There is a moderate number of rod- shaped and granular mitochondria (Wis- locki and Bennett, 1943) . In electron micro- graphs, the mitochondria of the Langhans cells are relatively few in number but larger than those in the syncytium. A moderate degree of succinic dehydrogenase activity is demonstrable in these cells (Figs. 15.37 and 15.38). The Golgi apparatus is situated on the side of the nucleus toward the syn- cytium (Baker, Hook and Severinghaus, 1944). The cytoplasm is faintly stained in the reaction for protein-linked sulfhydryl groups (Figs. 15.33 and 15.34). The acid and alkaline phosphatase activities are of a low order. 2. Trophoblastic Syncytium in the First and Second Trimesters of Pregnancy Free surface. The syncytium constitutes a broad layer of cytoplasm without cell boundaries and possesses small, irregularly shaped, darkly staining nuclei which are rather uniformly dispersed in its inner zone (Fig. 15.9). It possesses an outer surface, facing the intervillous space, which is ex- tremely variable in structure, ranging from a foamy, vacuolated border possessing deli- cate streamers and fronds, through various intermediate appearances, to regions where it bears a well defined brush border (Wis- locki and Bennett, 1943). In the earliest stages the former appearances predominate, but as gestation advances, the brush border increases in amount. It was suggested by Wislocki and Bennett that these variable surface appearances in fixed material indi- cate that the living syncytial cytoplasm is pleomorphic and plastic. The parts that are foamy and vacuolated and possess streamers are probably constantly moving and flowing, a physiologic activity that would promote the alisorption of fluid and metabolites from the intervillous space by the process of pinocytosis (Wislocki and Bennett, 1943). In confirmation of this, observations of explanted bits of placenta growing in tissue cultures show that the cy- toplasm of both the syncytial and cellular trophoblast moves quite actively, giving rise to a variety of streamers and thread- like processes (Friedheim, 1929; Jones, Gey and Gey, 1943) . The ultrastructure of the free surface of HISTOCHEMISTRY OF PLACENTA 895 the syncytium at 9 to 10 weeks typifies that of a pinocytotic membrane (Wislocki and Dempsey, 1955).^ A profusion of microvilli of various shapes reach into the maternal blood. Some microvilli are long and slender with enlarged tips; others are short and thick; and occasionally peninsulas of cyto- plasm studded with microvilli extend into the maternal blood space. Often in the marginal zone immediately beneath the microvilli there are large vesicles containing finely stippled or flocculent material. They are occasionally seen in the large tongues •of cytoplasm which protrude from the free surface. These vesicles are most likely formed as a result of pinocytotic activity. Basal surface. The inner surface of the syncytium is approximated to the surfaces of the Ijanghans cells or the subjacent stroma of the placental villus (Fig. 15.9). As mentioned previously, the Langhans cells gradually diminish in number so that the syncytium eventually comes widely in con- tact with the chorionic stroma. However, even early in gestation, there are occasional gaps between the Langhans cells where the syncytium is in direct contact with the sub- jacent mesenchyma. Through these gaps metabolites traversing the placental bar- rier can by-pass the Langhans cells. Cell organelles. Cytoplasmic basophilia is very intense in the broad inner zone of the syncytium, especially surrounding the nuclei (Figs. 15.7 and 15.10). Since this basophilia is abolished by ribonuclease, it has been attributed to the presence of ribo- nucleic acid (Dempsey and Wislocki, 1945). Further evidence supporting this identifica- tion is derived from: (1) the similarity of this basophilia to that of Nissl substance (Singer and Wislocki, 1948), (2) the meta- ohromasia of this region in young placentas (Wislocki and Dempsey, 1948), and (3) the <'oncentration of the endoplasmic reticulum (ergastoplasm) in the inner two-thirds of the .syncytium (Wislocki and Dempsey, 1955). This rich cytoplasmic basophilia, which has been shown to constitute the ^Bargmann and Knoop (1959) have also de- scribed the ultrastructure of the human placental barrier. They emphasize the syncytial nature of the outer trophoblastic layer, and offer further de- scription of the ultrastructure of the Langhans cells, Hofbauer cells, and stromal cells. microsomal fraction of the biochemist (Pa- lade and Siekevitz, 1956) points toward an active participation by these cells in pro- tein synthesis. In contrast to the inner zone, the outer zone of the syncytium is strongly acido- philic, although the narrow outermost zone corresponding to the brush border is less acidophilic (Singer and Wislocki, 1948). This acidophilia suggests the occurrence of basic proteins in the outer zone. The ultra- structure of this region shows that there are relatively fewer ergastoplasmic elements but there is a concentration of large vesicles which most likely are the products of pino- cytosis. Higher resolution electron micros- copy of this region is needed. It should be mentioned at this point that protein-bound sulfhydryl groups are concentrated espe- cially at the inner and outer borders of the syncytial cytoplasm (Figs. 15.33 and 15.34). Mitochondria are abundant in the syncy- tium, occurring as small granules and rods (Figs. 15.26 and 15.30). There is the indica- tion of high succinic dehydrogenase activity here at 6 weeks of gestation, although this histochemical determination was compli- cated by the presence of lipid in the syncy- tium (Figs. 15.37 and 15.38). With the electron microscope, it was observed that the mitochondria are smaller but more nu- merous than those in the Langhans cells (Wislocki and Dempsey, 1955). The Golgi apparatus forms a dispersed network in the syncytium. This organelle has been described in the various cells of the placenta by Acconci (1912), Wislocki and Bennett (1943), and Baker, Hook and Severinghaus (1944). Glycogen and other pas positive ma- terial. In the human placenta in the first month of gestation a moderate amount of glycogen is stored in the syncytial tropho- blast. It disappears almost entirely by the end of the second month. Similar early storage and loss of glycogen occur in the Langhans cells and stromal fibroblasts. PAS positive material which is resistant to digestion by saliva is conspicuous in the brush border and marginal cytoplasm of the syncytium (Figs. 15.29 and 15.54). A faint red stippling of reactive material is also visible in the deeper cytoplasm (Fig. 15.54). A dark red reaction occurs also in 896 SPERM, OVA, AND PREGNANCY tlie basement membrane upon whicli the Langhans cells or syncytium rest (Fig. 15.54 1 . Lipids. Birefringent, sudanophilic lipid droplets are abundantly present in both the inner and outer zones of the syncytium (Figs. 15.6 and 15.13). The droplets are acetone soluble. They react with phenyl- hydrazine (Wislocki and Bennett, 1943), give a positive Schiff reaction, exhibit yel- lowish green fluorescence (Dempsey and Wislocki, 1944; Rockenschaub, 1952), and give a positive naphthoic acid-hydrazide re- action (Fig. 15.18) (Ashbel and Seligman, 1949; Seligman, Ashbel and Cohen, 1951; Wislocki, 1952; Ashbel and Hertig, 1952). These histochemical reactions occur also in the lipid droplets of the gonads and adrenal cortex, and their occurrence in the syncy- tium suggests that it is the site of steroid hormonal synthesis in the placenta. Previ- ous and later paragraphs discuss this prob- lem more fully. Enzymes. Alkaline phosphatase (Figs. 15.4, 15.41, and 15.42) occurs in the syncy- tial trophoblast (Buno and Curi, 1945), where it is slight in amount at 6 weeks, but it increases tremendously as gestation advances (Dempsey and Wislocki, 1945). It varies in degree of activity according to the substrate used, the enzymatic reaction being most intense following the use of fructose diphosphate and nucleic acid, and less so with glycerophosphate and adenylic acid (Dempsey and Wislocki, 1947). The reaction occurs earliest and reaches its maximal intensity in the brush border, al- though, as gestation proceeds, it spreads throughout the syncytial cytoplasm and also involves the nuclei. However, the lo- calization of the enzymatic reaction within the nuclei may not represent the actual dis- tribution in the living state, for investiga- tions have shown that the reaction products are capable of migrating, especially from cy- toplasm to nuclei (Martin and Jacoby, 1949; Leduc and Dempsey, 1951 ; Herman and Deane, 1953). This enzyme is a distinguish- ing feature of the great absorptive surfaces of the small intestine, proximal convoluted tubule of the kidney, and syncytial tropho- blast of the placenta. Acid phosphatase (Figs. 15.39 and 15.40) occurs in great intensity in the syncytium in both cytoplasm and nuclei (Wislocki and Dempsey, 1948). The nuclear staining may Plate 15. V All of the drawings on tliis i)late (except Fig. 15.23) are of frozen sections of material fixed in 10 jier cent l^uffered formalin and stained by the Ashbel and Seligman method for car- bonyl groups. Comi^are the illustrations on this plate with those on Plate 15.IV. Fig. 15.18. The syncytial trophoblast of a human villus at 5 months of gestation, stained by the carbonyl method. The reaction is localized in the lipid droplets of the syncytium. Com- pare with Figure 15.13. X 7 ocular; X 90 objective. Fig. 15.19. A human placental villus at full term illustrating the positive carbonyl reaction of the minute lipid droplets in the syncytium as well as a diffuse reaction of the entire syncy- tial cytoplasm. Compare with Figure 15.14. X 7 ocular; X 60 objective. Fig. 15.20. The placental labyrinth of a cat (fetal crown to rump length, 75 mm.) showing an intense carbonyl reaction in lipid droplets located in the cytotrophoblasts of the placental lamellae. A diffuse lavender reaction is present in the contiguous trophoblastic syncytium. X 10 ocular; X 40 objective. Fig. 15.21. The carbonyl reaction in the placenta of a rat of 18 days of gestation. Compare with Figure 15.16 illustrating the distribution of sudanophilic lipids in the rat's placenta, and with Figure 15.17 illustrating the carbonyl reaction in the labyrinth of the mouse. X 10 ocular; X 40 objective. Fig. 15.22. The carbonyl reaction in the placenta of a pig (crown to rump length, 90 mm.). The reaction is localized in the uterine epithelium, whereas little staining is apparent in the chorion. The distribution of the reaction coincides with that of lipids revealed by sudan dyes (Fig. 15.15). X 10 ocular; X 40 objective. Fig. 15.23. The placenta of a pig (fetal crown to rump length, 235 mm.). Bouin's fixation. Masson's stain. This figure is shown to illustrate the detailed structure and relative thinness of the pig's placental barrier. It shows "intra-epithelial" maternal capillaries {m.c.) in the uterine epithelium (ep), and "intra-epithelial" fetal capillaries (/.c.) in the chorionic syncy- tium (c/i) ; the distance separating the two sets of capillaries varies between 6 and 8 /x. Com- pare with Figures 15.28 and 15.64 which also illustrate the extreme thinness of the trophoblast and the narrow distance separating the maternal from the fetal capillaries in the placenta of the pig. X 70 ocular; X 40 objective. * .. *a # 18 ^ > C\\J? if^^^lJ^ ^ /. 22 ;> 19 A.^^.;J :..-..^y^ "^ .-S ,« */ ^ I' .*•* .# s f^- •?• » "'^ i* »-^ ^ „ V ^* .v^ ■^'■ K .— «^ . k"^ ^ ■■-.i •- -' •, V. '^ ". * \ •• ,• ."k. i >.»\, .< > 2! .^r"^) ^^j ^^ m.c ^^*#&.^ ^ HI8T0CHEMLSTRY OF PLACENTA 897 not represent the true location of the en- zyme, because on localizing the enzyme by both histochemical and biochemical methods and comparing the results, Palade ( 1951 ) found in the case of the hepatic cells of the rat that the enzyme was confined al- most entirely to the cytoplasm. Further- more, recent biochemical evidence indicates that this enzyme is located in a particular cytoplasmic fraction, the lysosomes (De- Duve, 1959). High esterase activity is demonstrable in the troi)hoblast following the use of the method of Barrnett and Seligman ( 1952) on unfixed frozen sections ( Wislocki, 1953) . It could not be determined with certainty whether the intense but poorly localized crystalline reaction product was entirely in the syncytium or whether some was pres- ent in the Langhans cells (Fig. 15.36). With the method for esterase (Nachlas and Selig- man, 1949) carried out on sections of ace- tone-fixed, paraffin-embedded material, the 37 36 "-^ 1 3»; t^.>- Plate IS.VIII 912 SPERM, OVA, AND PREGNANCY evaluation of their ultrastructure. The transported substance next traverses the amorphous perivascular ground substance which varies in amount regionally, being abundant, moderate, or entirely absent. In areas in which this ground substance is lack- ing, the syncytium abuts on maternal endo- thelium. The endothelial margin of the ground substance is regular, but its opposite border, which is in contact with the syncy- tium, is irregular and seems to be sculptured by the syncytium. The lipid-rich syncytium sends branched processes between the cytotrophoblast to end in foot-like expan- sions on the trophoblastic basement mem- brane. Later in gestation, as the cytotropho- blasts diminish in number, these processes extend through large extracellular spaces. After crossing the two trophoblastic layers and the chorionic basement membrane, a substance then passes through a thin connec- tive tissue space containing fibroblasts and collagen fibers. From there it next passes through the basement membrane and endo- thelium of ordinary fetal capillaries. Thus, despite the loss of two uterine layers, the placental barrier in the cat is structurally complex. The paraplacental or brown border of the cat's chorion is a specialized region com- posed of cells rich in iron and caj^able of phagocytosing red blood cells, trypan blue, and other substances (Figs. 15.62 and 15.63). The absorptive surface of these cells j bears a striking resemblance to that of the visceral endoderm of the rodent yolk sac. The surface plasma membrane of these co- lumnar cells evaginates to form elaborate microvilli and invaginates to form a system of canals immediately beneath the cell sur- face. It is assumed that pinocytotic vesicles are farmed as ingested substances are seg- regated in the canalicular- system. In the paraplaQehtal cells, ingested erythrocytes in various stages of breakdown were frequently observed. The hemochorial type of placenta, in which extensive erosion of the uterine wall occurs, has been studied in the human and rodents by several investigators (Boyd and Hughes, 1954; Wislocki and Dempsey, 1955a, b; Wislocki, Weiss, Burgos and Ellis, 1957; Bargmann and Knoop, 1959; Schieb- ler and Knoop, 1959). In the "definitive" hemochorial placenta the trophoblast is bathed directly by circulating maternal blood. Observations on the ultrastructure of the human placenta (Wislocki and Demp- sey, 1955a) have been incorporated with the histochemical findings in Section III of this review. The syncytium of the human pla- centa resembles a pinocytotic epithelium having numerous pleomorphic microvilli and containing many large vesicles which prob- ably represent engulfed material (Wislocki and Dempsey, 1955a, Plates 1-4). Although the Langhans cells diminish in number as gestation proceeds, some flattened cyto- trophoblasts remain at term interposed be- tween the syncytium and the trophoblastic basement membrane. At term, when the pla- cental barrier is thinnest, a maternal sub- stance encounters the following successive layers in reaching the fetal blood: syncy- tium, trophoblastic basement membrane, fetal connective tissue, basement membrane, and endothelium of the fetal sinusoidal cap- illary. Many significant observations have been made on the fine structure of the chorio- allantoic placenta of the rat. To aid in ori- enting the reader, a brief description of the histology of the rat placenta follows. In the established chorio-allantoic disc of this spe- cies, three general zones are recognizable in the fetal portion: (1) a trophoblastic lab- yrinth which is served by both maternal and fetal blood vessels and is considered to be the principal area of transport; (2) a spongiotrophoblastic zone which partially surrounds the labyrinth and which, al- though it is not penetrated by fetal blood vessels, is perfused by maternal blood; and (3) a meshwork of giant cells which caps the spongy zone, is permeated by maternal blood, and forms the frontier of the fetal tissue. The first description of the ultrastructure of the labyrinth of the rat at 15, 17, and 21 days of gestation quickly established two important points (Wislocki and Dempsey, 1955b ) . It was demonstrated that the rat and rabbit placentas are hemochorial rather than hemoendothelial as had been proposed by Mossman (1926, 1937). Furthermore, it was shown that there is no syncytial tropho- HISTOCHEMISTRY OF PLACENTA 913 blast in the labyrinth, as had been described by Grosser (1908, 1909) ; the fetal blood ves- sels are clothed by two or three thin layers of overlapping individual cytotrophoblasts which together constitute a laminated mem- brane (Wislocki and Dempsey, 1955b, Plate 2). These trophoblasts are held together by small cytoplasmic pegs which fit into de- pressions in adjacent cell surfaces. Wislocki and Dempsey (1955b) observed that the labyrinthine lipid droplets are located mainly in the cytoplasm of the innermost trophoblasts. Schiebler and Knoop ( 1959) reported that there is a relatively wide space between the outer and middle layers which communicates with the maternal blood but that the deeper cells are closely apposed. The latter investigators also ob- served that pinocytotic vesicles occur in numerous trophoblasts and claimed that two kinds of trophoblastic cells can be dif- ferentiated in the labyrinth with the elec- tron microscope. According to Wislocki and Dempsey, the placental barrier in the labyrinth of the rat is composed of : ( 1 ) two or three sheets of laminated cytotrophoblast; (2) the base- ment membrane supporting the trophoblast; and (3j the basement membrane and endo- thelium of the fetal capillary. Schiebler and Knoop did not see two separate basement membranes and report the occurrence of a single basement membrane between the tro- phoblast and fetal endothelium. Schiebler and Knoop (1959) also pre- sented some interesting observations on the fine structure and histochemistry of the spongiotrophoblasts and giant cells. The cytotrophoblasts of the spongy zone have an intensely basophilic cytoplasm, and this is consonant with the presence of an exten- sive, highly oriented endoplasmic reticulum. This luxuriant endoplasmic reticulum is comparable in its arrangement and abun- dance to that of the pancreatic acinar cells and of the Nissl bodies of neurones. The function of the spongiotrophoblast remains unknown, but this observation points to- ward a special role in protein synthesis. Padykula (1958) reported a striking in- crease in acid phosphatase and adenosine triphosphatase activity in this zone during the last week of gestation. In the same report, Schiebler and Knoop offered much new information about the fetal giant cells, and their observations suggest a dynamic role for these strate- gically placed cells. The giant cells are contiguous with the spongiotrophoblasts, and with the aid of the electron microscope they can be differentiated into several types. The nuclei of the giant cells are invaginated in many places, and these recesses contain cytoj^lasm. In some planes of section, this morphologic arrangement gives the false impression of intranuclear inclusions, es- pecially when the invagination contains lipid or glycogen. However, the cytoplasmic mass enclosed by the nucleus maintains its connection with the main body of cyto- plasm. The cytoplasm proper is highly dif- ferentiated. It contains a great complexity and variety of vesicles and membranes, and resembles the cytoplasm of phagocytic cells in several aspects of fine structure. The surface of the giant cells presents a compli- cated interwoven array of microvillus-like projections to the intercellular space. This space is filled with a material which con- tains mucopolysaccharide, is fibrous, and appears to be continuous in some regions with Reichert's membrane. In some regions the maternal blood spaces among the giant cells are lined by a thin layer of cytoplasm which is judged to be endothelium by Schiebler and Knoop. In this location a subendothelial basement membrane seems to be lacking. Elsewhere the surfaces of the giant cells are in direct contact with the maternal blood. The fine structure of the granular cells of the metrial gland of the pregnant rat was described by Wislocki, Weiss, Burgos and Ellis (1957). The suggestion was offered that the basic protein granules of these cells contain relaxin. The fine structure of the hemochorial pla- centa of the nine-banded armadillo (Dasy- pus novemcinctus) was recently reported by Enders (1960). Some generalizations may be made con- cerning the fine structure of the placental barriers. Certainly the absorptive tropho- blasts resemble the cells of the proximal convoluted tubule of the kidney and the absorptive cells of the small intestine more 914 SPERM, OVA, AND PREGNANCY closely than the components of the pul- monary alveolar lining or the renal glo- merular membrane. The trophoblastic cells are characterized by microvilli and other surface projections which are pleomorphic and often branched. Many observations suggest that absorption by pinocytosis oc- curs in many types of placental cells. Whether or not erosion of the uterine wall occurs, the placental barrier is structurally complex. Along with the cellular layers, basement membranes are regularly inter- posed between the maternal and fetal blood- streams. Further discussion is presented in the section on yolk sac placentation where some experimental cytologic observations have been made during the process of ab- sorption. C. REDUCTION IN NUMBER OF THE LAYERS OF THE CHORIO-ALLANTOIC PLACENTA The successive elimination of the mater- nal layers of chorio-allantoic placentas as envisioned by Grosser's scheme has met with general acceptance. However, Wislocki and Dempscy (1946a I pointed out that the endotheliochorial type of placenta is prob- ably nonexistent, because in carnivores and sloths the endothelial-lined maternal blood vessels are surrounded by a basement mem- brane and in some species, as in the cat, large decidual cells are present in the laby- rinth. Consequently, in some carnivores the placenta is syndesmochorial rather than endotheliochorial. The hemoendothelial type of placenta postulated by Mossman finds no support in recent observations by Wislocki and Demp- sey (1955b) and Schiebler and Knoop (1960) with the electron microscope. These investigators found in the chorio-allantoic placental labyrinths of rat and rabbit late in gestation a complete trophoblastic mem- brane, consisting of 2 or 3 layers of flat- tened, imbricated trophoblastic cells. The presence of these layers is not detectable with the light microscope. These findings indicate that the placentas of these species are hemochorial and not hemoendothelial. D. REDUCTION IN WIDTH OF THE LAYERS OF THE CHORIO-ALLANTOIC PLACENTA The diminution in width of the tissues separating the maternal blood channels from the fetal capillaries, as postulated in Grosser's scheme of chorio-allantoic pla- centas, seems to be borne out by histologic observations of both his phylogenetic series and successive ontogenetic stages. How- ever, some have pictured the layers in a schematic way (Huggett, 1944; Arey, 1946) with no regard for their relative widths and relationships. Actually, in progressing from ei)itheliochorial to hemochorial placentas, the gradual reduction in width of the thin- nest areas is not nearly as striking as the theoretic concept of the removal of succes- sive layers implies. This is due partly to the fact that in all species the connective tissue layers at the sites of the thinnest places consist only of basement membranes. Fur- thermore, the capillaries in the thin areas of all animals are pressed against the ad- jacent epithelia and in some, for example in the sow (Figs. 15.23, 15.28 and 15.64) and many ungulates, the fetal capillaries follow intra-epithelial courses in the tropho- blast (Wislocki and Dempsey, 1946b: Amoroso, 1947, 1952). In addition, in the Plate 15. IX Fig. 15.39. Human placental labyrinth at 4 months, showing acid phosphatase activity in the syncytium and stroma of the chorionic villi. Gomori's method, using glycerophosphate as substrate at pH 4.7. X 140. (Wislocki and Dempsey, 1948.) Fig. 15.40. The basal plate of a human placenta at full term, showing the presence of acid phosphatase in the chorionic villi (above), its almost complete absence in the basal plate (center), and a marked reaction at the line of junction of the basal plate with the decidua (below). Gomori's method using glycerophosphate as substrate at pH 4.7. X 175. Fig. 15.41. Human placental labyrinth at full term, showing the activity of alkaline pho.s- phatase in the syncytium clothing the chorionic villi. Gomori's method using glycerophos- phatase as substrate at pH 9.4. X 220. Fig. 15.42. The basal plate of a human placenta at full term, showing an inten,se alkaline phosphata,se reaction in the chorionic villi (above), its nearly complete absence in the basal plate (center) and a slight reaction at the line of junction of the basal plate with the decidua (below). Gomori's method using glycerophosphate as substrate at pH 9.4. HISTOCHEMISTRY OF PLACENTA 9V • A ^;' c^ " r .^' Plate 15. IX 916 SPERM, OVA, AND PREGNANCY SOW, Amoroso (1952) has described the trophoblast at one period as sending actual processes between and past the maternal epithelium into the region of the underlying maternal capillaries, establishing an endo- theliochorial relationship. Despite the pres- ence of six theoretic layers in the sow, pro- visions exist which tend to by-pass or materially reduce the width of several of them, thus diminishing the distance between the two blood streams. Similarly, in the cat, in the last half of pregnancy, Amoroso (1952) observed "that the foetal capillaries come to lie so near the surface of the lamellae that only the thinnest laminae of syncytial trophoblast separate them from the mater- nal tissues." Measurements of the width of the mater- nal epithelium in the sow, made by Gell- horn, Flexner and Pohl (1941), show that its height changes from 18 fj. at midgesta- tion to 10 fi just before term. Nevertheless, in many of the thinner places between the capillaries by midgestation (Fig. 15.23 of the present study), the width of the inter- vening cytoplasm of the combined chorion and uterine epithelium is reduced to no more than 6 or 8 fj.. In the sheep at 100 days of gestation Barcroft (1947) reported that none of the fetal and maternal capillaries is closer than 20 ix and none is separated by more than 120 /j.. This offers no clue, how- ever, as to what the average distance may be. It is apparent, nevertheless, from some excellent figures of the sheep's placenta sub- mitted by Wimsatt (1950, Figs. 54 and 56) , that the numerous maternal capillaries arc about 10 to 20 /x from the fetal capillaries at 100 days and less at 133 days. In the cat at term, the distance between the two blood streams is narrowed in many places to 6 or 8 fji. In the human at term, the thinnest places vary between 3 and 6 /x in width. More extensive and careful measurements of the distances between the blood streams should be obtained in various animals at different stages of gestation, in order to pro- vide a better basis than now exists for com- parisons. In making such measurements the degree of shrinkage and separation of the layers in preparing the tissues should be carefully evaluated. A more important consideration than the actual diminution in width of the layers in the thinnest regions might be the apparent much larger extent of thin areas in hemo- chorial placentas than in other placental types. Thus, for example, although the thin- nest places in the sow's placenta do not seem to differ greatly from the human in respect to their actual widths, the relative extent of the thin areas is very much greater in the latter than the former. In this re- spect, hemochorial placentas differ greatly from epitheliochorial ones. This considera- tion, although possibly inherent in Gros- ser's doctrine, has never been clearly brought out and documented, but instead has been subordinated to the prevailing con- cepts of the phylogcnetic reduction in num- ber and widths of the layers. A further point of interest concerns the placentas of rodents. The physiologic ad- vantages obtained presumably by the re- duction in width of the trophoblastic mem- brane and the increased extent of the thin areas would seem to be offset by the func- tional disadvantage of the laminated ar- rangement of the trophoblastic cells as re- vealed by electron microscopy. Here, where a syncytium with only inner and outer sur- faces was believed to exist, the trophoblast is laminated, so that 4- or 6-cell surfaces extend across the placental barrier. Thus, with respect to cell surfaces and cell layers forming the placental barrier, the hemo- chorial placentas of rodents seem to be quite as complex as epitheliochorial and syndesmochorial placentas. The most im- portant difference between them would seem to lie in the relatively greater extent of the thin regions, rather than in any ex- treme reduction of the number of cell layers in the rodent's hemochorial placenta. Some degree of cytologic and histochemi- cal simplification is apparent in successive stages of gestation in any given species, but striking cytologic differences between the placental membranes of Grosser's phylo- genetic series at equivalent stages of gesta- tion are not very evident. Even the thinnest regions of the different types of chorio-al- lantoic placentas possess a far greater cyto- chemical complexity than the glomerular and pulmonary membranes. Several writers have postulated that toward the end of HISTOCHEMISTRY OF PLACENTA 917 pregnancy some of the human chorionic villi lose their syncytial covering entirely; the increase in placental permeability is at- tributed to this structural alteration. How- ever, it should be pointed out that the na- ture and degree of degeneration and loss of the syncytium in human villi have not been carefully analyzed. Moreover, it is not known whether such altered villi are func- tionally active or dead and functionless. However, the assumption that a fraction of the villi becomes functionless would be con- sonant with an observation of Flexner, Cowic, Hellman, Wilde and Vosburgh (1948) that there is a sharp terminal de- cline in placental permeability after the 36th week of gestation. V. Yolk Sac Placentation In those lower vertebrates, such as some fishes, amphibians, and reptiles which are either ovoviviparous or viviparous, the yolk sac plays the principal role as the fetal membrane subserving the transfer of meta- bolic materials (Amoroso, 1952) . An excep- tion to this is encountered in some reptiles (Weekes, 1935) in which chorio-allantoic placentation occurs. In marsupials, the yolk sac is very large, whereas the allantois is always relatively small, and in only three species does the latter vascularize a pla- centa. In accordance with Grosser's termi- nology, the chorio-allantoic placenta of Perameles is "endothelio-endothelial" in character, thus differing fundamentally from the types he defined. "In all other marsupials so far investigated the embryo is nourished exclusively through the yolk- sac and a definite yolk-sac placenta of somewhat complex character is present" (Amoroso, 1952). Thus it is apparent that Grosser's theory does not apply to placen- tation in the majority of lower placental vertebrates or to the Metatheria (marsu- pials). In eutherian mammals, on the other hand, the most typical structure subserving physi- ologic exchange between mother and fetus and which is constantly present, is the chorio-allantoic placenta (Hamilton, Boyd and Mossman, 1952). The yolk sac in these mammals is the most variable of the fetal membranes. It may occur as a primitive bi- laminar yolk sac, or as a vascularized tri- laminar yolk sac which develops early and is temporary. In some orders of mammals (rodents, bats, insectivores, armadillos), a very different and more complex structure, an "inverted" yolk sac placenta, develops. This usually increases in extent during ges- tation and in most species becomes covered with elaborately branched, vascularized villi which are in contact with the uterine mucosa (Amoroso, 1952; Hamilton, Boyd and Mossman, 1952). In ungulates, ceta- ceans, lemurs, sloths, and the Simiae (mon- keys, apes, man), it has been assumed that the yolk sac, although present as a vesicle, plays little or no role in the metabolic ex- change between mother and fetus. However, some histochemical findings on the human yolk sac challenge this assumption. In a histochemical study of 5-, 6-, and 7-mm. human embryos, McKay, Adams, Hertig and Danziger (1955a, b) localized the fol- lowing substances in the yolk sac endoderm : glycogen, glycoprotein, ribonucleoprotein, acid and alkaline phosphatase, 5-nucleo- tidase, and nonspecific esterase. These in- vestigators suggested that the large amount of glycogen in the yolk sac and its absence from the fetal liver may indicate that the yolk sac is supplying glucose to the embryo during the first weeks of embryonic life. McKay and his associates pointed out that there is no iron in the human yolk sac en- doderm, whereas the rodent yolk sac is rich in this substance (Wislocki, Deane and Dempsey, 1946). The inverted yolk sac placenta of rodents and lagomorphs has received more atten- tion since Brambell and his associates (1948, 1949, 1951, 1957) demonstrated that maternal antibodies are transferred exclu- sively by this ancient membrane. A short description of the histology of the inverted yolk sac of the rat follows for the purpose of general orientation. A good diagram of the histology of the rat placenta was pub- lished by Anderson (1959). The yolk sac placenta of the rat is divided into two mor- phologic zones. (1) An outer, nonvascular parietal wall (bilaminar omphalopleure) consists of scattered cuboidal endodermal cells w^iich form an incomplete lining on the interior surface of Reichert's membrane. 918 SPERM, OVA, AND PREGNANCY This thick and unusual basement membrane adheres externally to a meshwork of tropho- blastic giant cells. Maternal blood flows through the interstices of this meshwork of giant cells and presumably is a regional source of some of the substances which gain entrance to the vitelline circulation. A por- tion of the parietal wall of the yolk sac is firmly attached to the fetal surface of the chorio-allantoic disc. (2) An inner, vascu- lar, visceral wall (visceral splanchnopleure ) is composed of a simple columnar endoder- mal epithelium which rests on a mesenchy- mal layer which carries the vitelline blood vessels. A serosal basement membrane (Wislocki and Padykula, 1953) separates this mesenchymal layer from a narrow, basophilic layer of mesothelium which lines the exocoelom. As the allantoic vessels penetrate the placental labyrinth, portions of both visceral and parietal walls of the yolk sac are invaginated into the labyrinth, forming perivascular recesses which were called "endodermal sinuses" by Duval (1892). In the rat the parietal wall of the yolk sac breaks down on the 15th day of gestation, and this event makes the yolk sac cavity confluent with the uterine cavity and also puts the visceral endoderm into direct contact with the uterine contents. On the basis of histophysiologic studies on the absorption of dyes, Everett ( 1935 1 concluded that the yolk sac of the rat is a significant organ of exchange and that it is more permeable to dyes than the labyrinth. Vital dyes, such as trypan blue, which are relatively large molecules, find their way rapidly into the yolk sac where they are absorbed and stored by the visceral endo- derm. These dyes reach the yolk sac, ap- parently, either by way of the uterine mu- cosa or through that portion of Reichcrt's membrane covering the fetal surface of the allantoic placenta. Brambell and his co- workers (1948, 1950, 1951, 1957) have es- tablished experimentally in the rabbit and rat that antibodies find their way from the maternal circulation into the embryos, not by passage through the thin and supposedly more permeable layers of the chorio-allan- toic placenta, but by way of the yolk sac placenta, the latter mode of entry necessi- tating transfer across several layers of cells and tissues, including the structurally elab- orate vitelline epithelium. Histologic evi- dence in support of transport of antibodies and serum proteins by the yolk sac placenta comes from the localization of absorbed serum proteins labeled by fluorescent dyes (Mayersbach, 1958), and from autoradio- graphic studies (Anderson, 1959). Both in- vestigations substantiate the impermeabil- ity of the labyrinthine trophoblasts to these labeled proteins and demonstrate the accu- mulation of proteins in the visceral endo- derm. Brambell and Halliday (1956) and Alayersbach (1958) have suggested from different kinds of evidence that the endo- dermal sinuses of Duval may also partici- l^ate in antibody transport. Padykula (1958 » demonstrated a rise in the succinic dehydrogenase activity of the visceral com- ponent of the endodermal sinuses shortly before term in the rat. The absorptive visceral endodermal cells are interesting from the points of view of both cytology and placentation, since these cells are capable of transporting certain large molecules, such as antibodies and serum proteins, and withholding and segre- gating other colloidal substances, such as trypan blue. In the latter respect, they func- Plate 15.x Fig. 15.43. Semischematic drawing of a human placenta delivered at full term. Two cotyle- dons are illustrated, bounded above by the so-called chorionic or closing plate (c.p.) and on the sides and below by septa placentae (s.) and the basal plate (b.p.). Placental branches of the umbilical blood vessels are seen in the closing plate and in the anchoring villi (o.v.). Fig. 15.44. A section through a delivered placenta at full term, showing a darkly stained placental septum extending up from the base of the placenta and forming the boundary between two cotyledons. At the top of the figure, blood vessels in the closing plate are ap- parent. Buffered formalin fixative. Azan stain. X 3. Fig. 15.45. A photograph of a placenta at full term showing a portion of a placental septum at a higher magnification. Buffered formalin fixation. Periodic acid-Schiff stain. By this method the septum is seen to consist of darkly stained ground substance composed principally of fibrin in which there are numerous lacunae containing faintly stained individual tropho- blast.* or colonies of them. X 90. HISTOCHEMISTRY OF PLACENTA 919 '<% Plate 15.X 920 SPERM, OVA, AND PREGNANCY tion as phagocytes. The absorption of large molecules is believed to occur by the process of pinocytosis. The fine structure of these cells has been described in the guinea pig (Dempsey, 1953) and rat (Wislocki and Dempsey, 1955b). Some recent electron mi- crographs of the rat yolk sac (Figs. 15.80- 15.83) are presented in this review by Pady- kula. The free surface of these cells typifies that of a membrane engaged in pinocytosis. There are numerous surface projections or microvilli which are pleomorphic and branch frequently (Figs. 15.81 and 15.82). These projections form the brush border which has long been recognized with the light microscope, and which is rich in gly- coprotein and, at certain times in gestation, in alkaline phosphatase. In both the rat and guinea pig these surface projections l)c- come simpler and shorter near term. In ad- dition to these evaginations, the surface plasma membrane is invaginated in the form of minute anastomosing tubules which have a denser thicker wall than tiie micro- villi (Fig. 15.82). It seems fairly certain that during pinocytosis a local enlargement of such a tubule is produced and a pinocy- totic vesicle is formed. Vesicles fill much of the supranuclear cytoplasm, and there is considerable heterogeneity in the size and content of the supranuclear vesicles (Fig. 15.80). Filamentous mitochondria occur throughout the cytoplasm. The endoplasmic reticulum is most concentrated around tiio nuclei, although it is also diffusely distrib- uted throughout the cytoplasm. The typical agranular membranes and vesicles of the Golgi apparatus can be recognized near the nucleus (Fig. 15.83). Glycogen is stored in the lower half of the cell, especially in the infranuclear region. Lipid droplets occur throughout the cytoplasm, but the larger ones are usually infranuclear where they are often in close association with the basal surface of the nucleus (Figs. 15.81 and 15.83). The supranuclear lipid is often in the form of aggregations in complicated as- sociations with membranes (Figs. 15.79 and 15.81). Minute lipid droplets are also found within the nucleus (Figs. 15.78 and 15.80). The lateral cell boundaries of these cells are closely apposed early in gestation, whereas near term large lateral intercellular dilata- tions occur. Between these dilations, where the plasma membranes are closely apposed, desmosomes are evident. The bases of the cells rest on a narrow basement membrane. With the electron microscope, experimen- tal cytologic analyses of absorption by the ^•isceral cndoderm of the rabbit and mouse have been made by Luse and her associates (Luse, 1957; Luse, Davies and Smith, 1959; Luse, Davies and Clark, 1959). The follow- ing materials were injected into the uterus of the rabbit and mouse: colloidal gold, egg albumin, lii)ids, saccharated iron, bovine y-globulin, and salivary gland virus. All of these materials entered cytoplasmic pino- cytotic vesicles. However, more interest- ingly, iron, colloidal carbon, and salivary gland virus penetrated the nuclei. Further work suggests that pinocytosis by the nu- clear membrane is the method of nuclear ]ienetration. Similar nuclear inclusions arise in the nuclei of newborn rat duodenum after suckling. This amazing intracellular path- way of absorption i)robably occurs natu- rally as witnessed by the occurrence of lipid in the nuclei of normal visceral endoderm, duodenum, and liver. The inverted yolk sac placenta of the rat undergoes a striking differentiation during Plate 15. XI Fig. 15.46. A portion of a placental septum showing cytotroplioblasts surrounded by ground substance. Human placenta at 3% months of gestation. Masson's triacid stain. X 240. Fig. 15.47. A cell island containing a group of cytotioi)liol)last8 surrounded by ground sub- stance. Human placenta at 2 months of gestation. Azan stain. X 240. Fig. 15.48. Cytotrophobla.sts of the trophoblastic slioU stained by Baker's acid hematein method for phospholipids. Human placenta at 3V2 months of gestation. Observe the intense reaction in the cytoplasm of the tiophoblasts. Compare with Figure 15.25. X 160. Fig. 15.49. A portion of a placental septum of a human placenta at full term, stained by the periodic acid-Schiff method after exposure of the section to saliva. The cyto])l;ism of the cytotrophoblasts exhibits a faint reaction which should be compared with Figuio 15.55 which illustrates the cells at higher magnification. The clumps of cells are surrounded by masses of intensely stained fibrin. Compare with Figures 15.32 and 15.46 which illustrate the tropho- blastic shell at 3 to 3M montlis of gestation before a great deal of fibiin has appeared. X 300. HISTOCHEMISTRY OF PLACENTA 921 Plate 15. XI 922 SPERM, OVA, AND PREGNANCY its brief life history (Padykula, 1958a, b) . A major architectural reorganization occurs with the loss of the parietal wall shortly af- ter mid-gestation. A short period of lipid storage for from 10 to 15 days is succeeded by a phase of glycogen storage from 15 to 20 days. After the loss of the parietal wall, there is a sharp rise in certain enzymatic activities (alkaline phosphatase, adenosine triphosphatase, acid phosphatase, succinic dehydrogenase) . This burst of vitelline ac- tivity in this last third of gestation suggests greater functional activity after direct ex- posure of the visceral endoderm to the uterine contents. As in the case of the cho- rio-allantoic placentas, the reduction in the number of layers of the yolk sac probably increases the rate of absorption by the vas- cularized splanchnopleure. Shortly before term there is a sharp decline in glycogen content and certain enzymatic activities in the visceral endoderm. As these particular histochemical properties decline in the vis- ceral yolk sac, they appear in the fetal liver with good temporal correlation. If the yolk sac functions in part as a fetal liver, then the terminal decrease in enzymatic activity should not be interpreted as placental aging, but rather as a redistribution of the func- tional activities of the placental-fetal com- plex (Padykula, 1958). Further morpho- logic aspects of aging in the placenta were discussed by Wislocki (1956). With the ascendency of the chorio-allan- toic placenta in eutherian mammals and its postulated progression in the sense of Gros- ser's series from a simple epitheliochorial placenta to the physiologically more "effi- cient" hemochorial type, one might have expected that the mammalia would have abandoned yolk sac placentation altogether. But that is not the case, for paradoxically the greatest placental development of the yolk sac, involving inversion, is associated with hemochorial placentas (rodents, lago- morphs, bats, some insectivores), whereas the least developed yolk sacs occur in ani- mals possessing epitheliochorial placentas which are the most primitive, according to Grosser's scheme. The general adoption of Grosser's concept of the chorio-allantoic placenta has resulted in the almost complete exclusion of the yolk sac. For example. Needham (1931, Table 227) attributes the entire transfer of substances in mammals to the chorio-allantoic placenta, without ref- erence to other avenues of exchange. In view of the observations and experiments de- scribed here, it is evident that Grosser's doctrine will have to be re-evaluated and modified to include yolk sac placentation. VI. Histochemistry with Reference to Comparative Placentation It is beyond the scope of this chapter to describe in detail the structure and cytology of the placentas of various mammals. How- ever, certain histochemical observations on lipids will be presented because they afford some clues to the probable sites of localiza- tion of placental steroid compounds. In ad- dition, the localization of glycogen, other complex carbohydrates, and phosphatases in several types of placentas will be sum- marized. This histochemical information will serve as a basis for subsequent com- parisons of placental structures and func- tions. However, before proceeding to these mat- ters, attention should be drawn, for readers who may wish to familiarize themselves with comparative placentation, to the com- pendia of this subject by Grosser (1925b), Mossman (1937), and Amoroso (1952). Re- cent papers on placental histochemistry of various animals will be listed here. These include investigations of the chemical mor- phology of the placentas of the pig (Wis- locki and Dempsey, 1946b), sheep and cow (Wimsatt, 1950, 1951), shrews (Wislocki and Wimsatt, 1947), cat (Wislocki and Dempsey, 1946a), and bat (Wimsatt, 1948, 1949). In rodents histochemical observa- tions are more numerous, including earlier investigations of fat, glycogen, and iron in placentas of the rabbit (Chipman, 1902) and rat (Goldmann, 1912). More recent studies describe glycogen in the rat's pla- centa (Szendi, 1933; Krehbiel, 1937; Bridg- man, 1948; Bulmer and Dickson, I960), al- kaline phosphatase m the guinea pig's placenta (Hard, 1946; Nataf, 1953), and in the pregnant uterus of the rat (Pritchard, 1947), and multiple histochemical reactions in placentas of rats, mice, guinea pigs, and rabbits (Wislocki, Deane and Dempsey, HISTOCHEMISTRY OF PLACENTA 923 1946; Bridgman, 1948a, b; Wislocki and Padykula, 1953; Davies, 1956; Padykiila, 1958). The Ashbel-Seligman reaction for carbonyl groups has been briefly described in the placentas of various mammals (Ash- bel and Seligman, 1949; Wislocki, 1952), as has also the PAS reaction (Wislocki, 1950) . Many histochemical observations on a va- riety of placentas were summarized by Starck (1945-50). Present information on the histochemical localization of lipids in various placentas is fragmentary. The main effort has been di- rected toward localizing within the placenta the type of lipid droplets which occur in the steroid-producing cells of the adrenal cor- tex and gonads. These droplets are acetone soluble, birefringent, exhibit greenish fluo- rescence, and give positive Ashbel-Seligman and Schif! reactions for carbonyl groups. As has been discussed in the section on methods, these nonspecific reactions actu- ally reflect the degree of unsaturation in the compounds comprising the fixed lipid droplets. Certainly further work is needed to characterize more fully the various lipids of placentas, especially in relation to the storage of cholesterol and triglycerides. In the following descriptions it will be seen that the lipid reactions characteristic of steroid-producing cells usually occur in some part of the trophoblast. In the human placenta, only the syncytium contains the lipid droplets characteristic of steroid-pro- ducing cells. The cat possesses a so-called endotheliochorial type of placenta, consist- ing of sinusoidal maternal capillaries and "decidual" giant cells arranged in sheets alternating with lamellae of trophoblast, the latter enclosing the fetal stroma and capillaries. The trophoblast consists of an outer syncytial and an inner cellular layer. The cellular layer contains abundant lipid droplets of variable, but relatively large size. They are sudanophilic and birefrin- gent (Fig. 15.59), exhibit greenish fluores- cence, give an Ashbel-Seligman reaction for carbonyl groups (Fig. 15.20) and stain in- tensely with Schiff's reagent. The syncy- tium is negative in these respects, except for a mild diffuse coloration bv the Ashbel- Seligman carbonyl method (Fig. 15.20) and a greyish tint with sudan black B which is attributable probably to mitochondria. The "decidual" giant cells, generally regarded as of maternal origin, and the maternal endo- thelium give no lipid reactions beyond a delicate sudanophilia associated with the presence of mitochondria. In rodents two types of placentas, a cho- rio-allantois of the hemochorial type and a yolk sac placenta, function concurrently throughout gestation. Lipids occur in many placental constituents of the rat: labyrinth- ine trophoblast, giant cells, parietal endo- derm, visceral endoderm, decidua capsularis, and mesothelium lining the exocoelom. Bridgman (1948) pointed out that in the rat the labyrinthine trophoblast contains lipid from the 12th day onward and that it di- minishes shortly before term. This lipid in rats and mice is birefringent and gives an in- tense carbonyl reaction (Figs. 15.16, 15.17, and 15.21) (Wislocki, Deane, and Dempsey, 1946; Ashbel and Seligman, 1949; Wislocki, 1952). This cellular layer is a logical sus- pect as the site of steroid hormonal synthe- sis. However, these reactions are present also in the visceral endoderm of the yolk sac where lipid droplets occur in great abun- dance from 9 to 17 days (Figs. 15.74-15.77) . These lipids which occur principally as large infranuclear droplets (Figs. 15.78 and 15.83) are birefringent, strongly fluorescent, and give a strong carbonyl reaction with the Schiff reagents (Wislocki, Deane and Dempsey, 1946). Further work has con- firmed these observations and has also shown this acetone-soluble lipid gives a strong Ashbel-Seligman reaction and con- tains cholesterol. It immediately turns a brilliant blue-green in the Schultz test (Padykula, unpublished observations) . One difference between the labyrinthine and endodermal lipids is the color response in the Schultz test. The labyrinthine lipid turns red-brown but never the blue-green which indicates the presence of cholesterol. Lehner (1914) reported that intranuclear lipid droplets are abundant in the visceral endoderm of the mouse. This finding is con- firmed in the rat by electron microscopy (see Figs. 15.78 and 15.80). The significance of this lipid in the yolk sac is not clear, al- 924 SPERM, OVA, AND PREGNANCY though the findings of Luse (1958), Luse, Davies and Smith (1959), Luse, Davies and Clark (1959) suggest that it is absorbed lipid. Further discussion of the lipids of the yolk sac was given in the section on yolk sac placentation. In placentas of two species of shrews {Blarina brevicauda and Sorex fiimeus) Wimsatt and Wislocki (1947) described nu- merous coarse lipid droplets in the colum- nar trophoblastic epithelium forming the chorionic membrane. These droplets are su- danophilic and birefringent, exhibit green- ish fluorescence, and give an intense reaction with Schiff's reagent. In the cho- rio-allantoic placenta, minute sudanophilic particles are observed in the placental tra- becule, but birefringence and fluorescence are not evident and Schiff's reaction is feeble. In the placenta of the bat {Myotis luci- fugus lucifugus) Wimsatt (1948) observed sudanophilic lipids in nearly all placental constituents, but only those present in the columnar trophoblastic cells of the mem- branous chorion were birefringent, emitted a greenish-yellow fluorescence, and gave positive phenylhydrazine, Schiff's and Lie- bermann-Burchardt reactions. With respect to these reactions, it is apparent that the membranous chorion of shrews and bats is similar. In the placenta of the Virginia deer {Odo- coileus virginiayms borealis) in midgesta- tion, lipid droplets which are birefringent and give an Ashbel-Seligman reaction are present in a layer of epithelium lining the maternal crypts of the placentomas (Wis- locki, 1952) . A further interesting feature of the deer's placenta is an intense reaction for lipids (sudanophilia, birefringence, positive Ashbel-Seligman carbonyl reaction) in the withered, degenerating peripheral ends of the maternal septa. This reactive material is evidently attributable to degeneration of a portion of the epithelium covering the maternal septa. In the sheep, the epithelial layer clothing the maternal septa consists of syncytial tro- phoblast derived from the chorionic villi (Wimsatt, 1950; Amoroso, 1951, 1952). Al- though it has not been investigated, the epi- thelium lining of the maternal crypts of the deer's placenta may also be of fetal origin. In the sheep, Wimsatt (1951) remarks briefly that lipid droplets, which are bire- fringent and give positive Baker's acid- hematein and Liebermann-Burchardt reac- tions, are present in the columnar trophoblastic cells, but no mention is made of the reaction of the syncytial tro]ihoblast lining the maternal crypts. In view of the localization of these vari- ous lipid reactions in some part of the tro- phoblast, results obtained in the placenta of a pig (17 cm. crown to rump length) are an interesting exception (Wislocki, 1952). At this period of gestation, lipids are not en- countered in the chorionic epithelium, ex- cept phospholipids of mitochondria which are demonstrable by means of sudan black B (Fig. 15.15) and Baker's acid hematein Plate 15. XII Fig. 15.50. Human placental labyrinth at full term, .-stained by the periodic acid-Schiff (PAS) method after exposure of the section to saliva. Zenker's acetic acid fixative. The walls of the sinusoidal fetal capillaries and the (reticular) basement membrane upon which the syncytium rests are deeply stained. The outer zone of the syncytium is also noticeably stained. Although many of the capillaries deeply indent the syncytium producing so-called "syncytial" or "epithelial plates," a narrow rim of syncytium and the PAS-stained wall of the subjacent capillary always intervene between the intervillous space and the lumen of the capillary. Compare with Figures 15.14. 15.19, 15.25, and 15.41. X 280. Fig. 15.51. Degenerate placental villi at 2 months of gestation, illustrating their intense metachromatic staining with toluidin blue. Basic lead acetate fixative. Compare with Figure 15.57. X 240. (Wislocki and Dempsey, 1948.) Fig. 15.52. Human decidua vera at 2'/2 months of gestation, stained by the PAS method. Rossman's fixative. Observe the pronounced staining of ground substance encapsulating the poorlv stained decidual cells. Two arterioles are visible near the center of the figure. Compare with Figures 15.53 and 15.58 which illustrate decidua stained by other means. X 240. (Wislocki and Dempsey, 1948.) Fig. 15.53. Human decidua vera at 4 months of gestation, .showing the acid phosphatase reaction of the decidual cells. Gomori's method, using glycerophosphate as substrate at pH 47. X 240. (Wislocki and Dempsey, 1948.) HISTOCHEMISTRY OF PLACENTA 925 ».«^ ^ t'^'if^*^^*'- i% # % 53 Plate 15.XII 926 SPERM, OVA, AND PREGNANCY test. Instead, extremely minute, lipid drop- lets giving an Ashbel-Seligman reaction are present in the epithelium of the uterine mu- cosa (Figs. 15.15 and 15.22). Later in ges- tation, from about the 20-cm. stage on, large sudanophilic lipid droplets begin to appear in the basal ends of the columnar trophoblastic cells of the chorionic fossae (Wislocki and Dempsey, 1946b), but these have not been studied by other histochemi- cal reactions for lipids. B. GLYCOGEN AND CARBOHYDRATE CONTAINING MACROMOLECULES Since Claude Bernard suggested in 1859 that the placenta may perform the glyco- genic function for the embryo before the de- veloping liver has acquired this function, considerable attention has been given to lo- calizing this important metabolic reserve. In previous paragraphs, the localization and fluctuations in glycogen were reviewed for the human placenta. In this species, glyco- gen has a widespread distribution during the first 2 months, occurring in the syncy- tium, Langhans cells, stromal fibroblasts, Hofbauer cells, peripheral cytotrophoblasts, and decidual cells. After the second month, there is a sharp decline in glycogen storage in all these components, except in the de- cidual cells which retain glycogen until term. This decline has also been recorded biochemically by Villee (1953) whose meas- urements of glycogen content show a rapid drop after 8 weeks of gestation. Further- more, glucose production is possible early in gestation but not at term (Villee, 1953). Concerning Bernard's hypothesis, it is inter- esting to note Villee's observation that the glycogenic storage function of the human fetal liver is acciuired at 7 to 8 weeks of ges- tation. Thereafter, the glycogen content of the fetal liver rises sharply, as placental glycogen content falls. Glycogen storage in the rat placenta is also widespread, occurring in various parts of the maternal-fetal complex (Goldman, 1912; Krehbiel, 1937; Bridgman, 1948a, b; Padykula, 1958b; Bulmer and Dickson, 1960). Early storage during the first ten days is chiefly a decidual function. How- ever, the trophoblastic ectoplacental cone and its later derivative, the spongy zone, contain some glycogen from implantation until term, with peak storage occurring the 15th day of gestation. The vascularized tro- phoblast of the labyrinth and the visceral endoderm of the inverted yolk sac placenta initiate glycogen storage at 14 days, reach a peak at 18 days, and have released most Pl.\te 15. XIII Fig. 15.54. A portion of a human chorionic villus at 21/2 months of gestation, stained by the periodic acid-Schiff (PAS) method (exposed to saliva). Orth's fixative. Observe the marked reaction of the outer zone of the syncytium, the delicate stippling of the deeper layer, the chromophobic appearance of the Langhans cells, the intense staining of the base- ment membrane and the strong response of the large vacuolated Hofbauer cell. Compare with Figure 1529. X 7 ocular; X 90 objective. Fig. 15.55. Cytotrophoblasts from a human placental septum at full term, showing the cells partially surrounded by dark red stained fibrin. Orth's fixative. PAS stain. The cytoplasm of the trophoblasts contains a delicate stippling of PAS positive material as well as accen- tuated staining around the nuclear membrane. Compare with Figure 15.48. X 7 ocular; X 90 objective. Fig. 15.56. A lamella of a cat's placenta, stained by the PAS method. Orth's fixative. Treat- ment with saliva. Observe the intense reaction in a narrow zone located between the ma- ternal capillaries and giant decidual cells and the trophoblastic sjmcytium. In the tropho- blast occasional large intensely stained droplets of "colloid" are visible. Compare with Figures 15.60 and 15.61. X 7 ocular; X 60 objective. Fig. 15.57. A degenerating placental villus at 6 months of gestation, consisting of degen- erating stroma which has become intensely metachromatic (red), surrounded by a mantle of bluish green-stained, hyalinized syncytium. Basic lead acetate fixative. Toluidin blue stain. Compare with Figure 15.51. X 7 ocular; X 20 objective. Fig. 15.58. The decidua basalis of a human placenta at 2V2 months of gestation, illustrating the characteristic red metachromasia of the ground substance surrounding the decidual cells. A cleft in the decidua contains bluish green-stained fibrin. Basic lead acetate fixative. Com- pare with Figure 15.52, stained by PAS reagents. Toluidin blue stain. X 7 ocular; X 40 ob- jective. 56 _ HISTOCHEMISTRY OF PLACENTA 927 of this material by the 21st day. Concerning tliese fluctuations, it may be said that the glycogen content of the fetal placenta is highest immediately preceding the great terminal growth spurt of the embryo and fetal placenta. It should also be noted that, as in the human, placental glycogen content is decreasing during the period when the fetal liver is beginning to store glycogen (Padykula and Leduc, 1955). The distribution of glycogen in the rab- l)it placenta is drastically different from that of the human and the rat. In this spe- cies glycogen is localized exclusively in the decidua of the maternal placenta ; the fetal placental tissue, including the yolk sac, is devoid of glycogen (Bernard, 1859; Chip- man, 1902; Lochhead and Cramer, 1908; Loveland, Maurer and Snyder, 1931 ; Tuch- mann-Duplessis and Bortolami, 1954; Davies, 1956). Glycogen content reaches a peak near the 17th day of gestation, and decreases until term. Several investigators (Lochhead and Cramer, 1908; Tuchmann- Duplessis and Bortolami, 1954) have fur- ther substantiated Claude Bernard's obser- vation that the decline in placental glycogen correlates in time with the onset of the hepatic glycogenic function in the fetus. In the guinea pig a similar temporal correla- tion has been made for glycogen storage in the placenta and fetal liver (DuBois and Ducommun, 1955). Saliva-insoluble carbohydrates, such as glycoproteins and mucopolysaccharides, re- vealed by the PAS reaction are demonstra- ble in the placentas of all animals which have been examined (Wislocki, 1951). In the pig's placenta a positive reaction is given by minute droplets in the apical ends of the uterine gland cells, in the glandular secretion (Fig. 15.66), and in the uterine surface epithelium. An intense reaction is given by the secretion (uterine milk) in the lumens of the chorionic areolae. Numerous ]iositively stained, delicate droplets are present in the distal cytoplasm of the colum- nar epithelium lining the chorionic fossae and areolae (Fig. 15.65). In the basal part of the tall columnar cells lining the chori- onic fossae there are, in addition, large ''col- loid" droplets (Fig. 15.65) which are stained intenselv red (Wislocki and Demp- sev, 1946b). In the numerous trophoblastic binucleate giant cells of the sheep and cow, Wimsatt (1951) reported the presence of many PAS- positive cytoplasmic granules. The tropho- blastic giant cells of the Virginia deer react similarly (Wislocki, unpublished observa- tion) . In the chorionic lamellae of the cat's pla- centa, deeply stained PAS-reactive mate- rial is present between the fetal trophoblast and the maternal vessels (Figs. 15.56 and 15.61). In addition, large, deeply stained colloid droplets are located irregularly in the trophoblastic syncytium (Fig. 15.56). In the placental "brown" border of the cat, a reaction is present in the chorionic epithe- lium, as well as in the secretion in the uter- ine lumen and in the surface and glandular uterine epithelium (Fig. 15.62) . In the rat's placenta, the apical cyto- plasm of the uterine epithelium, amorphous material in the uterine and vitelline cavi- ties, the substance of Reichert's membrane and the apical cytoplasm of the vitelline epi- thelial cells (Fig. 15.72) all react strongly (Wislocki, and Padykula, 1953) . The tropho- blast of the chorio-allantoic placenta gives a relatively faint reaction (Fig. 15.72). C. METACHROMASIA None of the structures in the placentas of the various animals cited above, which are strongly PAS positive, exhibits any meta- chromasia (Wislocki, 1953) , except that the binucleate cells of the sheep react faintly under some conditions of staining (Wim- satt, 1951). Also the ground substance of the stroma of the chorionic rugae of the pig's placenta is moderately metachromatic. D. PHOSPHATASES Because the methods for alkaline and acid phosphatase were among the first his- tochemical procedures for localizing en- zymes, many observations have been made on this type of hydrolytic activity. The ab- sorptive surfaces of the small intestine, kid- ney, and placenta, which are characterized by brush borders, contain strong alkaline phosphatase activity. In many forms the syncytial trophoblast is rich in alkaline phosphatase activity. In the human syncy- tium, alkaline phosphatase activity which is low early in gestation increases greatly 928 SPERM, OVA, AND PREGNANCY later. This enzymatic activity is high in the syncytial trophoblast of the cat, rodents (Figs. 15.71 and 15.73), shrews, and bats. Alkaline phosphatase occurs in the visceral endoderm of the splanchnopleuric yolk sac of rodents (Fig. 15.70) (Hard, 1946; Wis- locki, Deane and Dempsey, 1946; Pritch- ard, 1947; Padykula, 1958a), shrews (Wis- locki and Wimsatt, 1947), and bats (Wimsatt, 1949). Thus, this enzyme is lo- cated at two major placental aljsorptive surfaces. In the pig's placenta, alkaline phospha- tase activity is high in the columnar epi- thelial cells of the chorionic fossae (Fig. 15.67) and in the stroma and blood vessel walls of the maternal endometrium (Fig. 15.68) (Wislocki and Dempsey, 1946b; Dempsey and Wislocki, 1947) . However, it is completely absent from the chorionic areolae and extremely low in the epithelium of the chorionic rugae. In the coiv and sheep, binucleate trophoblastic giant cells are ricli in alkaline phosphatase (Wimsatt, 1951). In the Virginia deer at midgestation, this enzyme was found in the binucleate giant cells, stroma and walls of the blood vessels of the maternal septa, epithelium clothing these septa, and at the surface of the tro- phoblast covering the chorionic \\\\\. In the cat, alkaline phosphatase occurs also in ma- terial surrounding the capillaries and de- cidual giant cells (Fig. 15.60). In the para- placental "brown" border of the cat, there is abundant alkaline phosphatase activity in the uterine glands and surface epithe- lium, in the uterine secretion, and in the outer parts of the columnar epithelial cells of the membranous chorion (Fig. 15.63). Acid phosphatase occurs in the human syncytium and also in the labyrinthine tro- phoblast of the i^at where it increases in activity in the last week of gestation (Pady- kula, 1958). In the rat placenta, this en- zyme appears in the cytotrophoblast of the spongy zone and in the giant cells on the 17th day and increases steadily until term. In the cat, acid phosphatase occurs in the trophoblast of the placental lamellae, in the uterine glands, in the uterine surface epi- thelium, and to some degree in the epithe- lium of the membranous chorion (Wislocki, 1953). In the pig, acid phosphatase activity is high in the uterine glands (Fig. 15.69), in the uterine milk occurring in the lumens of the chorionic areolae, and in the distal ends of the epithelial cells lining the area- lae (Wislocki and Dempsey, 1946b; Demp- sey and Wislocki, 1947) . The activity of the uterine surface epithelium is moderate. In Plate 15.XIV Fig. 15.59. Frozen section of the placental labyrinth of a cat, viewed under a polarizing microscope, to illustrate the strong birefringence encountered in the cellular trophoblast of the placental lamellae (fetal crown to rump length, 110 mm.). The birefringence is associated with numerous sudanophilic lipid droplets which exhibit greenish fluorescence and give a positive Ashbel-Seligman reaction for carbonyl groups. Compare with Figure 15.20 which illustrates the carbonyl reaction. X 280. (Wislocki and Dempsey, 1946a.) Fig. 15.60. A placental lamella of a cat, illustrating the presence of alkaline phosphatase in the interstitial matrix around the maternal blood vessels and decidual giant cells and ex- tending into the syncytial trophoblast (embryo length, 13 mm.). Gomori's method, using glycerophosphate as substrate at pH 9.4. X 800. (Wislocki and Dempsey. 1946a.) Fig. 15.61. Placental lamellae of a cat (fetal crown to rump length, 45 mm.), illustrating the periodic acid-Schiff (PAS) reaction which is localized in the interstitial matrix surround- ing the maternal blood vessels and decidual giant cells and intervening between them and the trophoblast. Compare with the similar localization of alkaline phosphatase in Figure 15.60 and also with Figure 15.56. Zenker's acetic acid fixative. Treatment with saliva. X 260. Fig. 15.62. The paraplacental endometrium ("brown" border) of a pregnant cat (fetal crown to rump length, 45 mm.), illustrating the intense PAS reaction in the secretion of the uterine glands which is poured into the uterine lumen. A reaction is present also in the cyto- plasm of the distal ends of the uterine epithelium. The paraplacental chorionic membrane (opposite the uterine epithelium at the extreme upper border of the photograph) consists of columnar cells the supranuclear cytoplasm of which contains PAS-stained droplets. Zenker's acetic acid fixative. Treated with saliva. X 260. Fig. 15.63. The paraplacental "brown" border of a pregnant cat (fetal crown to ru.mp length, 45 mm.), illustrating the intense alkaline phosphatase reaction in the endometrium (right) and lesser reaction in the chorion (left). Gomori's method, using glycerophosphate as substrate at pH 9.4. X 120. HISTOCHEMISTRY OF PLACENTA 929 Tlatk 15.XIV ^30 SPERM, OVA, AND PREGNANCY the trophoblast covering the chorionic ru- gae, acid phosphatase activity is low or completely absent. In the rat placenta the distribution of phosphatase activity toward adenosine tri- phosphate at alkaline pH has been de- scribed by Padykula (1958a). VII. Evidence of the Possible Site of Production of Placental Steroid Hormones In the section on methods, a procedure for characterizing lipids was outlined for localizing the sites of the synthesis of ster- oid hormones or their precursors in histo- logic sections. It was pointed out that none of the reactions involved in the procedure is specific for the identification of ketoster- oids, but lipids possessing all of the proper- ties enumerated have been found solely in those organs (adrenals, gonads, and pla- centa) in which steroid hormones are known to be produced. In formalin-fixed, frozen sections of hu- man placenta, birefringent, sudanophilic lipid droplets are abundantly present in the syncytial trophoblast throughout gestation. They are acetone soluble, react with phenylhydrazine (Wislocki and Bennett, 1943), give a positive Schiff reaction, ex- hibit yellowish-green fluorescence (Demp- sey and Wislocki, 1944; Rockenshaub, 1952), and also react positively with the Ashbel-Seligraan reagents for carbonyl groups (Ashbel and Seligman, 1949; Selig- man, Ashbel and Cohen, 1951; Wislocki, 1952; Ashbel and Hertig, 1952). The lipid droplets diminish in size and relative abun- dance as gestation advances, but are, never- theless, still apparent in the syncytium at full term (Wislocki and Bennett, 1943, Figs. 10 and 11). The decrease in droplet size with age may not necessarily indicate a reduction of functional activity, for in the adrenal cortex and ovaries a diminution in the size of the lipid droplets accompanies active secretion (Deane, Shaw and Greep, 1948; Barker, 1951). Furthermore, since the total volume of the syncytium must increase considerably as the placental villi grow and branch, it seems reasonable to assume that the absolute quantity of the lipids may not actually diminish. Thus, there is possibly no discrepancy between the increase in formation and excretion of steroid compounds in the course of gesta- tion and the total amount of lipids in the syncytium at term. Strands of syncytium which penetrate the trophoblastic shell and junctional zone in the first trimester of pregnancy (Wislocki and Bennett, 1943) and undergo degenera- tion are probably responsible for the pres- ence in these regions of occasional patches of lipids giving these reactions. The uterine glandular epithelium con- tains large sudanophilic droplets which are not birefringent, but stain with Schiff's reagent (Wislocki and Dempsey, 1945) and give a hydrazide reaction (Ashbel and Hertig, 1952). Wislocki and Dempsey (1945j erroneously equated Schiff's reac- tion with the "plasmal" reaction and specu- lated on its possible significance. It now seems more probable that Schiff's reagent, under the conditions of fixation utilized by them, reveals peroxides of unsaturated lipids (Nicander, 1951). Ashbel and Hertig (1952) attributed staining of the epithelium of the endometrial glands by the carbonyl procedure to "ketosteroids," a conclusion which Atkinson, in a discussion of their pa- per, found difficult to believe, since he ob- served that all of the cellular elements of the parietal decidua give a positive reaction for carbonyl groups. This objection seems valid, inasmuch as the hydrazide will react with the oxidation products of unsaturated groups. In mammals, other than man and the rhesus monkey, the observations cited in a previous section of this review indicate that lipid reactions characteristic of steroid- producing cells are also usually located in some part of the trophoblast (cat, shrews, bat, rodents) . However, the pig's placenta is exceptional in that the sudanophilic lipid droplets giving the Ashbel-Seligman and Schiff reactions are located in the uterine epithelium. It is doubted that the reactions in the sow's uterine epithelium are indica- tive of steroidal synthesis, inasmuch as Wislocki and Dempsey (1945) encountered no birefringence in the epithelium. Estro- gens have been detected by various means HISTOCHEMISTRY OF PLACENTA 931 of assay in the placenta of the sow, being excreted between the 20th and 30th days of gestation and thereafter diminishing, to in- crease again around the 10th or 12th week and continuing to do so until term (Cowie, 1948). As a possible source of estrogens, the sudanophihc lipid droplets, present in the columnar cells of the sow's chorionic fossae in the latter half of gestation (Wis- locki and Dempsey, 1946b), should be in- vestigated further. An histochemical method for visualizing steroid-3/3-ol-dehydrogenase activity by tetrazolium salts has proved useful in iden- tifying steroid-producing cells in the rat ad- renal, ovary, and testis (Levy, Deane, and Rubin, 1959) . Application of this technique to the rat placenta (Deane, Lobel, Driks, and Rubin, I960) has localized steroid-3/?- ol-dehydrogenase activity in the tropho- blastic giant cells. This activity is greatest between the 8th and 15th day, becomes low by 18 days, and is nearly absent by the 21st day of gestation. Further application of this technique to other placental types should be fruitful. VIII. Evidence of the Possible Site of Production of Placental Gonadotrophic Hormones A. HUMAN PLACENTA Friedheim (1929), Sengupta (1935), Gey, Jones and Hellman (1938), Jones, Gey and Gey (1943), and Stewart, Sano and Mont- gomery (1948) have grown human placental trophoblast in tissue cultures. It has been observed that the cytotrophoblast rather than the syncytium proliferates and that the latter, in so far as it arises, seems to be derived from the cellular form, and is small in amount and atypical in appearance. Friedheim observed no conversion of cyto- trophoblast into syncytium in actively grow- ing cultures. Furthermore, Gey, Jones and Hellman (1938), Jones, Gey and Gey (1943) , and Stewart, Sano and Montgomery (1948) demonstrated that tissue cultures ■containing actively growing cytotropho- blast produce appreciable quantities of chorionic gonadrotrophic hormone, even after repeated transplantation over several months. These observations indicate that the trophoblast, and more particularly the cytotrophoblast, is the Sburce of the hor- mone. Stewart, Sano and Montgomery reported their inability to grow trophoblast from mature placentas of the 8th and 9th months. Inasmuch as the syncytium does not divide mitotically and the Langhans cells are nu- merically much decreased at this period, the result is not surprising. However, if they had cultured tissue containing peripheral trophoblasts, obtained specifically from the placental septa or basal plate, growth might have been anticipated. Chorionic gonadotrophic hormone is, as a rule, al)undantly present in the urine of women suffering from hydatidiform moles or chorion epitheliomas (Tenney and Parker 1939, 1940; Rubin, 1941), and disappears promptly after the successful surgical re- moval of these tumors. Tenney and Parker noted that the amount of hormone corre- sponds roughly to the number of tropho- blastic cells in a mole or chorion epitheli- oma and that a mole with cystic villi and slight trophoblastic proliferation gives a low titer. These findings also indicate that pro- liferating cytotrophoblast is the source of the hormone and that the syncytium is of less or of no importance. Wislocki and Bennett (1943) emphasized that the curve of excretion of chorionic gon- adotrophic hormone corresponds very well with the period of active proliferation of the trophoblastic shell. Nevertheless, a discrep- ancy seemed to exist in that the cytotropho- blast has generally been believed to degen- erate and disappear in the last trimester, whereas the excretion of chorionic gonado- trophin continues throughout gestation (Venning, 1948). This apparent discrepancy is now understandable in the light of obser- vations reported here which demonstrate that, although the Langhans cells which are chromophobic diminish greatly in number, the peripheral cytotrophoblasts which are chromophilic survive until full term in large numbers in the septa placentas and basal plate as viable, functional cells (Wislocki, 1951). Baker, Hook and Severinghaus (1944) described blue granules in both the cyto- trophoblast and the syncytium of the hu- 932 SPERM, OVA, AND PREGNANCY man placenta, demonstrable by a trichrome stain devised by Severinghaus (1932). Ac- cording to them, these granules enlarge to- ward the surface of the syncytium and then seem to liquefy, forming vacuoles which liberate their contents through the brush border into the maternal blood stream. In late pregnancy, as the syncytium becomes thinner, these granules and vacuoles dis- appear. The investigators interpreted these findings as signifying that the trophoblast of early pregnancy performs a significant secretory function, and they emphasized that the period of activity was roughly con- temporaneous with the time of greatest ex- cretion of gonadrotrophin. Bruner and Witschi (1947) and Bruner (1951), investigating tlie distribution of chorionic gonadotrophin in the human pla- centa by biochemical means, reported that it is found at all stages of pregnancy, the concentration being highest in the fetal por- tion of the placenta. It is evident, they stated, that the major part of the hormone is "released from the plasmotrophoblast into the maternal blood, whereas only a small fraction passes the inner placental barrier, the cytotrophoblast, to enter the fetal blood stream." They apparently believed that the hormone is formed by the synctium. Similarities between the peripheral tro- phoblasts and the cells of the anterior lobe of the hypophysis believed to produce the hypophyseal gonadotrophic hormones, sug- gested to Dempsey and Wislocki (1945) that the peripheral trophoblasts produce chorionic gonadotrophin. They demon- strated that the cytoplasm of the syncytium and peripheral trophoblasts of the human placenta contains a basophilic substance similar to that in the true basophilic cells of the anterior lobe of the hypophysis, which in both instances is abolished by digestion with crystalline ribonuclease. That the hy- pophyseal basophilic cells contain ribonu- cleoprotein was first demonstrated by Desclin (1940). Since ribonucleoprotein is generally concentrated in cells in which pro- tein synthesis is actively taking place, Wis- locki, Dempsey and Fawcett (1948) sug- gested that its presence in the peripheral trophoblast might be related to the forma- tion tlierc of chorionic gonadotrophin. On the other hand, they thought that the ri- bonucleoprotein in the syncytium which is particularly abundant in the first months of gestation might represent the primary site of synthesis of fetal plasma proteins, a function taken over by the hepatic cells when the fetal liver becomes sufficiently dif- ferentiated. Wislocki (1951) observed that the basophilia of the peripheral trophoblasts persists until full term, coinciding with the Plate 15.XV Fig. 15.64. Cliorionic fold.s of a pig's placenta (fetal crown to rump length, 130 mm.), show- ing many intraepithelial blood capillaries (blood cells not visible), resulting in the formation of many extremely thin epithelial plates some of which appear to be quite as thin as the human "epithehal plates" seen at full term {cj. with Figure 15.50). Compare with Figures 15.23 and 15.28 which are also of pig's placenta. Zenker-formol fixative. Eosin and nifthylene blue stain. X 400. (Wislocki and Dempsey, 1946b.) Fig. 15. 65. A chorionic fossa of a pig's placenta (fetal crown to rump length, 120 mm.), illustrating the presence of finely dispersed periodic acid-Schiff (PAS) positive material in the distal ends of the columnar cells and coarse PAS stained droplets in their proximal ends. Orth's fixative. X 150. Fig. 15.66. Uterine glands in the maternal placenta of a pig (fetal crown to rump lengtli. 120 mm.), showing the strongly PAS-reactive secretion in the lumens. Orth's fixative. X 165. Fig. 15.67. The chorion of a pig's placenta (fetal crown to rump length, 120 mm.) to il- lustrate the intense reaction of alkaline phosphatase in the columnar cells of the chorionic fossae located between the chorionic rugae. Gomori's method, using nuclei acid at pH 9.6. X 200. Fig. 15.68. The endometrium of a pig's placenta (fetal crown to rump length, 120 mm.), to illustrate the intense alkaline phosphatase reaction in the endometrial stroma and blood vessel walls. The enzyme is essentially negative in the epithelium (ep) covering the endo- metrial folds as well as in the epithelium lining the uterine glands (g). Gomori's method, using fructose diphosphate at pH 9.4. X 200. Fig. 15.69. Uterine glands in a pig's placenta (fetal crown to rump length, 125 mm.), il- lustrating the intense acid phosphata.se activity of the glandular cells. Gomori's method, using glycerophosphatase at pH 4.7. X 170. (Wislocki and Dempsey, 1946b.) HISTOCHEMISTRY OF PLACENTA 933 ■.^-. 67 U2 J Plate 15.XV 934 SPERM, OVA, AND PREGNANCY continued production of chorionic gonado- trophin. The gonadotrophic hormones of both pituitary and placenta are known to be gly- coproteins (Bettelheim-Jevons, 1958). Pu- rified gonadotrophins of pituitary and pla- centa contain hexose and hexosamine. Catchpole (1949 J reported that he found a glycoprotein constituent of the basophil cells of the hypophysis of the rat, demonstrable by means of the PAS stain. On the basis of the increase of this reaction after castration, as well as from other physiologic correlates, he concluded that a part of the material represents the follicle-stimulating hormone. Pearse (1949) likewise observed a positive PAS reaction in the pituitary basophils which he ascribed similarly to the gonado- trophic hormone. Moreover, he described a particular type of vesiculated chromophobe which he suggested might represent a phase in the secretory cycle of the basophils. Purves and Griesbach (1951a, b) estab- lished that there are two categories of gly- coprotein-containing basophils in the rat pi- tuitary, the gonadotrophs and thyrotrophs. (See also discussion in chapter by Purves.) These two groups of basophils can be dis- tinguished on the basis of shape, geograph- ical distribution, nature of granulation, and responses to changes in hormonal environ- ment. Pearse (1949) noticed PAS-positive material in the form of granular masses, globules, and vesicles in "the trophoblast layer of the placenta and in the Langhans cells of chorionepithelioma." Inasmuch as the granular part of this material could be removed bv diastase, it seemed to consist of glycogen. However, the globules and vesi- cles which were saliva fast he regarded as being probably of "mucoprotein nature" and as representing chorionic gonadotrophin. Wislocki, Dempsey and Fawcett (1948) mentioned briefly that after fixation in Rossman's fluid and removal of glycogen they were unable to demonstrate any reac- tion in the cytotrophoblast by the PAS method. However, on further trials with other fixatives, including Zenker's acetic acid mixture and Orth's fluid, delicate granules were rendered visible (Wislocki, 1950) in the cytoplasm of some fraction of the periph- eral trophoblasts throughout gestation (Figs. 15.49 and 15.55). These fine, but often in- distinct, particles are more like those de- scribed in the pituitary basophils by Catch- pole (1949) than the globules and vesicles mentioned by Pearse (1949) which were not seen in these preparations. Although the re- actions observed in the peripheral cytotro- phoblast may bear a relationship to the presence of chorionic gonadotrophin, it should not be overlooked that the syncytial trophoblast also exhibits a delicately stip- pled, variable reaction and that its outer surface and brush border are quite strongly stained (Figs. 15.29 and 15.54). Further- more, it should be borne in mind that a variety of carbohydrate-containing sub- stances, including various mucopolysaccha- rides, glocoproteins glycolipids, and gly- coliproproteins react with the PAS reagents and that such reactive substances are widely distributed in cells and tissues. As a result of this, the possible identification of cho- rionic gonadotrophin by this single reaction Plate 15.XVI Fig. 15.70. The placenta of a guinea pig (fetal crown to rump length, 75 mm.), illustrating the distribution of alkaline phosphatase. The reaction is extremely intense in the placental cotyledons (fine-meshed s>'ncytium) and diminishes abruptly in the coarse, interlobular syn- cytium. The villous portion of the yolk sac (at top of figure above the placenta) is also rich in alkaline phosphatase. The subplacenta beneath the placental labyrinth is negative, but the junctional and decidual zones are quite reactive. Gomori's method, using gh^cerophos- phate as substrate at pH 9.4. X SVi. (Wislocki, Deane and Dempsey, 1946.) Fig. 15.71. The placental labyrinth of a mouse, on the 15th day of gestation illustrating the intense alkaline phosphatase reaction in the trophoblastic syncytium. Gomori's method, using glycerophosphate as substrate at pH 9.4. X 170. (Wislocki, Deane and Dempsey, 1946.) Fig. 15.72. The chorio-allantoic placenta and yolk sac of a rat on the 21st day of gestation, illustrating the periodic acid-Schiff reaction of the epithelium of the villous portion of the yolk sac and of Reichert's membrane. Orth's fixative. Treatment with saliva. X 175. (Wis- locki and Padykula, 1953.) Fig. 15.73. A detail of the guinea pig's placenta shown in Figure 15.70, illustrating the in- tense alkaline phosphatase reaction in the trophoblastic syncytium enclosing the maternal vascular channels. X 830. (Wislocki, Deane and Dempsey, 1946.) HISTOCHEMISTRY OF PLACENTA 935 Plate 15. XVI 936 SPERM, OVA, AND PREGNANCY must be accepted with considerable reserva- tion. From the foregoing summary of the evi- dence regarding the localization of chorionic gonadotrophic hormone, it seems probable that in the human the trophoblast is the site of its formation. However, it is not clearly established whether it is localized in the Langhans cells, syncytium, peripheral cy- totrophoblast, or in several of these ele- ments. Evidence favors the peripheral tro- phoblasts. The Langhans cells are a less likely site because they are chromophobic and decline perceptibly in size and number by the beginning of the last trimester, whereas chorionic gonadotrophin and the peripheral trophoblasts persist until full term. Involvement of the syncytium seems unlikely, because it is the probable site of formation of the placental steroid hormones, and because it gradually loses its cytoplas- mic basophilia, whereas the production of chorionic gonadrotrophin continues until full term. In attempting to evaluate the possible nature of granules, vacuoles, lipid droplets, mitochrondria, and other organelles in the syncytium, the entire role of the placental barrier must be kept in mind. The syncytial trophoblast is chiefly involved in the ab- sorption and transfer of metabolites from the maternal to the fetal blood stream, be- sides serving as a means of excreting certain waste products. Many of the organelles which its cytoplasm contains are related in some manner to these functions, although it is not possible at present to assign specific roles to many of them. Wislocki and Streeter (1938) and' Wislocki and Bennett (1943) suggested that a considerable number of the vacuoles seen in the syncytium, especially early in gestation, might be related to ab- sorption. They based their opinion on the probability that much of the syncytial cyto- plasm is in a state of motion and flux, with the likelihood that maternal plasma is ab- sorbed by a process of pinocytosis in the manner visualized by Lewis (1931) in cells growing in tissue culture. Recent observa- tions of the human placenta with the elec- tron microscope (Boyd and Hughes, 1954; Wislocki and Dempsey, 1955a) bear out this interpretation. On the other hand, waste products of fetal metabolism, such as cre- atine, creatinine, and urea, are so readily diffusible that their excretion would in all probability not be associated with the for- mation of granules which liquefied and formed vacuoles. There is considerable justification for associating the formation of placental steroid hormones with the sudan- ophilic, birefringent, lipid droplets present in the syncytium, but there is no evidence that steroid hormones are liberated from cells, in the adrenal glands or elsewhere, in a visible sequence of liquefying granules and discharging vacuoles. In regard to placental gonadotrophin, the slight evidence which can be assembled regarding its localization tends to place it in the peripheral tropho- blasts rather than in the Langhans cells and syncytium. On the other hand, early in gestation during implantation and subse- quent invasion of the uterine wall by the trophoblast, cytolytic substances and en- zymes are quite possibly released by the trophoblast and these probably account for some of the granules and vacuoles seen in parts of the trophoblast at that period (Wis- locki and Bennett, 1943). Considering all of the evidence, chorionic gonadotrophic hormone of the human is most likely produced by the peripheral trophoblasts. This opinion is based on the observed results of culturing trophoblast, as well as on several histochemical similari- ties of the basophils of the pituitary gland with the peripheral trophoblasts (cytoplas- mic basophilia, PAS reaction). Of impor- tance for this concept is the observation that, whereas the majority of the Langhans cells diminish in size and number by the 6th month of gestation, many of the peripheral trophoblasts remain viable and functional until full term (Wislocki, 1951), thus coin- ciding with the continued production of chorionic gonadotrophin. According to these observations, the Langhans cells, clothing the secondary villi and located in the proxi- mal ends of the trophoblastic cell columns, represent a germinal bed composed of chro- mophobic trophoblasts, from which the line- ages of the syncytium and the chromophilic peripheral trophoblasts are separately de- rived. A further parallelism with the pitui- tary is apparent here, in that in both organs HISTOCHEMISTRY OF PLACENTA 937 chrouiophobic cells are postulated as being the precursors of the chromoi:)hilic elements. Little is known about the site of placental gonadotrophin production in infrahuman mammals. The PAS reaction provides little information about possible sites of forma- tion of the hormone in various animals, be- cause the reaction occurs in many placental components. This undoubtedly reflects the presence of other carbohydrate-containing substances besides the gonadotrophic hor- mone. Several reports on the mare (Cole and Goss, 1943; Rowlands, 1947; Amoroso, 1952; and Clegg, Boda and Cole, 1954) indicate that the equine gonadotrophin is produced by special parts of the endometrium of the maternal placenta called endometrial cups. Observations of Clegg, Boda and Cole (1954) suggest that the glandular epithe- lium is the site of gonadotrophic hormone production. A recent detailed discussion of the comparative aspects of hormonal func- tions was presented by Amoroso (1960). IX. Significance and Relationships of Some Placental Constituents A. RIBONUCLEOPROTEIN Most of the cytoplasmic basophilia en- countered in the trophoblast, uterine sur- face epithelium, and cells of the endometrial glands is due to the presence of ribonucleo- protein. Because most embryonic or rapidly growing cells are rich in this nucleoprotein, it has been difficult to separate the ribo- nucleoprotein associated with grow^th from that related to the synthesis of specific proteins by placental cells. Intense cyto- plasmic basophilia occurs in the trophoblast of the pig (Wislocki and Dempsey, 1946b), cat (Wislocki and Dempsey, 1946a), ro- dents (Wislocki, Bunting and Dempsey, 1947), and man. Basophilia is extremely intense in the early part of gestation and diminishes in the second half of pregnancy, except in the pig in which it remains con- stant and in the bat in which, according to Wimsatt (1949), it becomes more pro- nounced. In the human, it has been pro- posed (Wislocki, Dempsey and Fawcett, 1948) that one of the functions of the ribo- nucleoprotein of the syncytium is to synthe- size the proteins of the fetal blood plasma before the fetal liver becomes sufficiently differentiated to assume that activity, whereas the ribonucleoprotein in the periph- eral trophoblasts might conceivably be re- lated to the formation there of gonado- trophic hormone. Consonant with the former thought is the gradual decline in cytoplas- mic basophilia as pregnancy progresses. Ribonucleoprotein is closely associated with the secretory activities of uterine glands. The surface epithelium and glands of the uterus, just before and during gesta- tion, are rich in basophilic substance, great- est in amount in the pregnant sow, interme- diate in carnivores, and least in rodents and in pregnant women (Wislocki and Dempsey, 1945). In the epitheliochorial placenta of the sow, all of the nutritive substances ob- tained by the fetus must either traverse or be secreted by the uterine surface epithelium or the uterine glands. The glands which are rich in ribonucleoprotein release a copious secretion which reacts intensely with PAS reagents (mucopolysaccharide), but is not metachromatic. It gives a strong reaction for acid phosphatase. This secretion, desig- nated as uterine milk, seems to be absorbed mainly by the cells of the chorionic fossae and areolae. In the cat, the markedly baso- philic paraplacental uterine glands release a secretion w^hich is rich in mucopolysac- charide, glycogen, and phosphatases and is absorbed l)y the columnar cells of the brown border of the chorion. The subplacental glands of the cat are strongly basophilic, but react only faintly with PAS reagents. In rodents, the uterine glands and surface epithelium also seem to secrete nutriment which is absorbed through the yolk sac pla- centa. These secretory cells are basophilic, and their distal cytoplasm and secretion react intensely for mucopolysaccharide with PAS reagents. In the human, at the time of implantation and for a considerable period thereafter, the paraplacental and subpla- cental glands are moderately basophilic and contain glycogen, PAS-reactive mucopoly- saccharide, lipids, and phosphatases. More- over, unlike the uterine glands of various animals, their secretion contains some meta- chromatic mucin (Wislocki and Dempsey, 1948). The secretion of the glands located in the basal decidua may supply the growing 938 SPERM, OVA, AND PREGNANCY peripheral trophoblast (trophoblastic shell and cell columns) with nourishment. B. ALKALINE PHOSPHATASE From the study of the placentas of man, cats, pigs, and rodents, Wislocki and Demp- sey (1945, 1946a, b) and Wislocki, Deane and Dempsey (1946) concluded that a layer of alkaline phosphatase intervenes between the maternal and fetal placental circula- tions. Wislocki and Wimsatt (1947) found this to be true also in shrews, and Wimsatt (1949) observed a layer located similarly between the maternal and fetal circulations in the placenta of the bat. In hemochorial and endotheliochorial types of placentas, the enzyme is usually present in the outer- most layer of the trophoblast, whereas in the epitheliochorial placenta of the pig it is present mainly in the stroma of the mater- nal placenta. Dempsey and Wislocki (1945) pointed out that many substances may de- pend on phosphatases for their transfer across cellular boundaries, and they sug- gested that a layer of different phosphatases located in the placental barrier may partic- ipate in the transfer of metabolites. Wim- satt (1949) remarked that this view is con- sonant with a variety of metabolic processes which must be carried out at the placental barrier and accords with the interpretation of the barrier as a "selective" membrane. This distribution of phosphatase in the outer zone and brush border of the human syncytium resembles very strikingly the location of the enzyme in the epithelium of the small intestine and in the convoluted tubules of the kidney. These three absorp- tive surfaces also have well developed brush borders composed of numerous microvilli. Hence in the placenta, as in these latter sites, alkaline phosphatase may be associ- ated in the microvilli with the absorption of phosphorylated compounds. Alkaline phosphatase is also a component of the barrier in the yolk sac placenta of rodents (Hard, 1946; Wislocki, Deane and Demp- sey, 1946; Padykula, 1958), although it fluctuates in the vitelline epithelium at dif- ferent periods of gestation. As in the human syncytium, the epithelium of the visceral layer of the yolk sac of rodents possesses a brush border. In various parts of the endometrium and fetal placentas of lower mammals and man, Dempsey and Wislocki (1947) observed differences in alkaline phosphatase reac- tions following the use of a variety of sub- strates. Some structures reacted with many substrates whereas others reacted with only one or two. It was concluded that the ob- served differences could be accounted for most reasonably by the assumption that the tissues contain multiple enzymes of varying specificity which frequently do not coincide in their distribution. In the placentas of man, cats, rodents, and shrews alkaline phosphatase increases Plate 15.XVII Lipids of the rat yolk sac All of the sections illustrated in Figures 15.74 to 1577 were fixed in 10 per cent buffered formalin and stained with sudan black B. Fig. 15.74. Parietal wall and nonvillous visceral wall of the yolk sac at 13 days of gestation. The parietal endoderm {p) contains minute lipid droplets and rests on an unstained Reichert's membrane (r). Across the yolk sac cavity larger lipid droplets occur in the non- villous visceral endoderm (v) ; however, the lipid here is less abundant than in the villous portion shown in Figure 15.75. In the left border of the photograph, lipid occurs also in giant cells and decidual cells of the capsularis. X 275. Fig. 15.75. Villous visceral splanchnopleure at 13 days of gestation. Peak storage of lipid by the visceral endoderm occurs at this time (12 to 13 days). Note that the position of the lipid droplets is principally infranuclear. Lipid is much less abundant in the mesenchyme and mesothelium of the visceral splanchnopleure. Compare with Figures 15.76 and 15.77. Higher magnification of these endodermal cells is provided in Figures 15.78 to 15.81. X 275. Fig. 15.76. Villous visceral splanchnopleure at 17 days of gestation. By the 17th day of gestation, the villi have elongated and branched, and the lipid content of each endodermal cell has decreased. The cells toward the tip of the villus tend to have more lipid than those at the base. The droplets remain infranuclear. Compare with Figures 15.75 and 15.77. X 275. Fig. 15.77. Villous visceral splanchnopleure at 19 days of gestation. The endodermal cells are quite free of lipid droplets. There is a background sudanophilia which is mostly concen- trated in the mitochondria. Compare with Figures 15.75 and 15.76. X 275. HISTOCHEMISTRY OF PLACENTA ,939 f i . 74 r-i. ^^,J^"^ ^..^«^ Plate 15.XVII 940 SPERM, OVA, AND PREGNANCY in the course of gestation as cytoplasmic basophilia decreases. In the human tropho- blastic syncytium, alkaline phosphatase first appears in the outer eosinophilic (alka- line) zone and seems to advance into the syncytium as the basophilia recedes. In the placentas of other animals listed above al- kaline phosphatase is also usually localized in regions which are acidophilic. Wimsatt (1949) reported an exception to this inverse relationship of basophilia and phosphatase, because in the bat both substances are plentiful during the first half of gestation, whereas, in the second half, phosphatase activity declines while basophilia persists. Wislocki and Dempsey (1945) observed from study of the placentas of various ani- mals that a layer of phosphatase intervenes between the maternal blood stream and re- gions where glycogen accumulates. Hard (1946) observed a similar spatial relation- ship in the placenta of the guinea pig be- tween alkaline phosphatase, on the one hand, and accumulation of glycogen and lipid on the other. Wimsatt (1948, 1949) re- ported similar relations between the two substances in the placenta of the bat. Whether or not these two phenomena are related has been debated (Wimsatt, 1949; Pritchard, 1947). In this connection it is of interest, as more has been learned about the localization of glycogen by the use of the PAS reaction, that the cellular elements of the secondary and tertiary villi of the hu- man placenta up to 6 weeks of gestation are moderately rich in glycogen, at a time when extremely little alkaline phosphatase has made its appearance in the placental bar- rier. And later, as alkaline phosphatase in- creases tremendously in amount, stainable glycogen disappears completely from the chorionic villi. Wimsatt (1949) observed that the troph- oblastic cells of the membranous chorion of the bat "contain heavy deposits of neu- tral fat, phospholipids, and cholesterids" and concluded that phosphatases, abun- dantly present in the adjacent decidua, "may be involved in the lipid metabolism and transport in this portion of the placental barrier." Regarding this he called attention to the i:)ossibility that phosphatases in the placental barrier provide a mechanism whereby ciruclating fats are phosphoryl- ated, thereby facilitating the absorption and transmission of lipids. In this connec- tion, it is interesting to note that Green and Meyerhof (1952) have shown that these enzymes can phosphorylate some com- pounds under certain circumstances. C. ALKALINE PHO.SPH.\TASE AND THE PERIODIC ACID-SCHIFF (PAS) REACTION Moog and Wenger (1952) reported that neutral mucopolysaccharide, as demonstra- ble by the PAS reaction, occurs at sites of high alkaline phosphatase activity and they cite examples in various tissues and organs. They suggested that the mucopolysaccharide may serve as a cytoskeleton for the enzyme. They described the placental labyrinth of the mouse as an illustration of this relation- ship, stating that the trophoblastic syncy- tium is rich in both substances. However, in the similarly constructed placental Plate 15.XVIII Visceral endoderm of the rat yolk sac Figs. 15.78 and 15.79. Cytologic distribution of lipids in the visceral ondoderm. Frozen section, sudan black B. In Figure 15.78 the large clear nuclei have tiny clinlcslciol containing droplets which are truly intranuclear. The infranuclear cytoplasm is packed uitli lar^irr diop- lets which are also rich in cholesterol. At the surface of these cells note I he ddicalc sudano- philia of the brush border and also the narrow sudanophobic band immediately beneath this border. The supranuclear cytoplasm contains many unstained vacuoles (Fig. 15.78) and also clusters of tiny lipid droplets (Fig. 15.79). In Figure 15.79 the plane of section runs obliquely through the apical cytoplasm. Three large lipid clusters are shown. X 1950. Fig. 15.80. Electron micrograph of a visceral endodermal cell at 12 days of gestation. A large homogeneous cholesterol rich droplet is located in the cytoplasm near the polymorphic nucleus. Two minute droplets (d), which are also rich in cholesterol, occur within the nucleus. The apical cytoplasm contains vacuoles which are heterogeneous with respect to size and content. The limiting membrane of these vacuoles is usually incomplete. The free surface of this cell is formed of numerous microvilli. Beneath the surface, many small canaliculi with dense walls can be seen. X 7600. HISTOCHEMISTRY OF PLACENTA 941 H « ^ .> -"w**^ "^ #1 79 y I Plate 15.XVni 942 SPERM, OVA, AND PREGNANCY labyrinth of the rat, it has been ob- served that the alkaline phosphatase reac- tion predominates in the trophoblast, whereas the PAS reaction occurs mainly in the adjacent basement membrane which supports the syncytium and encloses the fetal capillaries. Thus, although the two re- actions are closely associated, they are by no means located in the same tissue ele- ments. Despite this discrepancy, there is no question but that in many tissues the thesis Moog and Wenger have stated holds true. Indeed, numerous examples which bear out their conclusion can be found in the pla- centas of various animals. Thus, in the hu- man placenta, intense reactions for alkaline phosphatase and for PAS-positive sub- stances occur in the outer zone and brush border of the syncytial trophoblast. In the cat's placental labyrinth, both occur to- gether with great intensity in the perivascu- lar sheaths intervening between the mater- nal blood vessels and the trophoblast, as well as in the columnar chorionic epithelium of the paraplacental brown border. In the rat and guinea pig the two reactions are encountered in the epithelium of the vis- ceral layer of the yolk sac placenta. Finally, in the pig's placenta, the two reactions co- incide in the distal ends of the columnar cells lining the chorionic fossae. D. RELATIONSHIP OF LIPIDS TO THE PLACENTAL BARRIER In reference to the question of lipids demonstrable in the placental barrier, Wis- locki and Bennett (1943) emphasized, and Dempsey and Wislocki (1944) offered fur- ther substantiating evidence to show that, although lipids are demonstrable histologi- cally in great abundance in the human trophoblastic syncytium, they are probably, for the most part, lipids associated with mitochondria and with the production of steroid hormones, rather than lipids in process of transmission across the placenta. Huggett and Hammond (1952) summarized the question of the manner of transmission of fat from mother to the fetus. In their opinion the chemical results do not show how the fat actually traverses the placenta ; ■'they neither prove nor disprove the pos- sibility of hydrolysis at the placental mem- brane and subsequent resynthesis in the deep syncytium." All that the results show is "that particular or labeled fatty acids, originally on the maternal side, appear later in the fetal tissues." Regarding phospholipids, Popjak and Beeckmans (1950) concluded from a study of rabbits that the placenta does riot trans- mit unhydrolyzed phospholipid molecules to the fetus. Similarly, glycerophosphate which is a phosphorus-containing degrada- tion product of lecithin does not pass un- hydrolyzed. In a previous investigation Popjak (1947) showed that fetal phospho- lipids in rats, rabbits, and guinea pigs are formed by synthesis within the fetal tissues. Popjak and Beeckman's findings offer a situation where phosphatases might serve as the dephosphorylating agents. E. FIBRINOID Earlier in this review the histologic prop- erties of the ground substance of the human trophoblastic cell columns and shell were described. Grosser (1925a) called this ground substance "fibrinoid," adopting a term which had been introduced previously to describe substances occurring in a vari- ety of pathologic lesions. Fibrinoid was de- fined originally as a somewhat refractile, homogeneous, intercellular substance with an affinity for acid dyes and with histologic resemblances to fibrin (Neumann, 1880). Recently, Altshuler and Angevine (1949, 1951) maintained that fibrinoid stains met- achromatically with toluidin blue and re- acts with the PAS reagents. From this, they concluded that fibrinoid consists of an acid mucopolysaccharide containing mucoitin sulfuric acid. On the basis of metachromatic staining, they identified placental fibrinoid in Nitabuch's membrane, the subchorial plate, and degenerating chorionic villi. Wis- locki and Dempsey (1948) described meta- chromatic staining of the stroma of degen- erating villi (Fig. 15.57) and of the ground substance of the decidua (Fig. 15.58), but they did not observe metachromasia of the ground substance of the peripheral tropho- blast. However, it is specifically in the latter that Grosser (1925a) placed the placental fibrinoid, distinguishing it from fibrin and indicating that he did not believe it is re- HISTOCHEMISTRY OF PLACENTA 943 lated to fibrinoid elsewhere in the body. Altshuler and Angevine use the term "fibri- noid" differently from Grosser and other investigators. The staining seen by them in Nitabuch's layer is probably referable to metaehromasia of the ground substance of the decidua and is most likely physiologic rather than pathologic in nature, inasmuch as similar metachromatic ground substance occurs in the endometrium during the nor- mal menstrual cycle (Bensley, 1934; Wis- locki and Dempsey, 1948). Despite the fact that fibrin, placental fibrinoid, and collagen seem to be identical in reference to staining with acid and basic dyes and isoelectric points (Singer and Wis- locki, 1948; Sokoloff, Mund and Kantor, 1951) both fibrin and collagen are distin- guishable in a number of respects from placental fibrinoid. Moreover, although fi- brinoid of pathologic lesions may be meta- chromatic, the ground substance of the peripheral trophoblast is not and the only placental substance comparable to patho- logic fibrinoid is possibly the metachromatic ground substance of the stroma of degener- ating chorionic villi. X. Placental Permeability with Respect to Morphologic Types The evidence that the placental barrier in individual species becomes more permea- ble to some substances as pregnancy pro- gresses seems to be adequately established. Utilizing rabbits, it has been shown that permeability to antibodies (Rodolfo, 1934), phenolsulfonphthalein (Lell, Liber and Sny- der, 1932), neoarsphenamine (Snyder, 1943), and radioactive sodium (Flexner and Pohl, 1941a) increases during the course of gestation. In the rat, a similar increase in permeability to insulin (Corey, 1932) and radioactive sodium (Flexner and Pohl, 1941b) has been demonstrated, and the same is true for sodium in the guinea pig, sow, goat, and cat (Flexner and Pohl, 1941a-d; Pohl and Flexner, 1941). A pro- gressive increase in rate of transfer of heavy water has also been observed in the guinea pig and man (Gellhorn and Flexner, 1942; Hellman, Flexner, Wilde, Vosburgh and Proctor, 1948). Flexner and his associ- ates related their results to the progressive thinning of the chorio-allantoic placenta of the individual species studied with respect to the reduction of the number and width of the layers in the course of gestation. This is a natural conclusion in view of the mor- phologic observations reported in preceding passages which show that in individual species there is a diminution in both width and number of layers of the chorio-allantoic barrier in the course of gestation. For the human, this correlation is well illustrated by a series of drawings presented by Flexner, Cowie, Hellman, Wilde and Vosburgh (1948). Little comparative information exists with respect to the relative permeability of different types of placentas. What data are available concern mainly the over-all ex- change through the fetal membranes and give few, excepting inferential, clues to the exact regions and cytologic means of trans- fer. Moreover, most of the substances fol- lowed have been readily diffusible ones and proteins of various kinds which afford no histochemical means for their detection. The manifold combinations of placental structures in various animals and their cytologic complexities have already been outlined to some degree. However, the ma- jority of investigators, speculating on the comparative aspects of physiologic exchange across the placental barrier, have generally ignored all placental structures, excepting the chorio-allantois. The popularity of Grosser's morphologic scheme of the pro- gressive differentiation of the chorio-allan- toic placenta, to the almost complete ex- clusion of the consideration of all other routes of exchange, has been due doubtlessly to its relative simplicity and its adaptability to a concept, widely held until recently, that all placental transmission can be ex- plained on the basis of diffusion and filtra- tion. Moreover, the fact that little is known of the physiologic activity of the placental structures other than the chorio-allantois has also contributed to their neglect. It has been generally held that various proteins are readily transmitted by the hemochorial placentas of man and rodents, whereas their passage is slow or entirely prevented in epitheliochorial and syndes- 944 SPERM, OVA, AND PREGNANCY mochorial types of placentas (Needham, 1931, Table 227). To give a well known ex- ample, immune bodies are not transmitted through placentas of ungulates, whereas in animals possessing hemochorial placen- tas their transfer takes place readily (Kutt- ner and Ratner, 1923; Ratner, Jackson and Gruehl, 1927; Ratner, 1943). How- ever, the hemochorial placenta of the rat and rabbit is not involved in antibody transfer; the inverted yolk sac placenta handles this function exclusively in these species. In the monkey which lacks a yolk sac placenta, Bangham, Hobbs and Terry (1958) obtained experimental evidence that the hemochorial chorio-allantoic disc handles antibody transfer. Thus, the routes of antibody transfer are different in pri- mates and rodents. Flexner and his associates have related the variations in rates of transfer of sodium per unit weight of placenta in pigs, goats, cats, rodents, and man to the four morpho- logic types of Grosser. On this basis they found that approximately 320 times as much sodium passes across a unit weight of the rat's placenta per hour as across the sow's placenta, and that between the sow and the goat the difference is approximately 16 times, whereas between the goat and the cat, both of which possess syndesmochorial placentas (see Section IV C), the difference is slight (Gellhorn, 1943; Flexner, Cowie, Hellman, Wilde and Vosburgh, 1948j. The inference is that the fewer the layers of tis- sue intervening between the circulations the greater is the rate of transfer per hour of a readily diffusible substance across a unit weight of placenta. However, these results are based on transfer per unit weight instead of transfer per unit absorbing surface of the placental barrier. Weight as a unit of meas- urement would be more acceptable if the placentas of different animals were essen- tially alike in their internal structure, so that unit weights contained approximately similar amounts of transmitting surfaces. Actually a gram of pig's placenta contains a large amount of edematous chorionic stroma and much endometrium, including uterine glands, and hence cannot be truly compared or equated with a gram of human placenta which contains relatively closely packed chorionic villi. As a consequence, any representative part of pig's placenta by weight contains little effective absorb- ing surface and a large amount of extra- neous tissue, whereas a similar amount by weight of human placenta contains a rela- tively large amount of effective absorbing surface. Thus, comparisons of the relative rates of transmission of substances across various types of placental barriers based upon units of weight are relatively unsat- isfactory. Measurements based on the relative areas of the effective transmitting surface would be more significant. However, the rates of transmission of substances ex- changed b}'' diffusion would depend not alone on the surface area of the placental membrane but more exactly on the areas of contact with the maternal and fetal capillaries variously associated with the membrane. To obtain accurate measure- ments of these complex surfaces would be well nigh impossible and no attempts to do so have been made, with the exception of the surface area of the human placenta, for which crude measurements vary from Plate 15.XIX Fig. 15.81. Election micTograph of a \'isceral endodermal cell of the rat .yolk sac at 13 days. The junction of two endodermal cells occurs at J. Only a portion of the cell at the right is shown. Cholesterol rich lipid occurs in different forms both above and below the nucleus. A portion of a large homogeneous droplet (D) is seen in close association with the basal surface of the nucleus. Above the nucleus, lipid clusters {LC) of various sizes are conspicuous. The indi\idual lipid units which comprise the cluster are irregular in shape and are bound together by membranes. Long filamentous mitochondria (M) are oriented roughly parallel to the long axis of this columnar cell. The perinuclear cytoplasm is rich in typical elements of the endoplasmic reticulum, which are often longitudinally oriented. A vacuole (V) similar to those seen in Figure 15.80 occurs near the free surface. The supeificial cytoplasm is composed of pleomorphic, anastomosing microvilli. The plasma membrane of the microvilli {MV) is continuous with that which lines the canaliculi (C). These canaliculi which lie beneath the surface have dense walls, and they anastomose in a complicated fashion. The free surface is further enlarged in Figure 15.82. X 12,800. HISTOCHEMISTRY OF PLACENTA 945 I- ..m MV ^ § % M- am Platk 15.x L\ 546 SPERM, OVA, AND PREGNANCY six square yards (Dodcls, 1924; Rech, 1924) up to twice that figure (Christoffer- sen, 1934) . At present, there would seem to be no clear demonstration of a correlation between Grosser's four morphologic types and the relative rates at which placental exchange takes place. It is, of course, quite probable that read- ily diffusible substances, such as oxygen, water, and salts, are exchanged through the thinnest parts of the placental membranes which are usually confined to the chorio- allantoic placenta. However, it is probably not true, as previously generally believed, that almost all substances capable of trans- mission, including proteins, traverse the thinnest regions. It would seem likely that all of the substances requiring regulation, including most carbohydrates, lipids, and proteins, are exchanged through thicker and more specialized regions. Cunningham (1920, 1922) was one of the first to discern, from experiments on the differential permeability of the placental barriers of cats and rabbits to potassium ferrocyanide and iron ammonium citrate, that substances which traverse the placenta are divisible into three categories. (1) Those which are diffusible and which meet with no mechanism in the placenta capable of acting on them. These pass by diffusion from mother to fetus, or in the reverse direction, without any mediation on the part of the pla- centa. (2) Those which meet with a definite preformed, regulatory mechanism. These in- clude most of the substances which are de- signed for the fetal metabolism, including iron compounds. (3) Finally, those to which the maternal or fetal surfaces of the placental barrier are impermeable. These include most formed substances, such as cells and particulate matter. From all of these considerations it would seem most likely that readily diffusible substances, such as water, oxygen, and some salts, are exchanged through the thin- ner parts of chorio-allantoic placentas, whereas more complex substances are transferred mainly through thicker regions, including the paraplacental borders and yolk sac placentas of animals which pos- sess them. If it is true that readily diffusible substances traverse principally the thinner portions of the placental barrier, then with respect to them, the sequence of the several placental types defined by Grosser would continue to be significant. Nevertheless, it is apparent, as Huggett and Hammond (1952) and others have recently empha- sized, that the exchange of each substance will have to be individually investigated with reference to its mode of transfer and the factors affecting it, before a clear pic- ture of placental physiology can be drawn. In view of the results of Brambell and his associates which indicate that anti- bodies are transmitted through the yolk sac placenta of rodents rather than through the thin placental membrane of the allan- toic placenta, one wonders what prevents antibodies and some other proteins from traversing the syndesmochorial and epithe- liochorial placentas of ungulates. Is it so much that the placental barrier in ungu- lates is too thick to permit their passage, or is it perhaps mainly that the barrier lacks the particular provisions which in the ro- dent's yolk sac facilitate their passage? The human placenta is interesting in that it i)rovides only one general avenue for the transfer of substances from mother to fetus. The chorionic villi transmit both readily diffusible substances and those requiring chemical mediation of various kinds for their transfer. This raises the question as to whether, here, all substances follow the same morphologic route through the pla- cental membrane. This cannot be answered, except to suggest that possibly they do not, because of slight histologic differences be- tween the chorionic villi in various seg- ments of the villous tree and of possible differences in the relationships of the seg- ments to the maternal circulation. Simi- larly, the arrangement of the fetal blood vessels and the mode of circulation of the blood within them might also result in differ- ences in permeability and functional activity in different regions of the villous trees. These are questions which should be investigated further. It seems reasonable to anticipate that future biochemical investigations will define transport mechanisms in the placenta, as they have in the kidney tubules and small intestine. Another jioint of interest concerning the HISTOCHEMISTRY OF PLACENTA m: placental barrier is the fact that the troph- oblast of the human placenta forms a syncytial sheet completely devoid of inter- cellular spaces or cement. Unlike capillar- ies, in which diffusion and filtration of water-soluble substances are believed by some to occur solely or mainly through the intercellular spaces, this continuous sheet of cytoplasm affords the only possible route of placental transfer. In the sow, to take another example, it is interesting that the thinnest epithelium covering the chorionic rugae is syncytial in character; it is par- ticularly through these rugae that the transmission of gases, water, and salts is believed to occur. The relative inability of leukocytes to traverse the placental barrier may possibly be related to the absence of intercellular spaces. In contrast to many chorio-allantoic placentas, yolk sac placen- tas are composed of discrete epithelial cells with well defined intercellular spaces which appear with great prominence under the electron microscope (Dempsey, 1953). XI. Suiiiinarizing Reflections on Comparative Placentation and Placental Permeability The chorion, chorio-allantois, and yolk sac of mammals become variously apposed to the uterine mucosa to give rise to the "pla- cental barrier" which mediates the physio- logic exchange between the mother and the fetus. The chorio-allantoic placenta of eu- therian mammals undergoes changes in the course of gestation. This aging process is characterized by a gradual diminution in width and cytologic simplification of the var- ious layers of the placental barrier, and by a progressive elimination of one or more of the maternal layers in most groups of mam- mals. This gradual diminution in width of the chorio-allantoic placental barrier is be- lieved to account for the fact that placental transmission of some readily diffusible sub- stances, such as oxygen, water, and various salts, increases as gestation proceeds. In those groups of mammals possessing an inverted yolk sac placenta, an elimina- tion of several fetal, instead of maternal, layers occurs in the course of gestation. The principal remaining layer, the visceral endo- dermal epithelium, undergoes some degree of cytologic aging but does not diminish in width. Some mammals possess still other structures which mediate exchange between the mother and fetus. The principal of these, the various central and paraplacental hematomas of carnivores, persist through- out gestation, without apparent cytologic changes or any reduction in width or num- ber of layers. In mammals possessing only a yolk sac placenta, such as some marsupials, or solely a chorio-allantoic placenta, such as man, physiologic exchange must be mediated en- tirely through one type of placenta. In many groups of mammals both types of placentas develop and exist concurrently for varying periods of time, in which event pla- cental exchange seems to be divided between them. However, the respective functional roles of each of these very different pla- cental structures in the transmission of var- ious substances has not been extensively investigated. Until recently it was generally assumed that nearly all substances which traverse the placenta do so by diffusion through the thinnest parts of the chorio-allantoic pla- centa. However, it now seems more likely that most substances are regulated in their passage through the placental barrier and that possibly only some readily diffusible substances, such as oxygen, water, and some salts whose rates of placental exchange in- crease during the course of gestation, are transmitted mainly through the thinner parts of the chorio-allantoic placenta. Pro- teins, lipids, and carbohydrates which seem to be regulated in their passage are prob- ably transmitted in many different ways and are acted on variously by the enzymes and complex organelles present in the cyto- plasm of the cells of the barrier. Recent experimental observations indicate that in the visceral endoderm of the rodent yolk sac the nucleus may be involved in some transfers. In man and monkeys, which pos- sess only a chorio-allantoic placenta, this regulation must take place solely in the trophoblast, but in other groups of animals which possess, in addition, either a yolk sac placenta or placental hematomas, transfer of some substances evidently occurs through the latter structures. Recent investigations 948 SPERM, OVA, AND PREGNANCY indicate that in the rabbit and rat some proteins and dyes, administered experimen- tally, are transferred by way of the yolk sac. In those marsupials which possess only a yolk sac placenta nothing is known about the mode of physiologic exchange, except that it is sufficient to support fetal develop- ment. Grosser arranged the chorio-allantoic pla- centas of eutherian mammals in a phylo- genic series comprising four placental types dependent on a progressive decrease in the number of layers intervening between the maternal and fetal circulations. According to his theory, the most primitive placental barrier consists of six layers whereas the most highly developed barriers have been reduced to three layers. Although Grosser recognized yolk sac placentas and hema- tomas as supplemental means of transfer of some nutritive materials from mother to fetus, only his chorio-allantoic placental types have been generally adopted to ex- plain the passage of nearly all substances from mother to fetus. By the removal of a succession of three more or less functionally equivalent, maternal layers in a phylogenic sequence, the placental barrier has been en- visioned as becoming progressively nar- rower, or thinner, and increasingly more permeable to the passage by diffusion of an increasingly larger number of substances. This scheme of the phylogenic simplifica- tion of the placental barrier seemed to be repeated in an ontogenic sense by the ob- served thinning of the placental barrier in the course of gestation in individual species. According to this belief, substances of larger molecular size, particularly proteins, are the last which are enabled to diffuse across the placental barrier in the postulated phylo- genic and ontogenic series of stages. With the growing recognition that the passage of a great many substances across the placental barrier is chemically regulated (Huggett and Hammond, 1952) and that the cytoplasm of the barrier contains a host of enzymes and numerous organelles, it seems increasingly evident that Grosser's doctrine must be reevaluated and modified. It has become necessary to consider each substance individually, with respect to its place of passage and the fatcors regulating its exchange. Thus, the regional cytologic and histochemical organization of the bar- rier, of which Grosser's doctrine takes little cognizance, should assume much greater importance; and, in addition to the relative thickness and width of the barrier, the rela- tive extent and nature of its absorbing sur- faces will have to be more carefully ex- plored. The electron microscope should be of considerable value in ascertaining the struc- ture of the absorbing surfaces and interior of the barrier. Studies of the human pla- centa in early months of gestation, recently begun with the electron microscope, have revealed a multitude of microvilli on the surface of the trophoblast and vesicles in the syncytium. This suggests that absorption from the intervillous space takes place to a considerable extent by the process of pinocytosis. Indications of absorption by pinocytosis have also been reported by means of electron microscopy in the epi- thelium of the yolk sac of the guinea pig and rat. A major role in placental physio- logic exchange may eventually have to be assigned to pinocytosis. It seems ap- parent that pinocytosis is a process which predominates in the first part of human gestation but subsequently diminishes as the placental barrier becomes thinner and more simplified. It seems probable that as Plate 15. XX Electronmicrographs oj the rat visceral endoderm at 13 days of gestation Fig. 15.82. Free surface of the visceral endoderm. The junction of two cells occurs at J which is marked also by the dense cytoplasmic condensation of the terminal bar. Note that the microvilli (mv) are penetrated by tiny tubules. At point x, the plasma membrane of the microvilli is continuous with the denser membrane which forms the walls of the canaliculi. Near the center of the photograph, the canaliculi are in the form of a figure 8, showing the anastomosis of the system of superficial canals. X 28,000. Fig. 15.83. Basal surface of the visceral endoderm. The close association of a large lipid droplet with the concave basal surface of the nucleus is illustrated here. Two typical clusters of Golgi membranes (g) are located near the basal region of the nucleus. Mitochondria and elements of the endoplasmic reticulum are abundant. X 13.200. HISTOCHEMISTRY OF PLACENTA 949 I — mv -I-,- ' - ^ 82 Plate 15. XX 950 SPERM, OVA, AND PREGNANCY the rate of transfer of simple substances by diffusion across the barrier increases, the rate of absorption of more complex sub- stances by pinocytosis declines. In rodents' placentas, on the other hand, although the trophoblastic cells of the placental laby- rinth become thinner and their cytoplasm more simplified, pinocytosis seems to persist throughout gestation in the epithelium of the yolk sac thereby affording a continuous means for the absorption of substances of larger molecular size. Comparative studies of the placentas of animals with the elec- tron microscope are just beginning, so that, although it seems that pinocytosis might play an important role in placental phys- iologic exchange, no broader generalizations regarding its significance can at present be offered. 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W., and Dempsey, E. W. 1946. The histochemistry of the rodent's pla- centa. Am. J. Anat., 78, 281-345. WisLocKi, (i. B.. Dempsey, E. W., and Fawcett, D. W. 1948. Some functional activities of the placental tropliol^last. Obst. & Gvnec. Surv., 3, 604-614. WisLOCKi, G. B., Weiss, L. P., Burgos, M. H., and Ellis, R. A. 1957. The cytology, histochem- istry and electron microscopy of the granular cells of the metrial gland of the gravid rat. J. Anat.. 91, 130-140. Yamaua, E. 1955. The fine structure of the renal glomerulus of the mouse. J. Biophvs. Biochem. CytoL, 1, 551-566. Zancla, L. 1912. Contributo alio studio isto- chiniico del ferro i)lacentare. Folia Gynec, 6, 297. 16 GESTATION M. X. Zarrow, Ph.D. PROFESSOR OF ZOOLOGY, PURDUE UNIVERSITY, LAFAYETTE, INDIANA I. Introduction 958 II. Length of Gestation 958 III. Normal Reproductive Potential... 959 IV. Environment 962 A. Crowding 962 B. Body Temperature and Hypoxia. . . 962 V. Maternal Hormone Levels during Gestation 963 A. Estrogens 964 B. Gestagens 964 C. Sources of Gestagens 972 D. Relaxin 973 E. Sources of Relaxin 976 F. Adrenal Cortex 976 L Hydrocortisone 976 2. Aldosterone 979 G. Thyroid Gland 979 H. Growth Hormone 980 I. Prolactin 981 J. Placental Gonadotrophins 982 1. Human chorionic gonadotrophin (HCG) 983 2. Equine gonadotrophin (PMS). . . 985 VI. Pregnancy Tests 986 VII. Water and Electrolyte Balance.. 988 VIII. Plasma Proteins 993 IX. Renal Function 994 X. Enzymes 995 A. Histaminase 995 B. Carbonic Anhydrase 996 XI. Factors in the Maintenance of Ges- tation 997 A. Thyroid Gland 997 B. Adrenal Cortex 1000 C. Pancreas 1003 D. Ovarv: Progesterone, Estradiol, and Relaxin 1003 E. Pituitarv Gland 1005 F. Placenta 1006 G. Pelvic Adaptation 1008 H. Dilation of the Uterine Cervix .... 1011 XII. Uterine Myometrial Activity 1013 XIII. Parturition 1015 A. Progesterone 1015 B. Oxvtocin 1015 C. Relaxin 1016 D. Labor 1017 ^ Aided by grants from the Purdue Research Foundation. XIV. Conclusion. XV. References 1018 1018 I. Introduction Reproduction in the animal kingdom is accomiilished by a wide variety of methods, from simple budding and binary fission in the invertebrates to gestation in the mam- mal and the development of a new organ, the placenta. The development of vivipar- ity, which covers millions of years of evo- lution, brought with it many new prob- lems, and with each problem new factors came into play so that reproduction in the mammal is a highly co-ordinated series of events — a co-ordination that is both tem- poral and spatial, that requires certain events to occur in a proper sequential ar- rangement, and, above all, is dependent on the endocrine system. It is obvious that the maintance of ges- tation in the mammal is a complex phenom- enon. It involves directly or indirectly a major portion of the endocrine system with concomitant changes in the general meta- bolic state of the organism and in many of the enzymes present in the blood and the tissues. Finally, a new endocrine organ, the placenta, comes into being also to play its specific role in gestation. II. Length of Gestation The duration of gestation is highly vari- able and depends primarily on the species involved. In general, the longer the gesta- tion, the more self-sufficient and mature are the young at the time of birth. It is obvious, however, that this is not true under all conditions. The young of the guinea pig are highly advanced at birth, although the length of gestation is approxi- 958 GESTATION 959 iiiatcly 69 days, whereas in the iH-iniate, with a gestation period of 6 to 9 months, depending on the species, the young are helpless at birth. A partial summary of gestation length and tlie litter size of a representative but not inclusive list of mammals is presented in Table 16.1. The length of gestation appears to be rather constant for each species or at least within a strain. Even where the phenom- enon of delayed implantation is a natural event the length of pregnancy remains con- stant, although the quiescent period may vary. It is, however, possible for delayed implantation to occur in a species where this does not ordinarily appear, which could lead to a marked increase in the duration of gestation. Thus, an increase of 1 to 7 days has been reported in the rat or mouse if mated while lactating (Pincus, 1936). Recently, Bruce and East (1956) examined the effect of concurrent lactation on the number and viability of the young and the length of pregnancy in the mouse. They observed a wide variation in the delay of implantation for every size of litter studied, but, in general, the delay tended to be longer for the larger suckling litters. Smith, Albert and Wilson (1951) re- ported a 310-day pregnancy period in a human female. Gestation was confirmed early by pregnancy tests and a normal child with respect to body weight was born at 30 days after the expected parturition. Such phenomena seem to be rare in pri- mates and no explanation is possible at the present time. Although the lengths of the gestation periods are quite constant for a given strain, the length of gestation is inversely related to the litter size. This has been dem- onstrated in both a genetically pure strain and a heterogeneous strain of quinea pigs (Goy, Hoar and Young, 1957). An average gestation length of 69.9 days was obtained in the pure strain of guinea pigs with a litter size of 1, as compared with a gesta- tion length of 65.3 days for a litter size of 6. A sex difference has also been postulated in length of gestation. Although the dif- ference is very small, e.g., only a fraction ■of a day in man, the difference is signifi- cant. Recently, McKeown and MacMahon (1956) concluded that pregnancy is longer in the cow, horse, and possibly the sheep and camel when the offspring are male, and longer in man and possibly the guinea pig when the offspring are female. III. Normal Reproductive Potential The reproductive potential in the pri- mate is limited to the period from the menarche to the menopause. Hence, it is much shorter than the total life span of the female. Fertility studies as a function of age have l)ecn rather sparse for different species although it is generally agreed that fertility declines with age. A reproductive period considerably shorter than the life span of the animal has also been reported in certain strains of mice (Thung, Boot and Miihlbock, 1956) and in the rat (Ingram, Mandl and Zuckerman, 1958). Although Slonaker (1928) showed that the rat may remain fertile for 22 months, it is known that the average size of suc- cessive litters in both rats and mice first rises to a maximum and then falls (King, 1924; Ingram, Mandl and Zuckerman, 1958). The latter have shown both a de- crease in the number of fertile female rats with each successive litter and hence with age (Fig. 16.1) and a decline in the number of young with each successive litter (Fig. 16.2). These results indicate that the reproduc- tive potential of both the colony of rats and of the individual rat declines with age. Many factors may obviously be at work here, such as nutrition, size, and part played by the male. Ingram, Mandl and Zuckerman ( 1958 ) feel that none of the above factors is responsible for the decline in litter size and offer the following four possibilities: (1) the number of follicles which mature and ovulate declines with age, (2) the capacity of the ovum to be fertilized declines with age, (3) the number of fertilized ova that develop to term de- clines with age, and (4) the total number of available oocytes declines with age. Evidence from the pig (Perry, 1954) and rat indicates that factors 2 and 3 are certainly involved. Inasmuch as the num- ber of corpora lutea rises with age in the pig, the decline in size of litters can be TABLE 16.1 Length uf gestation and litter size in various species of mammals Species No. of Young Length of Gestation Reference*^ Common name Scientific name Armadillo Das y pus novemcinctus, L. 4 150 days (1), (2) Baboon Papio hamadrys, L. 1 183 days (1) Baboon, chacma Papio porcarius, B. 1 7 months (1), (2) Bat, common European Vespertilio miirinus, L. 1 50 days (1) Bat, common pipistrello Pipistrellus pipistrellus, S. 1 44 days (3) Bear, black Euarctos americanus, P. 1-4 208 days (1), (2> Bush baby Galago senegalensis, G. 1-2 4 months (1) Camel, batrachian Cameliis bactrianus, L. 1 370-440 da.ys (1) Capuchin Cebus apella, L. 160-170 davs (1) Cat Felis catus, L. 3.8 (1-8) 56-65 days^ (4) Cat, domestic Felis catus, L. 4 63 days (1) Chimpanzee Pan satyrus, L. 1 236.5 ± 13.3'' davs (1) Chimpanzee Pan satyrus, L. 1-2 226.8 ± 13.3" davs (17) Chinchilla Chinchilla laniger, B. 1-4 105-111 days (1), (2> Chipmunk Tanarios strialus, L. 3-5 31 davs (1) Cow Bos taurus, L. 1-2 277-290 days (1) Cow (Jersey) Bos taurus, L. 1-2 282.7 ± 5.4" days (5) Cow (Holstein-Friesian) Bos taurus, L. 1-2 278-280 days-^ (6) Coyote Canis latrans, S. 5.7 60-(J5 days (1) Deer, Virginia Odocoileus Virginian us, B. 2 7 months (1) Dog Canis familiaris, L. Multiple 58-63 days (1) Dog Canis familiaris, L. Multiple 61 davs (4) Echidna Echidna acn.leata 1 16-28 days (1) Elephant 1 20 months (8) Elephant, Indian Elephas maxim us, L. 1 607-641 days (1) Ferret Mustela furo, L. 5-13 42 days (1) Ferret Mustela furo, L. 5-13 42 days (4) Fox, red Vulpes fulfa, D. 1-8 52 days (1) Goat Capra hircxis, L. 1-2 21 weeks (7) Goat, domestic Capra hircus, L. 1-3 146-151 days (1) Gopher, pocket Geomys bursarius, S. 1-6 (1) Ground squirrel, thirteen- Citellus tridecimlineatus, N. 5-13 28 days (1> lined Guinea pig Cavia porcellus, L. 1-6 67-68 davs (1) Guinea pig Cavia porcellus, L. 65.3-70.5 days (7) Hamster, golden Cricetus auratus, H. 16-19 days (1) Hamster, golden Cricetus auratus, H. 5 10 days (9) Hare, snowshow Lepus americanus, E. 1-7 38 days (1) Hedgehog, European Erinoceus europaeus, L. 5 34-49 davs (1) Hippopotamus Hippopotamus amphibius, L. 1 237 ± 12 days (1) Horse Equus cabaUus, L. 1 330 davs (1) Hyena, spotted Crocuta crocuta, E. 1-2 110 days (1) Kangaroo rat Bettongia cuniculus, 0. 1 6 weeks (1) Lemur Lemur macaco, L. 1-2 146 days (1) Lion Felis leo, L. 2-6 105-113 davs (1) Macaque Macaca mulatta, Z. 1 163.7 ± 8 days (1), (10) Macaque 1 24 weeks (8) Man Homo sapiens, L. 1 280 ± 9.2 days (1) Marmoset Hopale jacchus, L. 1-3 140-50 davs (1) Marten, pine Martes americana, T. 3-5 220-265 days (1^ Mink Mustela vison, S. 4-10 39-76 days (1) Mink Mustela vison 4 51 days'* (40-75) (11), (12) Mole, common American Scalupus aquaticus, L. 2-5 6 weeks (1) Mouse Mus musculus, L. 19-20 days (4) Mouse, field Microtus pennsylvanicus , 0. 6-8 (1) Mouse, house Mus musculus, L. 4-5-7.5 19 days (1) Mouse, wild Peromyscus maniculatus 23 davs (13) Mouse, wood Peromyscus leucopus, L. 3-7 23 days (1) 960 GESTATION 961 TABLE m.l— Continued species No. of Young Length of Gestation Reference" Common name Scientific name Opossum, Australicaii Trichosunis vulpecula, K. 1 16 days Opossum, Virginia Didclphis rir(/itiiana, K. 8-12 12.5-13 days (1), (2), (8) Otter Lutra canadensis, S. 1-4 60 days Pig, domestic Sus scrofa, L. 4-12 112-115 days Pig, wild Sus cristatus, W. 4-6 4 months Porcupine ErclJiizan (lorsoliun, L. 1 16 weeks Puma Frlis ,„nr„ln,-, T. 1-4 90-93 days Rabbit, domestic Crycutaluyiis c/uiicidus. L. Multiple 30-32 days Rabbit, domestic Crycotalagus cnnicuhis, L. Multiple 31 (28-36 davs) Racoon Procyon lotor, L. 1-0 63 days Rat Rattus rattus, L. (J. 1-9.2'- 22 days Rat Rattus rattus, L. 21-23 days Reindeer Rangifer tarandus, L. 1-2 7-8 months Rhinoceros 1 18 months Rhinoceros, black Rhinocerus bicornuis, L. 1 530-550 days Seal, northern fur CaUorhinns ursinus, L. 1 Almost 1 year Sheep, bighorn Ovis canadensis, L. 1 180 davs Sheep, domestic Ovis aries, L. 1-2 144-152 davs-^ Shrew, common Sorex aranus, L. 6.45 13-19 davs Shrew, short-tailed Blarina brevicauda, S. 3-7 17-20 days Skunk Mephitis mephitis, S. 4-7 62 days Squirrel, red Sciurus hudsonicus, E. 3-() 40 days Stoat Mustela musleta 6 weeks'* (14) Tasmanian devil Sarcophilus ursinus, K. 4 31 days Vole Microtus agrestis 20-22 days (Ifi) Weasel Mustela nivalis 6-7 50 weeks'* (includes) lactation) (15) Whale, sperm Fhyseter catadon, L. 1 1 year (1) Wolf, timber Canis tycoon, S. 1-12 63 davs (1) Woodchuck Marmot a nionox, L. 4.07 28 days (1) Zebu Bos indicus, L. 285 davs (1) '- References. (1) Asdell, 1946. (2) Kenneth, 1947. (3) Deanesly and Warwick, 1939. (4) Farris, 1950. (5) Rollins, Laben and Mead, 1956. (6) Norton, 1956. (7) Goy, Hoar and Young, 1957. (8) Arey, 1946. (9) Selle, 1945. (10) Hartman, 1932. (11) Pearson and Enders, 1944. (12) Enders, 1952. (13) Svihia, 1932. (14) Deanesly, 1943. (15) Deanesly, 1944. (16) Chitty, 1957. (17) Peacock and Rogers, 1959. *" Standard deviation. " Depends on the strain. '* Excluding the quiescent period. 3l no. of litter -1^ Fig. 16.1. Decline in litter size with birth of successive litters. • mean of 35 litters; EI mean of 14 litters; A mean of 4 litters. (From D. L. Ingram, A. M. Mandl and S. Zuckcinian. J. Endocrinol., 17, 280, 1958.) 1 2 3 4 5 6 7 8 9 10 11 Serial no. of litter Fig. 16.2. Decline in number of fertile female rats with birth of successive litters. (From D. L, Ingram, A. M. Mandl and S. Zuckerman, J. En- docrinol., 17, 280, 1958.) 962 SPERM, OVA, AND PREGNANCY attributed only to failure of fertilization or to fetal death. A similar increased in- cidence of embryonic death or failure of fertilization has been described in the aged rat, although it should be noted that the number of corpora lutea present at parturi- tion in the rat is not an index of the number of ova released before conception because the corpora lutea in old age may persist for longer periods. Finally, a marked reduction in the number of oocytes with age has been shown in the rat, a drop from approximately 20,000 oocytes at age day 1 to approximately 2000 oocytes at age 250 to 300 days (Mandl and Zuckerman, 1951). In addition, Ingram (1958) showed that the litter size in rats declined markedly with the reduction in number of oocytes follow- ing graded doses of x-ray. This experiment tends to confirm the concept that the de- cline in fertility with age is due to a de- cline in the number of oocytes. IV. Environment A. CROWDING The factors concerned in the growth and survival of a population under natural con- ditions may obviously involve reproduction. Variations in population level have been of great interest to the mammalogist and student of wildlife for many years. De- creased productivity in mammalian popu- lations associated with increased density of the population has been considered a controlling factor in the regulation of wild- life population. Experimental analysis by Christian and Lemunyan (1958) indicated that a number of factors are involved. These authors ex- l)osed mice to excessive crowding and noted the number of implantation sites, embryos. and births. All the females became preg- nant but only 3 of the 10 bore litters during the period of crowding or later (Table 16.2). It would seem that the crowded females were unable to maintain normal pregnancies and that the environmental situation interfered with the endocrine bal- ance and resulted in a marked pregnancy wastage. The data reveal that in addition to a postimplantation loss, there was also a prc-implantation loss because the number of implantation scars in the uterus was markedly less in the crowded females. This could be due to a failure of the fertilized egg to implant or to a decrease in the number of available ova. Although direct data are not available, it is of interest to speculate as to whether this effect of crowd- ing is mediated by way of the pituitary- adrenocortical axis. B. BODY TEMPERATURE AND HYPOXIA Disturbances in reproduction have been noted in mammals exposed to high tem- peratures or chronic hypoxia. It has been known for some time that women moving to the tropics show a high rate of abortion (Castellani and Chalmers, 1919). Recently Macfarlane, Pennyamt and Thrifte (1957) reported a 30 per cent reduction in human conception rates in the summer as com- pared with the winter in Australia. The same authors reported a marked degree of fetal resorption in rats exposed to a tem- perature of 35°C. This confirmed the pre- vious observations of Sundstroem (1927) in rats and Oegle (1934) in mice where ex- posure to 31 °C. caused a reduction in litter size. In a similar manner, disturbances have also been reported in reproduction follow- ing exposure to decreased oxygen tension. TABLE 16.2 Productivity of mice crowded 10 pairs to a cage compared to their 10 control pairs Note that all of the females became pregnant but that the crowded females exhibited a marked intrauterine loss of young, reduction of implantation sites, reduction of litter size, and significant de- lay until the birth of first litters compared to the controls. Crowding produced a 75 per cent loss in the number of young born. (From J. J. Christian and C. D. Lemunyan, Endocrinology, 63 , 517, 1958.) No. of Pairs No. of Litters Born Mean No. Days to Litter Birth Mean No. Progeny per Litter ± S.E. No. Females with Placen- tal Scars Mean No. Scars per Female Crowded females Isolated females 10 10 3 10 40 ± 1.0 26 ± 1.5 7.67 ± 0.33 9.00 ± 0.75 10 10 6.90 ± 1.37 11.00 ± 0.47 GESTATOON 963 Monge (1942) reported a lack of repro- duction in the Spaniards for more than 50 years after residence in certain areas of Bolivia (14,000 feet or more above sea level). Many malformations have also been observed in the progency of mice, rats, and rabbits exposed to low atmosperic pres- sures. Exposure of mice on the 10th day of pregnancy for 2 hours to a 6 per cent oxygen-94 per cent nitrogen mixture at normal atmospheric pressure gave mal- formations in the young comparable with those found after exposure to a low at- mospheric pressure which was equivalent to the above with respect to the number of oxygen molecules per unit of air (Curley and Ingalls, 1957). Although these mal- formations involved the ribs and vertebrae, it is conceivable that more extensive mal- formations could result in death of the fetuses leading to resorption or abortion of the young. Vidovic (1952, 1956) made a very com- plete study of the effect of lowered body temperature on gestation in the rat using the technique of Giaja (1940) in which an hypoxic hypothermia is induced by cool- ing under reduced oxygen tension. The animal is placed in a sealed container which is surrounded by ice for a period of approximately 10 hours. Under these con- ditions a hypothermia of 3 to 4 hours' duration and body temperature of 14 to 18°C. can be induced. No deleterious ef- fects were noted in the rats cooled on or before the 13th day of pregnancy. However, the induction of hypothermia after the 13th day resulted in a marked increase in the disturbance of gestation. These dis- turbances consisted of an increased number of resorbed fetuses, an increased ratio be- tween stillborn and live young in that more stillborn occurred, a decreased body weight in the progency, and a delay in the onset of parturition. In addition, a marked increase in sensitivity to hypothermia was noted in the animals as pregnancy pro- gressed. Courrier and Marois (1953) cooled pregnant rats by exposure to a temperature of 0°C. for 2 hours. Thereafter the rats were placed in cold water for 3 to 4 hours and a body temperature of 15.5 to 17°C. was obtained. Exposure to the above treat- ment on the 7th to the 11th day of preg- nancy had no effect on the fetuses or the pregnancy. Treatment on the 12th to the 18th day of pregnancy led to resorption and abortion of the young. The authors concluded that the degree of deleterious effects following exposure to cold varied with the length of the pregnancy. Recently, Fernandez-Cano (1958a) ex- posed pregnant rats for 5 hours on 2 con- secutive days to one of the following three experimental procedures: (1) an environ- mental temperature of 103°F. that led to an increase in body temperature to 104°F.; (2) an environmental temperature of 26°F. that led to a decrease in body temperature to 94°F.; and (3) barometric pressure of 410 mm. Hg. Both temperature changes led to a marked decrease in the number of implantations and, to a lesser extent, to some embryonic degeneration after implan- tation (Table 16.3). Although some dele- terious action was seen before implantation, hyi^oxia was more harmful after implan- tation. Whereas these results are not in full agreement with Vidovic 's report, it must be remembered that Vidovic used a combination of cold and hypoxia to induce the effects that he observed. Adrenalectomy failed to increase embryonic degenerations in rats treated as above (Fernandez-Cano, 1958b). Inasmuch as adrenocorticotrophic hormone (ACTH) causes degeneration of the embryos in intact pregnant rats and not in adrenalectomized rats (Velardo, 1957 1 , it is apparent that these results are explain- able on the basis of an increased release of adrenal corticoids due to the stressor and/or a direct action of the corticoids on the development of the embryo. V. Maternal Hormone Levels during Gestation Proof that certain hormones are neces- sary for a successful pregnancy came from evidence involving ablation of the source of the hormone and replacement therapy. This was followed by quantitative analyses of the concentration of the hormone in the blood and urine throughout gestation. The increasing concentrations of the hormones as pregnancy advances can be used as a second argument for the role of hormones in the development and maintenance of pregnancy (Zarrow, 1957). Changes of this 964 SPERM, OVA, AND PREGNANCY TABLE 16.3 The effect of increase or decrease of body temperature and hypoxia on the pregnancy of the rat (From L. Fernandez-Cano, Fertil. & Steril., 9, 45, 1958.) Group Control High body temperature High body temperature High body temperature High body temperature Low body temperature . Low body temperature . Low body temperature . Low body temperature . Hypoxia Hypoxia Hypoxia Hypoxia Days of Treatment 1-2 3-4 6-7 10-11 1-2 3-4 6-7 10-11 1-2 3-4 6-7 10-11 Total No. Corpora Lutea 166 98 117 95 89 93 98 100 91 103 108 97 94 Percentage of Degener- ation Before implan- tation 2.4 52 28 2 2 25 33 3 2.1 21.3 25.9 0 2.1 After implan- tation 0 12 3 14 10 5 4 13 12.1 2.9 3.7 25.7 65.9 Total degener- 2.4 64 31 16 12 30 37 16 14.2 24.2 29.6 25.7 68.0 Means of Degenera- tion for Each Rat 0.2 8.3 4.6 1.9 1.3 3.5 4.5 2.0 1.6 3.1 3.8 3.1 8.0 Standard Error 0.11 1.6 2.6 0.5 1.8 1.0 1.4 0.2 0.5 0.4 0.3 1.0 1.4 Percent- age against Control >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 >0.01 kind have been observed for such steroids as the estrogens, gestagens, and the 17- a-hydroxycorticoids. In addition, certain nonsteroidal hormones such as the gonado- trophins human chorionic gonadotrophin (HCG) and pregnant mare's serum (PMS) and the polypeptide, relaxin, increase dur- ing gestation. Some evidence for a possible involvement of thyroxine, prolactin, and oxytocin will be included. The maximal concentration of these hormones in the blood of the female during pregnancy is given in Table 16.4. A. ESTROGENS The fact that large amounts of estrogen are excreted in the urine of pregnant women and mares has been known for a long time. Additional data (reviewed by Newton, 1939) indicate that this phenomenon occurs in all species studied, such as the chimpan- zee, the macaque, the cow, the pig, and the rat. In general, an increasing amount of estrogen is excreted as pregnancy pro- gresses. The estrogenic material in the urine of the pregnant woman appears mostly in the form of estriol with lesser amounts of estrone and estradiol (Fig. 16.3) . The estriol concentration increases only slightly in the urine of women for the first 100 to 125 days of pregnancy, but there- after it increases very rapidly until parturi- tion. Newton (1939) discussed the possible role of estrogen in pregnancy in great de- tail. He first asked whether the increased urinary concentration of estrogen indicates that this hormone is acting to a lesser de- gree as pregnancy advances or to a greater degree. He marshaled his facts pro and con and came to the conclusion that there is an increased production of estrogen through- out pregnancy and hence an increased ac- tivity of the hormone. In his analysis of the action of estrogen, five possibilities were suggested. (1.) Estrogen is involved in the growth of the uterus in pregnancy. (2) Estrogen is involved in the increased uterine contractility and sensitivity to oxytocin necessary for parturition. (3) Es- trogen is concerned with the continued se- cretion of progesterone by way of the pitu- itary glancl or acting directly on the corpus luteum. (4) Estrogen synergizes with pro- gesterone. (5) Estrogen stimulates mam- mary gland growth. A 6th possibility is that estrogen reverses the progesterone block (Csapo, 1956a). Several of these pos- sibilities will be considered later in con- junction with progesterone, the mainte- nance of pregnancy, and jiarturition. B. GESTAGENS The significance of the role of proges- terone during pregnancy stemmed from the historic work of Fraenkel who proved the validity of Gustav Born's suggestion that GESTATION 965 TABLE 16.4 Maximal hormone levels in the blood during pregnancy Hormone Species Type of Assay Hormone Amt./ml. Plasma Reference Estriol Man Chem. 0.0914 Mg. Aitkin and Preedy, 1957 Estriol Man Chem. 0.066 Mg • Loraine, 1957 PJstrone Man Chem. 0.0647 Mg. Aitkin and Preedy, 1957 E.strone Man Chem. 0.0305 Mg. Loraine, 1957 ]''stnuli()l Man Chem. 0.0144 Mg. Aitkin and Preedy, 1957 K.-^tradiol Man Chem. 0.0105 Mg- Loraine, 1957 ( lestajfen Rabbit Biol. 10 Mg." Zarrow and Neher, 1955 Cicstugen Mouse Biol. SMg." Forbes and Hooker, 1957 (Jostagen Ewe Biol. 12 Mg." Neher and Zarrow, 1954 Progesterone .... Ewe Chem. 0.0033 Mg. Short, 1957 Progesterone .... Ewe (ovar- ian vein Chem. 2Mg. Edgar and Ronaldson, 1958 Progesterone .... Cow Chem. 0.0086 Mg. Short, 1958b Gestagen Man Biol. 2Mg." Forbes, 1951 Gestagen Man Biol. 25Mg-" Fujii, Hoshino, Aoki and Yao, 1956 Progesterone .... Man Chem. 0.239 Mg. Oertel, Weiss and Eik-Nes, 1959 Prog(^stei'one .... Man Chem. 0.142 Mg. Zander and Simmer, 1954 Progesterone . . . . Sow Chem. 0.0034 Mg. Short, 1957 Progesterone . . . . Goat Chem. 0.0071 Mg. Short, 1957 Rela.\in Guinea pig Biol. 0.5G.P.U.'' Zarrow, 1947a Rela.xin Rabbit Biol. 10 G.P.U." Marder and Money, 1944 Relu.xin Man Biol. 2 G.P.U." Zarrow, Holmstrom and Salhanick, 1955 Relaxin Sow Biol. 2G.P.U.'' Hisaw and Zarrow, 1951 Rela.xin Cow Biol. 4 G.P.U.'' Wada and Yuhara, 1955 Hydrocortisone. . Man Chem. 0.22 Mg. Gemzell, 1953 Thyroxine Man Chem. 0.83 Mg.^ Peters, Man and Heinemann, 1948 STH Rat Man Man Horse Biol. Biol. Biol. Biol. 3.5-7-^ 120 I.U. 70 I.U. 50 I.U. Conlopoulos and Simjjson, 1957 Haskiiis and Slierman. 1952 Wilson, Allien and Randall, 1949 Cole and Saunders, 1935 HOG HCG PMS " Expressed as equivalents of progesterone. '' Guinea pig units. "■ Protein-bound iodine. 'Vg. equivalent of a purified bovine growth-promoting substance. the corpus luteum is necessary for the maintenance of pregnancy. Fraenkel dem- onstrated at the turn of the century that the corpus luteum of the rabbit is essential for the maintenance of pregnancy in the rabbit (Fraenkel and Cohn, 1901; Fraen- kel, 1903; Fraenkel, 1910). These observa- tions were confirmed by Hammond and Marshall (1925.) who found that castration before the 20th day of pregnancy led to the termination of pregnancy in 24 hours. Cas- tration later in pregnancy resulted in abor- tion approximately 2 days after the opera- tion. In 1928, Corner showed that an extract of the corpus luteum could induce a proges- tational endometrium in the castrated rab- bit. This was soon followed by the demon- stration that this extract could induce implantation of the fertilized egg in the rabbit and maintain pregnancy in the cas- trated animal (Allen and Corner, 1929; 1930). Purification of the extract of the corpus luteum led to the chemical identifica- tion of the active substance by Butenandt, Westphal and Cobler in 1934, and in the following year Allen, Butenandt, Corner and Slotta (1935) agreed to the name pi^o- gesterone for this hormone of the corpus luteum. These events were soon followed by tlu' discovery that progesterone is excreted in the urine as tlie glucuronide of pregnane- diol and i)regnanolone, metabolites of pro- gesterone. Studies of urinary products of progesterone were immediately undertaken and a marked increase in urinary preg- nancdiol was observed in the human female 966 SPERM, OVA, AND PREGNANCY throughout pregnancy, especially in the second half (Fig. 16.3). The discovery by Hooker and Forbes (1947) of a new assay for progesterone sensitive to a concentration of 0.3 /^g. per ml. led to many studies on the blood levels of this hormone during gestation. Subse- quent studies revealed a lack of specificity for the assay (Zarrow, Neher, Lazo-Wasem and Salhanick, 1957; Zander, Forbes, von Miinstermann and Neher, 1958) and a dis- crepancy between the values obtained by chemical and biologic techniques. It is ob- vious that the bioassay data possess signif- icance but a final evaluation can be made only when the identity of the compound or compounds measured in the blood of the animals by the Hooker-Forbes test has been established. The concentration of gestagen in the blood of pregnant sheep (Neher and Zar- row, 1954) , women (Forbes, 1951 ; Schultz, 1953; Fujii, Hoshino, Aoki and Yao, 1956), rabbits (Zarrow and Neher, 1955), and mice (Forbes and Hooker, 1957) has been determined by the Hooker-Forbes test and 80 70- o 2 16 60 ■12 //I 50- 40- e / > .'estriol / 30- PREGNANEDIOL r / 20 ■ 10 ■ 4 / / ESTRONE -t-ESTRADIOL Fig. 16.3. Urinary excretion of estrogens and pregnanediol throughout gestation in the human being. (From E. Venning, Macy Foundation, Con- ferences on Gestation, 3, 1957.) expressed as /^g. equivalents of progester- one. The data obtained from pregnant women by the different investigators are in marked disagreement. Whereas both Forbes (1951) and Schultz (1953) failed to observe any significant rise in blood ges- tation of pregnant women throughout ges- tation, Fujii, Hoshino, Aoki and Yao (1956) obtained a conspicuous rise during this period. The data reported by Forbes (1951) indicate an extremely low level for protein-bound progesterone (0.5 /^g. per ml. plasma or less) and a maximum of 2 /xg. per ml. free progesterone (Fig. 16.4). The concentration of the hormone in the blood showed a series of irregular peaks through- out gestation and varied from less than 0.3 /xg. to 2 /xg. per ml. plasma. In general, these results were confirmed by Schultz (1953) who assayed the blood from 46 women at 6 to 17 weeks of pregnancy. Again the results failed to reveal any con- sistent change with the length of preg- nancy. Both investigators (Forbes, 1951; Schultz, 1953) were led to question the importance of progesterone during gesta- tion in the primate. Fujii, Hoshino, Aoki and Yao (1956), on the other hand, re- ported a significant increase in the level of circulating progesterone throughout ges- tation. Again these investigators used the Hooker-Forbes assay but indicated that the plasma was not treated in any way except for dilution before the assay. The results obtained by this latter group re- vealed a rise from a level of 6 /*g. pro- gesterone per ml. plasma during the luteal phase of the cycle to a high of 25 /i,g. during: the last trimester of pregnancy (Fig. 16.5). The concentration showed a steady increase from the 4th to the 24th week of pregnancy,, and a plateau from the 24th week until term. A sharp drop occurred within 12 to 24 hours after parturition with zero values noted by 72 hours postpartum. Analysis of the urine for pregnanediol showed a rather good correlation between the two curves al- though the plasma levels rose sooner than the urinary pregnanediol. The curve for the concentration of pro- gesterone in the pregnant mouse is markedly different from those reported for other species (Forbes and Hooker, 1957). Again the Hooker-Forbes assay was used GESTATION 96/ S 2 .t=^t=^t^ ■J^ri. 19 21 23 25 27 H8 Weeks since start of L.M.P. ^^ 29 31 33 35 37 39 41 Caesarian I2!I5 A.M. Fig. 16.4. Free and bound ge.stagen in the plasma of the pregnant human female. (From T. R. Forbes, Endocrinology, 49, 218, 1951.) ■?6 nfl^ iTrHi -f-i ^ Z2 18 W" /•' o ° o'^i o ^wA^ -14 I'M o 0 GO % 2 °o '- 0 o o o % 0 ° 1 c L' 16 ^0 Z4 28 32 36 40 1 90H 70 50| 30 3 4 5 Weeks of Pregnancy Days after Delivery Fig. 16.5. Concentration of gestagen in the blood plasma and pregnanediol in the uterine of the pregnant human female. Gestagen levels were determined by the Hooker- Forbes test. (From K. Fujii, K. Hoshino, I. Aoki and J. Yao, Bull. Tokyo Med. & Dent. Univ., 3, 225, 1956.) as with the other species and the values expressed as activity equivalent to pro- gesterone. The values for the bound action were consistently low and, in general, less than 1 fjig. per ml. plasma (Fig. 16.6). The concentration of the free hormone showed marked variations on the first day or so of pregnancy. Actually a variation from 1 fig. per ml. plasma to 8 fig. per ml. plasma was seen on day 0. This type of fluctuation has also been seen in the rabbit and is with- out explanation at the present time. How- ever, such marked variations disappeared by the 4th day of pregnancy and the results became much more consistent. The average curve for the concentration of gestagen in the blood of the pregnant mouse showed two peaks, one the 7th to the 9th day and a second the 15th day. The concentration increased from 2 fxg. per ml. plasma the 4th day of gestation to an average of approxi- mately 8 fig. the 7th day. This level was maintained until day 9 and fell thereafter with a second peak occurring on day 15 and an immediate drop on day 16. Thereafter the levels remained low throughout the re- mainder of pregnancy. Although it may be assumed that the initial peak in the concentration of the gestagen is due to an increased activity 968 SPERM, OVA, AND PREGNANCY E 6 M o a- 5 u u UJ a. 3 2 I lii i fV^» 15 « ? i f T 0 12 3 4 5 6 Day* afl«r finding vaginal plug Fig. 16.6. Concentration of free and bound gestagen in the plasma of the pregnant mouse. Gestagen levels were determined by the Hooker-Forbes test. (From T. R. Forbes and C. W. Hooker, Endocrinology, 61, 281, 1957.) 15 17 Ttrm on the part of the corpora lutea, an ex- planation of the second peak and the drop between the two peaks offers more diffi- culty. The latter may reflect a diminished luteal activity. This could be assumed on the grounds that the corpus luteum is the only source of gestagen during this period of gestation and that the luteal cells show cytologic signs of regressive changes, al- though the drop in serum progestogen anti- dates the cytologic changes by several days. An explanation for the second peak would probably involve increased secretory activity !)y the placenta. Progestational activity has l)een found in i:)lacental ex- tracts and progesterone has been isolated from the placentae of human beings and mares (Salhanick, Noall, Zarrow^ and Samuels, 1952; Pearlman and Cerceo, 1952; Zander, 1954; Short, 1956). Thus, the drop in serum gestagen seen on day 10 could be due to loss in the activity of the corpora lutea and the second rise as a con- tribution from the placentas. It is of interest that the low levels on days 10 to 13 and be- tween day 16 to term appear to have no counterpart in other species. The physiologic significance of this is still unknown and will require further work on additional spe- cies and on the mouse before an explana- tion is forthcoming. It is of interest that the concentration of gestagen in the blood dur- ing the first 12 days of pregnancy corre- sponds with the intensity of the response to progesterone exhibited by the endometrium during the same period (Atkinson and Hooker, 1945). This w^ould suggest that the serum gestagen levels reflect the phys- iologic state of the animal. Serum gestagen levels in the rabbit re- veal a curve of increasing concentration throughout pregnancy (Zarrow and Neher, 1955). Initial values of 0.3 to 1 yug. per ml. serum were noted at the time of mating, with a sharp rise beginning on the 4th day of gestation. The concentration rose to a level of 6 to 8 /xg. per ml. by the 12th day and thereafter showed only a slight rise to a maximal concentration of 8 to 10 /xg. per ml. serum at parturition (Fig. 16.7). No drop in serum hormone level was observ- able at parturition or 1 hour later. The first significant drop occurred at 6 to 12 hours postpartum when the gestagen level had decreased 50 per cent. It is of interest that the serum progestagen levels did not fall until after the conceptus had been ex- pelled. castrated the 12th, of gestation aborted following removal of the ovaries (Zarrow and Neher, 1955). In all instances the serum gestagen levels fell before the abortion. Figure 16.8 shows the Pregnant rabbits 19th, or 24th day within 1 to 3 days GESTATION 969 ± 5 - 3 - NORMAL PREGNANCY RABBIT 25 o RABBIT 26 • RABBIT 31 e - RABBIT 27 x RABBIT 30 ^ 0 1 ] - - i:^ A •« 0 fc 0 - • A •xoe • 00 0 0 0» X ex 0 • - ©/iO 1 »XA • • • • 24 28 4 8 12 16 20 TIME IN DAYS Fig. 16.7. Concentration of gestagen in the blood of the normal pregnant rabbit as de- termined by the Hooker-Forbes test. (From M. X. Zarrow and G. M. Neher, Endocrinology, 56, 1, 1955.) INTERRUPTED PREGNANCY RABBIT 31 (CASTRATED) 10 - 0 9 - 8 - 0 7 - 6 - 0 0 5 - 4 V 0 |o 3 - 2 > 0 (♦(ABORTION) 0 1 1 j 1 0 0 0 0 0 0 12 16 TIME IN DAYS 20 Fig. tion in Zarrow 16.8. The effect of castration on serum progestogen levels and maintenance of gesta- the rabbit. Gestagen levels were determined by the Hooker-Forbes test. (From M. X. and G. M. Xeher. Endocrinology, 56, 1, 1955.) changes in serum gestagen levels before and after castration of a pregnant rabbit. The concentration increased from a level of 0.3 ing. per ml. at day 0 to 10 /xg. per ml. on day 24 when the rabbit was castrated. A 60 per cent drop in serum gestagen level is seen 12 hours after castration with a fur- ther drop at the 36th hour, when the animal aborted. Studies on the concentration of serum gestagen in the pregnant ewe (Neher and Zarrow, 1954) permit a comparison with the results obtained in the rabbit. Such a comparison is extremely valuable in view 970 SPERM, OVA, AND PREGNANCY 12 11 10 _ 9 DO I ^ ^ 5 ^ A 3 2 1 ^ Normal pregnancy Sheep SI ° Sheep S3 • Sheep S 2 « Sheep S2A x Additional sheep A I A ! I I -A ho I I I I I I I I I I JL_L 20 40 60 80 Time (days) 100 120 140 Fig. 16.9. Concentration of gestagen in the blood of the pregnant ewe. Gestation levels were determined bv the Hooker-Forbes test. (From G. M. Neher and M. X. Zarrow, J. Endocrinol., 11,323,1954.) of the fact that castration of the rabbit invariably leads to abortion whereas cas- tration of the pregnant ewe does not do so if the ovaries are removed during the sec- ond half of pregnancy. Again the proges- terone determinations were carried out on untreated serum and the samples assayed by the Hooker-Forbes technique using pro- gesterone as a standard. An initial rise in the serum gestagen level occurred soon after mating and seemed to level off at a concentration of 6 ixg. per ml. approxi- mately the 50th day of gestation (Fig. 16.9). Thereafter, the concentration re- mained unchanged for approximately 50 days, when a second rise to a level of 8 to 12 fj.g. occurred. These levels remained un- changed until at least 30 minutes after par- turition was complete. Castration at various times after the 66th day of pregnancy failed to influence the concentration of circulating gestagen or interfere with the pregnancy. The data in Figure 16.10 show a normal concentra- tion of 8 to 10 fxg. gestagen from the 114th day of gestation to parturition although the animal was ovariectomized the 114th day. Pregnancy was normal in all castrated ewes and the expected drop in scrum ges- tagen was observed following parturition. It can now be stated that the human being, the monkey, the ewe, the rabbit, the mouse, and probably the guinea pig (Her- rick, 1928; Ford, Webster and Young, 1951) have met the problem of a second source of progesterone supply with varying degrees of success. In the ewe, placental re- placement of the ovary as a source of pro- gesterone can be considered as complete by approximately the 66th day of preg- nancy. Castration at this time will neither interfere with the pregnancy nor with the concentration of the hormone in the blood. In the monkey, castration as early as the 25th day of gestation (Hartman, 1941) does not interfere with pregnancy and in the human being castration as early as the 41st day after the last menstrual period may not interfere with pregnancy (Melin- koff, 1950; Tulsky and Koff, 1957). One may conclude, therefore, that the placenta can adequately take on the role of the ovary in this regard. On the other hand, aspects of the situation in the human fe- male are still puzzling, especially the blood gestagen values; but despite this ambiguity GESTATION 971 10 9 J' w> 7 E 6 BO -* o 3 2 — p o Ovariectomy during pregnancy _ ] — k-(ovariectonny) 1 - 114 125 Time (days) Fig. 16.10. The effect of castration on gestagen levels in the pregnant ewe. Gestagen le\els were obtained by the Hooker-Forbes test. Note that castration failed to interfere with the pregnancy or the level of gestagen in the blood. (From G. M. Neher and M. X. Zarrow, J. Endocrinol., 11, 323, 1954.) it might be concluded that here also the placenta has successfully replaced the ovary. In the rabbit, on the other hand, castration at any time during pregnancy vvill cause a decrease in the level of the circulating hormone and terminate the pregnancy. Hence, in this species, the pla- centa has failed to replace completely the ovary. The mouse is another instance in which castration leads to abortion so that one can assume a failure on the part of the placenta to replace the endocrine activity of the ovary. In this case, however, the second peak of circulating gestagen has been as- cribed to the placenta and this presents the possibility of a partial replacement of the ovary by the placenta but a replacement that is not adequate since pregnancy is terminated by ovariectomy. As indicated above, a marked discrep- ancy exists between the bioassays and the chemical determinations of gestagens in the blood and other tissues. The chemical determinations of progesterone invariably give results that are far lower than those obtained by bioassay methods. Edgar and Ronaldson (1958) found a maximal con- centration of approximately 2 /xg. proges- terone per ml. ovarian venous blood during gestation in the ewe. This concentration was no higher than that seen in the ewe during a normal estrous cycle. The maxi- mal level reached during the estrous cycle was maintained when pregnancy super- vened and remained fairly constant until the last month of pregnancy. Thereafter the concentration fell and no progesterone was detectable at 15 days prepartum (Fig. 16.11). Inasmuch as no progesterone was found in the peripheral blood of the ewe, this poses again the following question: What was being measured in the peripheral blood by the bioassay procedure? In addi- tion, a second question is posed by the earlier discussion on the need of the ovary in the maintenance of pregnancy as to the relative contributions of the ovary and the placenta to the concentration of this hor- mone in the body. That the biologic methods are measuring more than progesterone is obvious from the many reports emphasizing the high levels obtained by bioassay and the low levels obtained by chemical techniques. In addi- tion to the above data. Short (1957, 1958a, 1958b) reported the presence of progester- one in the peripheral blood of the pregnant cow but onlv in the order of 0.0074 to 972 SPERM, OVA, AND PREGNANCY No. of observations 5 5 8 6 9 11 7 7 7 6 8 7 7 5 14 11 7 4 7 7 I 1 1 • Ewes under ■— 1 — r 2 years ■ 1 -T— T- — T" — r- — r~ T "T" 1 1 1 1 1 - » Ewes over 2 years 1' - •• Ji X X • i 3 - X , X * 5 . « > X • X , 1. 1^ X • X • ■X :/ r> ^ X 5 X ,^ '"^^ ^J__ X X J • X X . X /T^ SwV ^*^ «w--* "•x XJL, <*Ay M 1 -p '^ • X • . •• X XXX - X V> X X -f XX X X X T X s^ X • 0 - 1 \ — — i_ — 1— 1. . _L_ .1 . -.1 -i— J— i_ .,.,I.,„ 1 1 • 1 . ••xx^xxxxxx 1 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Weeks of pregnancy Fig. 16.11. The con«?ntiation of progesterone in the ovarian venous blood of the preg- nant ewe. Progesterone was determined by chemical methods. (From D. G. Edgar and J. W. Ronaldson, J. Endocrinol., 16, 378, 1958.) 0.0098 ixg. per ml. plasma. It is of interest that the level remained constant from the 32nd to about the 256th day of pregnancy and then decreased several days before parturition. In the human being values of 0.17 to 0.44 fig. per ml. during the final tri- mester of pregnancy have recently been reported (Oertel, Weiss and Eik-Nes ( 1959 ) . Numerous investigators have suggested that the discrepancy between the chemical and biologic assays is due to the presence of unknown gestagens in the blood. This has been validated in part by the discovery of 2 metabolites in the blood of the preg- nant human female (Zander, Forbes, Neher and Desaulles, 1957). They have been iden- tified as 20a-hydroxypregn-4-en-3-one and 20^-hydroxypregn-4-en-3-one and have been shown to be active in both the Clau- berg and Hooker-Forbes tests (Zander, Forbes, von IMiinstermann and Neher 1958 ) . The 20/?-epimer was twice as active as progesterone in the Hooker-Forbes test and the 20a-epimer one-fifth as active. It is likely that more unidentified gestagens occur in the blood and other tissues. C. SOURCES OF GESTAGENS The second question asked above con- cerning the role of the placenta versus the ovary as a source of progesterone probably cannot be answered in a simple manner. Wide differences exist between species (1) in the need of the ovary for maintenance of pregnancy, (2) in the concentration of the hormone in peripheral blood, (3) in the activity of the placenta in secreting pro- gesterone, and (4) in the presence of extra- ovarian and extraplacental sources of the hormone. The presence of progesterone in the pla- centa of the human being has been con- firmed (Salhanick, Noall, Zarrow and Sam- uels, 1952; Pearlman and Cerceo, 1952) and a high output of progesterone demon- strated. Zander and von ]\Iiinstermann (1956) and Pearlman (1957) independently GESTATION 973 reported the production of approximately 250 mg. progesterone into the peripheral circulation every 24 hours. This and other evidence tends to prove that the placenta is the major source of progesterone in the human species. However, with respect to other species, progesterone has been found only in the placenta of the mare (Short, 1957) although in amounts much less than in the human being. Placentas of the cow, ewe, sow, or bitch were all negative. Al- though the placenta of the mare contains progesterone and castration does not lead to abortion after day 200 of gestation, no progesterone was found in the peripheral blood or uterine vein blood. The ewe offers an even more intriguing problem inasmuch as (1) a discrepancy exists between the biologic and chemical values for progester- one in the peripheral blood, (2) the pla- centas contain no progesterone, and (3) no {progesterone is found in the uterine vein blood (Edgar, 1953). This has led to the conclusion that the maintenance of preg- nancy in the ewe may be dependent on an extra-ovarian, extraplacental source of pro- gesterone. If such a conclusion is correct, and it must be added that the evidence is still tenuous, then the adrenal cortex must be considered as a possible source. Beall and Reichstein isolated a small amount of pro- gesterone from the adrenal cortex in 1938 and Heehter, Zaffaroni, Jacobson, Levy, Jeanloz, Schenker and Pincus (1951) dem- onstrated from perfusion experiments that progesterone is an important intermediate metabolite in the synthesis of the adrenal corticoids. In addition, it has long been known that desoxycorticosterone possesses progesterone-like activity (Courrier, 1940) which is due to a conversion of the desoxy- corticosterone molecule to a gestagen. This has been shown by experiments in vivo in the monkey (Zarrow, Hisaw and Bryans, 1950), rat, and rabbit (Lazo-Wasem and Zarrow, 1955), and by an incubation experi- ment with rat tissue (Lazo-Wasem and Zarrow, 1955). In addition, Zarrow and Lazo-Wasem reported the release of a ges- tagen from the adrenal cortex of the rat and rabbit following treatment with ACTH. The substance was obtained from the peripheral blood and measured by the Hooker-Forbes test, but it was not identi- fied chemically. This was followed by the finding that pregnanediol is present in the urine of ovariectomized women, but not ovariectomized, adrenalectomized women (Klopper, Strong and Cook, 1957), and by the finding that progesterone is present in the adrenal venous blood of the cow, sow, and ewe (Balfour, Comline and Short, 1957). In all instances the concentration of progesterone in the adrenal venous blood was 10 to 100 times greater than the con- centration in the arterial blood. Thus the total evidence that the adrenal cortex can secrete progesterone is more than adequate. The question remains as to whether the adrenal cortex contributes to the proges- terone pool of the body during pregnancy and whether a species difference exists here. D. RELAXIN The initial discovery by Hisaw (1926, 1929) of the presence of an active substance in the blood and ovaries responsible for relaxation of the pubic symphysis of the guinea pig has led in recent years to a con- sideration of this substance as a hormone of pregnancy (Hisaw and Zarrow, 1951). Some doubt as to the existence of relaxin was raised in the 1930's by investigators who were able to show that pubic relaxa- tion in the guinea pig could be obtained with estrogen alone or estrogen and pro- gesterone (de Fremery, Kober and Tausk, 1931; Courrier, 1931; Tapfer and Hasl- hofer, 1935; Dessau, 1935; Haterius and Fugo, 1939). This matter was resolved by the demonstration that pubic relaxation in the guinea pig following treatment with the steroids or relaxin differed in ( 1 ) time re- quired for relaxation to occur, (2) histo- logic changes in the pubic ligament, and (3) treatment with estrogen and progesterone which induced the formation of relaxin (Zarrow, 1948; Talmage, 1947a, 1947b). Subsequent discoveries of additional bio- logic activities possessed by relaxin and fur- ther purification of the hormone has led to the conclusion that relaxin is an active sub- stance in the body, and that it plays a sig- nificant role during parturition. The hor- mone has been found in the blood or other tissues of the dog, cat, rabbit, sheep, cow, rat, and man. The specific action of this ^74 SPERM, OVA, AND PREGNANCY hormone varies with the species involved. Still unsolved is the question as to whether the water-soluble extract obtained from the ovary and referred to as relaxin is a single substance or a group of active substances (Friedcn and Hisaw, 1933; Sher and Mar- tin, 1956). The concentration of relaxin in the blood increases as pregnancy progresses until a plateau is reached. This has been demon- strated in the rabbit (Marder and Money, 1944), guinea pig (Zarrow, 1947), cow (Wada and Yuhara, 1955), and human being (Zarrow, Holmstrom and Salhanick, 1955). Relaxin has also been found to in- crease in the ovary of the sow (Hisaw and Zarrow, 1949). In general, the shape of the curve for the concentration of relaxin in the blood as a function of the length of pregnancy has been more or less the same for all species studied. Figure 16.12 indi- cates that the concentration of relaxin in the blood of the pregnant rabbit rises from a level of 0.2 guinea pig unit (G.P.U.) per ml. for the first trimester of pregnancy, i.e.. until day 12, to a level of 10 G.P.U. per ml. on day 24. This concentration was then maintained until parturition. After delivery of the young, the concentration of the hor- mone decreased 80 per cent in 6 hours. On the 3rd day postpartum no hormone could be detected. As indicated above, the concentration of relaxin in the blood of the pregnant cow and human being showed approximately the same type of curve. In the cow the con- centration rose gradually from a level of 1 G.P.U. per ml. to a maximum of approxi- mately 4 G.P.U. at 6 months (Fig. 16.13). Thereafter the level remained unchanged until parturition, wdien the level dropped at a rate comparable to that seen in the rab- bit. The curve for the concentration of re- laxin in the blood serum of the pregnant woman followed the general pattern de- scribed above (Fig. 16.14). The concentra- tion rose from a level of 0.2 G.P.U. per ml. the 6th week of i:)regnancy to a maximum of 2 G.P.U. the 36th week. Thereafter the level remained unchanged until delivery. Again the postpartum fall was precipitous and the hormone was not detectable at 24 lf\ 9 - yO ^0 ^ 8 . / 2 r 3 oc 7 / lij ' tn U.6 / o o5 / o O ccA 0 lU Q. 3 / iA t- §2 o/ < > 1 G i e , L 12 15 18 21 24 DAYS AFTER MATINO 27 30 35 Fig. 16.12. Concentration of relaxin in the blood of the rabbit during pregnancy. Parturi- tion (P) occurred 32 days after mating. Guinea pig units (G.P.U.) of relaxin are plotted against days pregnant. (From S. N. Marder and W. L. Money, Endocrinology, 34, 115, 1944.) GESTATION 975. 6 7 8 9 • — Pregnancy (in months) 2 6 10 15 After parturition (in days) — * Fig. 16.13. Concentration of I'elaxin in the blood of the cow during pregnancy. Partiui- tion is indicated by P. (From H. Wada and M. Yuhara, Jap. J. Zootech. Sc, 26, 12, 1955.) 6 12 18 24 30 36 LENGTH OF PREGNANCY - WEZKS HOURS POST- PARTURITON Fig. 16.14. The concentration of relaxin in the blood serum of normal pregnant women. (From M. X. Zarrow, E. G. Holmstrom and H. A. Salhanick, Endocrinology. 15, 22. 1955.) hours postpartum. Studies in the guinea pig revealed a marked rise in relaxin on day 21 of gestation to a maximal concentration of 0.5 G.P.U. per ml. serum on day 28 (Zarrow, 1948). Thereafter the level re- mained unchanged for approximately 4 weeks. Contrary to the results obtained in the rabbit, cow, and human being a drop in the concentration of the hormone in the pregnant guinea pig was noted before par- turition. The concentration of relaxin fell to 0.33 G.P.U. per ml. on the 63rd day of gestation and then dropped to nondetect- able levels within 48 hours postpartum. Although no studies have been carried out on the blood levels of relaxin in the sow as a function of the length of pregnancy, analysis of the ovary for relaxin has re- vealed a situation comparable to that re- ported for the blood in other species. The concentration rose from 5 G.P.U. per gm. ovarian tissue during the luteal phase of 976 SPERM, OVA, AND PREGNANCY the cycle to approximately 10,000 G.P.U. per gm. fresh ovarian tissue by the time a fetal length of 5 inches had been reached (Hisaw and Zarrow, 1949). E. SOURCES OF RELAXIN The ovaries, placentas, and uteri are possible sources of relaxin in different spe- cies. It seems from the extremely high con- centration in the ovary of the sow during pregnancy that this organ is the major site of relaxin synthesis at this time. However, studies on other species indicate that both the placenta and uterus may be involved. Treatment of castrated, ovariectomized rabbits with estradiol and progesterone stimulated the appearance of relaxin in the blood of the rabbit as indicated by the ability of the blood to induce relaxation of the pubic symphysis of estrogen-primed guinea pigs (Hisaw, Zarrow, Money, Tal- mage and Abramovitz, 1944). Similar ex- periments on castrated, hysterectomized rabbits failed to reveal the presence of the hormone in the blood of the treated ani- mals. Treatment with estradiol alone also failed to stimulate the release of relaxin. It is obvious then that, if the bioassay is specific for relaxin, the uterus is a definite source of this hormone. Comparable results were also obtained in the guinea pig (Zar- row, 1948). Treatment with estradiol and progesterone caused pubic relaxation and the presence of relaxin in the l)lood after approximately 3 days of treatment with progesterone. In the absence of the uterus relaxin was not demonstrable in the blood. The concentration of relaxin in the blood of the rabbit castrated the 14th day of i^reg- nancy and maintained with progesterone remained unaffected by removal of the ovaries, provided the pregnancy was main- tained (Zarrow and Rosenberg, 1953). Fig- ure 16.15 shows a typical curve for the relaxin content of the blood of such an animal. The concentration of the hormone rose between days 12 and 24 to a maximal concentration of 10 G.P.U. per ml. and was maintained till the time of normal parturi- tion. It is of interest that in those instances in which the placentas were not maintained in good condition, the concentration of the hormone fell. Analysis of the reproductive tract revealed concentrations of 5 G.P.U. per gm. fresh ovarian tissue during pseudo- pregnancy and approximately 25 G.P.U. during the last trimester of gestation. The uterus contained 50 G.P.U. per gm. fresh tissue during pseudopregnancy and an equal concentration the first 24 days of pregnancy. The 26th day of pregnancy the concentration fell to 15 G.P.U. per gm. The highest concentration was in the pla- centa which contained from 200 to 350 G.P.U. per gm. Some evidence indicated that after treatment with estradiol minimal amounts of relaxin, i.e., 5 G.P.U. per gm., were present in the vaginal tissue (Table 16.5). F. ADRENAL CORTEX 1. Hydrocortisone Initial studies on the possible role of the adrenal cortex in gestation involved the determination of the two urinary metab- olites of the gland, i.e., the 17-ketosteroids and the corticoids. Inasmuch as the 17-keto- steroids are believed to be associated with the androgenic activity of the adrenal cortex, bioassays for adrenogenic activity in the urine were carried out. Dingemanse, Bor- chart and Laqueur (1937) found no increase in urinary androgen by the 6th to the 8th month of pregnancy whereas Hain (1939) reported that pregnant women secreted even less androgen than nonjircgnant women. Pincus and Pearlman (1943) found no change in the urinary 17-ketosteroids of the pregnant and nonpregnant woman al- though Dobriner (1943), by the use of chromatograi)hic separation, showed a marked decrease in androsterone. Venning (1946) found no change in the urinary ketosteroids as measured by the antimony trichloride reagent described by Pincus (1943), but the ketosteroids measured by the Zimmerman reagent (dinitrobenzene) showed a significant rise in the latter part of pregnancy. The discrepancy between the two determinations can be explained by the fact that other ketonic substances besides 17-ketosteroids give a color in the Zimmer- man reaction. These are the 20-ketosteroids and to a limited extent the 3-ketosteroids. V^enning (1946) believes most of this in- GESTATION 977 RABBIT N0.80 ■e-e- 15 AFTE R 21 3:^ J3 MATING 9 12 DAYS Fig. 16.15. Concentration of relaxin in the blood of a pregnant rabbit castrated the 14th day of gestation and maintained with 4 nig. progesterone daily until the 32nd day. Post- mortem examination revealed 8 placentas and 2 dead fetuses. (From M. X. Zarrow and B. Rosenberg, Endocrinology, 53, 593, 1953.) TABLE 16.5 Relaxin content of the blood serum and tissue of the reproductive tract of the rabbit (From M. X. Zarrow and B. Rosenberg, Endocrinology, 53, 593, 1953.) No. of Rabbits Relaxin Concentration in G.P.U. Treatment Per ml. serum Per gm fresh tissue Ovary Uterus Placenta whole Placenta fetal Placenta maternal Pseudopregnant Chorionic gonadotrophin . . . 3 4 3 2 2 2 1 0.2-0.3 0.2 1.0 10.0 10.0 10.0 10.0 5 5 30 25 20 25 25 50 50 50 50 30 15 75 50 50 75 10 20 25 Pregnant 24 davs 250 Pregnant 25 days Pregnant 26 days 350 200 Pregnant 28 days •978 SPERM, OVA, AND PREGNANCY crease in ketosteroid excretion during preg- nancy is the result of increased output of the stereoisomers of pregnanolone: Measurement of urinary glucocorticoids by the glycogen deposition test showed an initial increase in the first trimester of pregnancy in the human being. After the initial rise, the urinary excretion level re- turned to normal with a second increase the 140th to 160th day of pregnancy. Values of 200 to 300 /xg. equivalent of 17,hydroxy- 11-dehydrocorticosterone per 24 hours of urine were obtained at days 200 to 240. In most instances the urinary outj^ut fell sev- eral weeks before parturition. ' Analysis of the blood levels for 17a- hydroxycorticosterone in the jiregnant wo- man confirmed the results obtained with the urine (Gemzell, 1953; Seeman, Varan- got, Guiguet and Cedard, 1955). Gemzell ( 1953) reported a rise from approximately 5 /xg. per 100 ml. plasma to an average of approximately 22 fxg. per cent (Fig. 16.16). A further rise to 36 /xg. per cent was noted at the time of labor. This has been con- firmed by McKay, Assali and Henley (1957) who found an average rise of ap- proximately 40 /xg. per cent during labor lasting more than 6 hours. Although Mc- Kay, Assali and Henley reported values still well above normal on the 4th to 6th day postpartum, Gemzell (1953) reported a drop to 1.99 /xg. per cent on the 6th day postpartum. E o o 20- Portus ©36t2,7 (n=J7) • 1 • • , ' ^ y^ • • • 10- ^ ^ • • • • • • ^/^ • , ^ ^ • • # • . • • • • ^'^ * ' , : • • • • 6 days after , • partus . • • . • (3,1,99 tost 0. • * (n-IO) ■n ^ ^^ T^ ^^ w T ^^^ n" ^ ^ ^ ^■^^ w^ . 1 .,,.,.. . to 20 Time, weeks JO AO Fig. 16.16. Correlation between the concentration of 17-hydroxycorticosteroids in the blood of pregnant women and the duration of pregnancy (in weeks). Conception at zero time. (From C. A. Gemzell, J. Clin. Endocrinol.,13, 898, 1953.) GESTATION 979 The mechanism whereby labor induces a marked stimulation of the adrenal cortex is still obscure. It is possible that labor is a stressful state and the stress induced by both the pain and the muscular work act to stimulate the increased release of ACTH resulting in increased adrenocortical ac- tivity. Some confirmation of this may be obtained from the fact that significant in- (■i'eas(> in }ilasma 17a-hydroxycorticoids is noted only if the labor lasts more than 6 hours. Analysis of the rise in plasma levels of hydrocortisone during pregnancy has sug- gested that the phenomenon is not simply the result of an increased rate of secretion from the adrenal cortex, but rather the re- sult of an increased retention and an altera- tion in the metabolism of the hormone < Cohen, Stiefel, Redely and Laidlaw, 1958). 2. Aldosterone The isolation for aldosterone by Simi)son, Tait, Wettstein, Neher, von Euw, Schindler and Reichstein (1954) and its identification as the hormone regulating fluid and mineral metal)olism stimulated marked interest in the role of this hormone. Among the items of interest was its significance in pregnancy and in the toxemia of pregnancy. Early studies by Chart, Shipley and Gordon ( 1951 1 revealed the presence of a sodium retention factor in the urine that increased from a normal pregnancy value of 36 to 106 fxg. equivalent of desoxycorticosterone ace- tate (DOCA) per 24 hours to a maximum of 1008 |U.g. equivalent in pregnancy toxemia. These results were confirmed by Venning, Simpson and Singer (1954) and by Gordon, Chart, Hagedorn and Shipley (1954). In addition a slight increase in the sodium re- taining factor was observed in gravid wo- men as compared to nongravid women. The discovery that the greater part of the aldosterone in urine is present in the conjugated fraction led to a repetition of the above work using both acid hydrolysis and incubation with /3-glucuronidase (Ven- ning and Dyrenfurth, 1956; Venning, Prim- rose, Caligaris and Dyrenfurth, 1957). The results show little change in the excretion of free aldosterone throughout pregnancy, but the glucuronidase and acid-liydrolyzed fractions increased markedly (Fig. 16.17). The urinary excretion values increased from a prepregnancy normal of 1 to 6 /xg. aldosterone (average for women was 3.8 ± 14 fig. per 24 hours; Venning, Dyrenfurth and Giroud, 1956) to approximately 25 /xg. per 24 hours. The first significant rise oc- curred about the third month of gestation and an increased concentration was ob- tained until after parturition, when there was a rapid fall to the nonpregnant values. G. THYROID GLAND Clinical data have long indicated a pos- sible involvement of the thyroid gland in gestation (Salter, 1940). In regions where the iodine supply is low this is demon- strated by an enlargement of the thyroid during pregnancy. The formation of a goiter has been interpreted as evidence for an increased need for iodine during gesta- tion. Scheringer (1930) and Bokelmann and Scheringer ( 1930) reported a rise in the iodine content of the blood of pregnant women during the first trimester of preg- nancy with a peak at the seventh month. The increased concentration is maintained until shortly after parurition. In the goat, however, Leitch (1927) reported no change in serum iodine during gestation until just before parturition. Analysis of umbilical vein blood revealed values that were nor- mal, i.e., lower than in the mother (Leipert, 1934). Increased thyroid secretion (Scherin- ger, 1931 ) and increased urinary excretion of iodine have been reported in pregnant women (Nakamura, 1932; 1933). However, Salter (1940) concluded in his review that no reliable evidence of increased thyroid hormone levels in the blood during jireg- nancy is available. Peters, Man and Heinemann (1948) re- ported a range of 4 to 8 fig. per cent of serum-precipitable iodine in the normal, nonpregnant woman with a rise to 8.3 fig. per cent (range 6 to 11.2 fig. per cent) in the pregnant woman (Fig. 16.18). It is of in- terest that the elevation in the protein- bound iodine (PBI) does not follow the course of changes in the basal metabolic rate. Whereas the former is already high by the second month of pregnancy the basal metabolic rate rises gradually after approx- 980 SPERM, OVA, AND PREGNANCY imately the 4th month of pregnancy (Rowe and Boyd, 1932; Javert, 1940). No other sym})toms of hyperthyroidism are seen in pregnancy which leads to the question of the significance of the rise in protein-bound iodine. A somewhat comparable paradox exists in the guinea pig in which a rise in the rate of oxygen consumption during pregnancy is not accompanied by an in- crease in heart rate (Hoar and Young, 1957). Recently, AVerner (1958) rcj^orted a de- crease in the I^-^^ up-take after treatment with triiodothyronine in both the normal and pregnant woman. From this and other data he ruled out any abnormal pituitary- thyroid relationship or marked secretion of thyroid-stimulating hormone (TSH) by the placenta and concluded that the increased PBI in pregnancy is due to an increased binding capacity of the serum protein. Feldman ( 1958) failed to find any increase in the level of serum-hutanol-extracted io- dine throughout pregnancy in the rat. Ac- tually the values were consistently lower than in the controls and similarly the total amount of PBI in the thyroid of the preg- nant rat was consistently lower. He did find an increase in the rate of excretion of V-''\ a diminished up-take of I^^^ by the thyroid, and a decreased half-life for thyroxine in the pregnant rats. It is obvious that these results are quite dissimilar from those obtained in the pregnant women. One can only conclude at this time that pregnancy has an effect on iodine metabolism and a species difference exists. H. GROWTH HORMONE Although it has been possible to demon- strate the presence of growth-promoting sub- stance (STH) in the blood plasma, there are few data bearing on the identity of the sub- stance and few ciuantitative measurements. Westman and Jacobsohn ( 1944) first showed the ]irescnce of a growth-]5romoting sub- ■z. '• 2 < r) 32. z 0 y- a: UJ ®^ < IT • "^^^ Q- cri 0=28- 0. 1- X z / -"(«) 0 ^ 0 X® Q. CM z ®' ® -^24. / ® UJ / ® o / ® S20. / / h- SO / 0 Q / • ® ^,6. / • / • a~ ^,2. / ® • • • 8- • • • 4. • • • 0 • 0 0 0 0 qO • • • MONTHS 2 DAYS 3 4 5 6 7 8 9 Fig. 16.17. Urinary excretion of aldosterone throughout pregnancy in the human being. O, free fraction only; •, free and acid-hydrolyzed fraction; O, free, enzyme and acid- hA'drolyzed. (From E. H. Venning and I. Dyrenfurth. J. Clin. Endocrinol,, 16, 426, 1956.) GESTATION 981 stance in the blood by cross transfusion be- tween a normal and hypophysectomized rat united in parabiosis. Gemzell, Heijkenskjold and Strom (1955), using the technique of adding exogenous growth hormone to the sample of blood, failed to find any growth- jiromoting substance in 23-ml. equivalents of blood. However, retroplacental blood from human beings gave a positive response at a level of 7- to 15-ml. equivalents of plasma without the addition of exogenous STH. In- crease in the width of the proximal tibial epiphysis of the rat was used as an end l)oint. A comparable concentration of 650 fxg. eciuivalent of the standard STH per 100 ml. plasma was also found in the blood from the umbilical cord. Contopoulos and Simpson (1956, 1957) measured the STH of the plasma in the pregnant rat, using the tibial cartilage, tail length, and body weight increase. No sig- nificant increase in plasma STH was noted on the 5th day of pregnancy, however, a sig- nificant rise was observed by the 9th day. An estimated 3-fold increase in plasma STH during pregnancy was reported from calcu- lations on both the tibial cartilage and the tail length tests. No changes were reported in the STH activity of the pituitary gland throughout pregnancy. Recently, the per- sistence of greater than normal amounts of growth-promoting activity was reported in the plasma of pregnant rats after hypophy- sectomy. Since the fetal pituitary probably does not contribute to the STH pool of the mother, at least in early pregnancy, it is likely that the placenta may be a source of the hormone. I. PROLACTIN Few data are available on the concentra- tion of prolactin during gestation. This has been due, in part, to the minute amounts of the hormone present in the urine and blood and to the inadequacy of the available as- says. Although Hoffmann ( 1936 ) failed to find any prolactin in the urine of women before parturition, Coppedge and Segaloff (1951) and Fujii and Schimizu (1958) re- ported measurable amounts of prolactin in the urine of pregnant women. Coppedge and Segaloff reported a gradual rise in the excre- FiG. 16.18. The level of protein-bound iodine in the pregnant woman. (From J. P. Peters, E. B. Man and M. Heinemann, in The Normal and Pathologic Physiology oj Pregnancy, The Williams & Wilkins Co., 1948.) 982 SPERM, OVA, AND PREGNANCY 28 30 32 3i 36 38 aO 0 1 23 45 6 78 2 4 6 8 10 12 14 28 Weeks of pregnancy Days post partum Weeks of lactation Fig. 16.19. Urinary excretion of prolactin throughout gestation in the human being. One pigeon crop unit (P.C.U.) is equivalent to 0.3 I.U. (From K. Fujii and A. Shimizu, Bull. Tokyo Med. & Dental Univ., 5, 33, 1958.) tion of prolactin throughout pregnancy and a gradual decline following parturition even though lactation was maintained. The num- ber of observations, however, was limited and the authors point out that the results were ecjuivocal. Fujii and Shimizu observed an initial drop in the prolactin output dur- ing the first month of pregnancy followed by a rise to approximately 32 P.C.U. (one pi- geon crop sac unit is equivalent to 0.3 I.U.) per 24 hours during the second trimester of pregnancy in women. (Fig. 16.19). This was followed by a drop to approximately 10 P.C.U. per^24 hours between the 30th and 38th wrecks of pregnancy and a marked rise to 64 P.C.U. per 24 hours during the lacta- tion period. J. PL.\CENTAL GONADOTROPHINS Placental gonadotrophins have been found in the monkey, chimpanzee, human being, mare, and rat (Hisaw and Astwood, 1942). The physiologic activities of these placental hormones differ among the three groups of niannnals and appear to represent divergent evolutionary steps in the adoption of pi- tuitary function by the placenta. The phys- iologic properties of the placental gonado- trophins differ not only among themselves but also from the pituitary gonadotrophins. The gonadotrophin from the rat placenta (luteotrophin) has been shown to be leuto- trophic with the ability to maintain a func- tional corpus luteum in the hypophysecto- mized rat (Astwood and Greep, 1938). The hormone has no effect on follicular growth or ovulation. Its function appears to be that of maintaining the secretory activity of the corjius luteum in the rat from the 10th day of pregnancy to term. The placental hormones of the human be- ing (HCG) and the mare (PMS) have been studied in much greater detail. These two hormones differ markedly in both chemical and physiologic properties. The presence of HCG in the urine and the absence of P]\IS in the urine would alone indicate a marked dif- ference in the size of the two molecules. Physiologically, PMS is highly active in pro- ducing follicular growth and some luteiniza- tion, whereas HCG has no effect on follicu- lar growth but will induce ovulation and a delay in the onset of menstruation. This would indicate a luteotrophic action. Al- though chorionic gonadotrophin has been re- ported in the macaque (Hamlett, 1937) be- tween the 18th and 25th day of pregnancy, and in the chimpanzee from the 25th to the 130th day of gestation (Zuckerman, 1935; Schultz and Snyder, 1935), little work has been done on the characterization and iden- tification of these substances except in man and horse. It is of some interest to note that the ap- GESTATION 983 MAX CONJCENTRATION CONTROP HORMONE lOOr n ,'-— X WOMAN MARE 10 20 30 40 50 7. OF DURATION OF PREGNANCY 60 70 80 90 100 Fig. 16.20. The relative time of appearance of placental gonadotropliins in the pregnant mare and the woman. (From E. T. Engle, in Sex and Internal Secretions, 2nd ed., The Williams & Wilkins Company, Baltimore, 1939.) pearance of the placental gonadotrophins in the blood and urine of horse and man occurs at approximately the same relative time in pregnancy (Fig. 16.20). The role played by these hormones in gestation is still not clear, but it is significant that their appearance corresponds with the time of implantation of the blastocyst and their disappearance roughly with the time when ovariectomy no longer interferes with the maintenance of the pregnancy. /. Human Chorionic Gonndotrophin (HCG) The discovery of the presence of a gonado- trophic hormone in human pregnancy urine by Aschheim and Zondek (1927j was soon followed by a description of its biologic ac- tivity and quantitative determinations of its concentration in the urine throughout pregnancy (Ascheim and Zondek, 1928). Recently a number of investigators have de- termined the titer of chorionic gonadotro- phin in the serum of pregnant women. These curves agree very well with the values ob- tained from the urine. Figure 16.21 is a typi- cal curve for the concentration of chorionic gonadotrophin in the blood of pregnant women (Haskins and Sherman, 1952). A peak value of 120 I.U. per ml. of serum was obtained on the 62nd day after the last menses and a rapid decline was noted to a low of approximately 10 I.U. per ml. of se- rum on day 154. A subsequent rise to 20 I.U. was noted by day 200 and this was maintained until the end of pregnancy. These results are in excellent argeement with those reported by Wilson, Albert and Ran- dall (1949) using the ovarian hyperemia test in the immature rat. These authors ob- tained a peak concentration of approxi- mately 70 I.U. per ml. of serum on the 55th day after the last menses. A gradual decrease occurred thereafter to a low of approxi- mately 20 I.U. per ml. of serum which re- mained unchanged from day 100 to par- turition although the data indicate a slight rise towards the end of pregnancy. The significance of the excretion pattern 984 SPERM, OVA, AND PREGNANCY o o o ii o X u o U. CQ o 5 ♦ ui 2 ■ MAXIMUM TITER /\ ^^\ /N, 20 60 100 140 180 ZZO 260 DURATION OF PREGNANCY DAYS AFTER THE LAST MENSES Fig. 16.21. Concentration of human chorionic gonadotiophin in the blood of the normal pregnant woman. The hormone levels were determineti b>' the male frog test. (From A. L. Haskins and A. I. Sherman. J. CUn. Endocrinol., 12, 385, 1952.) and concentration of the hormone in the serum is still a matter of conjecture. Browne, Henry and Venning (1938) suggested that the peak level of chorionic gonadotrophin in the blood reflects an increased production and a physiologic need in order to maintain a functional corpus luteum during early pregnancy. Recent evidence has tended to confirm this opinion in that HCG has been found to be active in the maintenance of the secretory activity of the corjius luteum in the primate (Hisaw, 1944; Brown and Brad- bury, 1947; Bryans, 1951 j. In addition, his- tologic studies reveal a direct proportion be- tween the number of Langhans' cells and the amount of hormone excreted (Stewart, Sano and Montgomery, 1948; Wislocki, Dempsey and Fawcett, 1948) . The possibility that the kidney plays a role in the changes in the concentration of HCG was investigated by Gastineau, Albert and Randall (1948) . The renal clearance was relatively constant throughout all stages of pregnancy although the urine and serum concentrations of the hormone varied as much as 20- fold. In addition, the mean, renal clearance found during pregnancy was not markedly different from that found in cases of hydatiform mole and testicular chorioma. Inasmuch as the renal elimination of the hormone remained constant, it was obvious that two possible explanations existed : these were (1) changes in the secretion rate, and (2) changes in extrarenal disposal of the hormone. Studies on the latter were con- tradictory. Whereas Friedman and Wein- stein (1937) and Bradbury and Brown (1949) reported an excretion of 20 per cent GESTATION 985 and higher of HCG following the injection of HCG, Johnson, Albert and Wilson (1950) found an excretion of 5.8 per cent in preg- nant women during the immediate post- partum period. Zondek and Sulman (1945) reported a 5 to 10 i)er cent elimination of HCG in the urine of animals. Thus Brad- bury and Brown felt that there is relatively little destruction or utilization of the hor- mone in the body; Wilson, Albert and Randall ( 1949) believed that 94 per cent of the circulating hormone is affected by extra- renal factors and that the fluctuating char- acter of hormonal level in serum or urine depends entirely on changes in rate of hor- mone production. An analysis of the distribution of chorionic gonadotrophin in the mother and fetus led Bruner (1951) to conclude that the ratio of maternal blood to urinary gonadotrophin is not constant although the ratio of gonado- trophin in the chorion to maternal blood is constant. Consequently, she concluded that the concentration of gonadotrophin in the urine does not depend entirely on the rate of production of the hormone and that the method of gonadotrophin elimination changes during pregnancy. She also pointed out that a significant amount of chorionic gonadotrophin is found in the fetus and that this is due to the fact that, although the chorion releases the hormone into the mater- nal blood, secondarily some of it passes the placental barrier and enters the fetal sys- tem across the wall of the chorionic vesicle. 2. Equine Goyiadotrophin (PAIS) The presence of a gonadotrophin in the blood of the pregnant mare was first de- scribed by Cole and Hart in 1930. The hor- mone appears in the blood about the 40th day of pregnancy and increases rapidly to a concentration of 50 to 100 rat units (R.U.) per ml. by the 60th day of pregnancy (Cole and Saunders, 1935). This concentration is maintained for approximately 40 to 65 days. By day 170 it has fallen to a nondetectable level (Fig. 16.22). Catchpole and Lyons (1934) suggested that the placenta is the source of the gonado- trophin and indicated that the chorionic epithelium is the probable source. Cole and leae/7(f • Co/7ce/7irct/o/? of oes^r/y?. — /re/7ral blood throughout pregnancy (Fig. 16.29). A marked rise is observed from the 10th to the 20th week of pregnancy, and thereafter the concentration plateaus until after parturition. 20 30 40 weeks Fif!. 16.29. Tlie histaminase activity of the peripheral blood of the human female during pregnancy (•) and at parturition (®). (From H. Swanberg, Acta scandinav., Suppl. 79, 23, 1950.) Both the maternal placenta and the decidual tissue have been identified as major sites for formation of the enzyme. Danforth and Gorham (1937) reported the presence of histaminase in the placenta of a series of patients at term. This was confirmed by Swanberg (1950) who, in addition, separated the placenta by a series of slices parallel to the surface of the organ and reported that the layer adjacent to the uterine wall, consisting of practically only the thin decidual membrane, contained a mean value of 614 /xg. per gm. per hr. of histaminase as compared to 38 for the fetal portion of the placenta. Confirmation of the concept that the maternal placenta is the main source of histaminolytic activity can be obtained from the finding of histaminase in decidual tissue of nonpregnant females and in the maternal placentas of animals. In cases in which maternal and fetal placentas can be separated easily, the maternal placenta contained from 14- to 100-fold the activity seen in the fetal placenta. Comparison of the histaminolytic activity in the decidual tissue of the sterile horn and the control pregnant horn of the uterus of a rabbit revealed 319 fig. per gm. per hr. and 222 fxg. per gm. per hr., respec- tively. Treatment with progesterone or in- duction of jiseudopregnancy caused a marked rise in the histaminase of the endometrium to upwards of 1000 fig. per gm. per hr. Nonetheless, histaminase was 996 SPERM, OVA, AND PREGNANCY not observed in the blood plasma of the progesterone treated rabbits whereas pro- gesterone treatment of two nonpregnant women caused a marked rise iti plasma histaminase. The physiologic significance of histami- nase is still unknown. A consideration of this problem must take into account not only the action of the enzyme and changes in its concentration under different physi- ologic conditions, but also the species prob- lem. In regard to the latter point, the data are extremely inadequate. Only two species have been studied in any detail and these are the human being and the ral)bit. One can conclude from the available data that histaminase is produced by the maternal placenta, decidua, and uterine endometrium. It increases with pregnancy in these tissues and its concentration may be correlated with the progestational hormone. It in- creases in the blood of tlie human being, I'at, and guinea pig during pregnancy but nut in the cat or rabbit (Swanberg, 1950; Carlsten, 1950). The obvious hypothesis that histaminase })rotects the uterus from the stimulating action of histamine has not been confirmed. But it is somewhat jiai'a- doxical to note that urinary histamine also increases during pregnancy. Kahlson, Rosengren and Westling (1958) reported a daily 24-hour excretion of 18 to 43 /xg. of histamine during the first 2 weeks of pregnancy in the human being. A marked increase was noted on the 15th day with a peak of 123 to 835 /xg. per 24 hr. at the peak of excretion which occurred 1 to 2 days before parturition. As yet no role can be attributed to this substance. It is of interest that the increased histaminase present during pregnancy can serve the role of protecting the uterus from the muscle- contracting action of this substance. Be- cause the amount of urinary histamine ex- creted is correlated with the number of young and no changes are apparent in the concentration of histamine in the tissues during pregnancy, it would seem that the excessive formation of histamine during the last trimester of pregnancy takes place in the uterus and its contents and the basic action of histaminase is protective. It was shown recently that the excessive formation of histamine during the last tri- mester of pregnancy in the rat is due to an increase in the rate of histidine decar- boxylase activity (Kahlson, Rosengren, Westling and White, 1958). Inasmuch as removal of the fetuses without other in- terference with the pregnancy abolishes the increased urinary histamine, it can l)e con- cluded that the site of formation is in the fetus. This histamine could escape into the maternal circulation and eventually be eliminated via the kidneys. Roberts ( 1954) reported that aminoguan- idine leads to a general disturbance of pregnancy in the rat; large doses tended to jiroduce death of the mother and smaller doses tended to kill all or part of the litters and some of the mothers. Again one could conclude a protective action on the part of histaminase dui'ing tlie latter i)art of i^reg- nancy. B. CARBONIC ANHYDRA.se Carbonic anhydrase was discovered by Aleldrum and Roughton in 1933 and soon shown to catalyze the following reaction, H,CO, z:± CO. + HoO. The enzyme was found to occur in many tissues and was generally located within the cell especially in cells possessing a secretory function. The discovery by Lutwak-Mann and Laser (1954) that carbonic anhydrase is present in tlu' uterine mucosa led to a thorougli study of the changes in the concentration of the enzyme and the factors controlling its presence (Lutwak-Mann, 1955; Lut- wak-Mann and Adams, 1957). The enzyme has been found to be present in the re- productive tract of a wide variety of mammals. In general, the uterine endome- trium, placenta, and Fallopian tubes are the main loci of activity although there are marked differences among different species. Carbonic anhydrase activity was found consistently in all the animals studied such as the rat, hamster, guinea pig, rabbit, pig, and ewe. No activity was noted in the uterine mucosa of the nonpregnant animal except the ewe and the rabbit. In several species, such as the cow, human being, and pig, carbonic anhydrase was also found in the Fallopian tube. A marked rise in carbonic anhydrase of the endometrium of the rabbit was noted during the first trimester of pregnancy GESTATION 997 (Fig. 16.30). The value rose from a pre- pregnancy level of 20 enzyme units (E.U.) per gm. of fresh tissue to a maximum of 100 E.U. per gm. at approximately the 8th day of pregnancy. This level was maintained until the 12th day and then declined to approximately the prepregnancy level by about the 20th day. Examination of the placentas at this time revealed marked ac- tivity, 68 E.U. per gm. of maternal placenta and 25 E.U. per gm. of fetal placenta. The curve for the concentration of carbonic anhydrase in the uterine mucosa during pscudopregnancy is essentially the same as that seen during pregnancy, although some minor differences exist. It is obvious from the above data and from the evidence involving the increased concentration of carbonic anhydrase in the uterine mucosa following treatment with progesterone, that the enzyme is probably under the control of the luteoid hormone. Indeed, an excellent correlation has been shown between the degree of {progestational proliferation in the uterus and the concen- tration of carbonic anhydrase. In the ewe, however, the carbonic anhydrase of the uterus is independent of the ovary. A pos- sible explanation for this discrepancy be- tween the two species has been offered on the basis of differences in the blood level of progesterone. However, no explanation is forthcoming for the failure to maintain the carbonic anhydrase level throughout pregnancy in the rabbit, even though the circulating progesterone remains liigh. The significance of this enzyme in the physiology of reproduction is still unknown. From the data on the rabbit, it miglit be inferred that the carbonic anhydrase con- tributes to the maintenance of bicarbonate in the blastocyst fluid. The universal pres- ence of the enzyme in placental tissue could also lead to the assumption that carbonic anhydrase is involved in fetal metabolism. Lutwak-Mann (1955) indicates that the enzyme might be involved in the trans- mission of calcium across the placenta. ^^'hether carbonic anhydrase is essential for fetal (Icvelopment and successful pregnancy is still unanswered. Treatment with car- bonic anhydrase inhibitors (Diamox) failed to affect adversely the pregnancy or fetuses in pregnant rats even though no Fid. 16.30. Carhonic anhydrase activity in the uteiu.s of the rabbit during pregnancy, i),seudo- pregnancy, large doses of gonadotrophin, and pregnant mare's serum (PMS). Pregnancy, • •; i^seudopregnancy, O O; gonado- trophin, D D: PMS, x" X. (From C. Lut- \val<-Mann. .1. Kndocrinol.. 13, 26. 1955.) enzyme acti\'ity was present either in the matei'nal blood or placenta. XI. Factors in the Maintenance of Gestation A. THYROID GLAND Several recent reviews have pointed out that the extract role of the thyroid gland in reproductive physiology is still in need of elucidation (Peterson, Webster, Rayner and Young, 1952; Reineke and Soliman, 1953). Numerous investigations over the past half century have definitely indicated that the thyroid gland is involved in reproduction but the site and manner of action are still not well known. In addition, contradictory reports indicate that each species and even each strain may have to be studied in- dependently (Alaqsood, 1952). Some evi- dence foi- the involvement of the thyroid gland in gestation has already been con- sidered. The increase in FBI at the onset of jiregnancy and the incidence of miscarriage in the human female when the FBI fails to rise tend to involve the thyroid hormone in the maintenance of pregnancy. Habitual abortion in women is usually associated with t'ithcr hypo- or hyperthyroidism (Litzenberg, 1926). Litzenberg and Carey (1929 » I'eported that in 70 married women with low basal metabolic rates appi'oxi- mately 45 per cent had one or more abor- 998 SPERM, OVA, AND PREGNANCY tions or stillbirthsrtf one eliminates the sterile woman from the group, the figure for women showing abortion or stillborn rises to approximately 35 per cent. However, the results are still controversial both with regard to data obtained within a single species and from different species. Hypothyroidism in the rat induced by the prolonged administration of thiouracil resulted in a resorption of the fetus in 100 per cent of the cases (Jones, Delfs and Foote, 1946). Rogers (1947) reported a re- duction in litter size following sulfaguani- dine and Krohn and White (1950) reported a reduction in litter size following thyroidec- tomy in the rat. Thyroidectomy early in pregnancy caused a resorption of the fetuses and if performed at a later stage in pregnancy resulted in the birth of still- born young (Chu, 1945). Following the in- duction of pregnancy in thyroidectomized rabbits, either a resorption of the young or abortion or prolongation of gestation was noted and the newborn young were usually dead. Chu concluded that the thyroid hor- mone was concerned with the vitality and growth of the embryos during gestation. In the pig the average duration of pregnancy was 114 days for normal gilts and 124.5 days for thiouracil-treated animals. In ad- dition, the controls farrowed an average of 8.67 pigs per litter compared with 3.25 per litter for the thiouracil-treated .sows ( Lucas, Brunstad and Fowler, 1958 ) . The difference was significant in both instances. Bruce and Fio. 16.31. Tlie effect of tliyioid deficiency on litter size. O, 422 litters from tliyroid-defieient mice; •, 423 litters from normal control mothers. (From H. M. Bruce and H. A. Sloviter, J. En- docrinol., 15, 72. 1957.) Sloviter (1957) pointed out that part of the conflicting reports on the role of the thyroid in gestation might be due to the different methods used in producing a thyroid-deficient state. Surgical removal of the gland generally results in the loss of the parathyroids which may be also im- portant in the maintenance of gestation (Krichesky, 1939), although adequate in- formation is lacking. The use of antithy- roidal substances offers more serious ob- jections because these drugs not only pass through the placenta but they are non- specific and interfere with other glands such as the adrenal cortex (Zarrow and Money, 1949; McCarthy, Corley and Zarrow, 1958), with nutrition, and with the general status of the animal. Consequently, Bruce and Sloviter preferred to establish a thyroidec- tomized state in mice by the use of radio- active iodine after establishing the dose necessary to induce total destruction of the thyroid without damage to the jiarathyroid or ganiete.s. Although ( lorbman ( 1950 ) rei)orted a complete loss of reproductive activity in the mouse following treatment with P'*\ Bruce and Sloviter (1957) reported no effect on the ability of the mouse to conceive or bear young. This discrepancy could be due in part to the strain differences in the sensi- tivity of the ovary to the I^^^. Bruce and Sloviter (1957), however, noted a decrease in the average litter size of thyroid-deficient mice (Fig. 16.31 ). The data indicate a max- imum of 6 young per litter in thyroid-de- ficient mice versus 10 young per litter for the normal mice. It is apparent that the entire curve for the litter size of thyroid- deficient mice is shifted toward a smaller size. This has also been observed in the rat following thyroidectomy (Nelson and To- bin, 1937). The thyroid-deficient mice also showed a prolongation of gestation as re- ported in rats, guinea pigs, and sows. Of the thyroid-deficient rats, 46 per cent showed a gestation period of more than 19 days whereas only 15 per cent of the normal controls showed a gestation period of more than 19 days whereas only 15 per cent of the normal controls showed a gestation pe- riod of more than 19 days (Table 16.9). Analysis of the data based on grouping according to litter size showed clearly an GESTATION 999 eifect of litter size on length of gestation. The smaller litter size gave a higher inci- (l(>nce of prolonged gestation. Studies on oxygen consumption in the guinea i)ig revealed a slight but significant rise of 8 per cent at the end of gestation (Hoar and Young 1957). The increase in oxygen consumption is consistent but slight for the first 60 days of pregnancy after which the significant increase occurs (Fig. 16.32). The rise continued until 5 days postpartum and then fell rapidly. In a sec- ond set of experiments oxygen consumption was measured in control, thyroidectomized, and thyroxine-injected, pregnant guinea pigs. Measurements were taken at the time of mating and at parturition. In all three instances, an increase in the oxygen consumption was noted at parturition as compared with the values at the time of TABLE 16.9 Ejfcct of thyroid-deficiency and litter size on length of gestation in mice (From H. M. Bruce and H. A. Sloviter, J. Endocrinol., 15, 72, 1957.) Thyroid-deficient Control No. of Young In Litter No. of pregnan- cies > 19 days No. of pregnan- cies > 19 days No. Per cent No. Per cent 1-5 6-9 10-14 Totals 36 40 20 96 24 13 7 44 67 33 35 46 16 38 28 82 7 3 2 12 44 7 8 15 mating (Fig. 16.33). Again the control guinea pigs showed a 7.9 per cent gain in oxygen consumption by the end of preg- nancy, but both the thyroidectomized preg- nant guinea pigs and the thyroxinc-treated guinea pigs also showed an increase in oxy- gen consumption of 11.9 and 16.2 per cent, respectively. The increase in oxygen con- sumption was not paralleled by increases in heart rate; actually the heart rate decreased in several instances. In addition, neither the weight of the thyroid gland nor the histology of the gland was changed during pregnancy. It is obvious then that an ex- planation for the rise in oxygen consump- tion during pregnancy may not involve the thyroid gland. On the basis of changes in its appearance. Hoar and Young (1957) suggested the possibility that the adrenal cortex is involved and that the increased oxygen consumption is due to an increased release of adrenal corticoids. More evidence is needed before this suggestion can be fully accepted. Further work from the same laboratory has led to the concept that one locus of action of thyroxine during pregnancy is at parturition (Hoar, Goy and Young, 1957). These investigators used an inbred strain of guinea pigs that is characteristically hy- pothyroid and a genetically heterogeneous stock in which the level of thyroid activity is presumed to be higher. It had been pre- viously shown that pregnancy wastage was high in the hypothyroid guinea pigs. Treat- ment with thyroxine reduced the percentage of stillborn from 40 to 13.6 in the hypothy- c 80 o ♦- a. £70 c o ^60 c o» >«50 40 71.012.2 58.7±1.4 59.411.3 59.6±l.5 -— *--63!410.9 63.811.9 ^ - Parturition Length of Gestation in Days 10 20 30 40 50 60 70 80 Fig. 16.32. Oxvgen consumption in the guinea pig during gestation. (From K. M. Hoar and W. C. Young, Am. J. Physiol., 190, 425, 1957.) 1000 SPERM, OVA, AND PREGNANCY 100 40 %(.._»( Thyroxin-injected females C— ^Control females 92.2±2.2 X — -XThyroidectomized females _.- .^¥. 63.4±0.9 59.6+1.8 o 5o[.53.3±i.4 Parturition Length of Gestation in Days 10 20 30 40 50 60 70 80 Fig. 16.33. Oxygen con.suinption in the pregnant guinea pig treated with thyroxine or thvroiclpctomized before mating. (From R. M. Hoar and W. C. Young, Am. J. PhysioL, 190, 425, 1957.) roid guinea pig!>, i.e., to a level !?een in the untreated heterogeneous group. Treatment of the heterogeneous group with thyroxine not only failed to reduce the percentage of stillborn but actually increased the abor- tion rate particularly in the 2nd and 3rd trimesters. The most consistent result, how- ever, was a decrease in length of gestation following treatment with thyroxine, and an increase following thyroidectomy. From these experiments it was concluded that the thyroid hormone facilitates parturition and need be present only late in gestation to exert its action. It is apparent that in some species the thyroid hormone is involved directly in pregnancy. In the absence of the hormone, certain species tend to resorb or to abort; or if pregnancy is maintained gestation tends to be lengthened. This is probably due to an interference with the mechanism of parturition. In certain species such as the guinea pig only a parturitional problem has been demonstrated; in others an entire galaxy of symptoms may be present. Re- duction in the size, number, and viability of the young give added emphasis to an es- sential role for thyroxine in the phenome- non of gestation. B. ADRENAL CORTEX Removal of the adrenal cortex without further treatment invariably leads to dis- turbances in rejM'oductive i)hysiology and the termination of pregnancy. Although the early results were controversial in that some investigators reported that adrenal- ectomy failed to affect gestation in the rat (Lewis, 1923; Ingle and Fisher, 1938), others reported that adrenalectomy led to abortion (Wyman, 1928; Dessau, 1937) or to some other disturbance of gestation (Mc- Kcown and Spurrel, 1940). Davis and Plotz (1954) adrenalectomized two groups of pregnant rats on the 4th to 6th and the 14th to 16th day of pregnancy. Abortion occurred in all 12 rats adrenalectomized during the first half of pregnancy whereas only 1 of the 12 adrenalectomized during the second half of pregnancy aborted. How- ever, even in those adrenalectomized during the second half of gestation, an effect on jiregnancy was observed. A significantly higher incidence of stillborn and sickly young (14.4 per cent) and a marked de- crease in the weight of the fetuses were noted (Table 16.10). Early results indicated that extracts of the adrenal cortex could readily replace the absent adrenal gland and maintain suc- cessful pregnancies. Within recent years it has been demonstrated that many steroids such as cortisone and 9a-chlorohydrocorti- sone at 10 /xg. per day (Llaurado, 1955) permit fecundation and successful mainte- nance of pregnancy. Successful maintenance GESTATION 1001 TABLE Ki.lO Effects of adrenalectomy on the character of the litter, and on fetal body weight and adrenal weight (From M. E. Davis and E. J. Plotz, Endocrinology, 54, 384, 1954.) Pregnant Controls Adrenalectomy 2nd Half of Pregnancy Per- centage versus Preg- nant Con- trols No. of litters .... 21 11 Dead and "sickly" young. Vigorous voung . . . 5 182 13 78 <0.01 Fetal hodv weight (gm.) 6.13 5.22 <0.01 (=b0.10)« (±0.30)" Fetal adrenal weight (mg.). . . . 0.49() 0.554 >0.3 (±0.017)" (±0.041)« Fetal l)()dv weight/ fetal adrenal weight X 1000. . . 12.35 9.42 <0.01 (0.34±)'' (±0.31)" Calculation of standard error of the mean: S.E. / Ed-^ ]/ nin - 1) of a i)ivgnancy has also been reported in an adrenalectomized human female main- tained on hydrocortisone 9a-fliiorohydro- cortisone (Laidlaw, Cohen and Gornal, 1958). In this instance measurements of urine excretion of aldosterone revealed an increase to 4.4 fxg. per 24 hours during the last trimester of pregnancy and a postpar- tum value of 0.5 fig. Inasmuch as the value is only 1/10 of that seen in a normal preg- nancy the authors concluded that the ad- renal cortex of the mother is the major source of aldosterone during pregnancy and that a high output is not a major prereq- uisite for a normal pregnancy. Treatment with either 0.9 per cent saline drinking water or with cortisone increased the number of successful pregnancies fol- lowing adrenalectomy during the first half of gestation. Pregnancy was normal in 8 of 11 adrenalectomized rats (Davis and Plotz, 1954). Treatment with 2 mg. of corti- sone acetate resulted in successful preg- nancies in 13 of 14 rats adrenalectomized on the 4th to 6th day of gestation and 12 of 12 rats adrenalectomized on the 14th to 16th day of gestation. However, complete main- tenance was not obtained. The body weight of the mothers and the weight of the fetuses were significantly lower than in the con- trols, and the number of stillborn and sickly young was increased. A comparison of the pregnancy-mainte- nance activity in a number of adrenal cor- ticoids indicated that a combination of a glucocorticoid and mineralocorticoid pro- vides the best protection in the adrenalecto- mized rat (Cupps, 1955). Nulliparous rats were adrenalectomized, placed on treat- ment, and mated. Under these conditions the adrenalectomized controls and the rats treated with desoxycorticosterone acetate failed to become pregnant inasmuch as no implantation sites were obtained (Table TABLE 16.11 Effect of adrenal steroids on reproduction in adrenalectomized female rats (From P. T. Cupps, Endocrinology, 57, 1, 1955.) Daily Treatment Control Cortisone acetate ^i mg Cortisone acetate ^i mg Cortisone acetate 1'^ mg Cortisone acetate 2,4 mg Hydrocortisone acetate Vi mg.. . , Cortisone acetate Vi mg. plus Desoxycorticoster- one acetate ^ mg Desoxycorticoster- one acetate ^^ mg Desoxycorticoster- one acetate ^ mg Desoxycorl icoster- one acflate 1 mg , Adrenalectomized control No. of Rats No. Born Alive (aver- age) Implan- tation Sites (average) 7 8.2 11.0 6 3.5'> 5.6^ 6 3.6* 6.2'^ 5 3.5 8.6" 5 5.8 10.0 7 5.0 8.5" 5 9.0 9.6 4 0 0 5 0 0 4 0 0 5 0 Weight Change during Preg- nancy (average)" gm. 46.4 -30. S'' -1.5« 17.2^ 12.8^ 30.7 44.6 "Weight change of mother from day of breed- ing to day after parturition. " Significant at 0.05 level. '= Significant at 0.01 level. 1002 SPERM, OVA, AND PREGNANCY 16.11). Treatment with 2.5 mg. cortisone acetate per day was partially effective in restoring reproductive capacity. Injections of 1.25 mg. hydrocortisone acetate per day gave results comparable with those ob- tained when cortisone was given, although the ratio of young born alive to implanta- tion sites indicated that hydrocortisone acetate was more effective. It was definitely more effective than cortisone acetate in maintaining the body weight of the mother. However, reproduction was completely re- stored to normal in the adrenalectomized rat following treatment with desoxycorti- costerone acetate and cortisone acetate. Interference with gestation in the normal animal has been reported by several in- vestigators following treatment with ACTH or adrenal corticoids (Courrier and Co- longe, 1951; Robson and Sharaf, 1952; Velardo, 1957). This is taken to indicate that there is a finely balanced requirement for adrenocortical hormones during gesta- tion ; and that suboptimal or supra-optimal amounts of the hormone interfere with pregnancy. Courrier and Colonge found that cortisone administered to intact rab- bits in the second half of pregnancy inter- fered with gestation. Robson and Sharaf treated both pregnant rabbits and mice with ACTH and reported a marked effect on gestation. Abortion or resorption oc- curred in 8 of 9 mice and in 8 of 11 rabbits. Contamination by posterior pituitary hor- mones or gonadotrophins can be excluded. A subsequent experiment with cortisone also caused marked interference with preg- nancy in the rabbit when 20 mg. were given ; 10 mg. were without effect. Administration of cortisone to castrated or hypophysecto- mized pregnant rabbits maintained with progesterone also caused damage to the pregnancy. Since the hormone was not act- ing by way of the ovary or pituitary gland, the authors felt that cortisone was acting directly on the uterus and the uterine con- tents. In the rat, however, ]Meunier, Duluc and Mayer (1955) observed an effect on preg- nancy only when cortisone acetate was in- jected at the time of mating. Rats injected with 10 to 25 mg. cortisone acetate daily for 5 to 6 days beginning on day 12 or day 14 of gestation had a normal pregnancy. Velardo (1957) reinvestigated the prob- lem in the rat and reported a marked re- duction in litter size and an increase in the number of stillborn following ACTH treat- ment. Although quantitative differences appeared, a significant decrease in litter size w^as observed only when the hormone was given (1) before mating, (2) immedi- ately after mating, or (3) between the 11th and 15th day after mating. However, the greatest effect was noted when the ACTH was administered immediately after mating. Surprisingly enough, litter size was mark- edly reduced only if adrenalectomy was performed on day 7 of gestation. Adrenalec- tomy on day 8 to 14 of gestation had no effect on live litter size. However, a total of 6, 9, and 13 stillbirths were obtained following adrenalectomy on days 8, 9, and 11. It is interesting that the number of still- births decreased from 21 following ad- renalectomy on day 7 to none following ad- renalectomy on day 14. It is apparent that the adverse effects of adrenalectomy on gestation decrease as pregnancy progresses. It is also apparent from these and other experiments that the action of ACTH is mediated by the adrenal cortex. From these results and others described above, it seems likely that the adrenal corticoids may be acting on the uterus. Mayer and Duluc (1955) found that adrenalectomy of the I'at on the 14th to the 16th day of pregnancy led to variable results. In 17 pregnant adrenalectomized rats, gestation was terminated in 8, but no interference was observed in 9. The rats that failed to maintain pregnancy died witiiin 2 to 3 days. Again it would appear that delicate hormonal balances are in- volved. In a further investigation of this problem Aschkenasy-Lelu and Aschkenasy ( 1957) reported that a diet adequate in salt and proteins would prevent interference with pregnancy in rats adrenalectomized before mating. On a low protein diet, preg- nancy could be maintained only in the in- tact rat (80 per cent) and then only if daily injections of progesterone were given. These authors believe that the role of the GESTATION 100.^ adrenal corticoids in pregnancy is con- cerned with stimulation of appetite and mobilization and degradation of proteins to amino acids. The latter action would permit the replacement of body protein in the absence of a normal jirotcin intake. C. PANCREAS The impact of diabetes mellitus on the course of pregnancy has been of interest to the clinician for many years. In a recent review of the subject, Reis, DeCosta and Allweiss (1952) came to the conclusion that "the carefully controlled diabetic aborts no more frequently than the nondiabetic." On the other hand, it has been well known for many years that uncontrolled diabetes and pregnancy are basically incompatible (Eastman, 1946). Studies in the rat have given contro- versial results with regard to the influence of insulin on pregnancy. Davis, Fugo and Lawrence (1947) reported that in the al- loxan diabetic rat pregnancy was normal for the first 12 days. Thereafter death of the fetuses occurred followed by resorption. Sinden and Longwell (1949) and Levi and Weinberg (1949) reported no detrimental effect from diabetes on the course of i^reg- nancy. The latter group obtained 12 preg- nancies from 25 rats made permanently dia- betic with alloxan. Eleven of the 12 rats went to term and delivered normal fetuses and 1 died during pregnancy. Recently, Wells, Kim, Runge and Lazarow (1957) reported a 14 per cent loss in fetal weight, an increase in gestation length from a nor- mal of 538 to 563 hours, and an increase in fetal or neonatal mortality in the pregnant rat made diabetic by pancreatectomy or treatment with alloxan. In general, the clinical data indicate that uncontrolled diabetes has a detrimental ef- fect on pregnancy, but that the abortion rate in the controlled diabetics approaches that seen in the ''normal" population. Since the crux of the matter seems to hinge on the severity of the diabetes, one might conclude that the effect of insulin is an indirect one by virtue of its action in maintaining a good metabolic state. The conflicting reports from animal experimentation may be due to the differences resulting from uncon- trolled environmental and dietary factors. D. ovary: progesterone, estradiol, AND RELAXIN Marshall and Jolly (1905) were probably the first to point out that ovariectomy dur- ing pregnancy leads to abortion or resorp- tion of the fetuses in the rat. Subsequently, a number of investigators repeated these experiments and confirmed the findings in all species tested thus far, provided ovariec- tomy is performed before implantation. Re- moval of the ovaries after gestation is well under way, however, does not disturb the course of pregnancy in all species. The hu- man being, monkey, horse, ewe, and cow are examples of species not dependent on the ovary for the maintenance of pregnancy once it has been well established. Species such as the rabbit and the rat require the presence of the ovary throughout preg- nancy. The importance of progesterone for i)reg- nancy was established by Allen and Corner (1929) who first showed that an extract of the corpus luteum will maintain preg- nancy in the castrated rabbit. Identification of the active substance in the extract as progesterone led to the use of the hormone in many other species. Allen (1937) reported that crystalline progesterone was inferior to the crude luteal extract in the mainte- nance of pregnancy in the castrated rabbit. From these and other data, such as the en- hancing action of estrogen on the proges- terone-induced progestational reaction, he inferred that a combination of estrogen and progesterone should be superior to proges- terone alone in the maintenance of preg- nancy. However, he pointed out with proper caution that the dosages would have to be carefully regulated because estrogen could also antagonize progesterone. Although Rob- son (1936) failed to enhance the action of progesterone with estrone in the pregnant hypophysectomized rabbit, Pincus and Werthessen (1938) obtained enhancement with both the androgens and estrogen. Whereas the early work indicated that a pregnancy maintenance dose of progester- one varied from 0.5 to 2 mg. (Allen and Corner, 1930), later experimentation indi- 1004 SPERM, OVA, AND PREGNANCY cated that the dosage varied with the stage of pregnancy. An adequate dose of approxi- mately 1 mg. progesterone in the early stages of pregnancy needs to be increased to 5 mg. in the later stages (Allen and Heckel, 1939; ComTier and Kehl, 1938a, b). These investigators also revealed that an optimal effect could be obtained by using a progesterone-estrogen combination in the ratio of 750 to 1. Chang (1951) transferred ova to nonovulated intact rabbits and noted that massive doses in the order of 25 mg. macrocrystalline progesterone injected for three times were required to obtain a 50 per cent maintenance of pregnancy. He also re- ported that under the conditions of his ex- periment an initially high dose was needed for the passage of the ova, implantation, and early maintenance. Since then, further experimentation, especially on other spe- cies, has revealed a significant role by estro- gen in enhancing the pregnancy-maintain- ing action of progesterone. A vast literature exists for the human being on the prevention of threatened abor- tion by progesterone which is beyond the scope of this review. Variation from nega- tive results to excellent maintenance is re- ported. It is obvious that a great deal of variability exists here and, to some extent, this is explained by a need for more objec- tive criteria in evaluating threatened abor- tion and the therapy (Guterman and Tul- sky, 1949). It is obvious that if the TABLE 16.12 Maintenance of pregnancy in the rat castrated on the 12th day of gestation (From J. Yochim and M. X. Zarrow, Fed. Proc, 18, 174, 1959.) Progester- one No. Estra- diol Daily Implan- tation Site No. of Fetuses No. of Fetuses Alive Preg- Rats Daily dose No. daily nancy Index mg. Mg. 4 40 37 37 0.925 4 2 1 47 32 27 0.574 7 1 2 76 12 2 0.026 9 1.5 2 99 61 49 0.495 7 2 2 85 65 63 0.741 6 1 2 0.1 69 50 48 0.696 5 1.5 2 0.1 51 48 46 0.900 5 2 2 0.1 60 55 54 0.900 threatened abortion were the result of some disturbance other than progesterone, that progesterone therapy might be without suc- cess. Indirect evidence for the need for progesterone to maintain a successful preg- nancy in the human being and for the lack of need for the corpus luteum once preg- nancy is established has been presented by Tulsky and Koff (1957). Corpora lutea were removed from day 35 to day 77 of pregnancy in 14 women. Two of the women exhibited spontaneous abortion and a marked drop in pregnanediol excretion. The remaining 12 maintained a normal preg- nancy and pregnanediol excretion. The data can be interpreted to indicate a need for progesterone during pregnancy and that this need can be met by a nonovarian source, i.e., the i)lacenta. In both the rat and mouse, successful maintenance of pregnancy after castration has been obtained with progesterone or a combination of progesterone and estrogen. However, partial maintenance following castration can be obtained in the rat under special circumstances. Haterius (1936) re- moved all the fetuses except one and left all placentas intact. Under these conditions the remaining fetus was carried beyond term. Alexander, Fraser and Lee (1955) found that castration of the rat on the 9th day resulted in 100 per cent abortion, whereas 60 per cent of the fetuses were retained until term if castration was on the 17th day. Dosage of progesterone as high as 5 to 10 mg. daily following castration the 9th day gave only partial maintenance. It is possible that better results would have followed multiple daily injections. Yochim and Zar- row (1959) castrated rats on day 12 of ges- tation and obtained a pregnancy index (no. of fetuses alive at day 20 h- no. of implanta- tion sites at day 12) of 0.741 when 2 mg. progesterone were gi^'en in two divided daily doses and 0.495 when 1.5 mg. proges- terone was given (Table 16.12). However, the addition of 0.1 /^.g. estradiol daily markedly enhanced the action of the proges- terone so that a pregnancy index of 0.9, i.e., equivalent to the normal controls, was ob- tained with 1.5 mg. progesterone. Finally, Hall (1957) has indicated that relaxin synergizes with estradiol and pro- gesterone in the maintenance of jiregnancy GESTATION 1005 in the castrated mouse. One nig. progester- one per day maintained pregnancy in 83 per cent of the mice castrated on day 14 of gestation, but 0.5 mg. maintained preg- nancy in only 30 per cent of the animals. The addition of 1.5 ^g. estradiol per day was without effect. On the other hand, the addition of relaxin to the estradiol and 0.5 mg. progesterone gave pregnancy mainte- nance in over 80 per cent of the mice as compared with 30 per cent when progester- one alone was given. Smithberg and Runner (1956) induced ovulation and mating in prepubertal mice (age 30 to 35 days) and obtained 100 per cent implantation with 0.5 to 1 mg. proges- terone daily and approximately 90 per cent successful pregnancies when 2 mg. proges- terone were given. A comparison of the amount of progesterone required for main- tenance of pregnancy in the normal and castrated prepubertal mouse is given in Fig- ure 16.34. In an interesting application of the information available on the induction of ovulation and maintenance of pregnancy, Smithberg and Runner (1957) were able to obtain successful pregnancies in genetically sterile, obese mice. Haterius (1936) observed that distortion of the fetus occurred following ovariectomy in the rat. This has been confirmed by Zeiner (1943) in the rat and by Courrier and Colonge (1950) in the rat and rabbit. It was noted that castration greatly com- pressed the fetuses and eventually caused death. Courrier and Colonge (1950) in very elegant experiments showed that removal of the rabbit fetus into the peritoneal cavity prevented the distortion and death which ordinarily followed castration. Frazer (1955) obtained similar results in the rat and concluded that fetal death after cas- tration of the mother follows a rise in intra- uterine pressure which is associated with an increased tone of the circular uterine mus- cle fibers. Consequently the increased sur- vival of the extra-uterine fetuses following ovariectomy in the mother is the result of the removal of this pressure by the circular muscle of the uterus. Many investigators have demonstrated that gestation can be prolonged by inhibit- ing parturition. Both the injection of large doses of progesterone or the formation of 0.25 0.5 1.0 2.0 PROGESTERONE (mg) Fig. 16.34. Daily dose of progesterone required to maintain pregnancy in the normal and cas- trated prepubertal mouse. (From M. Smithberg and M. N. Runner. J. Exper. Zool., 133, 441, 1956.) a new set of functional corpora lutea during pregnancy will prevent parturition. The in- jection of an ovulating dose of HCG on the 25th day of pregnancy in the rabbit delayed parturition for 15 days after the injection, i.e., until the 40th day of gestation (Snyder, 1934). The fetuses survived in utero for only 3 days and grew to greater than nor- mal size during this period. The placentas persisted until day 41 of gestation. Com- parable results were obtained following daily injections of progesterone into preg- nant rabbits (Zarrow, 1947a). Haterius (1936) obtained prolongation of pregnancy in the castrated rat by removing all the fetuses except one, leaving all placentas intact. Recently a comparable experiment was performed in tlie rabbit with intact ovaries (Hafez, Zarrow and Pincus, 1959). In 2 of 10 rabbits, live fetuses were obtained l)y cesarean section on day 36. However, in 8 of the 10, delivery was delayed beyond day 36, although some degree of fetal re- sorption was present in all instances. Pro- longation of pregnancy in the rat was ob- tained by the injection of prolactin (Meites and Shelesnyak, 1957), but only if the ova- ries were present. E. PITUITARY GLAND In general, hypophysectomy before mid- pregnancy leads to resorption. This is es- pecially true of the rat and mouse. On the 1006 SPERM, OVA, AND PREGNANCY other hand, hypophysectomy at midpreg- nancy or later does not interfere in the maintenance of gestation in these species (Pencharz and Long, 1933; Selye, Collip and Thompson, 1933a, b; Pencharz and Lyons, 1934 ) . In the dog, ferret, and rabbit, hypophysectomy leads to abortion (Asch- ner, 1912; McPhail, 1935a; White, 1932), whereas the results in the cat seem contra- dictory (Allan and Wiles, 1932; McPhail, 1935b) . Hypophysectomy of the rhesus monkey does not always interfere with pregnancy. Smith (1954) obtained normal pregnancies in 10 of 20 hypophysectomized rhesus mon- keys. The remaining animals aborted. Al- though more data are needed, it seems that the pituitary gland can be removed very early in gestation without disturbing the pregnancy. Whereas hypophysectomy be- fore midterm invariably leads to abortion or resorption in the rat or mouse, 1 of the 4 monkeys hypophysectomized between the 29th and 34th day of gestation carried its young to term. Inasmuch as Hartman and Corner (1947) showed that the placenta se- cretes sufficient progesterone by the 25th day of gestation to maintain pregnancy, it is apparent that the placenta in the monkey is able to maintain its endocrine secretory activity independent of the pituitary and at a sufficiently high level to replace the ovary. Little, Smith, Jessiman, Selenkow, van't Hoff, Eglin and Moore (1958) reported a successful pregnancy in the 37-year-old woman hypophysectomized the 25th week of pregnancy. The mother w^as maintained on thyroid, cortisone, and pitressin tannate replacement therapy. The excretion of cho- rionic gonadotropin and pregnandiol was not markedly different from that seen in normal gestation. Estrogen excretion was slightly reduced and the 17-hydroxy corti- costeroids dropped to zero when cortisone therapy was discontinued. It would seem that this phase of adrenocortical activity was reduced and that ACTH or corticoid- like substances from the placenta were in- adequate. No interference in aldosterone output was observed. Hypophysectomy on the 10th day of gestation in mice terminated the pregnancy in only 3 of 19 animals (Gardner and Allen, 1942). Sixteen mice carried their litters to term although 7 of the 16 had a difficult and prolonged parturition. Body weight curves were normal and the corpora lutea appeared unaffected by the loss of the pitui- tary gland, indicating either the independ- ence of the corpus luteum or the presence of a placental luteotrophin. Marked involu- tion of the adrenal cortex was noted in all instances. Simultaneous measurements of the con- centration of cholesterol in the adrenal gland and ACTH in the pituitary of the rat revealed a drop in adrenal cholesterol and pituitary ACTH on the 15th day of ges- tation (Poulton and Reece, 1957). This was followed by a marked increase of both sub- stances on the 21st day of pregnancy and a sharp drop at parturition. The authors con- cluded that a gradual increase occurs in the secretory activity of the adrenal cortex which reaches a peak on the 15th day of pregnancy in the rat. Thereafter the ac- tivity decreased until parturition when a marked increase was observed. The initial decrease in pituitary ACTH potency fol- lowed by an increase after day 15 is inter- preted as an initial increase in ACTH re- lease followed by a decreased release. The decrease in pituitary ACTH potency at parturition is compatible with the marked increase in adrenocortical activity at this time if the decreased pituitary ACTH ac- tivity is interpreted as indicative of ACTH release. Maintenance of pregnancy in rats hy- l')ophysectomized early in pregnancy was obtained with prolactin by Cutuly (1942), although Lyons, Simpson and Evans (1943) reported negative results with a purified prolactin. However, a partial maintenance of pregnancy was obtained with purified prolactin and estrone. F. PLACENTA The placenta is not only involved in the synthesis of hormones during pregnancy but also in the transfer of substances between mother and fetus. It is obvious that the transfer of substances is limited and the l^lacenta does offer a barrier. This problem bears not onlv on the matter of fetal GESTATION 100/ nutrition, but also on the fetal environment and as such is important in the sexual de- velopment of the fetus (see chapter by Burns) . The presence of estriol in the urine of newborn male infants has led to the con- clusion that estrogens can pass through the placenta because of their low molecular weight (Diczfalusy, Tillinger and Westman, 1957). Studies on the transfer of estrogens across the placental barrier in the guinea pig with C^'^-labeled estradiol revealed an extremely rapid disappearance of radio- activity from the maternal blood following intravenous injection of the hormone into the mother, and the appearance of large amounts of water-so.luble radioactivity in the fetal plasma (Dancis, Money, Condon and Levitz, 1958). However, no estradiol was found in the fetal plasma. Replacement of fetal circulation with a perfusion system indicated that estradiol did not j^ass the placenta although estriol was readily trans- ferred in both directions. These authors re- ported that the placenta was relatively im- permeable to the water-soluble estrogens found in the urine, wliich are essentially glucuronides. The discovery in 1927 of large amounts of estrogens and gonadotrophins in the blood and urine of pregnant w^omen led to the cjuestion as to whether the placenta is a gland of internal secretion. This can be answered with an uneciuivocal yes. Never- theless, several questions are still unan- swered: (1) the number of hormones pro- duced by the placenta, (2) the quantities, and (3) the secretory activity of the pla- centa in different species. Data on the presence of gonadotrophins in the placenta have already been discussed. At least three different types of gonado- trophins have been extracted from the pla- centas of the human being, mare, and rat. These have been defined physiologically and appear to be different in the three species. Cole and his co-workers have identified the endometrial cups as the source of PJVIS in the mare, whereas the elegant experiments of Stewart, Sano and Montgomery (1948) indicate that HCG in the human being is secreted by the Langhans cells. These in- vestigators grew human placental cells in tissue culture and obtained ^ gonadotrophin in the culture. They also noted a direct cor- relation between the growth of the Lang- hans cells and the production of gonado- trophic hormone (see also the discussion of this subject in the chapter by Wislocki and Padykula). The initial discovery of a progressive rise in the secretion of adrenal corticoids in pregnancy (Venning, 1946) has been con- firmed by numerous investigators. Gemzell ( 1953) attributed the steady rise to a stimu- lation of the adrenal glands by excessive amounts of estrogen present during preg- nancy and to hyperactivity of the fetal adrenals. The hypertrophy of the fetal ad- renal cortex in the rat following adrenalec- tomy of the pregnant mother was first re- ported by Ingle and Fisher in 1938 and confirmed by Walaas and Walaas (1944), and Knobil and Briggs (1955). However, the 17-ketosteroid and corticoid level of fetal urine is very low (Day, 1948; Jailer and Knowlton, 1950) as are the 17-hydroxy- corticosteroids in the blood of the newborn infant (Klein, Fortunato and Papados, 1953). ACTH-like activity has been found in extracts of the placenta (Jailer and Knowlton, 1950; Tarantino, 1951; Opsahl and Long, 1951) and corticoid activity has been found in the placenta of horses and human beings, as demonstrated by the gly- cogen deposition and growth-survival test in adrenalectomized rats (Johnson and Haines, 1952). Berliner, Jones and Sal- hanick (1956) isolated 17a-hydroxy corti- coids from the human placenta. Pincus (1956) reported that ACTH can stimulate steroidocorticogenesis in the per- fused placenta. Using the ascorbic acid de- pletion test, Assali and Hamermesz (1954) assayed the blood in the intervillous space and the chorionic villous tissue for ACTH. Good activity was observed in the blood from the intervillous spaces and in the tis- sue of the chorionic villi. Corticotrophic ac- tivity was also obtained by Lundin and Holmdahl (1957) from placentas obtained at full term, but the activity was small com- pared with that obtained from the pituitary gland. The possible role of the fetal pituitary was investigated by Knobil and Briggs 1008 SPERM, OVA, AND PREGNANCY (1955) who noted that hypophysectomy of the mother prevented the fetal adrenal weight increase observed following adrenal- ectomy of the pregnant mother. However, complete atrophy of the adrenal gland was not observed in the pregnant mother if the conceptus was present. It was concluded that ACTH can cross the placental barrier and that the fetus or placenta or both pro- duce a sufficient amount of ACTH, to influ- ence the maternal adrenal gland in the absence of the maternal hypophysis. It is still questionable, however, whether these sources, i.e., placenta and fetal pituitary, are of sufficient magnitude to account for the increased release of adrenal corticoids. Hofmann, Knobil and Caton (1954) showed that the ability of the hypophysectomized nonpregnant rat to secrete a water load is not greater than that of the hypophysecto- mized pregnant rat. Hence the contribution of the fetal pituitary or j^lacenta to the corticoid pool is not of sufficient magnitude to influence water balance. As with the gonadotrophins, the increased amounts of estrogen ancl pregnanediol dur- ing pregnancy were thought to be derived from the placenta. In 1933, Selye, Collip and Thompson presented evidence to indi- cate that the placentas of rats jiroduce both estrogen and gestagen. Many physiologic data have been accumulated to prove this point, but completely convincing evidence was obtained only when these hormones were identified in placental extracts and in fluid perfused through the placenta. Dicz- falusy and Lindkvist (1956) identified es- tradiol in the placenta and the presence of progesterone was described by Salhanick, Noall, Zarrow and Samuels (1952) and by Pearlman and Cerceo (1952). Perfusion experiments on human placen- tas have revealed that this organ secretes a number of steroids (Pincus, 1956). These include progesterone, desoxycorticosterone Cortisol, and a number of unidentified ster- oids. Addition of ACTH to the perfusate had no effect on the concentration of Corti- sol, but it did increase the concentration of the reduced corticosteroids, namely, the tetrahydro derivatives of cortisone and Cor- tisol. This was interpreted as a stimulation of the placenta by ACTH resulting in an in- creased release of the corticoid as demon- strated by the increase in the degradation products. The identification of the placenta as a source of both sex steroids and certain gon- adotrophins clarifies the manner by which jiregnancy can be maintained in certain species in the absence of the pituitary gland or ovary (see sections above on ovary and pituitary gland). Newton and Beck (1939) and others showed the hypophy- sectomy of the pregnant mouse does not pre- cipitate abortion. Studies of the ovary re- veal that, if the placentas are retained, the corpora lutea remain normal but removal of the placentas causes immediate degenera- tion of the corpora lutea (Deanesly and Newton, 1940). A comparable situation ap- pears to exist in the rabbit and rat ; it is as- sumed, therefore, that the placenta takes over control of the corpus luteum in preg- nancy in those species that require the ovary for successful gestation. In other spe- cies, such as man, sheep, cattle, and guinea pig, it seems that the placenta can supplant the ovary after pregnancy has progressed to a certain stage. G. PELVIC ADAPTATION The discovery that pelvic changes are under hormonal control in certain species was the result of extensive studies on pelvic adaptations associated with parturition (see reviews by Allen, Hisaw and Gardner, 1939; Hisaw and Zarrow, 1951). It has been ar- gued that, in general, a narrow pelvis is present in mammals living in burrows. This would have the advantage of permitting an animal to turn within narrow confines, but a narrow pelvis would also interfere with the delivery of the young at parturition. As Hisaw pointed out in his extensive studies, this problem has been met by special adap- tations on the part of different species. This has varied from a resorption of the carti- laginous pubic arch in the male and female mole iScalopiis aquaticus machrinus, Raf.) which is independent of the endocrine sys- tem (Hisaw and Zilley, 1927) to elongation of the pubic ligament which is directly un- der hormonal control (Hisaw and Zarrow, 1951). The symphysis pubis of the pocket go- pher, Geomys bursarius (Shaw), behaves as a female secondary sexual character so that GESTATION 1009 a sex dimorphism exists in this species. The pubic cartilages ossify in both sexes and unite to form a complete pelvis with a rigid symphysis pubis. At this stage, the pelvis is too small for the passage of the young, but with the first estrus in the female, the pubic bones are gradually resorbed, leaving the pelvis open ventrally. The pelvis in the male remains intact (Hisaw, 1925). Treatment with estrogen alone can readily bring about the resorption of the pubic bones. A third type of adaptive mechanism has been described in great detail in the guinea pig and led to the discovery of the hormone, relaxin. A sex dimorphism of the pelvis ex- ists in the guinea pig, as in the pocket go- pher, but in addition parturition is further facilitated by marked relaxation of the pubic ligaments and of the sacroiliac joint. Thus far extensive pelvic relaxation has been described in the guinea pig (Hisaw, 1926, 1929 », mouse (Gardner, 1936; Newton and Lits, 1938; Hall and Newton, 1946a), women (see review by Hisaw and Zarrow, 1951), and rhesus monkey (Straus, 1932; Hartman and Straus, 1939). No relaxation of the pubic symphysis has been reported in the ewe but a relaxation of the sacroiliac joint and an elongation of the sacrosciatic ligament was noted the 2nd to 3rd month of gestation. These changes increased as preg- nancy progressed (Bassett and Phillips, 1955). Treatment with stilbestrol alone caused a marked loosening of the sacroiliac joint and the sacrosciatic ligament. The ad- dition of relaxin to the treatment was with- out effect (Bassett and Phillips, 1954). The role of relaxin in the relaxation of the pubic symphysis has been studied most extensively in the guinea pig and mouse. The work before 1950 was reviewed by Hi- saw and Zarrow in 1951. The controversies (de Fremery, Kober and Tausk, 1931 ; Ha- terius and Fugo, 1939) as to whether such a hormone exists need not be discussed here, in detail, except to point out that the evi- dence supporting this opinion is more than adeciuate. Zarrow ( 1946, 1948) showed that pubic relaxation could be induced by es- tradiol alone, by a combination of estra- diol and progesterone, or by relaxin in an estrogen primed animal (Table 16.13). The difference in the time required to in- duce relaxation, i.e., 23 days for estrogen alone, 13 days for estrogen and progesterone, and 6 hours for relaxin, and data indicating that progesterone caused the presence of relaxin in the blood of guinea pig only if a uterus was present led to the concept that pubic relaxation may be produced independ- TABLE 16.13 Relaxation of the symphysis pubis and relaxin content of blood, urine, and uteri of castrated and castrated, hysterectomized guinea pigs after treatment with moderate doses of estradiol and progesterone (From M. X. Zarrow, Endocrinology, 42, 129, 1948.) Treatment, Daily Average Relaxation Time Relaxin Content No. of Guinea Pigs Estradiol Progesterone Total After pro- gesterone treatment Blood serum Urine Uterus MS- mg. days days G.P.U./ml. G.P.U./ml. G.P.U./gm. Castrated 9« 10 1 from day 11 13.5 (13-14) 3.5 0.5 0.3 10 10 10 2 from day 11 13.0 3 0.5 0.5 10 10 10 (12-14) 23.7 (16.31) Negative at 4 ml. Negative at 5 ml. Negative Castrated, hys- terectomized 11 10 1 from day 11 23.7 (17-30) 13.7 Negative at 4 ml. Negative at 8 ml. 10 10 25.6 (18-32) Negative at 4 ml. Negative at 4 ml. One guinea pig not included in the table refiuired 22 days of treatment for pubic relaxation. 1010 SPERM, OVA, AND PREGNANCY ently by estradiol (prolonged treatment) or relaxin (single injection). It is also possible to conclude that the action of progesterone is indirect and due to the formation of re- laxin in the uterus (Zarrow, 1948; Hisaw, Zarrow, Money, Talmage and Abramovitz, 1944) . In the mouse, however, progesterone inhibits the action of relaxin on the pubic symphysis (Hall, 1949). Further evidence that two hormones are involved in pubic relaxation was provided by histologic examination of the pubic liga- ment. Symphyseal relaxation following es- trogen appeared to be due to a resorption of bone and a proliferation of loose fibrous connective tissue with an increase in mucoid alkaline phosphatase and water content (Talmage, 1947a, 1947b, 1950; Heringa and van der Meer, 1948). Relaxin produced a breakdown and splitting of the collagenous fibers into thin threads and a similar change was noted with progesterone (Talmage, 1947a, 1950). Histochemical and biocliemical studies of the pubic symphysis have recently been re- viewed (Frieden and Hisaw, 1933) and tend to show that relaxin produces specific changes. These include loss of metachro- masia (Heringa and van der Meer, 1948) , accumulation of Evans blue m vivo, and in- creased solubility of the glycoproteins in the McManus-Hotchkiss reaction, all of wiiich indicate that a depolymerization of the ground substance and basement mem- brane glycoproteins had occurred (Perl and Catchpole, 1950) . Frieden and Hisaw (1951) found an increase in water content of the symphyseal tissue, but failed to find a de- crease in the water-soluble hexose and hex- oseamine following a single injection of re- laxin. On the basis of a depolymerization of ground substance, a decrease should have occurred. However, repeated injections of relaxin led to a decrease in the insoluble hexoses and hexoseamines. In addition, con- sistent decreases in collagen content and trypsin-resistant protein content were noted. No hyaluronidase was found, but ^-glucu- ronidase was increased during relaxation. Gersh and Catchpole (1949) reported the presence of a collagenase from histochemi- cal studies, but no confirmation has been forthcoming. Relaxin also has a protein anabolic effect which occurs in the absence of pubic relaxation (Frieden, 1956). This action was demonstrated by the increased up-take of labeled glycine by the connective tissue proteins of the pubic symphysis. Re- cent experiments indicate that relaxin not only acts in conjunction with the female sex steroids but can also act alone (Bren- nan and Zarrow, 1959). However, it is ap- parent that the available data are still in- adequate for a clear understanding of the mechanism of action of relaxin. Relaxation of the pubic symphysis of the mouse has been studied in great detail by Hall. In a series of reports she showed that pubic relaxation occurs in the mouse dur- ing pregnancy and following treatment with estradiol and relaxin (Hall and Newton, 1946a, b). This was later confirmed by Kli- man, Salhanick and Zarrow (1953). Con- trary to the results reported following work on the guinea pig, progesterone not only failed to influence the effect of estrone on the pubic symphysis of the mouse, but pro- gesterone also inhibited the action of re- laxin. It was suggested that this inhibition is the result of an antagonism by progester- one on the action of relaxin and that a true species difference exists (Hall, 1949, 1955). Histologic studies revealed that changes in the pubic symphysis during pregnancy and after treatment with relaxin and estradiol are similar (Hall, 1947) . These changes con- sist of proliferation of articular hyaline car- tilage, resorption of the medial ends of the pubes, lengthening of the pubic ligament by formation of new cartilage, and reversion of the cartilage to collagenous connective tis- sue. Hall (1956) suggested that estradiol causes a depolymerization of the mucopoly- saccharides through enzymatic action re- sulting in a matrix sufficiently pliable to respond to the tensions set up by relaxin. Evidence presented in support of this con- cept was the loss of metachromasia and the increase in water. In addition, a two-step effect was seen with relaxin: (1) complete degradation of the matrix, and (2) the ap- pearance of a gap in the cranial part of the cartilage produced by stretching of the sym- physeal cleft. Some data in support of the latter part of this concept were presented by van der Meer (1954) who showed that in- GESTATION 1011 hihition of pelvic muscle tension inhibited relaxation in the guinea pig. In a similar type of experiment Crelin (1954) tied to- gether the innominate bones of a mouse be- fore pregnancy and obtained some dorso- ventral displacement of the pubic symphysis but normal relaxation was inhibited. H. DILATION OF THE UTERINE CERVIX Dilation or softening of the uterine cervix in the pregnant woman at the time of labor has been known for a long time. This reac- tion has been used to determine whether delivery can be anticipated. Within recent years this phenomenon has been described in a number of animals and some analysis of the hormonal control of the reaction has been attempted. Relaxation of the uterine cervix of the rat during pregnancy was first reported by de Vaal in 1946 and confirmed by Uyldert and de Vaal in 1947. Relaxation was meas- ured by the insertion of a gauging pin into a cervix that had been removed and the diameter determined at the point where re- sistance is first felt. The measurements re- vealed a marked rise from approximately 3.5 mm. on the 17th day of pregnancy to 10 mm. at parturition. Recently, both Hark- ness and Harkness (1956) and Yochim and ZaiTow (1959) have taken in vitro measure- ments of the relaxation of the uterine cervix of the rat and observed marked relaxation during the latter part of gestation and at parturition. Yochim and Zarrow (1959) re- moved the cervix, suspended it from a rod and measured the stretch due to weights added at fixed intervals until the cervix broke. The amount of relaxation of the cervix was determined by the amount of stretch obtained with a weight of 50 gm. Under these conditions, the curve for re- laxation of the cervix showed two sloi^es as pregnancy progressed (Fig. 16.35). The ini- tial slope between day 12 and day 20 showed a rise of approximately 4 mm., with an ex- tremely abrupt rise of 14 mm. on day 21. By 24 hours after parturition the degree of dilation had fallen to 3 mm. It is of interest that the curve for the tensile strength of the cervix (expressed in grams force necessary to tear 1 mg. cervical tissue in a rat weigh- ing 100 gm.) was the opposite to that seen for cervical dilation. The tensile strength fell from approximately 50 gm. force to a low of 3 gm. at parturition and then rose during the postpartum period. The drop in tensile strength preceded the changes in the dilation of the cervix and was essentially completed 5 to 6 days before parturition or when the abrupt increase in dilatability of the cervix occurred. Similar changes have been described in the dilatability of the cervix of the mouse (Steinetz, Beach and Kroc, 1957) with in- creased dilatability progressed beyond the 15th day (Fig. 16.36). The diameter of the cervix increased from a])proximately 2 mm. to about 5 mm. at delivery. It is apparent that the rate of the reaction, i.e., dilation, is much more rapid in the rat, although it is possible that the method of measurement is responsible for the differences. The induction of cervical dilation by re- laxin was reported by Graham and Dracy (1953) in the cow, and by Zarrow, Sikes and Neher (1954) in the sow and the heifer. Treatment with stilbestrol followed by re- laxin caused a dilation of the uterine cervix of the gilt from % or % inch to 1 inch (Zarrow, Neher, Sikes, Brennan and Bul- lard, 1956). Measurements were made by the passage of aluminum rods, and, al- though the technique is not too exact, the differences are significant. Stilbestrol given alone or in combination with progesterone had no effect on the cervical dilation. On the other hand. Smith and Nalbandov (1958) have recently reported that estrogen treatment constricted the uterine cervix of the sow and that relaxin was without effect. A cue with respect to the mechanism of ac- tion of relaxin is given by the similarity of the action of relaxin on the pubic sym- physeal ligament and the uterine cervix. In both instances, an increase in water content and a marked dei)olymerizatioii occurs. Cullen and Harkness ( 1958) observed re- laxation of the uterine cervix of the rat with estradiol alone, or with estradiol and pro- gesterone, or with estradiol and relaxin, but maximal dilation was obtained only with a combination of estradiol, progesterone, and relaxin. In general Kroc, Steinetz and Beach (1959b) obtained comparable results in the rat. Estrogen alone caused some in- 1012 SPERM, OVA, AND PREGNANCY 14 PREGNANCY Fig. 16.35. Dilation and ten.sile strength of the uterine cervLx of the rat during estrus, pregnancy, and 2 days postpartum. The dihition of the cervix in mm. of stretch per 50 gm. of added weight. The tensile strength is expressed in grams force necessary to tear 1 mg. cervical tissue in a rat weighing 100 gm. E = estrus; P = parturition. (From J. Yochim and M. X. Zarrow, Fed. Proc, 18, 174, 1959.) crease in dilatability when 5 fxg. estradiol cyclopentylpropionate were given, and a de- crease when 50 /Ag. were given. Progesterone had no consistent effect either alone or in estrogen-primed animals. Relaxin alone caused some softening of the cervix, but gave a maximal effect only when given with progesterone in estrogen-primed animals. Normal cervical dilation was also obtained in pregnant rats castrated the 15th day of gestation and maintained with progesterone, estradiol, and relaxin (Kroc, Steinetz and Beach, 1959; Yochim and Zarrow, 1959). Data on dilation of the uterine cervix of the mouse are rather sparse; nevertheless, sof- tening of the cervix with relaxin has been reported (Kroc, Steinetz and Beach, 1959). It is not the purpose of this review to evaluate the data on cervical softening in the human female. The nature of the action of relaxin in the human female is contro- versial. Nevertheless, softening of the cervix following treatment with relaxin has been reported (Eichner, Waltner, Goodman and Post, 1956; Stone, Sedlis and Zuckerman, 1958) although McGaughey, Corey and Thornton (1958) reported no effect on the cervix following relaxin. GESTATION 1013 cr UJ X t— O . c cr •= , <=• UJ -J UJ t^'- ir ^ CD UJ < < > O I- ^ ^ ^ 111 ^ Q O cn 03 _i UJ Z) < CO Q. (J ^s > UJ r: cr U. 5 UJ u. - o o d z 6.0. 5.0- 4.0- 3.0 2.0 1.0 0.0. o o CERVICAL DILATABILITY (5-12 MICe/POINT) ^^ - INTERPUBIC LIGAyCNT • • (5-12 MICE/POINT) □ D RESPONSE TO OXYTOCIN ( 3-25 MICE/POIMT) STANDARD ERROR OF THE MEAN DIESTRUS ESTRUS IS 16 17 18 19 20 CYCLE DAYS PREGNANT 1 DAYSP 4 OSTPA RTUM PREGNANCY PARTURITION Fig. 16.36. Increased length of the pubic ligament, inciea.sed cervical dilatability, and increased responsiveness to oxytocin with the length of pregnanc.y in the mouse. L = lactating; NL = not lactating. (From B. G. Steinetz, V. L. Beach and R. L. Kroc, En- docrinology, 61, 271, 1957.) XII. Uterine Myometrial Activity The classical and well known description of uterine muscular activity has been more than adequately reviewed by Reynolds (1949). Since then Csapo and his colleagues have reported a series of elegant experi- ments involving the action of estrogen and progesterone on the uterine myometrium and have evolved the concept of "i)rogester- one block" in the control of uterine activity (1956a, 1956b). It has been shown that the ovarian steroid hormones regulate myo- metrial activity and that the uterine con- tractions are dependent on the relative amounts of the two hormones. Contractility is dependent basically on the concentration of the high energy phosphates which are maintained by estrogen w^iich in turn is involved in the synthesis of these substances (Csapo, 1950; Menkes and Csapo, 1952). Discovery of the staircase phenomenon in the uterine myometrium similar to that ex- hibited by cardiac muscle led to a marked difference between the action of estrogen and progesterone (Csapo and Corner, 1952 ) . With decreasing freciuency of electrical stimidation in an isometric arrangement, tension decreased if the uterus was domi- nated by estrogen and increased if it was dominated by progesterone. Uteri from cas- trated rabbits were insensitive to the fre- quency of electrical stimulation. Thus estro- gen induced a "positive staircase" response and progesterone a "negative staircase" re- sponse, although in the latter instance some estrogen is also present. These staircase re- sponses have been used successfully as a measure of hormone domination and have been shown to be a function of the Na+ and K+ gradients across the myometrial cell membrane. Uterine motility during estrus, the di- estrum, and pregnancy has been described by many investigators in great detail (for a review see Reynolds, 1949). The diestrous uterus shows extremely slow, feeble, unco- ordinated movements. The contractions may arise in any part of the uterus and extend in any direction. At estrus, the uterine con- tractions become rhythmic and sweep over 1014 SPERM, OVA, AND PREGNANCY STAIRCASE Negative 9 § o p o o o o 0 ox 8 ox o 8 ox o K 0 1^ ox X Transient O o o o o 0 o o o o X X X X X X X Positive ox o X X X X X X X X X X ox X X X X X X X X X X 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Hours after mating Fig. 16.37. Change from a positive to a negative staircase as the hormone dominance of the myometrium moves from the estrus to the progestational state after mating. X and O indicate the two strains of rabbits used. (From B. M. Schofield, J. Physiol., 138, 1, 1957.) the uterine horn in a wave starting at the tubal end. Both amplitude and rate are in- creased. During pregnancy the uterus be- comes relatively quiescent. In general this pattern of myometrial activity has been re- produced with both hormones, estradiol and progesterone. Recently Schofield 11957), using the Csapo technique, has studied, in vivo, myo- metrial activity in the rabbit. In a series of experiments she was able to show in several strains of rabbits that, when mating occurs during estrus, the uterine myometrium is dominated by estrogen. Within 20 to 28 STAIRCASE Negative Transient Positive o o o o o 8x OX o ox X X X o o o ox X 26 27 28 29 30 31 Day of pregnancy 32 Fig. 16.38. Change from negative through transient to positive staircase as the hormone dominance reverses at the end of pregnane}', indi- cating estrogen dominance. X and O indicate the two strains of rabbits used. (From B. M. Schofield, J. Physiol. 138, 1, 1957.) hours after mating, the positive staircase effect passes through a transient effect to a negative effect indicating the development of progesterone dominance (Fig. 16.37j. This condition remained in effect through- out pregnancy until 24 hours before parturi- tion when a reversion to estrogen domina- tion was indicated by the positive staircase response ( Fig. 16.38) . Thus the progesterone- dominated uterus is maintained throughout pregnancy and the uterus is nonreactive to oxytocin. Csapo (1956a) and others have shown that labor cannot be induced by oxytocin in the rabbit before day 30 of ges- tation, but 24 hours later, on removal of the progesterone block, 96 per cent of the rab- bits delivered following treatment with oxy- tocin. He believes that the specific action of progesterone involves a blocking of the ex- citation-contraction coupling which is a consequence of the disturbed ionic balance in the myometrial cell. Thus a block is set up to the propagation of the contraction wave which can be removed only by a de- crease in the level of progesterone. The role of the water-soluble extract, re- laxin in myometrial activity, is still un- certain. That an inhibition of estrogen-in- duced uterine contractions is obtained in certain species, such as the rat, mouse, and guinea pig, with relaxin preparations is un- (luestionable. However, we still have not answered the questions as to w'hether this hormone plays a role in uterine contractions under normal physiologic conditions and whether the uterine contraction-inhibiting GESTATION 1015 substance is relaxin or a contaminant of the relaxin extract. Krantz, Bryant and Carr (1950) reported than an aqueous extract of the corpus lu- teum would produce an inhibition or de- crease of uterine activity in the guinea pig and rabbit previously primed with estrone. This has been amply confirmed with both in vivo and in vitro preparations involving spontaneous contractions measured isomet- rically in the guinea pig (Felton, Frieden and Bryant, 1953; Wada and Yuhara, 1956; JMiller, Kisley and Murray, 1957) , rat (Saw- yer, Frieden and Martin, 1953; Wada and Yuhara, 1956; Bloom, Paul and Wiqvist, 1958), and mouse (Kroc, Steinetz and Beach, 1959). However, Miller, Kisley and Murray (1957) failed to show any action of relaxin on uterine motility in the rabbit and the human being in vitro. Thus, the in- formation on the rabbit is contradictory and a similar situation exists with regard to the human female for whom both positive and negative results have been reported follow- ing treatment with relaxin for threatened abortion (McGaughey, Corey and Thorn- ton, 1958; Stone, Sedlis and Zuckerman, 1958; Eichner, Herman, Kritzer, Platock and Rubinstein, 1959). In briefly summar- izing the action of relaxin on the uterine myometrium it should be pointed out that ( 1 ) relaxin inhibits uterine motility in an estrogen-primed animal, (2) the action may be species-limited, and (3) relaxin treat- ment docs not interfere with the action of pitocin. XIII. Parturition A. PROGESTERONE A number of theories have been suggested to explain the hormonal control of parturi- tion. The most popular is that parturition is due to a decrease in the level of progester- one which allows oxytocin to exert its ef- fect on the uterus. Evidence has already been presented indicating that pregnancy can be maintained in the castrated rabbit by an extract of corpora lutea, or progester- one, and even prolonged in rats (Nelson, Pfiffner and Haterius, 1930; Miklos, 1930), mice (Mandelstamm and Tschaikowsky, 1931), and rabbits (Zarrow, 1947a). Snyder (1934) and Koff and Davis (1937) pro- longed gestation in rabbits by inducing the formation of new corpora lutea during the last trimester of pregnancy. Knaus (1930) originally noted a marked antagonism between posterior pituitary ex- tract and the corpus luteum hormone and Koff and Davis (1937) reported that in pro- longed gestation induced by progesterone, posterior pituitary extract was ineffective until two days after the last injection. Csapo (1956a) performed a series of elegant experiments and concluded that progester- one blocks the uterine contractions, and that premature labor could not be induced with oxytocin before the 30th day of gesta- tion in the rabbit except for a very small percentage of animals. This has been con- firmed by Fuchs and Fuchs (1958). Zarrow and Neher (1955) found the se- rum gestagen levels in the pregnant rabbit fell only after parturition was under way. Hence the problem arose as to how parturi- tion could begin while a high blood concen- tration of gestagen was present. A partial answer was obtained in experiments by Csapo (1956b) and Schofield (1957) who showed that the progesterone-dominated uterus of the pregnant rabbit becomes es- trogen-dominated and responsive to oxy- tocin 24 hours before parturition. Hence the concentration of progesterone in the serum is meaningless by itself and it could be theorized that the significant point is the ratio of estrogen to progesterone. Csapo (1956a), however, offered an alternative so- lution. He observed a local effect of pla- cental progesterone on the myometrium so that the myometrium closest to the placenta is under a greater progesterone-dominance than that portion of the myometrium lying more distant. Hence the local level of pro- gesterone would be the significant factor in the onset of parturition and not the systemic level. B. OXYTOCIN It is now generally believed that parturi- tion is the result of the action of the pos- terior pituitary hormone on the myome- trium of the uterus sensitized by estrogen. The development of this hypothesis followed from the well known fact that oxytocin pro- 1016 SPERM, OVA, AND PREGNANCY duces uterine contractions and induces labor and delivery of the young. It is apparent, however, that a mass of contradictory data exist and the hypothesis is still in need of better evidence before it can be fully ac- cepted (for review of early literature see Reynolds, 1949) . Some of the evidence supporting the above hypothesis is the fact of the presence, to a limited degree, of a deficiency syn- drome in parturition following removal of the posterior pituitary gland. The data, however, are still equivocal. Labor is ap- parently prolonged in the monkey (Smith, 1946) and guinea pig (Dey, Fisher and Ranson, 1941 ) after total hypophysectomy. Nevertheless, parturition will occur nor- mally after removal of the pituitary gland in the rabbit (Robson, 1936), cat (Allen and Wiles, 1932), mouse (Gardner and Al- len, 1942), and rat (Smith, 1932). Even where there is some indication of interfer- ence with labor, delivery occurs. However, the lack of consistent results and species differences may be due to the recent finding that the posterior pituitary hormones are synthesized in the hypothalamus and that removal of the posterior pituitary is only effective under limited conditions because the source of the hormone is still present. These experiments have also been criti- cized on the ground that the anterior pitui- tary was also removed and hence inter- ference with many other hormones occurred. Additional evidence in favor of a role for the neurohypophysis in the delivery of the young is the increase in uterine motility following stimuli that bring about release of the posterior pituitary hormones, and the lack of an effect on the uterus when release of the hormone is blocked. Positive evidence for the release of oxy- tocin at the time of parturition is still lack- ing as are measurements of the concentra- tion in the blood. Fitzpatrick (1957) takes the view that oxytocin is liberated as an es- sential part of normal parturition and cites the following evidence. (1) A superficial similarity exists between spontaneous labor and that induced by oxytocin. Harris (1955) also stresses the similarity in the uterine re- sponse to oxytocin and to electrical stimula- tion of the supraoptic hypophyseal nucleus. (2) Mechanical dilation of the uterus or cervix evokes an increase in uterine con- tractions presumably by way of a nervous reflex release of oxytocin (Ferguson, 1941). (3) Oxytocin is decreased in the posterior pituitary gland of the rat and the dog after labor (Dicker and Tyler, 1953). Evidence from the attempts to measure the concentration of oxytocin in body fluids at the time of parturition is inadequate. The early reports of higher concentrations in the urine (Cockrill, Miller and Kurzrok, 1934) and blood (Bell and Morris, 1934; Bell and Robson, 1935) during parturition are questioned because of the inadequate methods of extraction and lack of specificity in the assay. Recently, Hawker and Robert- son (1957, 1958) reinvestigated the problem and concluded that two oxytocic substances are present in the blood and hypothalamus of cats, cows, and rats and blood of women. However, they found that the concentration of oxytocin in the blood fell during labor from a high during pregnancy. It is ap- parent that this presents a paradoxical situ- ation in view of the fact that the concentra- tion of oxytocin is low at the time of parturition; a time when the hormone is supposedly exerting its greatest effect. The situation is further complicated by the pres- ence of two oxytocic factors and the pres- ence of an oxytocinase in the blood and l)lacenta (von Fekete, 1930; Page, 1946; Woodbury, Ahlquist, Abreu, Torpin and Watson, 1946; Hawker, 1956). Although more work is required on this problem and esi)ecially with regard to the specificity and concentration of the oxytocinase, there is some indication of a fall in enzyme level before parturition. Tyler (1955) reported a decrease in the blood level of the enzyme towards the end of pregnancy and Sawyer (1954) reported a decrease in oxytocinase activity in rat tissues at the end of preg- nancy. C. RELAXIN Recently, the discovery of the action of relaxin on the pubic symphysis, uterine cervix, and uterine motility has raised the question of the role of this hormone in par- turition. Certainly in the species that nor- mally show pubic relaxation, relaxin would appear to play a significant role. However, this phenomenon is a special adaptation and GESTATION 1017 the question of cervical dilatability becomes more important because it seems to occur in all species examined thus far. It would seem that relaxin can induce cervical di- latability in conjunction with the sex ster- oids and that cervical dilation is a necessary event in parturition, but whether relaxin controls this event under physiologic con- ditions is still unknown and direct evidence is unavailable. It is also apparent in some species that relaxin can inhibit uterine con- tractions w'ithout interfering with the action of oxytocin. Kroc, Steinetz and Beach (1959) reported that relaxin actually re- stored responsiveness to oxytocin in mice treated with progesterone. Again the ques- tion is raised as to whether this is merely a good experiment or a part of the normal physiologic events. In a general way the events leading to labor may be summarized as follows. As pregnancy approaches term, the uterus be- comes subject to increasing pressure from within, due to a differential change in the growth rates of the fetus and the uterus (Woodbury, Hamilton and Torpin, 1938). Concurrently, a reversal from progesterone to estrogen domination occurs, which also contributes to an increase in uterine tonus. As intra-uterine tension increases, spon- taneous contractions acquire a greater ef- ficiency and forcefulness. Because the radius of curvature in the human uterus is greater at the fundus than at the cervix, and be- cause the myometrium is thicker at the up- per pole (by a factor of 2) the contractile force is stronger at the fundus than at the cervical end. This contractile gradient i^ro- duces a thrust toward the cervix. Utilization of a type of strain gauge, the tokodynamometer, has afforded informa- tion on the rate and strength of contraction of the various parts of the parturient uterus simultaneously (Reynolds, Heard, Bruns and Hellman, 1948). These measurements have indicated that, during the first stage of labor, the fundus exerts strong contractions of rather long duration. The corpus exhibits less intense contractions, usually of shorter duration, which frequently diminish in force as labor advances. The lower uterine seg- ment is almost inactive throughout the first stage of parturition. According to Reynolds (1949), both the fundus and the midportion contract at the same time, but the fundus remains contracted for a longer period of time than the corpus beneath, thus building up a force downward. If cervical dilation has not occurred, the three areas of the uterus will continue to contract. As cervical dilation begins, the contractions in the mid- portion of the uterus decrease in intensity and the contractions in the lower segment disappear. Cervical dilation has been ob- served only when there is a preponderance of rhythmic activity of the fundus over the rest of the uterus. When amniotic fluid is lost after the rup- ture of the membranes, the absolute tension within the wall of the uterus is reduced so that the ratio of force between fundus and cervix is increased. Thus rupture of the membranes decreases the tension in the cer- vix more than the fundus and the net effect is an increased force from the fundus. This change tends to precipitate the parturition more rapidly. As pregnancy nears term, both increased tonus of the myometrium and rapid growth of the fetus cause a rise in intra-uterine pressure. This rise results in a decrease of effective arterial blood pressure in the pla- centa. During this period also, thrombosis is observed in many of the venous sinuses of the placenta and many of the blood ves- sels become more or less obstructed by giant cells. During parturition, the systemic blood pressure of the mother rises with each con- traction, but, due to the increased intra- uterine pressure produced by the contrac- tions, the effective maternal arterial blood pressure in the placenta decreases to zero. Thus maternal circulation is cut off from the fetus. Measurements of intra-uterine pressure at term show that the human uterus con- tracts with a pressure wave which varies from 25 to 95 mm. Hg (Woodbury, Hamil- ton and Torpin, 1938). The uterine wall is subjected to an average tension of 500 gm. per cm.- and, during delivery of the head, may, with the aid of abdominal muscula- ture, develop as much as 15 kg. force. In animals giving birth to multiple young (rat and mouse) evacuation of the horn pro- ceeds in an orderlv fashion beginning at the 1018 SPERM, OVA, AND PREGNANCY cervical end. As evacuation of the lowest implantation site starts, changes occur in the periods of contractions of segments of uterine artery near its entrance into the uterine wall (Knisely, 1934; Keiffer, 1919). Gradually the constriction phase becomes proportionately longer than the dilation phase until the arterial lumen is obliterated. The myometrium in the area of the con- stricting segments becofes more active and, after long intense local contractions of the uterine muscle, the fetuses and the placen- tas separate and are discharged through the dilated cervix. After evacuation, a relaxa- tion of the contracted segment of uterus occurs and the process is repeated at the next implantation site. Recently, Cross (1958) re-examined the problem of labor in the rabbit. He concluded that (1) oxytocin in physiologic amounts can induce labor that is comparable to the events normally seen, (2) oxytocin is re- leased during a normal labor, and (3) oxy- tocin can induce delivery without sup- plementary mechanisms. He noted that straining movements involving reflex ab- dominal contractions initiated by distention of the vagina and cervix aided in expulsion of the fetus. It is also possible that this might cause reflexly an increased secretion of oxytocin. Other reflex mechanisms have been suggested, but evidence is inadequate. Cross cites a report by Kurdinowski pub- lished in 1904 in which the entire process of labor and delivery in an isolated full-term rabbit uterus perfused with Locke's solution is described. In these experiments orderly delivery of the viable fetuses was affected by the contractile efforts of the uterus and vagina in absence of any hormonal or nerv- ous stimuli. XIV. Conclusion Although we have garnered much infor- mation, no major conclusions can be drawn at this time concerning gestation in the mammal. This is probably true because of the vastness of the subject and the lack of sufficient data, especially that of a com- parative nature. It is probably fitting to close this chapter with the final statement written by Newton in the second edition of Sex and Internal Secretion, "It seems rather that the investigation of endocrine rela- tionships during pregnancy is still in the exploratory stage and that the time is not ripe for systematization." It is true that many data have been ac- cumulated in the last two decades since the publication of the second edition of this book. It is also probably true that some sys- tematization can now be started. 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Zarrow, M. X., and Neher, G. M. 1955. Concen- tration of progestin in the serum of the rabbit dining pregnancy, the puerpurium and follow- ing castration. Endocrinology, 56, 1. Zarrow, M. X., Neher, G. M., Lazo-Wase.m, E. A.. and Salhanick, H. A. 1957. Biological activ- ity of certain progesterone-like compounds as determined by the Hooker-Forbes bioassay. J. Clin. Endocrinol., 17, 658. Zarrow. M. X., Neher, G. M., Sikes, D., Brennan. D. M., and Bull-ard, J. F. 1958. Dilation of the uterine cervix of the sow following treat- ment with relaxin. Am. J. Obst. & Gynec, 72, 280. Z.ARROW, M. X., AND RosENBERG, B. 1953. Sources of relaxin in the rabbit. Endocrinology, 53, 593. Zarrow, M. X., Sikes, D., and Neher, G. M. 1954. Effect of relaxin on the uterine cervix and vulva of young castrated sows and heifers. Am. J. Physiol., 179, 684. GESTATION 1031 Zakroav, M. X., AND Zarrow, I. G. 1953. Anemia in the rabbit dvu'ing pregnancy and following treatment with water soluble o\arian extracts. Endocrinology, 52, 424. Zeiner, r. N. 1943. .Studies on the maintenance of pregnancy in the white rat. Endocrinology, 33, 239. Zondek, B., and Sulmax, F. 1945. The mecha- nism of action and metabolism of gonadotro- phic hormones in the organism. Vitamins & • Hormones, 3, 297. ZrcKERMAN, S. 1935. The Aschheim-Zondek di- agnosis of pregnancy in the chimpanzee. Am. J.Phvsiol., 110, 597. Aclcleiitluin Several reports on an exteroceptive block to pregnancy in mice appeared since this manuscript was completed. In a series of three articles, Bruce (Nature, London, 184, 105, 1959; Science, 131, 1526, 1960; and J. Reprod. Fertil, 1, 96, 1960) has shown that exposure of newly mated, female mice to strange males caused an inhibition of preg- nancy that ran as high as 80 per cent. Prior removal of the olfactory bulbs abolished the reaction. The pregnancy block in tliis in- stance consisted in a failure of the blasto- cysts to implant. SECTION E Physiology of Reproduction in Submainmalian Vertebrates 17 ENDOCRINOLOGY OF REPRODUCTION IN COLD-BLOODED VERTEBRATES Thomas R. Forbes, Ph.D. SCHOOL OF MEDICINE, YALE UNIVERSITY, NEW HAVEN, CONNECTICUT I. Introduction 1035 This chapter resulted from an effort to bring II. Testis and Spermatogenesis 1035 together some of this information. In gen- Vv. ExcuTrent R™a."s''f^^^^ 1039 ^ral, the approach has been to discuss as- V. Secretory Specializations OF Mes- pects of the structure and function of the oNEPHRos AND Metanephros. . . 1040 rcproductive tract and later to outline what VI. Intromittent Organ 1041 ig known regarding their endocrine control. ^'"' ^^^ll^J'"'''-^'''^^'' Structures in ^^^^ ^j^^, attempt has been both to correlate what VIII. Effects' OF Orchiectomy.;......'. 1044 is known and to indicate what must still IX. Effects OF Androgens 1045 be explored. Several possible topics have X. External Transport of Eggs and been omitted, either because they are dealt ^ ouNG 1047 with elsewhere in this volume (see chapters XI. Ovary; Ovogenesis; Ovulation.. 1048 ^ ^ ^ ^^j^j Blandau, Bishop, XII. Sources of Estrogens 1050 ■' , ^' ' '. ' ^' XIII. Oviduct; Egg Transport 1051 doling) or because, arbitrarily, they were XIV. Other Specializatio.ns IN Females 1052 considered to be outside the scope of this XV. Effects of Ovarikc tdmy 1052 review. References in most cases have been XVI. Effects of Estrogens AND Proges- ^^^^j ^^ ^ representative but not total XVII. terone 1053 u ■ r^ ■ ■ j j Fertilization; Sperm Storage in ^asis. Generic names since superseded are XVIII. Females 1054 retained, although usually in parentheses, Oviparity AND Ovovi viPARiTY . . 1056 to asslst tlic reader who seeks out the origi- XIX. Viviparity 1057 y^^\ paper XX. Corpus Luteum .^ ^ 1058 Special" attention is directed to the par- XXI. Developmental Basis for Sexual ^- \ ^ ^ , ,• • , . ' „ XXII. Dimorphism 1060 ticularly useful discussions and reviews of Spontaneous Adult Hermaphro- Cunningham (1900), Bridge (1910), Noble XXIII. ditism AND Sex Reversal 1062 (1931), Regnier (1938), Vols0e (1944), Experimental Sex Reversal 1065 Bretschneider and Duyvene de Wit (1947), ^xxv! References. :;::::::::::.:; 1070 po^^e (1949) , Hoar ( 1955, 1957a) , Marshall (1956), Harrison ]\Iatthews and Marshall I. Introduction (1956), and Pickford and Atz (1957). To The cold-blooded vertebrates, particu- t^!^''^^ ^."^ ™^y. ^^h^r sources and to the larly fish and reptiles, are forms studied l)v ^^■^•^;: "^^*/f .«Vf "^^^^^^^"'^1^^ ^^'^ ^''^^'^' '' relatively few investigators of reproductive l>''oioundly indebted, endocrinology. Even so, enough information H. Testis and Spermatogenesis has accumulated to emphasize that prob- Icnis ill the biology of reproduction of the '^ lowci- \'ertebrates are varied, phylogeneti- The six'rmatogenetic process is seasonal cally significant, and altogether fascinating, in some fish and more or less continuous in 1035 103(5 SUBMAMMALIAN VERTEBRATES others. The testis of the perch is smallest from late June to late August (northern hem- isphere). Spermatogenesis then proceeds rapidly enough so that by early November the gonad has reached its greatest size (Turner, 1919). In the stickleback, Gaster- osteus pungitius, spermatogenesis occurs in the winter and spring (van Oordt, 1924) , but it extends through the autumn, winter, and spring in G. aculeatus (Courrier, 1921c, 1922a, b; Craig-Bennett, 1931). Testicular volume, an indicator of spermatogenetic rate, in the top-minnow, Gambusia affinis, in summer is eight times as great as in winter (Geiser, 1922). In Fundulus sperma- togenesis is at its height in late May, June, and July (Matthews, 1938). The process l)t'gins in June or July in the salmon parr, Salmo solar, and ripe sperm have accumu- lated by October or November (Jones, 1940; Jones and Orton, 1940). (A parr is a young salmon which has not yet migrated to the sea.) This fish, incidentally, may be pacdogenetic, i.e., it may spawn before be- coming adult (Jones and Orton, 1940). In Brachijraphis episcopi, related to Gambusia, on the other hand, reproductive cycles may occur in any month (Turner, 1938a). In higher orders of fish spermatogenesis occurs in testicular lobules which are the homologues of the seminiferous tubvdes of the amniotes (Oslund, 1928). It is common for the ripe sperm to form balls or cysts within the testes (Conel, 1917; Turner, 1919; Okkelberg, 1921; Geiser, 1922; Os- lund, 1928; Vaupel, 1929; Matthews, 1938). In the basking shark, Cetorhinus maximus, cilia rotate the masses of sperm into balls while testicular epithelial cells secrete con- centric layers of investing material. The resulting spermatophore is 2 to 3 cm. or more in diameter (Harrison Matthews, 1950). The sperm ball of Lebistes reticu- latus contains thousands of germ cells; their tails are directed toward the center and intertwine to hold the ball together (Vaupel, 1929 ) . The cephalic end of the mesonephros of the elasmobranch Chimaera secretes a substance which similarly agglutinates the sperm into a spermatophore (Parker and Burlend, 1909). Sperm storage in the male has been re- ported in Gambusia. This fish breeds from spring until September, and again in No- vember, spermatozoa remaining in the testis for release the following spring (Self, 1940). Spawning occurs in the early spring in the three-spined stickleback, Gasterosteus aculeatus aculeatus (Ikeda, 1933), and in the spring and early summer in the carp and goldfish (Scruggs, 1951), top-minnow (Car- ranza and Winn, 1954), perch (Turner, 1919), and striped bass (Pearson, 1938). In Fundulus, sperm are shed in June, July, and even August (Matthews, 1938). Fertili- zation is internal in the basking shark, Cetorhinus maximus, the clasper being used as an intromittent organ; mating occurs in late spring and early summer (Harrison Matthews, 1950). Eels spawn in the spring and summer. The wonderful story of their migration from as far away as Europe to the breeding grounds in the western Atlantic Ocean and of the eventual return of the young, sometimes again to rivers 3000 feet above sea level in Switzerland, is one of the most remarkable known to naturalists. The eel's migration was described by the Danish biologist, Jobs. Schmidt (1922) ; the reader is urged to consult his paper. Gerbil'skii's (1955) studies of sturgeons {Acipenser giildenstddti, A. stellatus, and Huso huso) reveal that within each species there may be several "biologic races." The time of spawning of the race depends on the river system in which the race lives and may vary by several months from the spawning time of other races of the same species. Temperature is believed to be one of the factors involved. Atrophy of the testis in old age has been reported in the myxinoids Bdellostoma and Mijxine (Conel, 1917) and in a teleost, Astyanax mexicanus (Rasquin and Hafter, 1951). Amphibians In Salamandra, the female may be in- seminated at any time between February and October (Baylis, 1939). Spermatogene- sis occurs in Tritunis in summer and early fall. The testes reach their maximal size in July and August. Actual mating, how- ever, is deferred until the period from April through June, the mature sperm being stored over the winter (Adams, 1940). Eurycea COLD-BLOODED VERTEBRATES 1087 breeds in late March and tlie first part of April. By the latter date, the store of ma- ture sperm is exhausted. In June the masses of spermatogonia in the testicular lobules have begun to transform into primary sper- matocytes. Spermatids are seen in late July, and mature spermatozoa are present before the first of September (Weichert, 1945). Spermatogenesis in Necturus extends from April through August, with shedding of sperm from late August to early October. Thereafter the testes are inactive until the following spring (Aj^lington, 19421. The toad, Bujo bujo, breeds in the sjiring, and the testes are largest then. Spermato- genesis begins in the summer in preparation for the next spring (Ting and Boring, 1939). Sperm production is also cyclic in Rana (Aron, 1926; van Oordt and van Oordt, 1955). Control of spermatogenesis has been proven to be largely through the pituitary gland (see chapter by Greep). Release of spermatozoa has also been effected in Rana by the injection of epinephrine, even after hypophysectomy or total section of the spinal cord. Bufo, however, did not make this response to the administration of epi- nephrine (Li and Chang, 1949). Except for two primitive families, all salamanders enclose masses of their sper- matozoa in sacs called spermatophores. The latter are formed by specialized male eloacal glands (Dunn, 1923; Noble, 1931) which presumably are under endocrine control. Reptiles The organization of the reptilian testis is in general like that of higher vertebrates. True seminiferous tubules are present. The period of active spermatogenesis is evident on macroscopic examination, because the testis increases in size as the germ cells multiply and accumulate. In Testudo orbic- ularis, the musk turtle, Sternotherus odo- ratus, the box turtle, Terrapene carolinensis, and the Algerian Emys leprosa, spermato- genesis begins late in the spring and con- tinues until early in the fall (Pellegrini, 1925; Risley, 1938; Hansen, 1939; Altland, 1951; Combescot, 1954a). These turtles mate in the spring, whereas the musk turtle mates in the spring and fall (Risley, 1938; Combescot, 1954b). In lizards, active sper- matogenesis may, depending on the species, take place at almost any time of year, with a peak of spermiogenic activity, fol- lowed by mating, appearing perhaps most commonly in the spring and early summer.^ In the snake, Thamnophis, spermatogenesis begins late in the spring and is most active in June and July. Spermiogenesis is con- spicuous in August and October, and mat- ing occurs in the spring (Cieslak, 1945; Fox, 1952). Spermatogenesis is continuous in Vipera berus except during hibernation, and mating begins in late April or early May (Volspe, 1944). The alligator ap- parently breeds in the spring (Reese, 1915), but information on the mating activities and spermatogenesis in the Crocodilia is surprisingly scanty. III. Sources of Male Hormone Fish Fish testes produce androgen. Hazlcton and Goodrich (1937) and Potter and Hoar (1954) prepared extracts of salmon testes which when bioassayed showed male hor- mone activity. (The effects of castration will be described later.) Which testicular cells produce androgen is controversial. In the higher vertebrates, the interstitial cells (Ley dig cells) are usually believed to se- crete male hormone, and it was natural to search for these cells in fish. Champy (1923a, b) and van Oordt (1923, 1924, 1925) concluded that male hormone does not come from interstitial cells in Tinea indgaris, Pho.rinus laevis, Gasterosteus pungitius L., and Xiphophorns helleri. Courrier (19211)) and Wcisel (1949) did not find interstitial cells either in primitive elasmobranchs or in two teleost species. Such cells have, however, been noted in the testes of other species (Courrier, 1921b, 1922c; Kolmer and Scheminzky, 1922a). Further- more, interstitial cells undergo maximal development (thus seeming to signal secre- tory activity) just before and during the breeding period; their presence may be 'Pellegrini. 1925; Frankenberger, 1928; Cour- rier, 1929; Altland, 1941; Breckenridge, 1943; Rey- nolds, 1943; Dutta, 1944; Kehl, 1944b; Woodbury and Woodbury, 1945; Miller, 1948, 1951; Kitada, 1951; De8.sauer, 1955; Fox, 1958. 1038 SUBMAMMALIAN VERTEBRATES partly obscured, before spawning, by the crowding and distention of the testis with sperm.- Follenius (1953) showed that if prepubertal Lebistes are exposed to x-rays the testes become completely sterile and Sertoli cells are also destroyed, but second- ary sex characters nevertheless develop on schedule. Since the interstitial cells are the only functional tissue remaining, this ex- ])criment is strong evidence for origin of androgen from these cells. In several tele- osts in which intersitial cells are not seen, there are specially developed cells in the wall of the testicular lobule or crypt (Hoar, 1957b; Marshall and Lofts, 1956). When the spawning season approaches, these "lobule boundary cells" acquire fat inclu- sions, give a positive test for cholesterol, and generally resemble the interstitial cells of higher vertebrates. Amphibians There are conflicting opinions as to wiiether androgen is produced by the in- terstitial cells of the amphibian testis. In- deed, it is likely that the term "interstitial" is not appropriate in this case. The urodele testis does not have seminiferous tubules, l)ut rather seminiferous lobules or cysts (Perez, 1921). Between the lobules there are only thin layers of connective tissue which, according to Perez, is not at all homologous with the interstitial tissue seen between the seminiferous tubules of higher vertebrates. Aron (1924a, 1926), on the other hand, refers to testicular interstitial tissue in Bana and says that its cyclic evolution is correlated directly with the seasonal de- velopment of secondary sex characters, par- ticularly the growth of glands in the callos- ity of the thumb. This has been confirmed in Discoglossus, another anuran, in which interstitial tissue is said to be abundant (Bcnoit, Kehl, and Leportois, 1941; Kehl, 1 944a ) . Because the administration of tes- tosterone also induces development of the callosity of Discoglossus, it is concluded that the interstitial cells produce andro- gen. The male secondary sex characters of -Couirier, 1921a, c, 1922a, b; Craig-Bennett, 1931; Stephan and Clavert, 1938; Potter and Hoar, 1954 ; Marshall and Lofts. 1956. Triturus, a newt, are highly developed dur- ing the fall, winter, and spring; at the same time a variety of mature cells— sperm, in- terstitial cells, and Sertoli cells — is also abundant (Adams, 1940). Noble (1931) has much support for his conclusion that in the amphibian testis male hormone is derived from sperm or Sertoli cells rather than from interstitial cells. In the urodele some of the testicular stromal cells develop into interstitial cells, but only after the breeding season, when, of course, the secondary sex characters have regressed. Thus, although the interstitial cells increase in number, size, and lipid con- tent, thereby coming to resemble mamma- lian interstitial cells, there is no direct evidence for their endocrine function, and the reason for their development is unex- plained. In addition, these cells are en- tirely undeveloped at the time when the sex characters indicate by their prominence that male hormone is being released.-^ Fur- ther research on the source of testicular androgen is required. Reptiles The rei)tilian testis produces male hor- mone, as shown by the results of castration. Valle and Valle (1943) extracted the testes of the rattlesnake, Crotalus t. terrificus, and of the related viper, Bothrops jararaca; 10 mg. testicular tissue contained enough androgen for positive results in two non- quantitative biologic tests. The role, if any, of interstitial cells in the production of androgen in reptiles has not been established. Interstitial cells have been seen in turtles of the genera Emys, Terra- pene (Cistudo), and Testudo; the cells in- crease in size and lipid content before and during the breeding season.'* Since inter- stitial cell volume decreases in June when the epididymis is undergoing seasonal de- velopment, the interstitial cells are prob- ably not responsible for epididymal changes (Dornesco, 1926a). In Terrapene the in- crease in cell size may be related to the se- =* Humphrey, 1921, 1925; Champy, 1922a, b, 1923c, 1924, i932; Harms, 1926a; Oslund, 1928. 'Ganfini, 1902; Pellegrini, 1925; Dornesco, 1926a; Stieve, 1933; Altland, 1951; Combescot, 1954a. COLD-BLOODED VERTEBRATES 1039 orction of male hormone (Altland, 1951). In the musk turtle, Sternotherus, on the other hand, interstitial cells are present but show no seasonal variation (Risley, 1938). Franz von Leydig (1821-1908), the Ger- man anatomist and zoologist whose name enters into the eponym for the interstitial cells, observed them (1857) in the testis of a lizard, Lacerta agilis. Since then, the cells have been seen in the testes of several lac- ertilians.^ Herlant (1933) and Regamey (1935) state unequivocally that in the liz- ards they studied the development of the interstitial gland and of the secondary sex characters is correlated. Interstitial cells have also been noted in the testes of several snakes.^ Herlant (1933) reported no sea- sonal variation in number, size, etc., of the interstitial cells in Vipera and Tropido- yjof.us, but Vols0e (1944) observed maximal development of the cells in Vipera in the early spring, shortly before the mating sea- son. Fox (1952), reviewing the situation for reptiles generally, concluded that "the maximal interstitial cell size occurs during the breeding period although in certain spe- cies the cells may be relatively large during other seasons as well." Since secondary sex characters become conspicuous during the breeding season and the interstitial cells give indication of activity at this period or a little earlier, the presumption is strong that the two phenomena are related. Interstitial cells are absent from the testis of the innnature alligator (Forbes, 1940a). There seems to be no information on whether interstitial tissue occurs in adult Grocodilia. ""AjioHs (Fox, 1958), Phrynosorna (Blount, 1929), Uromasiix (Kehl, 1935, 1944b), Hemidacty- bis (Dutta, 1944), Xantusia (Miller, 1948), Scincus (Kehl, 1935, 1944b), Acanthodactylm (Kehl, 1935, 1944b), Takydromus (Takewaki and Fukuda, 1935a), Lacerta (Frankenberger, 1922; Reiss, 1923a, b; Pellegrini, 1925; Herlant, 1933; Stieve, 1933; van den Broek, 1933; Regamev, 1935), Aiiguis (Ganfini, 1902; Dakq, 1920; Herlant. 1933). Helo- derma (Pesce, 1935), and Varanus (Hoberer, 1930; Kehl, 1935, 1944b). "Boa (Ganfini, 1902), Matrix (Tropidonotus) (Herlant, 1933), Coluber (Zamenis) (Ganfini, 1902; Pesce, 1935), Thamnophu (Eiitcnin) (Cies- lak, 1945; Fox, 1952), and Vipcm (Herlant, 1933; Vols0e. 1944). IV. Excurrent Pathways for Sperm Fish In cyclostomes the testicular cysts rup- ture and release mature sperm into the coe- lom, from whence they escape to the ex- terior by way of a genital papilla (Conel, 1917; Hoar, 1957a). In higher forms sperm are carried in "vasa deferentia," but these, according to Eggert (1931), are not homol- ogous with the structures of the same name in amniotes. ''Vasa deferentia" and testes may be paired or single (Geiser, 1922; Oslund, 1928; Matthews, 1938; Chavin and Gordon, 1951). Courrier (1922b) observed seminal vesi- cles in the stickleback, as did Eggert (1931) in Periophthalmus, but the latter believes that the seminal vesicles are not homolo- gous with those of mammals. In Gillichthys the vesicles are large structures caudal to the testes proper. Their function is uncer- tain, but may simply be to store sperm. They do not change their appearance sea- sonally, are not affected by the administra- tion of testosterone, and presumably are not under endocrine control (Weisel, 1949). The sperm ducts are secretory in the tele- ost, Astyanax (Rasquin and Hafter, 1951). The goby and the blenny have glandes an- nexes at the caudal ends of the testes; when the interstitial tissue of the testis is in- creased in volume, the tubes of the glandes annexes are dilated with colloid secretion. These are glands of external secretion ; their product has been compared to that of the prostate, but the function of the secretion is uncertain (Vivien. 1938; Coujard, 1941a, b). Amphibians The duct systems whereby sjierm are col- lected and conveyed to the exterior have been comprehensively described by Noble (1931) in his admirable book on the am- phibians. Several fine tubules, the vasa ef- ferentia, join the testis directly or indirectly to the mesonephros, which in amphibians is also the functional kidney. In most genera the vasa efferentia empty into glomerular capsules. The sperm then traverse the uri- nary collecting tubules and the meso- nephric (Wolffian) duct to reach the cloaca. 1040 SUBMAMMALIAN VERTEBRATES This pattern is somewhat modified in dif- ferent genera. In Rana and Alytes (Noble, 1931), and in Eurycea (Weichert, 1945) and Triton (Aron, 1924b), the terminal part of the mesonephric duct is distended by accumulated sperm and thus functions as a kind of seminal vesicle. Reptiles In all the amniotes, the mesonephros is eventually supplanted as a urinary organ by the metanephros, or permanent kidney. The mesonephros, however, is immediately ad- jacent to the gonad and in the adult male serves as a collecting and conveying mech- anism for the mature germ cells. In rep- tiles a few rete tubules or canals join the terminal ends of the seminiferous tubules to some of the mesonephric glomerular capsules; in many lizard species the rete canals are reduced to one (Alverdes, 1926, 1928; Regamey, 1935). The glomeruli them- selves disappear after they cease to func- tion, but the excurrent i)athway i)rovided by the mesonephric tubules and the meso- nephric duct persists, affording the sperm a continuous route into the cloaca. In the adult reptile, as in higher vertebrates, the mesonephros is often referred to as the epi- didymis, and the mesonephric duct as the vas deferens. V. Secretory Specializations of the Mesonephros and Metanephros Fish Fishes lack the metanephros of the am- niotes. Instead, most of the mesonephros continues to function as a kidney. The an- terior end of the mesonephros may be mod- ified in the male as a collecting organ for the sperm. In various species of stickleback {G aster osteus) a further and very inter- esting specialization has occurred. The male cares for the young. One of his functions is to build a nest, collecting and assembling plant debris, and sticking it together with a special secretion, a kind of waterproof cement. In the spring, as the time for nest- building approaches, the diameter of the tubules in the secretory portion of the mes- onephros increases considerably. The epi- thelial cells of the tubules become three or four times as tall as before and fill with eosinophilic secretory granules which liqu- efy to produce the glutinous cementing substance for nest-building. This specializa- tion does not occur in the female.'^ Amphibians Elevated, granule-filled epithelial cells lining the mesonephric collecting tubules and duct are a male sex character in Triton alpestris and T. cristatus (Aron, 1924bj. Their function is not apparent. Reptiles Cyclic changes in the height and ap- parent secretory activity of the epithelium investing the epididymal tubules have been noted repeatedly (van den Broek, 1933). In the turtle Terrapene (Cistudo), epithelial cell height rises steadily from 14.5 fx in Feb- ruary to 37.6 /x in July, declining thereafter to 11.8 fji in October. The increase in cell height correspondingly reduces the internal diameter of the tubule (Dornesco, 1926b). Van der Stricht (1893) apparently was the first to note the glandular nature of the epididymal epithelium of a lizard, Lacerta vivipara, and of the legless lacertilian, An- guis fragilis. In Lacerta, Hemidactylus, and Anguis there are four well defined seasonal periods for the epithelium lining the epi- didymal tubules: repose (autumn and win- ter), presecretion (February and March), secretion (April to June), and reconstruc- tion (late June and early July). During the secretory phase tlic tul>ules are distended with a milky liciuid containing sperm (Henry, 1900; Reiss, 1923b; Regamey, 1935). Secretory activity has been observed during the breeding season in the epididy- mides of lizards.^ When an epididymis of Takydromus was surgically isolated, the contained sperm retained their activity for at least 70 days. This finding implies the possibility of prolonged epididymal sperm "Mobius, 1885; Borcea, 1904; Hess, 1918; Counier, 1921c, 1922a, b; van Oordt, 1924; Craig- Bennett, 1931; Ikeda, 1933; van den Broek, van Oordt, and Hirsch, 1938. ^ Anolis (Fox, 1958), Emneces (Reynolds, 1943), Hemidactylus (Dutta, 1946), and Takydromus (Takewaki and Fukuda, 1935a). COLD-BLOODED VERTEBRATES 1041 storage. Readers interested in the detailed anatomy of the hzard eiiididyniis arc re- ferred to the accounts of Alverdes (1926, 1928). There are no true seasonal changes in the ductuli effcrentes (tubules connecting testis and epididymis) of the viper, Vipera berus (Vols0e, 1944). Each epididymal tu- bule has high, secretory epithelial cells in one portion and low, nonsecretory epithelial cells in the other. Chromophilic secretion granules accumulate in the apices of the secretory cells at all seasons and are even- tually released into the lumen of the tubule. There seems to l)e no seasonal variation in the quantity of granules. Epithelial cells of the ductus epididymidis show consider- able seasonal variation in height (low in summer, high in winter) but little or no secretory activity at any time. The ductus deferens also lacks secretory activity. In the garter snake, Thamnophis, epithelial cells are low in April and May and high in sum- mer, fall, and winter (Fox, 1952). Gampert ( 1866) first described what later came to be called the sexual segment of the metanephros. In the nephron of the com- mon water snake, Tropidonotus natrix, a special segment is interposed between the distal convoluted tubule and the collecting tubule. Tribondeau (1902) described a tube intermedaire in the kidneys of T. viperinus and in Vipera aspis. He called attention to the segment's distinctive secretory granules and speculated as to its function. Later it was determined that in Tropidonotus, Col- uber {Zamenis}, and Vipera, the segment in question is not the last but next to the last, or preterminal, preceding the final portion of the nephron which in turn empties into the collecting tubule (Regaud and Policard, 1903a, b, c, d). Cells from the segment could be kept alive in vitro for 3 days in salt solution and could be stained supra- vitally. In some tubes, the cells were accu- mulating granules, whereas elsewhere gran- ules were being discharged. It was further noted that a large preterminal segment oc- curs in male but not in female Lacerta and Anguis. The term segment sexuel was therefore applied to the structure. Finally, it was discovered that the sexual segment shows seasonal variations at the same time as does the testis, bespeaking androgenic control. Many studies have since been made of the sexual segment in lizards. General agreement emerges that its epithelial cells in the adult male become so tall during the breeding season that the segment is macro- scopically hypertrophied. The apices of the cells are crowded with protein-like secre- tion granules which push the small round nucleus to the base o^the cell.'' In Taky- dromus, not only the sexual segment but the terminal segment, collecting tubules, and even the ureters of the adult male are lined with epithelial cells showing conspic- uous secretory activity during the breeding period (Takewaki and Fukuda, 1935a). Among adult male snakes, the pretermi- nal segment lacks seasonal variation in Natrix {Tropidonotus) and Thamnophis (Eutenia) sirtalis but does show seasonal secretory activity in T. r. radix, T. elegans terrestris, and Vipera.^*' Curiously enough, the sexual segment has not been found in turtles (Regaud and Po- licard, 1903c, d; Herlant, 1933). Informa- tion is lacking to as to a possible sexual segment in the Crocodilia. VI. Intromittent Organ Fish In viviparous fish, sperm are transferred to the female, at least in some species, with the aid of an intromittent organ. Male elasmobranchs have modified pelvic fins which are used as "claspers" during copu- lation. The further modification of the clasi)er as a phallus-like organ for internal fertilization has been described in Scijlliiim, Acanthias, Raia (Leigh-Sharpe, 1920), and Cetorhinus (Harrison Matthews, 1950). The specialization of this organ in the frilled shark, C hlamydoselachus anguineus, is remarkable. The male discharges into a groove on the medial side of the clasper a mixture of sperm from the urogenital pa- jiilla and sea water from the siphon sacs. "Zamik, 1910; Reiss, 1923b; Dornesco, 1925; Cordier, 1928; Courrier, 1929; Matthev, 1929; Kelil, 1935, 1944b; Regamev, 1935; Forbes, 1941; Reynolds, 1943; Fox, 1958. '"Coi-dier, 1928; Waters, 1940; Takewaki and Hatta, 1941; Vols0e, 1944; Fox, 1952. 1042 SUBMAMMALIAN VERTEBRATES At copulation the mixture is ejected into the oviducts of the female (Gilbert, 1943). The anal fin of species of Xiphophonis, Molliensia, Gambusia, and Fundidus be- comes specialized after puberty into a gono- podium which is used as an intromittent organ (van Oordt, 1925; Vaupel, 1929; Turner, 1941a, b; Cummings, 1943a). In some poeciliids this fin, although elongated, is not used for intromission, but instead is employed to "fan" the ejected sperm toward eggs which also have just been discharged (Newman, 1907). The male's anal fin is employed for in- ternal fertilization by at least one oviparous fish, Apogon imberbis. The fertilized eggs are then deposited in a clump which the male picks up, carrying the mass in his mouth during most of their incubation (Garnaud, 1950). One wonders how hungry the father becomes and how lie resists temptation. Amphibians The majority of urodeles fertilize their eggs externally in the water, whereas nearly all female salamanders obtain sperm for internal fertilization by picking up sperma- tophores with their cloacal labia. According to Noble (1931), both Ascaphus, a very primitive frog, and the Gymnophiona, an order of limbless, burrowing amphibians, practice internal fertilization with the aid of an intromittent organ which is simply a muscular, highly vascular extension of the cloaca. The rectus abdominis muscles of Ascaphus draw this copulatory tube an- teriorly so that it can be thrust into the cloaca of the female. Since N ectophryno'ides occidentalis is viviparous (see below) it must also practice internal fertilization, but the mechanism is unknown (Angel and La- motte, 1944). Reptiles In lizards and snakes the paired penial sacs are posterior diverticula of the cloaca and lie caudal to it in pouches under the skin of the tail. Before mating the sacs are drawn by muscle action into the cloaca and then, as they become erect, evert themselves like the fingers of a glove through the cloacal outlet. The semen passes through a spiral furrow in each penis. The latter is then withdrawn into its recess by an elon- gated retractor muscle. In turtles and crocodilians the single, arched phallus is a solid, cavernous struc- ture. It is attached to the ventral wall of the proctodeum (the terminal chamber of the cloaca), from which it is everted on erection. The semen is carried in a deep groove along the convex penial surface, the groove being converted into a canal during erection." In these reptiles the phallus of the female is morphologically similar to, but much smaller than, that of the male. VII. Other Specialized Structures in Males Fish Evidence will be presented later that the dimorphism in size and color seen in many species is at least partly under the con- trol of the sex hormones. Sometimes the di- morphism is extreme. For example, late in his life the male pink salmon, Oncorhynchus gorbuscha, grows a huge hump on his back, and his head becomes large and bizarre in appearance (Davidson, 1935). The adult male sockeye salmon, 0. nerka, develops the dorsal hump, an elongated jaw, and a thickened skin (Weisel, 1943). The ''sword," a greatly elongated portion of the caudal fin which gives Xiphophonis its name, is an- other conspicuous male secondary sex char- acter (van Oordt, 1925). Amphibians Many male frogs and toads have wart- like excrescences on their fore limbs in the breeding season. These "nuptial callosities" help the male to cling to the female during amplexus. Bles (1905) described the "nup- tial asperities" (a delightfully ambiguous phrase) of Xenopus laevis, an African toad. Other species have similar structures, often spiny. Braun (1878) reported "large, round, black-colored warts" on the hind limbs of male Triton viridescens and erroneously supposed that these growths also made pos- sible a firm grip on the female during mat- "Wiedersheiin, 1886: Gadow, 1887, 1923; Coe and Kunliel, 1905; Moens, 1912; Reese, 1915; Nicholson and Rislev, 1941. COLD-BLOODED VERTEBRATES 1043 inp;, although he admits that he never oh- served this iirocess. Siniihir structures have l)ecn described on the medial surfaces of the hind limbs of Diemectiihis^ iTriturus) (Jordan, 1891). A dorsal crest or skin fold is connnon on adult male urodeles during the breeding season. The crest usually extends along the back and tail and in profile makes the ani- mal look much larger. Special masculine skin {)igmentation may occur. Secretions from hedonic glands on the chin and ad- jacent areas in Eurycea and Tritnrus per- \vdps attract females; these glands begin to accumulate their product in November and are distended by the start of the breeding season in March. Cloacal glands are a dis- tinctive male characteristic of Desmogna- thus, Triturus, and other salamanders. Sea- sonally, the epithelial cells of these glands become tall and accumulate granules. Pos- sibly the glandular secretion of Desmogna- thus may form the spermatophores.^- The male D. fuscus has a monocuspid premaxillary tooth which differs from that of the female (Noble, 1926). The linea masculina is a curious and dis- tinctive band of connective tissue lying at the dorsal and ventral edges of the obliquus abdominis muscles in various adult male ranid frogs, but not in toads (Liu, 1935; Davis and Law, 1935; Schmidt, 1938). Fore limb muscles (fiexor carpi radialis, extensor carpi radialis, abductor pollicis longus) are much larger in adult male than in female toads such as Bufo a. ainericaniis, ap- parently aiding the powerful and prolonged grasping of the female during amplexus. As Howell (1935), who described this in- teresting specialization, has remarked, it would be desirable to study the effects of gonadectomy and gonad transplant on these muscles. Reptiles The lizard's cloaca shows sexual speciali- zation. It is divided into an anterior cop- rodeum, receiving the small intestine, an intermediate urodeum, and a posterior proc- todeum. Adjoining chambers are separated ^-Jordan, 1891; Biesca, 1910; Anm, 1924b: Humphrey, 1925; Noble, 1926, 1931; Adams, 1940; Weiflioit, 1945. * l)y sphincter muscles. From the urodeum arises a dorsal urogenital diverticulum of varying size ; into this diverticulum or fossa usually open conjointly the ureter and vas deferens in the male and, separately, the ureter and oviduct in the female (Gadow, 1887; Regamey, 1935; Forbes, 1941). In the male Lacerta agilis (Regamey, 1933, 1935) the urogenital diverticulum, urodeum, and anterior proctodeum are lined in the spring with tall, columnar, stratified ( two or three layers ) epithelium containing many mucous cells. The total depth of the epithelium is 35 /x. In June and July, dur- ing the breeding season, the epithelium thickens to 40 to 45 ju,. The urodeum, an- terior proctodeum, and terminal portion of vas deferens (a urogenital fossa is lacking) of the adult male Sceloporus spinosus flori- darnis has a bilaminar epithelium (Forbes, 1941). Its inner stratum is formed of a single layer of columnar cells. Peripheral to them is a second layer, in most areas one cell thick, of cuboidal and low columnar cells. The regularly arranged nuclei of the two layers present a striking appearance. Opening into the anal vestibule, or ter- minal portion of the proctodeum, in male Lacerta and Sceloporus are cloacal glands. One pair lies anterior and ventral, the others (a single gland in Lacerta, a pair in Scelop- orus) lie i)osterior and dorsal, to the proc- todeum. The glands lack definitive capsules and consist of numerous lobules separated by thin but dense connective tissue septa. Each lobule forms an acinus-like epithelial pouch, sometimes containing a little secre- tion. Several lizard genera have femoral glands which are developed as male accessory sex structures.^-^ In Lacerta there are 16 to 21 glands on the anterior surface of each thigh. The glands lie just under the skin and open by way of a short duct which penetrates a conical elevation of the skin. The duct open- ings are visible macroscopically. The golden- yellow secretion of the glands is abundant dui'ing the bi'eeding season.'^ "von Leydig, 1872; Braim, 1877; Schaefer, 1901, 1902; Cohn, 1904; Tolg, 1905; Mahendra, 1936, 1953; Forbes, 1941. ''Felizet, 1911; Reiss, 1923b; Matthev, 1929; Suchow. 1929; Padoa, 1933; Regamey, 1935. 1044 SUBMAMMALIAN VERTEBRATES Few species of turtle show external sexual dimorphism. An exception is Teirapene Car- olina, in which the eye of the male is red whereas that of the female is brown (Blake, 1921). The male tuatara, Sphenodon punctatum, the sole living representative of the order Rhynchocephalia, is much larger than the female and has a conspicuous dorsal crest with white spines. In the breeding season the male's crest may become erect and swollen (Thomas, 1890). Remarkably little is known about the reproductive system and its function in this fascinating reptile. The gular skin fold which the lizard Anolis can erect from its throat is a striking mas- culine characteristic. Male lizards of some species distinguish themselves by dorsal crests and bright colors. Among the snakes, certain scales or scaly tubercles near the anus may show sexual di- morphism, and, as in the lizards, the slight bulging of the penial sacs on external ex- amination distinguishes the male from the female (Blanchard, 1931; Noble, 1934; Le- derer, 1942). The tip of the tail of the male fer-de-lance, Bothrops atrox, may be a bright yellow. There is reason to think that this wriggling, twisting tail-tip may lure lizards, toads, and other food within striking distance (Burger and Smith, 1950) . The male Paraguayan anaconda is smaller than the female, and his pelvic spurs are better devel- oped (Lederer, 1942) . Males of Engyrus car- inatus, a New Guinea bold snake, have pelvic spurs, whereas these structures are missing in most adult and all juvenile fe- males (Stickel and Stickel, 1946). Male boas and pythons use their spurs to scratch the body of the female during mating (Davis, 1936). Tubercles on the chins of some colu- brid snakes are tactile organs; if the tuber- cles are covered with tape, the male will not court the female (Noble, 1934). Davis (1936) has reviewed the role of spurs, tu- bercles, etc., in the courting beliavior of snakes. Although evidence will l)e presented later for endocrine control of the adult develop- ment of some accessory sex structures, it must be remembered that genetic factors also ])lay tlieir role. Relative degrees of con- trol exercised by genes and hormones con- stitute a significant area for research. VIII. Eflfects of Orchiectomy Fish Ablation of gonads helps to disclose the structures, physiologic processes, and be- havior which are partly or completely under the control of testicular or ovarian hormones ( see Pickford and Atz, 1957, for review ) . The most frequently observed result of the cas- tration of male fish has been a loss of the special, occasionally brilliant, skin color in those species with color dimorphism. Some- times there is a seasonal pigmentary change in the chromatophores, or pigment-bearing cells. Such change is associated with the time of spawning, and is referred to as Hoch- zeitskleid, or nuptial coloration. If castra- tion is performed before the breeding season, the nuptial coloration fails to appear; if during the season, the color may rapidly fade. Removal of only one testis has little or no effect; apparently the remaining gonad can release enough androgen to maintain normal coloration. Loss of male coloration due to castration has been demonstrat(>d in several genera. ^'^ The male Japanese bitterling also has pearl organs, 5 to 10 small dermal excres- cences like white warts on the anterior part of the head. Orchiectomy interferes with their development ( Tozawa, 1929) , as it does with the growth of the gonoj^odium in Gam- busia (St. Amant, 1941; Turner, 1941a). Amphibia)is Orchiectomy has shown that many male secondary sex characters are under the con- trol of testicular hormones. Steinach (1894) found removal of the Samenbldschen, a sac- like appendage of the mesonephric duct, from a frog had no effect on the sex charac- ters, but castration before the breeding sea- son resulted in the failure of amplexus to occur. Among the urodeles, orchiectomy in Discoglossiis is followed by prompt regres- '•'Thc stickleback, Gasterosteus (Bock, 1928; Becker and Lehmensick, 1933; Ikeda, 1933), tlie bitterling, Acheilognathus (Tozawa, 1929), and Phuxinus (Kopec, 1927), Halichoeres (Kinoshita, 1935), Oryzia.s (Niwa, 1955), and Amia (Zahl and Davis, 1932). „ COLD-BLOODED VERTEBRATES 1045 sion of the nuptial pad (Kehl, 1944a) and in male Xenopus by a much delayed drop in serum calcium (Shapiro and Zwarenstein, 1933) . Removal of the testes is succeeded by regression of sexual accessories in Bufo, but removal of Bidder's organ does not have this effect (Ponse, 1922a, b). Nuptial excres- cences and other sex structures regress after orchiectomy in Bombinator (Moszkowska, 1932) . In Rana, removal of the testes results in diminution in size of seminal vesicles and thumb pads and in failure of nuptial color- ing and other sex characters to appear ( Aron, 1926; Christensen, 1931; Kinoshita, 1932), but the linea masculina is not affected (Davis and Law, 1935) . It is suggested that once the latter is established it does not re- quire hormonal support from the testis. In Triton {Tritunis) and Desmognathus, two familiar genera of Salientia, orchiec- tomy of adults results in regression or dis- appearance of the dorsal crest, of the spe- cialized epithelium of the Wolffian duct and cloaca, and of male skin coloration. The masculine premaxillary tooth is replaced by a tooth like that seen in the female (Bresca, 1910; Aron, 1924b; Noble and Davis, 1928; Noble and Pope, 1929). Reptiles Castration of the box turtle, Terrapene Carolina, caused great reduction in the size of the epididymis. Motile sperm could still be obtained from the epididymis 74 days after orchiectomy (Hansen, 1939). (The ability of the surgically isolated epididymis of the lizard to store sperm has already been mentioned.) Sanfelice (1888) removed part of a testis from one snake, Natrix {Tropidonotus) na- trix, and one lizard, Lacerta agilis, and ob- served some testicular regeneration. The lat- ter phenomenon perhaps represents early evidence of sorts for a gonadotrophic hor- mone in these reptiles. In the blindworm, Angitis fragilis, seasonal development of the renal sexual segment and of other sexual ac- cessories was prevented if the castration was performed before the breeding season. If done during this season, atrophy of the ac- cessories followed in about 15 days (Herlant, 1933). Orchiectomy of Lacerta caused in- crease in the size of the fat bodies, disap- pearance of the green body color, persistence of the femoral glands in the quiescent phase, involution of the epididymis, and absence of epididymal and femoral gland secretion. The sexual segment of the kidney remained in the nonsecretory phase, and the epithe- lium of the urogenital fossa was low and non- glandular (Matthey, 1929; Regamey, 1935; Padoa, 1929, 1933). Seasonal development of sex accessories was prevented by castra- tion of Takydromus (Takewaki and Fu- kuda, 1935a). Sperm viability in the epidid- ymis was much less than in normal lizards (Takewaki and Fukuda, 1935b; c/. Hansen's results, above, in the box turtle). Involution of sexual accessories after orchiectomy also occurred in Eumeces (Reynolds, 1943) and Uromastix (Kehl, 1935, 1944b). Removal of the testes had no effect on the renal sexual segment in the snake Tham- nophis r. radix (Waters, 1940) but did in Natrix (Takewaki and Hatta, 1941). IX. Effects of Androgens Sex hormones have been given experimen- tally in a variety of ways. These hormones are freely soluble in certain vegetable fats such as sesame oil and in alcohols and are slightly soluble in water. Solutions can be in- jected subcutaneously, intramuscularly, or intraperitoneally. Solid pellets made by compressing the crystalline hormone can be imi:)lanted in various body sites for slow, continuous absorption. Sex hormones can even be fed. Solutions can be applied to the skin; in fish and amphibians, percutaneous absorption can be achieved if the hormone is dissolved in the aquarium water. Finally, in one of the oldest techniques, testes or ovaries can be grafted into experimental animals. Fish Administration of testicular hormone to intact carp and of testosterone propionate to hypophysectomized killifish accelerated the rate of spermatogenesis (Castelnuovo, 1937; Burger, 1942). Intraperitoneal injec- tion of androgens in Fundulus heteroclitus stimulated increase in weight of the testis even after hypophysectomy (Pickford and Atz, 1957). Prcgneninolone (ethinyl testes- 1046 SUBMAMMALIAN VERTEBRATES terone) when fed to Lebistes interfered with normal body growth in both sexes (Scott, 1944). The liver of Oryzias is sexually di- morphic (luring the breeding season; if a testosterone propionate pellet is implanted in the female at this time, her liver acciuires a typical male appearance (Egami, 1955a). It has already been shown that skin color may be under the control of androgens, and it is therefore not surprising that adminis- tered male sex hormone may conspicuously affect coloration. Thus, the injection of tes- tosterone propionate into hypophysecto- mized Fundulus or into Fimdulus during the nonbreeding period evoked the yellow body color typical of the spawning season (Bur- ger, 1942). Nuptial coloration was produced in Rhodeus, Acheilogriathus, Oryzias, and an unspecified genus of bitterling by adminis- tration of male hormone in various forms; the androgen was given during the nonbreed- ing season, or to castrated males, or to fe- males.^*^ The addition of pregneninolone to a(|uarium water induced the ai)pearance of male coloration, and also caused the disajv pearance of the black "gravid spot" from the tail fin of the female, in Lebistes (Ever- sole, 1941; Regnier, 1941). Gonopodia and other male secondary sex characters have also been studied as indi- cators of androgen action. Gonopodia have been produced in female Platypoecilus, Le- bistes, Gmnbusia, and Molliensia by injec- tions of testosterone propionate or by the addition to the aquarium water of pregneni- nolone (in some cases, 1 mg. in 14,000,000 ml. water) or methyl testosterone ( 1 mg. in as much as 25,000,000 ml.)i" Treatment of castrated male Gmnbusia with pregnenino- lone or with testis grafts permits the normal development of the gonopodium (St. Amant, 1941). If the anal fin, the female homologue of the gonopodium of adult female Platy- poecilus maculatus, is amputated and if any of several androgens (androsterone and tes- tosterone propionate are most effective) is then injected, a male-like but atypical go- '" Gliiser and Haempel, 1932 ; Owen, 1937 ; Glaser and Ranftl, 1938; Havas, 1939; Niwa, 1955. '"Eversole, 1941; Regnier, 1941; Turner, 1941b, 1942a, b, c; Cummings, 1943b; Hamon, 1945; Gallien, 1948; Hopper, 1949; Tavolga, 1949; Egami, 1954a. noiiodium is regenerated (Grobstein, 1940, 1942a, b, 1947). When adult lampreys become sexually mature, ducts develop for the escape of ma- ture germ cells from the mesonephros to the exterior, and the cloacal labia undergo vas- cular distention. Administration of either testosterone or of estrone, a female sex hor- mone, to adult but sexually immature 1am- i:)reys will evoke the same changes (Knowles, 1939). Further evidence for the control of sexual accessory structures by androgens is sum- marized by Pickford and Atz (1957). A)nphibians Androgen has been supplied experimen- tally by injection and by the transplantation of testes. Testicular homotransplants failed to restore male sex accessories in castrate Rana in an early experiment (Smith and Schuster, 1912), perhaps because the grafts did not survive. Amplexus occurred promptly after injection of a suspension of dried bull testes into the dorsal lymph sacs of adult male frogs ( Brossard and Gley, 1929) . Ponse (1922a, b, 1930) restored male sex charac- ters by testis grafting in castrate Bujo vul- garis, as did Moszkowska (1932) in Bombi- nator. The injection of androgens caused development of the nuptial pads in Disco- glossus (Kehl, 1944a) but not in Xenopus (Berk, 1939). Curiously, ovulation could be induced in the latter genus both by injecting the intact animal with any of several andro- gens or other steroids (Shapiro, 1939) or by adding testosterone and androstenedione to a frog Ringer solution in which an excised ovary was suspended (Shapiro and Zwaren- stein, 1937). Implantation of testosterone propionate tablets into castrate male and fe- male toads (Bufo vulgaris) resulted in the development of male accessories in both sexes (Harms, 1950). Similar treatment be- fore the breeding season of both male and female cricket frogs {Acris gryllus) evoked male skin coloration and hypertrophy of Wolffian ducts (both sexes), oviducts, and seminal vesicles (Greenberg, 1942). Injec- tions of the same hormone into castrate male Rana pipiens resulted in growth of the nup- tial pads and vestigial oviducts (Wolf, 1939) . Bresca (1910) made an interesting ob- COLD-BLOODED VERTEBRATES 1047 servation in Triturus {Triton ) . Transplanta- tion of testes to females was not followed by masculinization of accessory sex organs, but if a secondary female sex structure was itself transplanted to a normal male, the sexually appropriate transformation oc- curred, e.g., the middorsal skin stripe of the female became a dorsal crest when grafted on the male. In later experiments with Triton, castration and successful re-im- plantation of testes (autotransplantationi, and the injection of testosterone in oil into adults during the nonbreeding season both resulted in the development of male acces- sory structures (Aron, 1924b; Fleischmann and Kami, 1936). In Desmognathus, testis transplants into spayed females evoked the development of masculine premaxillary and maxillary teeth and of abdominal, pelvic, and cloacal glands (Noble, 1926; Noble and Davis. 1928; Noble and Pope, 1929). Eeptiles In the juvenile box tortoise, Terrapene Carolina, testosterone propionate pellets stimulated the growth of claws almost as large as those of adult males (Evans, 1951a) . The administration of androgens to imma- ture Chelydra and to two species of Pseu- demys resulted in acceleration of claw growth and growth of the jienis (Evans, 1946, 1951b, 1952a, b». The first successful effort to graft testes into lizards seems to have been that of Re- gamey (1935) in castrate Lacerta. His grafts did not survive in males but were successful in 3 of 25 females; in these 3 the mesonephric rudiment was transformed into an epididy- mis-like structure, and the mesonephric duct became a vas deferens. Kehl (1944b) in- jected androsterone benzoate into female Uromastix during the sexually quiescent pe- riod. This resulted in conspicuous growth and glandular development of the oviducts. (Other evidence will be presented below for a "bisexual" action of the androgens in some cases.) A second result (Kehl, 1938) was the development of the renal sexual segment to a stage resembling that of the male's sexual segment at the height of the breeding season. Treatment of Sceloponts with testosterone and testosterone propionate stimulated slight development of the mesonephric rest and duct in females and conspicuous enlarge- ment of the male epididymis, femoral glands, and hemipenes. Both the oviduct and the male Miillerian duct segments also grew re- markably (Gorbman, 1939; Forbes, 1941; Altland, 1943) . The administration of andro- gens caused growth of the epididymis and sexual segment of the kidney of Eumeces (Reynolds, 1943) and of the dorsonuchal crest of Anolis (Evans, 1948). In the snake, Thamnophis, injections of testosterone propionate stimulated pro- nounced development of the sexual segments in castrate adult males and females and in intact immature snakes of both sexes (Wa- ters, 19401. Administration of testosterone to the im- mature alligator, Alligator mississippiensis, evoked striking growth of the oviducts in the females and of the penis in the single ex- perimental male. The mesonephroi and Wolffian ducts in both sexes and the phal- luses of the females did not respond (Forbes, 1938b). Injection of older but still immature alligators with testosterone propionate caused conspicuous development of the ovi- duct and of the male and female phallus (Forbes, 1939). Other effects of administered androgens in embryonic and immature reptiles are dis- cussed in the section on "Experimental Sex Reversal." X. External Transport of Eggs and Young Fish Most fish appear to be indifferent to their eggs and offspring. However, male pipefish and seahorses carry the developing eggs in skin pouches, whereas the eggs adhere to the surface of the belly of a catfish, Platystacus. The male sea catfish Galeichthys and Cono- rhynchos protect the developing eggs by car- rying them in their mouths (Jordan, 1905; Strawn, 1958). The transport period lasts 60 to 80 days in Galeichthys (Ward, 1957). Amphibians In general, amphibians abandon their eggs after they are laid. It is an interesting vagary of nature, however, that a number of species 1048 SUBMAMMALIAN VERTEBRATES do transport their eggs and larvae by one means or another. In most cases, this is a matter not only of specialized behavior but of some morphologic modification. Aiytes obstetricans, a European form, is popularly called the midwife toad. Actually, it is the male of this species which manages to wind about his hind legs the strings of eggs which the female has laid. Here they stay, encased in their sticky jelly, until they hatch. The female Hyla goeldii, a South American frog, carries a mass of incubating eggs on her back with the aid of a low skin fold which helps to form a kind of shallow receptacle (Boulenger, 1895 ) . At least four other genera of South American tree frogs similarly trans- port incubating eggs as a mass, partly or completely covered by a flap of dorsal skin, or individually, each egg in a separate pocket of skin on the back (Noble, 1931). Bartlett (1896) reported to the Zoological Society of London how the eggs are placed on the back of the Surinam toad, Pipa ameri- cana. He and a keeper observed the process in the reptile house at the Zoological Gar- dens. Late in April two pairs of the frogs were seen in amplexus. The male had firmly grasped the lower abdomen of the female, his body extending caudal to hers. Her "ovi- duct" was protruded more than an incli, arching between the belly of the male and the back of the female. (Subsequent dissec- tion by Boulenger of a female which died during oviposition showed that the "oviduct" was actually an "ovipositor, formed by the cloaca.") The male squeezed the sac-like ovipositor and moved it about, thus direct- ing the even placement of the eggs. The lat- ter stuck to the back, and the ovipositor later was retracted. It is known (Parker and Haswell, 1921) that the eggs eventually sink into cavities which develop in the spongy dorsal skin, that lid-like structures form over the cavities, and that the larvae remain in these convenient, individual containers until they metamorphose. What regulates the modification of the dorsal skin so that, at the proper time, each egg can "implant" in this strange site? Tadpoles of the genera Dendrobates and Phi/llobates attach themselves by buccal suckers to the back of an adult male (Bou- lenger, 1895). The larvae of Arthroleptis seychellensis, a frog found in the Seychelles Islands of the Indian Ocean, swim to the back of the adult. Here they attach them- selves, not by buccal suckers but by a sticky ectodermal secretion. Contact is always be- tween the ventral skin of the larva and the dorsal skin of the adult (Brauer, 1898). Reptiles There appear to be no reports of morpho- logic specialization for the external trans- port of reptilian eggs or young. XI. Ovary; Ovogenesis; Ovulation Fish The ovaries of fish range from primitive structures to rather complex organs which may combine functions of the mammalian ovary, oviducts, uterus, and even, to an ex- tent, the mammary gland. On the other hand, detailed knowledge regarding the geni- tal systems of most species of fish is sur- IH'isingly scanty, particularly in terms of histologic detail. Surely this is an impor- tant and fascinating area for research. The brook lamprey, Entosphenns wilderi, which has been carefully studied by Okkel- l)(>rg (1921), attains its full body length while still a lar\-a. Then it metamorphoses. Now, in xAugust or September, comes the climactic stage of its life. The gonads ma- ture, growing swiftly to the point where they occupy all the body cavity. The digestive tract atrophies. Thereafter the lamprey eats nothing. It l)reeds the following April, and then dies. There is a single testis or ovary in the sexually mature animal. The eggs are enclosed in follicles; the latter are believed by Okkelberg to be homologous with the cysts of the testis. The mature ova are, as in cyclostomes generally (Hoar, 1957a), shed into the coelomic cavity and then escape to the exterior by way of abdominal pores, urogenital sinus, and urogenital pa- pilla. In the basking shark, an elasmobrancli, only the right ovary develops (Harrison Matthews, 1950). It may contain about 6,000,000 ova, 0.5 mm. or more in diameter and possessing some yolk. When the ripe follicles rupture, the ova escape into a pouch inside the ovary, then are propelled into the COLD-BLOODED VERTEBRATES 1049 oviduct and uterus. In the dogfish, Scylio- rhinus canicula, abdominal cilia, present only in the adult female, move the ova from the ovary to the oviduct (Metten, 1939). The varieties of female reproductive tract in the bony fish were long ago categorized by Brock (1878) on the basis of his study of 57 species in almost as many genera. In one class the solid ovary has no excurrent duct, eggs being discharged into the coelomic space and escaping by way of the abdominal pore. The ovary may consist of a single layer, as in the eel Anguilla, or of several layers, as in the Salmonidae. In a second category of fish the ovary is a sac, closed anteriorly and ending posteriorly in an oviduct. Only a lit- tle of the ovarian wall may contain eggs, as in Scorpaena, Lepadogaster, and Ophidmm, or most of the wall may be filled with eggs carried in masses which are knob-shaped in cross section, as in Lophobranchis and Blen- nius, or in lamellae. MacLeod (1881) also recognized these two classes of ovary. He pointed out that in the first, or solid, type the medial surface {face vasculaire) is smooth, invested with endothelium, and lacks germinal epithelium. The lateral sur- face, or face germinative, is covered with egg-bearing folds and germinal epithelium. The solid type of ovary is seen in salmonid and murenid fish. In all other bony fish, the paired ovaries are hollow ; their external sur- face is vascular, whereas the interior is in- vested with germinal epithelium. The ovar- ian sac ends posteriorly in an oviduct. Right and left oviducts join to form a common canal which opens through the body wall between anus and urinary orifice. Part or all of the internal, or ovigerous, surface of the ovary is folded in a variety of patterns. The ovary of Fundulus as described by Matthews (1938) belongs in the second cat- egory. This ovary is single but bilolx'd an- teriorly. (Ovigerous lamellae project into its central cavity. Follicles may begin to ma- ture at any time of year. The ovary is small- est in July, increases slowly in size until April, then grows rapidly until o\'uhition in June. The ovaries of Neotoca and Oryzias also belong in this class (Mendoza, 1940; Robinson and Rugh, 1943). Oviposition necessarily coincides with the shedding of sperm by the male in those fish in which fertilization is external. A))ip}ubiatis Ovulation and fertilization occur during the spring in most amphibians (Smith, 1955). In Rana pipiens the the cyst-like fol- licle bulges as it matures (Rugh, 1935). Eventually it ruptures at the stigma, not abruptly but in less than a minute. The ovum slowly emerges, being forced through an aperture smaller than the egg itself. The fol- licles of the frog's ovary can be rui^tured ar- tificially by pressure or by the application of a pepsin-hydrochloric acid mixture (Rugh, 1935; Kraus, 1947). Smooth muscle fibers are a normal component of the am- phibian ovary. Kraus and others have noted that the muscle fibers are larger in the ma- ture than in the immature ovary. Tlie fibers contract rhythmically, not only during ovu- lation but at other times. Their role, if any, is not clear. Rugh believes that normal fol- licular rupture follows, and is due to local changes induced earlier by pituitary hor- mones. It is interesting that if ripe follicles are excised they still may ovulate up to 12 hours later. Thus the final release of the ovum would seem to be an autonomous proc- ess. Reptiles Important early descriptions of the adult reptilian ovary include those of von Leydig (1853, 1872)"^ Waldeyer (1870), Braun (1877), and Loyez (1905-1906). In turtles and lizards the ovaries are round and plump ; in snakes, elongated; in the immature alli- gator, flat and ratlier long (Reese, 1915; van den Broek, 1933; Forbes, 1937). The musk turtle, Sternotherus odoratus, breeds in April and early May and ovulates usually between May 15 and 20. The eggs are carried in the oviducts for 20 to 35 days, then laid (Risley, 1933a). The box turtle, Terrapene Carolina, ovulates in .June and July and lays its eggs soon afterward (Alt- land, 1951). Munson (1904) says that the ovaries of the tortoise, CleniDu/s niannorata, fill most of the abdominal ca\ity when the eggs have acquired all of their yolk. In an Algerian tortoise, Emys leprosa, the eggs ap- 1050 SUBMAMMALIAN VERTEBRATES parently reach their maximal size and are ovulated in June (Combescot, 1954b). Waldeyer (1870) mentions an investing layer of germinal epithelium on the ovary of Lacerta and the numerous layers of epi- thelium in the mature follicle. Regamey (1935) adds details. The ovary is supported by a mesovarium developed from the dorsal abdominal wall. Young ovocytes lie close to the hilus. The ovary is invested with cuboi- dal epithelium, but only the epithelium close to the hilus is considered to be germinative. Ovogenesis continues throughout the year. Ripe follicles are seen in April and May, and ovulation occurs at this time. Uromastix fol- lows the same schedule (Kehl, 1935), as do Eumeces (Breckenridge, 1943), Hemidacty- lus (Dutta, 1944), and Sceloporus (Wood- bury and Woodbury, 1945). It is believed that Anolis ovulates alternately from the right and left ovaries (Noble and Green- berg, 1941) ; the ovaries are largest between March and September (Dessauer, 1955). This lizard produces single eggs at intervals of about two weeks during a breeding season lasting from midspring until the end of sum- mer (Hamlett, 1952) . Usually only one ovum matures at a time in Xantusia vigilis (Mil- ler, 1948) . It seems that the snake Tropidonotus viperinus mates in October or November and ovulates in the following June or July, whereas Coronella laevis mates in August and September and ovulates in May and June (Rollinat, 1898). The viviparous prai- rie rattler, Crotalus v. viridis, ovulates late in the spring or early in the summer. The young are born in August or September. The evidence is strong that the snake does not ovulate the next year, but only in the spring two years after the previous ovulatio;< (Rahn, 1942). Alligators lay their numerous ( 100 to 200) eggs in April or May after maturing to a length of at least six feet (Cope, 1900; McU- henny, 1935 ) . The immature alligator ovary is lobulated (Reese, 1915). It consists of a well defined cortex and a medulla (Forbes, 1937, 1940a). The cortex is invested with germinal epithelium and contains numerous immature follicles with eggs of various sizes. The underlying medulla consists chiefly of connective tissue strands (the remains of the medullary cords), between which are large lacunae. The posterior third of the ovary is composed of solid medullary tissue. This is a "medullary rest"; it persists with little change from the embryonic period, lacks germinal epithelium, and resembles primi- tive testicular tissue. XII. Sources of Estrogens Fish A few investigators have searched for es- trogens in fish gonads. Assay of an extract of 10 pounds of swordfish, Xiphias gladius, ovaries showed less than 6 rabbit units of estrogen (W^eisman, Mishkind, Kleiner and Coates, 1937) . Ovaries of the flounder, Pseu- dopleuronectes americanus, contain small amounts of estrogen, as indicated by assay of extracts in rats (Donahue, 1941). Chemi- cal assay of 420 cc. pooled urine from 25 male and female Lophius piscatorius, the angler fish, revealed 0.7 mg. folliculin, 1.5 mg. total phenolic steroids, 0.055 mg. 11- oxy steroids, and 0.35 mg. 17-ketosteroids (Brull and Cuypers, 1954). The mature ova of the dogfish, Mustelus canis, contain a large amount of estrogen (Hisaw and Abramowitz, cited bv Pickford and Atz, 1957). The production of estrogens by fish ova- ries deserves vigorous study. The results might well shed light not only on morpho- logic changes but on reproductive behavior. Perhaps migration itself is stimulated in part by sex hormones. Amphibians The writer has found only one pertinent report (Grant, 1937), and that without de- tails: an "estrogenic substance can be ex- tracted from amphibian ovaries." Further research is desirable, but no doubt has been impeded by the problem of obtaining enough ovarian tissue to yield detectable amounts of sex hormone. Reptiles Injection daily for four days of 0.1 cc. follicular fluid from the ovaries of Crotalus terrificus provoked vaginal estrus in castrate mice (Fraenkel and Martins, 1938). Alco- holic extracts of the ovaries of crotalid COLD-BLOODED VERTEBRATES 1051 snakes on bio-assay contained the equivalent of 200 estrone units i)er kilogram of fresh ovaries (Valle and Valle, 19431. XIII. Oviduct; Egg Transport Fish According to Stronisten (1931), Rathke (1824) discovered the oviduct of the fish. This renowned old anatomist and embryolo- gist also recognized that the oviduct is ab- sent in the lamprey, Petromyzon, in the eel, and in some salmonid fish, a deficiency since noted in additional species (Brock, 1878; MacLeod, 1881). In such fish the eggs are released into the coelom and then reach the exterior through an abdominal pore. As al- ready stated, in most bony fish the ovary is hollow, and the eggs or young pass succes- sively into the ovarian cavity, oviduct, and (in some viviparous forms) uterus. Informa- tion is needed as to how the inert eggs are moved. Eggert ( 1931 ) has concluded that the oviduct is not homologous with the Mullerian duct of higher vertebrates. The oviduct of the Japanese medaka, Oryzias latipes, has been described in some detail (Robinson and Rugh, 1943) , but there is little information on seasonal variation in the fish oviduct and on the role, if any, of sex hormones in oviducal development. Amphibians In most and })erhaps all adult females, but not in males or in inmiature females, the ven- tral and lateral coelomic peritoneum is cili- ated (Donahue, 1934; Rugh, 1935). The cilia roll the liberated eggs into the ostium of the oviduct, about two hours being reciuired for the journey. If transplanted eggs or even buckshot are introduced into any part of the coelom, ciliary action will eventually deliver them to the oviduct, although as Rugh remarks, the heavy buckshot are moved very slowly. The amphibian oviduct may increase in size during the breeding season, then regress (de Allende, 1939). The jelly-like coating characteristic of extruded amphibian eggs is contributed by oviducal glands. After ovi- position this coating takes up water and swells I Noble, 1931) to form a sticky layer which protects the egg and often attaches the egg mass to underwater debris or even to the adults in those species which transport the eggs externally. Reptiles Lataste described in 1876 the histology of the oviduct of a turtle, Cistudo [Terrapene) europaea. The three layers of the ostium consist of partly ciliated mucosa, connective tissue, and investing peritoneum. The mid- dle portion of the duct also has glandular and muscular layers. In the final, or cloacal, portion there are two muscle layers, and the gland cells contain chromophilic granules. Argaud (1920) saw both granule-producing and mucin-producing gland cells in the ovi- duct of an unidentified turtle. The oviduct of the immature Testudo is lined with non- ciliated cuboidal epithelium, and the epithe- lium is also low in the sexually inactive adult. During the breeding season the ovi- duct is enlarged and the mucosa contains muciparous cells interspersed among cells in which the cytoplasm is crowded with large secretion granules (Argaud, 1920; Kehl, 1930). A classic study of the oviduct, including that of the turtle Chrysemys, was made by Parker (1931). He described longitudinal pro-ovarian bands of cilia wiiich sweep the sperm toward the ovary. Abovarian bands of cilia beat in the opposite direction. If the oviduct were opened longitudinally and par- ticles of coal dust were scattered on it, both types of band demonstrated their ciliary ac- tion. Eggs, on the other hand, were believed to be moved down the oviducts by peri- stalsis. Van den Broek (1933) confirmed Parker's observations regarding the regular longitudinal folds of the mucosa. The former states that the albumen of the egg is pro- duced by the oviduct proper, whereas the egg shell, the final investment, is secreted by the "uterus" or last part of the oviducal tube. Seasonal development of the mucosal cells of the ostium occurs in Terrapene (Hansen and Riley, 1941). In adult Emijs, on the other hand, the mucosal cells do not change throughout the year (Combescot, 1954b, 1955). The mucosa of the oviduct of Hatteria (Sphenodon ) is folded and during pregnancy is glandular in its terminal portion (Osawa, 1052 SUBMAMMALIAN VERTEBRATES 1898) . Ciliated cells and goblet cells are also present (van den Broek, 1933). For the liz- ards, the general picture which emerges is of seasonal development of the oviducal mu- cosa in most forms, of a specialized area for the secretion of albumen, and of a "uterus" or "incubation chamber" for the oviparous species. ^*^ Crowell (1932) has seen in Phrij- nosoma and Sceloporus, as has Dutta ( 1946) in Hemidactylus, a tract of pro-ovarian cilia like that described by Parker for the turtle. Specialization of the uterus for viviparity is discussed below. In April to July the oviducal glands of the snake Xatri.v t. tigrina are conspicuous and their cells are crowded with secretion gran- ules (Takewaki and Hatta, 1941). Seasonal development of the uterine portion of the praii-ie rattler's oviduct also occurs (Rahn. 1942). The immature alligator's oviduct has been described (Reese, 1915; Forbes, 1937. 1940a), but information on the adult struc- ture seems to be lacking. XIV. Other Specializations in Females Reptiles During the i^eriod of sexual inactivity the stratified epithelium lining the urogenital fossa of female Lacerta is about 30 /x in thickness. In the spring breeding period, however, due to a remarkable increase in stratification the epithelium becomes 210 to 260 fjL thick. 0]:)ening through the stratified epithelium are long, sinuous tubules produc- ing an amorphous secretion. Regression of the stratified layer starts in July, continues gradually, and is not yet complete when hi- bernation begins in November. As Regamey (1933, 1935) points out, the glands at the height of their development remind one of the uterine glands of mammals. Dantchakoff (1938) i-emarked that the highly developed '" Flinjuosuma and Sceluporits (Crowell, 1932), Vromastix (Kehl, 1935), Hoplodactylus (Boyd, 1942), Hemidactylus (Dutta, 1946; Mahendra, 1953), Xantusia (Heimlich and Heimlich, 1950), Lygomma (Wcckes, 1927b), Lacertn (Sacchi, 1888; Regamey, 1935; Jacobi. 19?6), Anguis (Coe and Kunkel, 1905; Ja.obi. l!):!fi), Anniella (Coe and Kimkel, 1905), Am plnxlui, no, Anops, and Tro- gonophis (Coe and Kunkel, 1905), unspecified genu.s (Giersberg, 1922). stratified epithelium of the urogenital fossa and cloaca in the lizard has a good deal of resemblance to the stratified vaginal epithe- lium of a rodent in estrus. In male, immature, and nonestrous female colubrid snakes of a viviparous species of Natrix, calcium, magnesium, and protein levels in the plasma were relatively low and showed little variation throughout the year. The same was true in Thamnophis. How- ever, concentrations of all three substances rose very conspicuously while females of both genera were in estrus, with the highest values of all being attained in Thamnophis just after ovulation (Dessauer, Fox, and CJil- bert, 1956). XV. Effects of Ovariectomy Fish ( )variectoniy has little effect on female coloration in the bowfin, Amia calva (Zahl and Davis, 1932) in Halichoeres poecUop- terus (Kinoshita, 1935), or in the stickle- back, ddstcrosteiis aciileatus (Bock, 1928). AiHphihidtis If R(ina pipiens is ovariectomized in Se]i- tember, the oviducts degenerate by Decem- ber (Wolf, 1928). The glandular cells de- crease in size, and few secretory granules are observed. This is said to be the only species of frog in which the IMiillerian ducts (corresponding to the oviducts of the fe- male) are cjuite well formed in the male (Christensen, 1931). In immature animals of both sexes the ducts are similar and small ; with the onset of sexual maturity, the ovi- ducts grow further in females. That this is due to ovarian hormones was proved by ovariectomy. Removal of the ovaries from adult BkJo arenarum also results in oviducal atrophy (Galli-Mainini, 1950). In the female, as in the male, of Xeuopus laevis castration causes a drop in the serum calcium level (Shapiro and Zwarenstein, 1933). Reptiles Alalc and female Lacerta casti'ates are indistinguishable externally. The oviduct atrophies to the nonbreeding stage (evidence COLD-BLOODED VERTEBRATES 1053 that ovarian hormones are secreted chiefly or only during the breeding season), as does the ei)itheliinn of the urogenital fossa, and seasonal development of all structures there- after, of course, fails to occur (Regamey, 1935). Ovariectomy of the snake Natrix causes rapid oviducal atrophy (Takewaki and Hatta, 1941). XVI. Effects of Estrogens and Projjesterone Female sex hormones when experimentally administered are usually very effective in modifying the reproductive systems of fish, amphibians, and reptiles. Fish Estrogen is reported to have stimulated spermatogenesis when injected into imma- ture male carp (Castelnuovo, 1937) but to have suppressed spermatogenesis in Platy- poecilus (Cohen, 1946). In the loach, Mis- gurnus, the injection of estrone or estradiol benzoate caused discharge of sperm and in- hibition of spermatogenesis in the male, whereas ovarian development was inhibited in the female. Suppression of the release of gonadotrophin from the pituitary was sug- gested as the underlying mechanism (Egami, 1954b, c). The liver of Oryzias and Gaster- osteus during the breeding season is sexually dimorphic in structure, color, and weight. Administration of estrogen to males at this time results in transformation of their livers to the female type (Egami, 1955a; Oguro, 1956) . Curiously, in Platypoecilus estradiol and estradiol benzoate had opposite effects. In males less than 18 mm. long, estradiol did not affect the testes but the anal fin grew slightly. In older males, large gonopodia de- veloped and the testes were stimulated. The same hormone caused ovarian degeneration and growth of gonopodia in females. Estra- diol benzoate, however, inhibited testes and ovaries and caused no gonopodial develop- ment (Ta Volga, 1949). Pregneninolone, sometimes regarded as a "bisexual" hormone, induced partial mas- culinization of immature female Platypoe- cilus, and evoked typical female body size and bodv index in immature males (Cohen, 1946). In the years before World War II much interest centered on some experiments on the bitterling, Rhodeus amarus. At breeding time the urogenital papilla of the European cyprinid hypertrophies into a rather lengthy ovipositor; with the latter, eggs are depos- ited in fresh water mussels (Bretschneider and Duyvene de Wit, 1947 ) . In 1932 Fleisch- mann and Kann reported that the injection of follicular hormone into the female bitter- ling during the nonbreeding period resulted in lengthening of the ovipositor. Injections of salt solution or anterior lobe hormone did not give this result. Further, implantation of the bitterling ovary in a castrate female mouse, it was stated, produced estrus. This seemed to show that ovipositor growth was due to estrogen from the fish's ovaries. Ehr- hardt and Kiihn (1933, 1934) found that the addition to 1 liter aquarium water of 5 ml. pregnancy urine, one tablet (150 mouse units) ovarian hormone, or urine extracts also caused ovipositor lengthening. Injec- tion, or addition to aciuarium water, of Pro- gynon (estradiol) produced ovipositor growth in females outside the breeding sea- son and in castrate males (Fleischmann and Kann, 1934). The idea developed that the response might be sensitive and specific enough to provide the basis for a bio-assay for estrogens or progesterone. There is little doubt that the ovipositor responds to rather small amounts of these hormones. ^'•' How- ever, it was discovered that ovipositor growth may also be evoked by adrenal cor- tical hormones, purified male hormones, and male urine, as well as by various alcohols and other solvents and at least one inor- ganic comiiound.-" de Groot and Duyvene de Wit (1949), who have vividly described the oviposition of Rhodeus, found that the ovipositor rapidly elongates in response to copulin, a male hormone which, they postu- lated, is released into the aquarium water by the male bitterling. The bitterling assay ^'' Floischinanu and Kann, 1935; Duyvene de Wit, 1940; Bretschneider and Duyvene de Wit, 1947; van der Veen and Duyvene de Wit, 1951. -"Sziisz, 1934; Barnes, Kanter and Klawans. 1936; Kleiner, Weisman and Mi.?likind, 1936; Duyvene de Wit, 1938, 1939; Glaser and Ranftl, 1938; Fleischmann and Kann, 1938; van Koersveld, 1948. 1054 SUBMAMMALIAX VERTEBRATES appears not to have been used in recent years. Atriphibians Diethylstilbestrol provoked hyi)ertrophy of the rudimentary oviducts and atrophy of the Wolffian ducts in adult normal and cas- trated Triturus (Adams, 1946). Injection of mammalian follicular extract or of estrone into ovariectomized Rana pipiens prevented atrophy of the oviducts and sometimes caused oviducal hypertrophy (Wolf, 1928; :March, 1937 ) . Estrone had little effect on the oviducts of normal toads (de Allende, 1940) , but estradiol helped to prevent ovitlucal cas- tration atrophy (Galli-Mainini, 1950). The injection of estrone into male toads caused growth in the summer but not in the winter of the vestigial Miillerian ducts; growth was due to development of mucus-secreting glands (van Oordt and Klomp, 1946). Pen- hos and Nallar (1956) determined the rate of oviducal secretion in Bufo arenarum by ligating both ends of the oviduct, treating the toad, then removing and weighing the oviduct and accumulated secretion. Proges- terone administration stimulated secretion; this action of the hormone was enhanced by concurrent administration of estradiol ben- zoate and testosterone propionate, but not by desoxycorticosterone and folic acid, whereas preliminary treatment with hydro- cortisone had an inhibitory effect. Proges- terone, on the other hand, had little or no effect on the accessory sex structures of tad- poles of Bufo bufo (Lugli, 1955 ) . Although peritoneal cilia do not normally occur in male Eana pipiens, the intraperi- toneal injection of theelin (estrone) into males for 30 days resulted in the appearance of patches of cilia on the coelomic perito- neum (Donahue, 1934) . In the toad Xenopus the hyperemia of the cloacal labia which is typical in females during the breeding sea- son could be produced at other times by the administration of pituitary hormones, methyl testosterone, testosterone propionate, or progesterone. These steroids also produced oviducal hyperemia (Berk and Shapiro, 1939). Reptiles The injection of folliculin, an estrogen, into immature female Testudo iberica, a tur- tle, every other day for 3 or 4 weeks resulted in oviducal hypertrophy to more than nor- mal adult size. The mucosal cells became columnar, and some acquired cilia (Kehl, 1930) . Estrogens and testosterone propionate both caused moderate growth of foreclaws in immature Pseudemys elegans (Evans, 1946, 1952a). It is surprising that estrogens should have the same effect as an androgen on this accessory sex structure. Theelin injections provoked conspicuous growth of the oviduct of the female, reduced epididymal diameter in males, and increased the number of mitotic figures in the male vas deferens in Scelopor^us (Gorbman, 1939). In another lizard, Anolis, administration of the same estrogen resulted in major atrophy of the testis, lesser atrophy of the ovary, and hypertrophy of oviducts, ductus deferens, and cloacal epithelium in both males and fe- males (Evans and Clapp, 1940). Treatment of male Eumeces with estradiol benzoate had little effect (Reynolds, 1943), but injection of estradiol diproprionate into sexually qui- escent female Uromastix caused develop- ment of the reproductive tract equal to that seen in the breeding season (Kehl, Leportois and Benoit, 1941; Kehl, 1944b). Progester- one caused conspicuous growth of the ovi- duct in Uro IN astir (Kehl, 1941, 1944b). XVII. Fertilization; Sperm Storage in Females Fish External fertilization, of course, takes place in the water. Internal fertilization oc- curs in various sites. Sometimes the eggs are shed into the ovarian cavity and there en- counter the sperm (Stuhlman, 1887; Turner, 1938c). In the poeciliids Lebistes, Xipho- phorus, Heterandria, and Glaridichthys and in Xeotoca and Jenynsia the ovum is ferti- lized while still in the follicle. Shortly before arrival of the sperm the follicular cells sep- arating the mature ovum from the central ovarian cavity thin out to form a funnel- shaped invagination or delle. At the apex of the latter there is a minute pore or propyle that permits entrance of the sperm. The em- COLD-BLOODED VP^RTEBRATES 10,^ hiyo develops in the follicle, which even- tually ruptures at the site of the i)roi)yle and releases the young fish for birth.-^ Some female fish are able to store sperm in the oviduct or ovary for long periods, making possible the fertilization of the eggs months after contact with the male. Sperm storage is reported for sharks and rays (Lo Bianco, 1908-19091, and is common in the Embiotocidae and Poeciliidae.-- Xothing is known regarding the endocrine control of fertilization and sperm storage. Amphibians Salamander sperm are transported in spermatophores (Noble, 1931). Spallanzani knew in 1785 (Jordan, 1891) that several European salamanders somehow practice in- ternal fertilization, although he was not aware that the female usually picks up with her cloacal labia the spermatophores which the male has just shed. Among the urodeles, Cryptobranchoidea and ^iren are excep- tional in that the males do not form sper- matophores, the females lack spermathecae (see below), and fertilization is external (Dunn, 1923). Most female urodeles have special semi- nal receptacles, or spermathecae, in which the sperm are stored after the spermato- phores disintegrate within the cloaca ( Dunn, 1923; Noble, 1931). The spermathecae are actually cloacal glands, in the depths of which the sperm congregate. As Noble points out, de Beaumont ( 1928) proved the homol- ogy of these glands with the cloacal glands of the male. He transplanted testes into fe- male Triton (Triturus) cristatus, and 6 to 10 months later found that the cloacal glands liad assumed the secretory appearance char- acteristic of the male. (It will be recalled that the homologous male glands form the spermatophores. I Noble (1931) has traced the jihylogeny of the spermatheca. -^Wyman, 1854; Philippi, 1908; Scott, 1928; Bailey, 1933; Purser, 1938; Eraser and Renton, 1940; Mendoza, 1940. -- Cymatogaster (Eigenmann, 1892a; Turner, 1938b), Amphigonopterus (Hubbs, 1921), Xipho- phorus (Vallowe, 1953), Platypoecihis (Tavolga and Rugh, 1947), Glaridichthys (Philippi, 1908; Winge, 1937), Lebistes (Vaupel, 1929; Purser, 1937; Clark and Aron.son, 1951), Gambusia (Dulzetto, 1928), and Heterandria (Eraser and Renton, 1940). Baylis (1939) kept a female *S. .sa/a/zia/^r/ra in an acjuarium, isolated from all other sal- amanders. In 2 weeks she produced a brood of motile larvae, and did so again almost 2 years later. Baylis feels that it is usual for the female to store sperms in her spermathe- cae from impregnation in the summer at least until after a brood is born the next spring. The stored sperm then fertilize in- ternally the next batch of eggs. The long "gestation period" for this brood includes, incidentally, several months of hibernation. Adams (1940) found sperm in the sperma- thecae of the newt, Triturus viridescens, during every month of the year, but in greatest quantity in the fall and spring. Reptiles Female reptiles may store sperm which re- tain their fertilizing capacity for months or even years, possibly an advantage in that members of some species are slow moving and relatively scanty, with conseciuent re- duction in the opportunities for mating. Iso- lated diamond back terrapins, Malaclemmys centrata, have been known to lay fertile eggs (as indicated by the presence of an embryo) as long as 4 years after the last mating (Barney, 1922; Hildebrand, 1929). A similar record has been established by the box tur- tle, Terrapene c. Carolina (Ewing, 1943). An instance of sperm storage in a chame- leon, Microsaura p. pitmila has been re- ported (Atsatt, 1953) Prolonged sperm storage has been ob- served most frequently in female snakes, some of which have been proven capable of keejjing sperm alive and functional for up to 5 years (see Fox's review, 1956, for all reptiles) .^^ '^ Agkistrodon contortrix, 11 days (Gloyd, 1933) ; Causus rhombeatus, 5 months (Woodward, 1933); Crotnlus v. viriditi, throughout winter (Rahn, 1942; Ludwig and Rahn, 1943); Vipera aspis, through- out winter (Rollinat, 1946); Boiga multimaculata, 1 year (Kopstein, 1938); Coronella austriaca, throughout winter (RoUinat, 1946); Drymarchon cornis couperi, 4 years and 4 months (Carson, 1945); Lcimadophis viridis, delayed fertilization (Mertens, 1940); Leptodeira albojusca, 1 year (Kluth, 1936); L. annulata polysticta, 5 years (Haines, 1940); Nntrix natrix, throughout winter (Rollinat, 1946; Petter-Rousseaux, 1953); A^. sub- miniata, 5 months (Kopstein, 1938); A'', vittata, VA years (Kopstein, 1938); Storeria dekayi, 4 1056 SUBMAMMALIAN VERTEBRATES The prairie rattler's reproductive system includes, in anteroposterior succession, ovary, oviduct, uterus, and vaginal pouch, all of these being paired, and finally the single cloaca. A careful study of sperm dis- tribution during their storage in winter hi- bernation showed that the germ cells are concentrated in the anterior extremity of the vagina and in the posterior end of the uterus. It is believed that in the spring the sperm migrate into the oviduct in order to fertilize the eggs (Rahn, 1942; Ludwig and Rahn, 1943). Live sperm can be demonstrated in a uter- ine smear from the garter snake for a month or more after mating (Rahn, 1940a). The female garter snake stores sperm between the uterus and the most anterior portion of the oviduct (designated the infundibulum) in a short, thick segment with specialized alveolar glands. The latter communicate with the oviducal cavity by branched, cili- ated ducts. In this species sperm from a fall mating spend most of the winter in disorgan- ized masses in the oviducal cavity. In Feb- ruary or March the germ cells move into the lumina of the alveolar glands. Here in strik- ing fashion the sperm are ranked side by side, their heads against the alveolar epi- thelium and their tails projecting into the lumen. Finally, at ovulation, the sperm move on to fertilize the eggs (Fox, 1956). Very little seems to have been published on physiologic aspects of sperm storage in reptiles. One wonders how the metabolic re- quirements of the sperm are met for months or years, how the sperm are guided to the storage site, and how they are "released" at the proper time. It seems safe to assume that these processes are at least partly under the control of sex hormones. This is an area much in need of study. XVIII. Oviparity and Ovoviviparity Oviparous animals release their eggs to de- velop outside the body. In ovoviviparous animals, fertilization is internal, and the embryo undergoes at least part of its devel- opment within the mother. However, in this months (Trapido, 1940); Thamnophis sirtalis, several months (Rahn, 1940a; Blanchard, 1942); Tropidoclonion lineatutn, probably throughout winter (Gloyd, 1928; Force, 1931); Xenodon mer- remi, 1 year (Graber, 1940). case the fertilized ovum acciuires a definite investing membrane or shell. There is enough yolk for the nutrition of the growing embryo so that no food materials need be supplied by the mother. In viviparity at least part of the embryo's nourishment is of maternal origin. Intermediate stages also exist. The evolution of viviparity is ably discussed by Harrison Matthews (1955). Fish Oviparity of course is common. Ovovivi- l)arity also occurs (see, for example. Turner, 1937a; Hisaw and Albert, 1947) ; in poecihid fish the embryos develop within the follicles and maintain gas exchange with the mother. Because more than one (sometimes up to nine!) brood, each of a different age, may develop in the same ovary, a kind of super- fetation exists (Turner, 1937a, 1947). Aittphibidns Nearly all amphibians are oviparous. A few salamanders, including Oedipus, Hydro- mantes (Noble, 1931), and Salaniandra (Bay lis, 1939) , bring forth living young and are regarded as oA'oviviparous. Reptiles Reptiles, (lei)en(ling on species, may be oviparous, ovoviviparous, or viviparous. Oviparity has been reported, for example, for lizards of the genera Amphibolurus, Ly- gosoma, and Egernia (Weekes, 1934). Phry- nosoma cornutum, the horned toad, is ovipa- rous, whereas P. douglassi is viviparous (Edwards, 1903). Hemidactylus flaviviridis, the Indian house gecko (Mahendra, 1936), and Anniella pidchra, the American legless lizard (Coe and Kunkel, 1905) are ovovivip- arous. Jacobi (1936) has described in detail the reproductive anatomy of the ovovivipa- rous lacertilians Lacerta agilis and Anguis frag His. Some snakes, the turtles, and the croco- dilians bury their eggs or lay them in nests for extended incubation. Other snakes ap- pear to be ovoviviparous, i.e., the egg is well supplied with yolk and acquires a shell, but is retained in the oviduct for at least part of the incubation period. Examples are Trachy- boa (Barbour, 1937), the colubrid snakes Xatri.r and Thamnophis (Bragdon, 1946),. COLD-BLOODED VERTEBRATES 105 and the sea snakes Aipysurus, Enhydrina, Hydro-phis, Lapeinis, Laticcuida, and Tha- lassophis (^mith, 1930; Smedk-y. 1930, 1931 ; Bergman, 1943). XIX. Viviparity Fish ^'i^'il)al■ity in some fish and in mammals has, it will he seen, mueh in common. Hor- mones from the glands of internal secretion initiate and regulate many aspects of rei)ro- duction in the higher vertebrates, and there is reason to believe that similar controls are important in fish. Although actual evidence for such endocrine regulation is scanty, some phenomena of gestation in this class should hv briefly reviewed. For details, see Turner (1933, lb40c, 1947), Mendoza (1937), and Needham (1942). The finding of an eml^ryo within an egg case in a specimen of Rhineodon typus, the whale shark (this adult exceeded 65 feet in length), was taken as strong presumjitive evidence that this species is oviparous ( Baughman, 1955 ) . However, specializations of the uterus of the basking shark, Ceto- rhinus maximus, suggest that it is viviparous (Harrison Matthews, 1950), as is Spinax (Wallace, 1903) and the dogfish, Mustelus (Te Winkel, 1950). It is thought that ovar- ian tissue and immature eggs in another shark, Lamna, disintegrate into the oviduct, are passed into the uterus, and are swallowed as food by the embryo. The gut of the em- bryo becomes greatly distended with this yolk-like material; the actual yolk sac is separate and very small (Shann, 1923). In three genera of ray, Myliobatis, Ftero- plataea and Trygon, the uterine mucous membrane gives off long paj^illae, or tro- phonemata, which secrete a fluid rich in al- bumen. The secretion escapes into the uter- ine cavity and is swallowed by the fetus, as proven by finding the same material in the fetal intestine. In Pteroplataea the uterine pai)illae extend into the fetal spiracle, ac- tually a kind of suckling. The fetuses are not otherwise attached to the mother (Wood- Mason and Alcock, 1891; Alcock, 1892). Viviparity in teleosts was known to Aris- totle (Thompson, 1910) and has interested biologists ever since. Embryos develop within the ovarian follicles in representa- tives of the Poeciliidae, Anablepidae, and (roodeidae.-^ In the Ernbiotocidae, Zoarci- d(ie, (ioodeidae, and Jenynsiidae the ferti- lized ovum is released promptly into the ovarian cavity and develops there. -^"^ The ovary may contribute to the food sup- ply of the embryo by the seci'etion of nu- tritive fluid into the follicular or ovarian ca\-ity ; the embryo is bathed in and swallows or absorbs this fluid through the skin.-*' The food in addition may consist of dead si)erm, dead embryos, and disintegrated ova. Ab- sori)tion is facilitated in Annblepidae, Goodeidae, and Poeciliidae by villi (tro- photaeniae) extending from the yolk sac or gut opening, by specialization of the tips of the embryonic fins, by ovarian wall proc- esses which extend into the branchial cham- ber of the embryo, or by development of the pericardial membrane as an absorptive sur- face.-" In certain Anablepidae and Poecilii- dae there is actually a pseudoplacenta (Fraser and Renton, 1940; Turner, 1940a, 1) ) . Respiratory exchange and the removal of waste are effectefl by the same mecha- nisms as is nutrition. Mendoza (1940) noted that epithelium of the ovary of the Goodeidae secretes not only when embryos recjuiring nutrition are pres- ent but also in virgin females; he concluded that these changes are cyclic and independ- ent of the presence of embryos. The analogy with the uterine epithelium of mammals is evident. Turner (1937a, 1940c, 1947), com- menting on the regulation of successive broods in those poeciliid fish in which super- fetation occurs, hyi)othesizes that follicle- stinndating hormone from the intuitary may be responsible. In Lebistes an increase in thyroid activity is correlated with the pe- riod of rapid growth and differentiation of theem})ryo (Stolk, 1951a). Amphibians Vivii:)arity has been described for the Af- rican frog Xectophrynoides. Internal ferti- "Kiintz, 1913; Tiiiner, 1933, 1937a, 1940a, c; I^irspr, 1938. "■'■Stuhlman, 1887; Eigenmann, 1892b; Scott, 1928; Mendoza, 1936, 1937, 1940; Turner, 1937b. '"Eigenmann, 1892a, b; Hubbs, 1921; Scott, 1928; Turner, 1933, 1938c, 1940a, c, 1947; Mendoza. 1936, 1937, 1910; Fra8er and Renton, 1910. -■Turner, 1933, 1937b, 1938c; Mendoza, 1937. 1910. 1936; Tavolga and Rugh, 1947. 1058 SUBM A M M ALIAN VERTEBR ATES lization is somehow accomplished without an intromittent organ. In .V. vivipara as many as one hundred larvae may develop in the bicornuate uterus. The larvae have long, vascular tails which possibly maintain con- tact with the uterine wall to permit respira- tory exchange (Noble, 1931). In N. occiden- talis (Angel and Lamotte, 1944; Lamotte and Tuchmann-Duplessis, 1948 1, each slim oviduct is dilated posteriorly to form a uter- ine horn. The two horns, which join caudally, are muscular, well vascularized, and lined with columnar epithelium. In pregnancy the oviduct does not change, but the horn en- larges as its one to ten embryos grow. There is no placenta; the fluid in which the em- bryos rest may supply nourishment and oxy- gen. The young are retained until after metamorphosis. When born, they may be two-fifths the length of the mother. A", occidentalis has been found only at an elevation of 1200 meters, in fields on top of Mount Nimba in French Guinea. The gesta- tion period is from September to June (La- motte and Rey , 1954 ) . During the dry season (December to February) no specimens were found, and it is believed that the adults may aestivate at this time. It may be that vivi- parity represents an adjustment to an en- vironment which not only lacks bodies of water for incubation of eggs but which has the added hazard of a dry season. This frog is also typically viviparous in that only 10 to 30 ova are found in an animal and that the eggs have almost no yolk (Lamotte, Rey and Vilter, 1956) . Reptiles Many snakes and lizards are viviparous. Several types of placentae occur, and pla- cental exchange takes place. -'^ Fraser and Renton (1940) call attention to the simi- larity between the uterine epithelium under- lying the allantoplacenta in the lizard Lygo- soma ocellatum, as described by Weekes (1930), and the expanded pericardium through which respiratory and nutritional exchange take place in the embryo of the viviparous fish, Heterandria jonyiosa. '^Flvnn, 1923; Harrison and Weekes, 1925; Weekes, 1927a, 1929, 1930, 1935; Rahn, 1939; Boyd, 1942; Dutta, 1946; Heimlirli and Heimlicli, 1950; Kasturirangan, 1951; Bellaiis, Giiffit)is and Bel- laiis. 1955; Clark, Florio. and Huiowitz, 1955. XX. Corpus Luteum Fish Structures resembling corpora lutea occur in widely divergent types of fish — the myx- inoids Bdellostoma and Myxine (Conel, 1917), the elasmobranchs Spinax (Wallace, 1903), Squalus (Hisaw and Albert, 1947), Cetorhinus (Harrison Matthews, 1950), Myliobatis (Giacomini, 1896a), and several teleosts, including Lebistes (Stolk, 19511)), Rhodeus ( Bretschneider and Duyvene de Wit, 1947 ) , and Fundulus (Matthews, 1938 ) . The corpora persist during pregnancy in vi- viparous species. Mendoza (1943), however, feels that the corpus luteum of Neotoca, be- cause it lacks any indication of endocrine ac- tivity, cannot be compared to the mamma- lian corpus luteum. Physicochemical assay of blood drawn from carp, Cyprimis carpio, in October, after the spawning season, did not reveal the presence of progesterone (Bondy, Ui)ton and Pickford, 1957). Hisaw and Abramowitz (cited by Pickford antl Atz, 1957) failed to find progesterone in the cor- jiora lutea of the dogfish, Mustelus earns, and removal of the corpora lutea during pregnancy did not affect the embryos. It is possible, as suggested by various authors, that acciuisition of an endocrine function by the corpus luteum was a somewhat belated evolutionary development. This area de- serves fui-tlicr investigations. Amphibians Corpora lutea have l)een reported in Rana, Xectophrynoides, Bufo, Triton, Sala- mandrina, and Salamandra (Giacomini, 18961); Hett, 1923; Lamotte and Rey, 1954), although Duscliak (1924) denies that cor- poi'a occur in Rana. Reptiles Altland (1951) has described the cori)us luteum of the box turtle, Terrapene c. Carolina, and Rahn (1938), that of the snapping turtle. In general, the corpora re- semble those of mammals. A curious fea- ture of the reptilian corpus luteum is the absence of blood vessels in the central epi- thelial portion. The box turtle's corpora are present in June, the month when the eggs COLD-BLOODED VERTEBRATES 1059 arc rt'tainecl in the o\-idu('t, and atrophy after oviposition. Corpora lutea occur in both ox'iparuu.s and viviinirous lizards.-" The f^uggestion that a hormone from the corpus luteum })revents further follicular growth and ovulation was made by Cunningham and Smart (1934) and Panigel (1951b). Boyd (1940), calling attention to the corpora lutea of oviparous reptiles, offers the hypothesis that in this vertebrate class the corpus luteum may have evolved before the i^lacenta and only subsequently acquired an endocrine func- tion related to viviparity icf. discussion of corpus luteum function in fish and amphibi- ans). The gestation period in Xantusia lasts about three months; by histologic criteria the corpus luteum appears functional for the first two months (Miller, 1948, 1951). Bilateral ovariectomy or destruction of the corpora lutea at the beginning of preg- nancy in Lacerta vivipara does not inter- rupt gestation or embryonic development (Panigel, 1953, 1956). Hypophysectomy also fails to affect embryonic growth, but both this operation and ovariectomy make parturition difficult, as does the administra- tion of progesterone (Panigel, 1956). Corpora lutea have been found in the ovaries of many genera of snakes.-^*^ Corpora may persist during part or all of gestation. Ovariectomy during early and middle preg- ■" Oviparous: Amphiholunis (Weekes, 1934), Hemidactylus (Dutta, 1944), and Lacerta viridis (Cunningham and Smart, 1934). Viviparous: Hoplodactylus (Boyd, 1940), Xatitusia (Miller, 1948, 1951, 1954), Egernia, Lygosoma, and Tiliqua (Weekes, 1927a, 1934), Lacerta (Zuotoca) vivipara (Hett, 1924; Regamey, 1935; Panigel, 1951a), and Anguis (Lucien, 1903; Cunningham and Smart, 1934). *' Oviparous: Xeiiodon (P'raenkel and Martins, 1939). Ovoviviparous: Coronella (Rollinat, 1898) and Dryophylax and Tomodon (Valle and Souza, 1942). Viviparous: Natrix {Tropidonotiis) (Rol- Hnat, 1898; Rahn, 1939, 1940b; Bragdon, 1946, 1951 ; Bragdon, Lazo-Wasem, Zarrow and Hisaw, 1954). Potamophis (Rahn, 1939), Storeria (Ralin, 1939), Thamnophis (Rahn, 1939, 1940a, b; Cieslak, 1945; Bragdon, 1946, 1951, 1952; Bragdon, Lazo-Wasem. Zarrow and Hisaw, 1954), Enhydrina (Samuel, 1944; Kasturirangan, 1951), Hydropliis (Samuel, 1944), Bothrops and Crotrdus (Fraenkel and Mar- tins, 1938, 1939; Fraenkel, Martins, and Mello, 1940; Porto, 1941; Rahn, 1942; Valle and Valle, 1943). Natrix, Thamnophis, and Crotalus are re- garded as ovoviviparous tiy some investigators. nancy of \'arious species of the colubrid snakes Xntrix, Thamnophis, and Storeria was followed by resorption of the embryos or the birth of dead embryos. The injection of progesterone after ovariectomy did not prevent death of the embryos. Ovariectomy late in jiregnancy seemed to have no effect. Hypophysectomy interfered with pregnancy at any stage. Injection of posterior pituitary extract did not influence early or middle pregnancy, but induced delivery thereafter (Clausen, 1940). Removal of all corpora lutea from pregnant Bothrops and Crotalus resulted in cessation of embryonic develop- ment (Fraenkel Martins and Mello, 1940), but, as Bragdon ( 1951 ) suggests, the high postoperative mortality leaves some doubt whether embryonic death was due to loss of the corpora lutea or to other factors. On the other hand, Rahn (1939, 1940b) cas- trated 23 pregnant Thamnophis and Natrix at various stages of jiregnancy and found live young at autopsy up to 25 days later. In Xatrix and Thamnophis neither hy- pophysectomy as early as the first week af- ter ovulation nor ovariectomy interfered with pregnancy (Bragdon, 1942, 1946, 1951. 1952). Both operations did interfere with parturition. Bragdon concludes that in these snakes corpora lutea are not essential for the maintenance of pregnancy. Panigel's ex- tensive study of the viviparous lizard iZoo- toca vivipara (1956) supports this opinion. Bio-assay of alcoholic extracts of the corpora lutea of Bothrops and Crotalus re- vealed progestin (Porto, 1941), as did bio- assays of plasma from Natrix and Tham- nophis {Bragdon, Lazo-Wasem, Zarrow and Hisaw, 1954). In the latter study, the results, expressed in Progesterone Equiva- lents, were as follows. In two donors with inactive ovaries and two with pre-ovulatory follicles, plasma levels were 0.3, 1.0, 0.3, and 0.3 /xg./ml. Other assays showed, at early ovulation, 2.0 /i,g./ml.; end of first third of pregnancy, 4.0 and 4.0 /xg./ml.; end of second third of jiregnancy, 4.0 and 6.0 fig./ml.; full term, 8.0 /xg./ml. It is also of interest that bio-assay of plasma from two snakes with testes in full development gave Progesterone Equivalent values of 0.3 and 1.0 /xg./ml. (Bragdon, Lazo-Wasem, Zarrow and Hisaw, 1954). 1060 SUBMAMMALIAN VERTEBRATES This may be compared to the observation that progestin can be detected in tlie blood of roosters but not of capons (Fraps, Hooker and Forbes, 1949). XXI. Developmental Basis for Sexual Diniorphisni Fish Most embryologists believe that in am- phibians and amniotes the functional ovar- ian cortex develops from the primitive cor- tex of the fetal gonad and that the testis is derived from the embryonic medulla. Ponse (1949), in an extensive and careful discussion of the embryology of the gonad, concludes that a cortex and medulla cannot be distinguished in the developing gonad of the fish. D'Ancona (1950) shares this opin- ion. Studies of the brook lamprey, Ento- sphemis irilderi (Okkelberg, 1914, 1921). and of the closely related Petromyzon (Lu- bosch, 1903) reveal that male and female gonads are at first indistinguishable mor- phologically (undifferentiated gonad stage). Later (bisexual stage) a distinction can be made on the basis of the relative numbers of spermatocytes and oocytes; both kinds of germ cells appear in both sexes. Pro- longed juvenile bisexuality resulting from arrest of sexual develoiiment at the end of the second stage is probably the basis for much of the hermaphroditism in fish. In a third phase (stage of sexual differentia- tion ) , the heterosexual germ cells disappear from the lamprey gonad or persist in rudi- mentary form, and the sex of the lam})rey finally becomes apparent. D'Ancona (1950) summarizes evidence for similar develop- ment of the gonad in several other jilagio- stomes. Much, or even all, of the development of tlie o\'ary and testis in both lower orders of fisli and teleosteans may occur after hatching or birth. In Gobius the gonads are said not even to appear until 15 days after birth ( MacLeod, 1881 ) . Knowledge of the development of the reproductive system of the teleosts, as of the cyclostomes, is fragmentary. D'Ancona (1950) divides the teleosteans which have been studied into two groups. In the first, the gonads gradually differentiate from an indifferent to a bisexual stage and then into ovaries or testes. The viviparous teleost Cymatogaster is an example (Eigenmann, 1897). The gonadal anlage in the 8-mm. lar^'a is a simple fold of peritoneum on either side of the mesentery. Each fold ac- quires a core of germ cells and stromal cells, and the two folds, or germinal ridges, fuse posteriorly. In 15- to 17-mm. larvae the ovaries can be distinguished because they are shorter than the testes and have a lon- gitudinal groove which will later invaginate further to form the central cavity of the ovary. The testes develop internal lobules and a central, branched collecting tubule. At 22 mm. the ovaries have become tubular; the walls are thick superiorly and medially where "oviferous folds" will develop, and ai'e thin inferiorly and laterally. Later the two ovarian cavities join posteriorly. The oviducts develop much as in higher verte- brates: a plate is grooved into two parallel ridges, and the latter bridge over the in- tervening space to form a tube which is then extended caudally. Ovaries and ovi- ducts are continuous. The vas deferens, which is not homologous with the oviduct, is lined with stromal cells from the testis. Jolmston ( 1951 ) gives a similar descrip- tion for another acanthopterygian, Mi- cropterus, the black bass. He adds that the testis also becomes tubular; the cavity proper, or testocoel, functions as a primary collecting duct. Branches from the testocoel complete the collecting system. A similar development of the gonads and gonoducts appears to occur in several other genera.^^ D'Ancona's (1950) second group of tele- ost fish includes representatives of the Sparulae and Serranidae (Dantchakoff, 1936; Kinoshita, 1936; Lavenda, 1949; D'Ancona, 1950). Although these are also acanthopterygian families (see above) the embryonic gonads are persistently ovotes- tcs. Both ovarian lamellae and testicular lobules may protrude into the central cavity of the same gonad. In Spams longispinis "Anna (D'Ancona, 1955). eel (Grassi, 1919), the trout (Mrsic, 1923, 1930; Asliby, 1952), Xiphu- phorus (Essenberg 1923; Regnier, 1938; Vallowe, 1957), pipe fish, sea horse, and goby (MacLeod, 1881), goldfish (Stromsten, 1931), guppy (Dildine, 1936), and Cottns (Hann, 1927). COLD-BLOODp]D VERTEBRATES 1061 tliere are two oviducts and two ^'aJ^a def- erentia. When a body length of 100 nnn. is reached, either the testicuUir component and vasa deferentia degenerate, or the ovar- ian conii^onent and the oviducts disajipear (Kinoshita, 1936). Ainphihians Sec chapter by Burns. Reptiles Probably the first i)ublislied observations on the embryology of the reproductive sys- tem in the turtle were those of Rathke (1848). Risley has summarized the sub- sequent literature on the subject and has carefully analyzed the origin of the germ cells (1933b) and the embryology of the reproductive system (1933c) in Sterno- therus odoratus, the musk turtle. He rec- ognized three stages in gonadal de- velopment— indifferent, bisexual, and differentiated. During the indifferent phase the primitive germinal epithelium, a spe- cialized area of peritoneum investing the ventromedial surface of each mesonephros, dorsally proliferates sex cords containing germ cells. Stromal elements grow between the epithelial sex cords. The gradually thickening mass of tissue thus formed is known as the genital ridge. Extension of the epithelial elements of the sex cord beyond the gonad and to a nearby Malpighian cor- puscle in the mesonephros marks the estab- lishment of the rete cord connection. In the bisexual period there is a further proliferation of the germinal epithelium and its germ cells to form an investing cortex. Internal to the latter is a medulla composed of the original sex cords and their gametes. Cortex and medulla are both well developed, and all embryos at this stage also have mesonephroi (potential epididy- mides), mesonephric ducts (potential vasa deferentia), Miillerian ducts, and phalluses. This bisexual condition, sometimes referred to as juvenile or rudimentary hermaphrodi- tism, persists for a relatively long time. Late in embryonic development, morpho- logic sex differentiation ensues as a third stage. In the male, the medullary cords begin to acquire lumina and to become seminiferous tubules and the cortex and Miillerian duct begin to regress. In the fe- male, the cortex develops further, the med- ullary cords regress to connective tissue strands, and the growth of the phallus is checked. It must be emphasized, however, that at hatching heterosexual vestiges are still conspicuous. The development of the reproductive tract in most other reptiles is also relatively slow as compared to that of higher verte- brates. The process may in fact never really be completed, so that the adult frequently or regularly retains definite heterosexual remnants, e.g., a vestigial mesonephros in the female and a part of the Miillerian duct in the male. Risley's account for the turtle in general confirms Braun's early (1877) account of the development of the reproductive system in lizards and snakes (chiefly Lacerta, Anguis. Tropidonotus, and Coronella). Mihalkovics' (1885) excellent study (chiefly of the lizard) added important information. He reminds his readers, for example, that Braun "was struck by the proximity of the adrenal anlage to that of the gonad in rep- tiles ... so that one would be led to think of a relationship between the two struc- tures." This is in reference to the fact that the adrenal cortex takes origin from spe- cialized epithelium lying between the root of the mesentery and the germinal epithe- lium. The anlage of the adrenal cortex (medial) and the anlage of the genital ridge (lateral) i^roliferate epithelial cords dor- sally at about the same time. Actually, it is diflficult to determine the dividing line be- tween the two anlagen at this early stage except on the basis that the germ cells normally are confined to the develop- ing gonad. This physical continuity in the embryo is sometimes overlooked, but is of interest in view of the demonstrated ability of the adult mammalian adrenal cortex to produce sex hormones, an ability which may well be shared by some of the lower vertebrates. Other accounts are those of Hoffmann (1889), Peter (1904), Simkins and Asana (1930), and Forbes (1956). Regamey (1935) mentions a male Lacerta embryo of advanced development which retained both oviducts. Dantchakoff (1938) describes male and female lizard embryos 1062 SUBMAMMALIAX VERTEBRATES at hatching. The testis, clearly recognizable as such, contains sex cords with numerous germ cells and is invested with a thin coelomic epithelium. The ovary is also well differentiated, consisting of outer cortex and central medulla. The two components are clearly delimited. Most of the germ cells are in the cortex. The metanephros is well developed. Both sexes have mesonephric ducts, but Miillerian ducts are present only in the female. The development of the re- productive tract in Anolis is similar, al- though in this genus the Miillerian ducts may persist after hatching in the male (Forbes, 1956). Probably the earliest study of the em- bryology of the snake's gonads was that of Rathke (1839). Hartley's modern (1945) account calls attention to the brief and early (16th to 19th day) i)eriod of sex- ual differentiation in the garter snake and to the relative scarcity of heterosexual vestiges at birth. Both phenomena ap- parently are unique for the snake among reptiles. The writer (Forbes, 1940a) has described the embryology and post-hatching develop- ment of the gonads, adrenal cortex, and Miillerian duct of the alligator, Alligator mississippiensis. Prolonged indifferent, bi- sexual, and sexually differentiated stages much like those reported in the turtle by Risley (see above) were observed. A period of pronounced bisexuality is succeeded by sexual differentiation, but heterosexual structures regularly persist until well after hatching. The degree of adult bisexuality is unknown, but the male alligator 18 months after hatching still has irregular patches of cortex (potential ovarian tissue) on his testis. The male Miillerian ducts are fully formed at hatching, and their anterior extremities may still be present 18 months later. Females at this age and earlier have in the posterior third of each gonad a solid mass of medullary cords (potential semi- niferous tubules ) similar to those seen in the embryonic testis, but lacking germ cells. A mesonephros and mesonephric duct are always present. Thus again there is a con- siderable retention of heterosexual struc- tures. XXII. Spontaneous Adult Herniaphrotlitisni and Sex Reversal Fish Aristotle recognized hermaphroditism in the channe, a perch-like form, and in two other varieties (Thompson, 1910), as did Ovid (Ovidius Naso, 1911). All three fish are thought to have been serranids (Du- fosse, 1856; Thompson, 1910). The classical writers apparently shared the popular belief that the fish are herma])hroditic because all specimens caught contained eggs, i.e.. no males were ever seen. A chapter title in a book by Guilaume Rondelet (1558) con- cerns fish "which give birth without the assistance of the male," evidently a refer- ence (the text is not enlightening) either to hermaphroditism or to parthenogenesis. Du- fosse (1856) states flatly that in three serranid species the same fish which lays the eggs also fertilizes them. Cavolini (1792) found ovotestes in the Potsch and Blutstrieme] there is reason to think that these were two of the fish about which Aristotle had conmiented. Yarrell ex- hibited to the Zoological Society of London in 1845 a herring "having a lobe of female, or hard roe, on one side, and a lobe of male, or soft roe, on the other." The note adds that the same phenomenon had been seen in several other kinds of fish. In the 1880's a friend of Carl Vogt, the German naturalist, was about to eat a smoked herring when he noticed that the reproductive tract was ab- normal. The friend carefully removed the tract, sent it to Vogt with a letter, and, one hopes, ate the rest of the herring. In spite of the unconventional fixation of the tissues it was possible to confirm histologically that both gonads were ovotestes; in addition, an excretory duct on each side was so arranged as to carry off both sperm and ova (Vogt, 1882). A number of examples of adult hermaph- roditism are summarized in Table 17.1. Ad- ditional references and details may be found in books by Gemmill (1912) and Dean (1923) and in D'Ancona's article (1956). Two types of adult hermaphroditism can be recognized. The basis for the first lies COLD-BLOODED VERTEBRATES 10G3 TABLE 17.1 Spontaneous adult pseudvhennaphrodiliHiii and licniiaphroditistn in fish Genus Gonads Accessory Structures Reference Myxine Bdellostoma (Scyllium)* Ovotestis Ovotestis Ovotestis Ovotestis Testis and ovary Testes Testes Testes Testis and ovary; ovotestes Ovotestes Ovotestis ; testis and ovary Ovotestes Testis and ovary; ovotestes Testis and ovary Ovotestis Ovotestes Testis and ovary Ovotestis Ovotestes Ovotestis Ovotestes Testis and ovary Ovotestis Ovotestis Ovotestis Testes Ovotestes Ovotestes Ovotestes Ovotestes Testis and ovary Testis and ovary; ovotestes Ovotestis Ovotestes Ovotestes Ovotestes Ovotestes Ovotestes Ovotestes Gonopodium, oviducts Gonopodium, oviducts Oviducts, male pelvic fins Oviduct Oviduct Oviduct Oviduct, vas deferens Male genital ducts Male gonoducts and pigmentation Oviduct, vas deferens Female external ap- pearance Vas deferens Oviduct, male colora- tion Female coloration, gonopod Intersexual accessories Male to female type anal fin Cunningham, LS8Ga; Nansen, 1887; Cole, 1905 Cunningham, 188Gb Vayssiere and Quintaret 191-1 Scyliorhinus Murray and Baker, 1924; Ar- thur, 1950 Rowan, 1929 Matthews, 1885 Acipensei' MaschkowzefT, 1934 Pseudoscaphirhynchus.. Clupea MaschkowzefY, 1934 Smith, 1870; Smitt, 1882; Vogt, 1882; Southwell, 1902; Grimpe, 1922-1925; (ialiler, 1930; Bullough, 1940a Andreu, 1955 A losa Fowler, 1912 Sal7no Stewart, 1894a; (iilihs, 195G "Trout" de Beer, 1924; Mrsic, 1930 Oncovh ynchus Crawford, 1927 Esox Kolmer and Scheminzkv, 1922b Phoxinus Bullough, 1940b N otropis Reed, 1954 Carassius Kinoshita, 1933 Kobavashi, 1955 Grassi, 1911, 1919 (Morrhua)* Smith, 1870 Howes, 1891 FunduJus Newman, 1908; Chidester, 1917 Blacher, 1926; Spurway, 1957 Essenberg, 1926; Harms, 1926b; Glaridichthys Friess, 1933; Regnier, 1938 Philippi, 1904 Bullough, 1947 SevTanus Dufosse, 1856; van Oordt 193()- Ceniropristes van den Broek, 1933; Dant- chakolT, 1936 Lavenda, 1949 Bishop, 1920 Roccus Schultz, 1931 James, 1946 "Perch" Turner, 1927 Boleosotna Lagler and Chin, 1951 Stephan, 1901; van Oordt, 1930 Paoellus Gomez Larraneta, 1953 Taius Aoyama, 1955 Coris .... Bacci and Razzauti, 1958 Scomber Stewart, 1894b Synonymous with following genu 1064 SUBMAMMALIAN VERTEBRATES in the fact that, as has been stated, the de- veloping gonads of most fish pass through an indifferent and then a bisexual stage. In the latter phase, both ovarian and tes- ticular elements are present and there is, in effect, a transient and juvenile hermaph- roditism. Subsequently, the gonad usually becomes a testis or ovary. In exceptional cases, functional gonadal tissue of both sexes persists into adulthood (Table 17.1). In Sargus and Serranus such hermaphrodit- ism appears to be normal and frequent. In many individuals of these genera, gross and microscopic study has proven the co-exist- ence of normal ova and sperm (van Oordt, 1930; van den Broek, 1933; Dantchakoff. 1936; D'Ancona, 19501. Hermaphroditism in Lebistes is not regu- lar. However, functional bisexuality was ob- served in 1 fish and in 18 of its young; parent and offspring, although virgin and individually isolated, bore young (Spurway, 1957 ) . Parthenogenesis was discarded as an explanation in view of the co-existence of functional testicular and ovarian tissue and the likelihood of self-fertilization. The oc- currence of the latter, although claimed in other cases, seems not previously to have been supported by the evidence; in Spur- way's series self-fertilization seems proba- ble. The offspring of the 18 fish, inciden- tally, were almost all females. In a second type of hermaphroditism the adult fish at first is morphologically of one sex but subsequently undergoes sjiontaneous reversal to the other. If the testis develops first, the condition is known as protandry. Cunningham (1886a) and Nansen (1887) believed that Mijxine is usually a protan- drous hermaphrodite, but Cole (1905) and Conel (1917) were not convinced. Cole was of the opinion that every adult Myxine has "either a mature testis and a rudimentary ovary, or a mature ovary and a rudimen- tary testis." Hermaphroditism due to sex reversal does occur during adulthood in widely divergent teleostean genera (Table 17.1). It is significant that, wuth the excep- tion of the doubtful case mentioned above, all instances of spontaneous sex reversal seem to be protogynous (ovary first, testis later) rather than protandrous. Bullough (1947) has reviewed Liu's al- most inaccessible report (1944) on the Ori- ental species Monoptenis javanensis, which regularly "functions as a female during the first half of its life and as a male during the second." The same phenomenon is re- ported for Pagellus (Gomez Larrafieta, 1953) and for Coris (Bacci and Razzauti, 1958). In C entropristes the males retain ovarian, and the females testicular, tissue. Age can be determined by examination of the scales. The females are more numerous in the younger age groups, but disappear by the tenth year, whereas all fish surviving during the next 10 years are males. On the basis of this evidence, Lavenda (1949) be- lieves that there may regularly be sponta- neous female to male sex reversal after the fifth year. Aoyama (1955) found that 22 of 3291 individuals of the yellow sea bream, Taius tuniifrons, had hermaphroditic gon- ads in which a transition from ovary to testis was in jjrogress. Similar transforma- tion was observed in 10 of 414 minnows (Bullough, 1940b). Although exceptional, female to male sex reversal has been described repeatedly and in full detail for Xiphophorus (Essenberg, 1926; Harms, 1926b; Friess, 1933; Regnier, 1938). Before sex reversal the females give birth to young; after reversal the newly formed males demonstrate the completeness of their transformation by successfully im- pregnating virgin females. The oviduct is converted to a sperm duct, male coloration replaces that of the female, and the gono- podium and characteristic "sword" develop. No pathologic tissues have been found, a point of possible significance, as develop- ment of a testis in a hen, for example, is known to follow tubercular destruction of the functional ovary. Oviposition is arrested and the female's characteristic "pregnancy spot" (Trdchtigkeitsfleck) between the pel- vic and anal fins fades. Then, as the gono- podium and sword begin to grow, the ova disappear, the follicles become atretic, and most of the ovary disintegrates, leaving the epithelium of the ovarian cavity. Leuko- cytes eventually dispose of the masses of ovarian debris. From the residual epithe- lium radial sex cords rapidly proliferate to form a testis, and germ cells multiply quickly. Eventually the new testis is in- COLD-BLOODED VERTEBRATES 10(55 (listingiii!shal)le in moi'iihology, location, and function from the testis of a typical male. The process of reversal may re(iuire 3 or 4 months. It is possible that co))i})lete adult sex re- versal is more common than is realized. Un- less by observation or autopsy it were detected while in progress, or unless subse- quent genetical studies could prove that sex reversal had occurred, the phenomenon might easily be unrecognized. The difficult question of the role, if any, of the chromosomes in regulating sex re- versal appears to be very imperfectly un- derstood. Consideration of this and related genetical problems is outside the scope of this chapter. Amphibians See chapter by Burns. Reptiles Cases of postembryonic retention of het- erosexual accessory structures and of her- maphroditism in reptiles are summarized in Table 17.2. The conditions described for Hatteria {Sphenodon) (Osawa, 1897) ap- parently, and for Malaclemmys (Risley, 1941b) and Alligator (Forbes, 1938b, 1940b) definitely, are normal rather than excep- tional. One of the most remarkable cases was that of Hansen's (1943) turtle. Histologic sections showed two ovotestes. The anterior and posterior extremities of each gonad were testicular and contained seminiferous tubules. The central ovarian portion con- tained mature and immature eggs. Vasa efferentia joined the testicular areas to the epididymides. The latter contained sperm, and were connected by vasa deferentia to the cloaca. There were two normal and aj)- parently functional oviducts and a normal penis. External characteristics were mascu- line. Tayler's hermaphroditic lizard (1918) had bilateral ovotestes with ova, seminifer- ous tubules, and interstitial tissue. Epididy- mides were normal except for the absence of sperm and of vasa efferentia. Oviducts were incomplete, and there was a penis. The writer (Forbes, 1940b) has described the medullary "rest" at the j^iosterior end of each ovary in the immature alligator. The rest is composed of medullary cords con- taining germ cells. The cords may have lumina. Rete canals join the medullary tu- bules to a persistent mesonephros, the duct of which extends to the cloaca. The female thus seems to have potential testicular tis- sue served by a complete excurrent system. The medullary rest is bilateral but is quite similar to the vestigial right gonad of the hen (Erode, 1928) ; the latter contains germ cells for three weeks after hatching. Rete tubules and a right mesonephros and meso- nephric duct also occur in the hen. Retention of heterosexual gonoducts is not unusual. For an unknown reason, in such cases all or nearly all of the meso- nephric duct persists, whereas only part of the ]\Iullerian duct is retained. XXill. Experimental Sex Reversal Fish Some effects of androgens and estrogens on gonoducts, coloration, and other acces- sory sex structures have been described. Attention will now be directed primarily to the effects of administered sex hormones on the gonads themselves and to the sex-re- versing action of castration. When the fighting fish, Betta splendens, was ovariectomized, a testis regenerated from the severed end of the oviduct in 7 of 150 spayed fish (Noble and Kumpf, 1937). In time the 7 fish showed male fins, sperma- togenesis, male behavior, spawning, and fer- tilization of eggs. Nine males and 12 females from these matings grew to maturity. If males, on the other hand, were castrated, they always regenerated testes. Testosterone propionate influenced the genital ridge and accessory sex structures in the elasmobranch Scyliorhinus to develop in the female condition (Chieffi, 1954). The same compounrl when given to female Fhoxinus minnows caused ovarian disinte- gration and the appearance of mascu- linization pigment (Bullough, 1940b). In Xiphophorus feeding (by adding it to aquarium water) of testis powder or testos- terone propionate to pregnant females and to the young when they were born had the result that all the young which grew to TABLE 17.2 Spontaneous postembryonic pseudohermaphroditism and hermaphroditisui in reptiles Genus Gonads Accessory Structures Reference Chelonia Mesonephros and duct in fe- Evans, 1939 Chrysemys male Ovo testes, epi- Risley, 1941a didymides, oviducts Malaclemmys Masculinizing (?) medullary tumor of ovary Risley, 1941a Male Miillerian ducts; meso- Risley, 1941b nephros and ducts in fe- males* Pseudemys Ovotestes Oviducts, vasa deferentia, epi- didymides, male claws, penis Hansen, 1943 Em ys Mesonephric ducts in female; van Wijhe, 1881; Wied- oviducts in male ersheim, 1886; Hoff- mann, 1890 Ovotestis, testis Mesonephric duct and oviduct Matthey, 1927 Miillerian ducts in male Nicholson and Risley, 1940 Female had penis. Male had Combescot, 1954b Mullerian duct. Testudo Male had Mullerian duct Gadow, 1887 Ovotestis, testis Oviduct Fantham, 1905 Mullerian ducts in male van den Broek, 1933 Mesonephric ducts in female Alverdes, 1928 Chelonia Female had vas deferens van Wijhe, 1881 Male hadMidleriaii duct. Mes- Hoffmann, 1890 onephric duct in female Trionyx Mesonephric duct in female van Wijhe, 1881 Rhynchocephalia Hatteria iSphenodon) Mullerian ducts in male* Osawa, 1897 Lacertilia Sceloporus Mullerian ducts in male Forbes, 1941 Amphibolurus Mullerian ducts in male Hill, 1893 Stellio Mullerian ducts in male Schoof, 1888 Uromastix Mesonephros and duct in fe- male Schoof, 1888 Chamaeleo Mesonephros and duct in fe- male Schoof, 1888 Hemidactylus Mesonephros and duct in fe male Mahendra, 1953 Flatydactylus Mesonephric duct in female Braun, 1877 Gongylus Mesonephros and duct in fe- male Schoof, 1888 Lacerta Miillerian duct in male; meso- von Leydig, 1853, 1872; nephros and duct in female Braun, 1877; Howes, 1887; Schoof, 1888; Jacquet, 1895; Lantz, 1923; Regamev, 1931 Ovotestes Penial sacs, oviducts, epididy- mides Tayler, 1918 Anquis Miillerian duct in male; meso- Leydig, 1853, 1872; nephros and duct in female Braun, 1877 Anniella Mesonephric ducts in females Coe and Kunkel, 1905 Ophidia Callopeltis Mesonephric ducts in females Braun, 1877 Coronella Mesonephric ducts in females Braun, 1877 Tropidonotus Mesonephric ducts in females Braun, 1877 Zamenis Mesonephric ducts in females Braun, 1877 Pelias Mesonephric ducts in females Braun, 1877 COLD-BLOODED VERTEBRATES 10t)< TABLE n .2— Continued Genus Gonads Accessory Structures Reference Crocodilia Alligator ALilleriaii ducts in males; mes- onephric ducts in females (iadow 1887 Ovarian meduL Rete system, mesonephros and Forbes 19401) larv rest; tes- duct in females; Miillerian ticular cortex ducts in males.* Occur regularly. maturity were males. Intramuscular injec- tions of testosterone propionate into preg- nant females, immature fish, and adult nonjiregnant females caused complete mas- culinization of some but not all fish. Male sex characters were conspicuously developed in all cases (Regnier, 1938, 1939). Baldwin and Goldin (1939), Querner (1956), and Vallowe (1957) in carefully controlled ex- periments carried out a somewhat similar series of injections and obtained masculini- zation, in some cases with spermatogenesis, of about half the experimental fish. Treat- ment of female Lebistes and Platypoecilus with androgens had little or no effect on the ovaries except to suppress ovogenesis but did stimulate the growth of male accessory sex structures such as the gonojiodium (Eversole, 1939; Regnier, 1939, 1942; Tay- lor, 1948; Querner, 1956). Mohsen (1958) more recently, however, gave relatively low doses (0.015 or 0.03 mg. thrice weekly) of pregneninolone to Lebistes for 6 months after hatching and observed not only the development of gonopodia in all treated fish but the transformation of ovaries to ovo- testes. Testes of treated males were normal, except that the higher dose stimulated sper- matogenesis. Testosterone propionate, para- doxically, elicited the appearance of ova at the base of an otherwise normal testis in adult male Oryzias (Egami, 1955c). Not only androgen but estradiol benzoate, progesterone, and desoxycorticosterone ace- tate, if injected into the embryonic yolk sac, feminized the genital ridges of Scyliorhinus (Chieffi, 1954). Immature trout were parti- ally feminized by keeping them in water to which estrogen had been added (Padoa, 1939a). Injection of estrin into the body cavity of the minnow Phoxinus laevis caused l)reakdown of tlie testes and assump- tion of female coloration (Bullough, 1940b). Administration of female sex hormones to Lebistes males transformed the testes to ovotestes and feminized the secondary sex characters of immature, but not of mature, males. Estrogen had no effect in females (Berkowitz, 1938, 1941; Querner, 1956). Various estrogens when given to Oryzias caused the appearance of testis-ova (Egami, 1955b, c; Okada, 1943). Similar treatment api)arently had the same result in immature but not in mature male Xiphophorus (Val- lowe, 1957). In Hepatus (Sey'raynis) hepatus, treatment with estrogen stimulated the growth of the hermaphroditic gonad (Pa- doa, 1939b). Thus, the accessory sex structures of fish apparently may be influenced by adminis- tration of heterosexual sex hormones, par- ticularly androgens, at any time, and the sex of gonads of embryonic, immature, and sometimes mature fish may be partially or completely reversed by the same agents. Further experiments w^ith special attention to the histologic details of each stage of reversal would be valuable, as would the measurement of endogenous sex hormone levels in blood and gonads. Amphibians See chapter by Burns. Reptiles Risley (1940) injected 0.25 mg. testos- terone propionate in oily solution into the eggs of the turtle, Chrysemys marginata belli. The embryos were in the gastrula stage. The mortality rate was high, and only one injected male embryo survived. Of the five surviving injected female embryos, two had gonads with slight and three with more advanced modification toward testes 1068 SUBMAMMALIAN VERTEBRATES in that the ovarian cortex was thinner, and the medullary cords were larger and more numerous, than in the controls. No acces- sory structures were affected. Juvenile diamond-back terrapins, Mala- clemmys centrata, were also injected with testosterone propionate (Risley, 1941b ». In the males there resulted cavitation of the medullary cords and slight growth of Miil- lerian ducts. In the females ovarian and follicular size were less than normal, and Miillerian ducts grew conspicuously. Wolf- fian ducts and ureters hypertrophied in both sexes. Estradiol dipropionate injections caused reduction of testis size but growth of its cortical remnant and of Miillerian ducts, Wolffian ducts, and ureters in both sexes. Classic studies on sex reversal in lizard embryos of an unidentified species have been made by Dantchakoff (1938). Fertile eggs were removed surgically from the mother, 8 to 12 being obtained at each operation. An injection of 0.05 to 0.15 mg. folliculin (an estrogen) in sesame oil was made into the egg under the blastoderm. Treated eggs and their controls were opened and examined 2 weeks after injection, ap- proximately 2 weeks before hatching, 1 week before hatching, or 3 to 5 days after hatching. Estrogen treatment converted the testes to "hypo-ovaries." The medulla was inhibited, and a true cortex was developed, although it was thinner than in a normal female. In treated females the ovarian cor- tex and medulla were hypertrophied. In experimental embryos of both sexes large Miillerian ducts were present, although the ducts were often incomplete in the males. (Miillerian ducts were always absent at hatching in control males.) Estrogen did not appear to affect the mesonephric ducts. The cloacal epithelium of treated males and fe- males was transformed, as Dantchakoff points out, very much in the manner de- scribed by Allen and Doisy (1923) for the vaginal epithelium of rodents in estrus. The epithelium had increased from 2 or 3 to 25 or 30 cell layers in thickness. The ureters were unaffected, but the cloacal extremities of the Wolffian ducts and the bladder-like diverticulum of the cloaca showed epithelial hypertrophy and metaplasia. Dantchakoff concluded that folliculin helps to control the normal development of the female repro- ductive system in the lizard, particularly in regulating Miillerian duct growth and in the "feminine orientation" of the indifferent gonad. From this she reasoned that follicu- lin is not present in the normal male em- bryo. Implantation of testosterone propionate pellets into gonadectomized and intact, im- mature and adult Anolis of both sexes (Noble and Grcenberg, 1940, 1941) resulted in oviduct hypertrophy in all females as compared to untreated controls. Pellets of testosterone propionate or es- tradiol dipropionate were implanted sub- cutaneously in gonadectomized and intact, male and female, immature and adult Anolis (Noble and Greenberg, 1940, 1941). In the females both hormones caused conspicuous oviduct hypertrophy and extreme keratini- zation of urodeal cloacal epithelium. The androgen also produced hypertrophy of the ovaries. Wolffian ducts, and "sexual seg- ments" of the kidneys. Both hormones caused a similar keratinization of the cloaca in males, whereas androgen evoked hyper- trophy of the Wolffian duct and sexual seg- ment and maintained in normal condition the epididymis and vas deferens of adult castrate males. Forbes (1941) implanted either testosterone or estrone in adult male Sceloporus. Treatment was followed by some reduction in testicular volume, ac- celeration of spermatogenesis, hypertrophy, and mucosal hyperplasia of persisting seg- ments of JNIiillerian duct, apparent stimula- tion of the femoral glands, and hypertrophy of the epididymides and vasa deferentia. Absorption of estrone resulted in reduction of testicular volume, and spermatogenesis almost ceased. Seminiferous and cpididymal tubules and femoral and cloacal glands be- came atrophic, as did the cells of the sexual segments. On the other hand, there was great hypertrophy of the Miillerian duct vestiges and stratification and cornification of the urodeal and proctodeal mucosa. It is believed that in mammals the administra- tion of either sex hormone depresses the re- lease of pituitary gonadotrophin, thus re- moving endocrine support for the gonad, but that testosterone also acts directly to stimu- late both spermatogenesis and accessory sex COLD-BLOODED VERTEBRATES 1069 structures. Such an interpretation would seem applicable as well to the results ob- tained in Sceloporus. In the garter snake (Hartley, 1945) sex differentiation, as previously stated, nor- mally occurs between the 16th and 19th day of pregnancy. To study the effect of sex hormones, testosterone propionate or es- tradiol di[)ropionatc was injected during the first month of pregnancy, either into the peritoneal cavity of the mother or into the amniotic sacs. For some reason, intra- peritoneal injections had no effect on the young. Intra-amniotic androgen increased the rate of spermatogonia! mitoses and growth of seminiferous tubules. This hor- mone prevented regression of the ovarian medulla but checked development of the ovarian follicles. Estrogens reduced the size of the seminiferous tubules but caused no change in the ovary. Miillerian duct hy- pertrophy was seen during the last month of gestation in females injected with estro- gen and androgen. Parturition did not occur if estrogen had been injected; the reason for this failure was not clear. Sex hormones have also been injected into the immature alligator (Forbes, 1938a, b, 1939). Estrone injections for 80 days evoked conspicuous hypertrophy not only of the ovarian cortex but also of the cortical ves- tiges on the testis. The oviducts of the treated females became so huge as to oc- cupy most of the abdominal cavity, and persistent Miillerian duct segments in the injected males also showed much growth. Treatment with testosterone and testoster- one propionate was followed by hypertro- phy of the oviducts, penes, and clitorides. Curiously enough, neither hormone affected the mesonej)hroi or their ducts. As Dantchakoff (1938) points out, sex hormones may play an imjiortant role in the differentiation and development of the reproductive system of reptiles, both during and after embryonic life. Whereas experi- mental treatment by no means duplicates the normal release of endogenous sex hor- mones, it is likely that the latter are produced in significant quantity during de- velopment. This hypothesis should be in- vestigated in the reptile, perhaps by tech- niques which have already shown their value in comparable studies of other ver- tebrate classes. Discovery of the mecha- nisms of sex reversal and of normal morpho- logic sex differentiation still challenges the l)iologist. XXIV. Addenclum Fish Seasonal development of the gonads of the Japanese cyprinodont Oryzias reaches its peak in June and July, and spawning ensues; by September the gonads are of minimal size, recovering during the winter (Egami, 1956). Spawning occurs in late September to early March in the sea gar- fish, Reporhampus, of southern Australia; neither sex usually attains maturity until the age of three years (Ling, 1958). The sea horse, Hippocampus, breeds off the Florida coast from February to October on days having more than eleven hours of sun- shine (Strawn, 1958). The Black Sea mer- ling, Odontogadus, spawns the year around (Burdak, 1955). In the whiting (Gadus merlangus L. ) and Norway pout {G. es- markii Nilsson ) , seasonal development of the gonads occurs from January to May, and spawning takes place in May and June. Cells resembling interstitial cells can be seen in the spent testis, and a corpus lu- teum-like structure has been detected in the ovary (Gokhale, 1957). The selachian cor- pus luteum appears to be functional ( Chieffi and Rattazzi, 1957). Administration of methyl testosterone to juvenile male Lebistes provokes early ma- turation of the testis, promptly followed by its degeneration. In adults this compound causes hypertrophy of the sperm duct, in- hibition of ovogenesis, and enlargement of the lumen of the ovarian excurrent duct (Geske, 1956). Testosterone treatment pro- motes growth of the vas deferens and of the dorsal fin rays of both sexes of the file fish, Monocanthus (Ishii and Egami, 1957). Im- plantation of testosterone propionate pellets into females, or of estrone pellets into males, of Oryzias promotes growth of male or female type, respectively, in the inter- neural and interhemal spines and the basip- terygium (Egami and Ishii, 1956). Unhatched eggs of Fundulus confliientus, 1070 SUBMAMMALIAN VERTEBRATES stranded in moist plant debris three months after flooded lowlands had dried up, were placed , in tap water. Normal hatchlings emerged from the eggs in 15 to 30 minutes (Harrington and Haeger, 1958), another instance of successful adaptation of the em- bryo to an unfavorable environment. Ovar- ian eggs of the dogfish, Squalus suckleyi, contain estradiol-17/3 (Wotiz, Botticelli, Hisaw, Jr., and Ringler, 1958). Estrogen administration to Lebistes damages the testis, inhibits ovogenesis, and is associated with reduction in height of the epithelium of the sperm duct and with enlargement of the lumen of the ovarian excurrent duct (Geske, 1956). Treatment of the goldfish with estradiol was followed by a rise in the serum level of total protein and of non- ultrafilterable phosphorus and calcium, but not, as in the bird, by concomitant hyperos- sification (Bailey, 1957). In the selachians Torpedo and Sci/lior- hinus the primitive gonad consists of both cortex and medulla. During development, the germ cells remain in the cortex if the gonad is to become an ovary; if it is to be a testis, they migrate to the medulla. In either case, the gonad of the female is briefly bi- sexual in appearance. The interrenal body and gonadal medulla have a common origin. As in birds, ovarian asymmetry appears to be genetically fixed (Chieffi, 1950, 1952, 1955). Embryos of l)oth sexes of Scylior- hinus have Miillerian ducts, but they per- sist only in females (Thiebold, 1954). Two adult female Lebistes reticulatus and one adult female Xiphophonis helleri, individually and continuously isolated from birth, gave birth to litters of 22, 14, and 28 young, all females. The mothers showed the characteristic "pregnancy spot," a pig- mented area near the base of the tail. Mi- croscopic study of the gonads showed them to be entirely ovarian and revealed the total absence of sperm, ruling out the possibility of self-fertilization by gametes from testic- ular tissue. Parthenogenesis seems the only other explanation. It is suggested that par- thenogenesis may have resulted from stimu- lation of the mature ova by the toxin of a phycomycete with which all three mothers were infected (Stolk, 1958). Ova were ob- served in a testis of Barbus stigma (Sath- yanesan, 1957). True hermaphroditism has been reported for the cutthroat trout (Turner, 1946; Benson, 1958). Injection of testosterone propionate into Scyliorhinus embryos was followed by hy- pertrophy of the Wolffian ducts of females and testicular inhibition in males. Treat- ment of male embryos with estradiol benzoate resulted in persistence and hyper- trophy of the Miillerian duct and conver- sion of the testis to an ovotestis. In a strain of the medaka, Oryzias, the female is white due to a recessive, sex-linked color gene (X'"X''), whereas the male is orange-red due to a dominant color gene linked to the Y chromosome (X''Y^\). Thus color reveals the genetic sex. If the third-generation offspring of such fish were reared from hatching to 8 months on a diet containing estrone or stilbestrol, both the white and the red fish were morphologically females. (An exceptional red fish had both ovarian and testicular tissue.) The red fish, geno- typic males, clearly had undergone sex re- versal. Normal diet had no effect on sex. If the sex-reversed fish were mated to normal males, they had normal offspring (Yama- moto, 1953, 1957). If methyl testosterone were fed, beginning at hatching, to the off- spring of normal parents, development of both testes and ovaries was inhibited. At certain dosage levels of this androgen, fe- male to male sex reversal also occurred (Yamamoto, 1958). Beptiles Stefan (1958) studied immature male and female tortoises, Eniys leprosa. Even after hatching, testes still retained cortical rests, medullary cords persisted in the ova- ries, and Miillerian ducts were present in both sexes. 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Naturwiss., 46, 113-224. 18 ENDOCRINOLOGY OF REPRODUCTION IN BIRDS Art van Tienhoven, Ph.D. ASSOCIATE PROFESSOR OF AVIAN PHYSIOLOGY, DEPARTMENT OF POULTRY HUSBANDRY, NEW YORK STATE COLLEGE OF AGRICULTURE, CORNELL UNIVERSITY, ITHACA, NEW YORK I. Introduction 1088 II. The Male 1088 A. The Testis 1088 1 . Anatomy 1088 2. The interstitium and its secre- tions 1089 3. Seminiferous tubules 1092 4. Vasa deferentia 1095 B. Endocrine Reguhition of Testicu- lar Activity 1097 1. The pituitary ghmd 1097 2. The pineal body 1103 3. The adrenal gland 1103 4. The thyroid gland 1104 5. Progesterone 1105 6. Estrogen 1106 7. Nutrition 1107 8. Drugs 1108 C. Fertilization and Sperm Physiology. 1109 1. In vivo ". 1109 2. In vitro 1110 III. The Female 1111 A. The Gonads 1111 1. The right (rudimentary) gonad. . 1111 2. The ovary 1114 3. Function of the ovary 1115 4. The oviduct '. 1122 B. p]ndocrine Regulation of Ovarian Activity 1126 1. Anterior pituitary 1126 2. Estrogen ." 1130 3. Androgen 1131 4. Progesterone 1131 5. Corticosteroids 1133 6. Epinephrine 1133 7. Thyroid hormone 1133 8. Nutrition 1134 C. Regulation of Breeding Cycles of Seasonally Reproducing Birds. . . 1134 1. Hypothalamic-pi^uitary system. 1134 2. Light " 1137 3. Temperature 1141 4. Rainfall 1142 5. Food 1142 6. Vocalizations 1142 7. Nesting site 1142 8. Psychic factors 1143 D. Regulation of the Reproductive Cycle of the Fowl 1145 1. Use of birds in bioassays 1152 IV. References 1154 I. Introduction The fascination of avian reproduction with the accompanying migration over great distances, the majestic flight of a flock of geese, the reproduction of penguins at low temperatures in uninhabitable wastes of the antarctic, all these justify a con- sideration of the endocrinology of avian re- production. So little is known, however, about the factors controlling the migrations and of the maturation of the gonads of wild birds. Most of the author's experience has been gained by the study of the chicken, which by selection has become almost a zoologic monstrosity, but nevertheless has been a useful experimental animal. In writ- ing this chapter, the author has tried to con- sider the literature on the subject of avian- sexual-endocrinology both for domestic and nondomestic birds. His aim has been to give both their fair due. The absence of con- siderations of the behavioral aspects of re- production has been deliberate in view of the chapters in this book devoted to that subject. II. The Male A. THE TESTIS 1. Anatomy ^ Aristotle made the observation that the avian testis has a remarkable capacity for 1088 REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1089 growth, as may be deduced from his state- ment: "iSo also the testicles of birds are either small or entirely invisible when not excited, but when urged by desire they be- come very large; this is so remarkable in pigeons and partridges that some persons have supposed that they had no testicles during winter." Unhke most female birds, the avian male has two well developed gonads, of which the left is usually the larger one. This fact was observed in 1789 by Tannenberg and later confirmed for the sparrow. Passer domesti- cus (Etzold, 1891), and many other species (Domm, 1939). Exceptions are the pigeon, Columba livia (Riddle, 1918), and the tur- key, Meleagris gallopavo (Law and Kosin, 1958) , in which the right testis is larger. The difference in testicular size is not a reflec- tion of lesser activity, for Macartney (1942) established that the number of mitotic di- visions was larger in the right than in the left testis of the fowl. The testes are sus})ended in the body cavity from the body wall by the short mesorchium. The ex})osure to the high internal temperature (40.5 to 41.6°C. ac- cording to Williams, 1958a) does not affect spermatogenesis adversely, although in mammals such temperatures would cause de- generation of the seminiferous tubules (Moore, 1939). In order to account for this phenomenon Cowles and Nordstrom (1946) proposed, without experimental evidence, that the abdominal air sacs might act as a cooling mechanism for the testes. However, Williams (1958a) found that the surface temperature of the testes (41.30°C.) was the same as the mean body temperature, and, what was new, that the exposure of the testis to lower temperatures (38.60° to 40.26°C.), by transplantation under the skin, caused an acceleration in spermato- genesis in the testes of young cockerels. On the other hand, if pieces of testes were transferred to saline with temperatures of 44.4° and 43.3°C., before transplantation, destruction of the germinal epithelium oc- curred (Williams, 1958b), the destruction being roughly proportional to the degree of heat used. Exposure to saline of body temperature liad no effect. These results seem to indicate that lower-than-body temperatures are beneficial for si)ermato- genesis, as in the mammal, but that the threshold at which higher temperatures in- terfere with spermatogenesis is higher in birds. Indirect evidence for the beneficial effect on spermatogenesis of lower tempera- ture is also found in Riley's (1937) investi- gation of the diurnal rhythm of mitotic activity in the testes of the house sparrow. Maximal mitotic activity coincided with the lowest body temperature. Macartney (1942) could, however, not confirm these observations for the domestic fowl. The more direct experimental evidence of Wil- liams (1958a, b) seems to confirm Riley's hypothesis. The testis is surrounded by the tunica albuginea. In seasonally breeding birds this is replaced at the end of each breeding season by a new tunica which forms under the old one. Enclosed by the tunica albu- ginea are the seminiferous tubules and in the spaces between them the interstitial cells. 2. The Interstitiuin and Its Secretions The cells of Leydig originate in the sex- ual cords of the embryonic gonad and mi- grate to the intertubular spaces (Benoit, 1950a). Several lines of evidence show that the Leydig cells secrete the androgenic hor- mone. In brief the evidence is that: (1) In hypophysectomized cocks the comb can be maintained only when the Leydig cells are histologically active (Nalbandov, Meyer and McShan, 1946, 1951). (2) Selective destruction of the germinal epithelium by x-rays does not affect comb size (Benoit, 1950a). (3) When parts of testes regenerate after ca.stration, one sometimes finds tissue without Leydig cells but w^ith Sertoli cells; in other cases Leydig cells are encountered but there are no tubules. On the basis of such dissociation, Benoit (1950a) concluded that Sertoli cells do not secrete androgens, whereas Leydig cells do. (4) Kumaran and Turner (1949a) observed the presence of birefringent crystals in the Leydig cells, suggesting the production of the hormone or its precursor in these cells. The concen- tration of the birefringent material in- creased with increasing age of young cockerels. Such observations are consistent with the increased rate of secretion of the 1090 SUBMAMMALIAN VERTEBRATES hormone as shown by gradually increasing comb size. Breneman and Alason (1951) estimated the cumulative androgen secretion of White Leghorn cockerels between the age of 10 and 40 days as ecjuivalent to 614.90 /x.g. testosterone propionate (TP) for the 30- day period. Such a high rate of secretion in an immature male indicates early secre- tory activity of the Leydig cells and sup- ports the obser^'ations of Kumaran and Turner (1949aj. The response of the comb to androgen administration is influenced by a number of variables : 1. Dorfman (1950) investigated the sen- sitivity of the comb of baby chicks to vari- ous doses of androgen and found that, if the sensitivity of the White Leghorn is taken as 100 per cent, the Rhode Island Red had a sensitivity of 10 per cent and the Barred Rock of 1.8 per cent. Jaap and Robertson (1953) established that inbred lines within a breed may vary in their coml) response to androgen. Campos and Shaffner (1952) selected males and females from a nonpedigreed stock and established that the offspring of such matings differed be- tween sire families as well as between dam families within sire families. It is thus im- portant to randomize thoroughly any chicks used for androgen bioassay. 2. Forced exercise of cockerels reduced the response of the comb to a given dose of testosterone without affecting body or adrenal weight ( Wong, Lavenda and Haw- thorne, 1954). This experiment suggests that differences in the results of the bio- assay between laboratories might in some cases be explainable by differences in the voluntary exercise the birds obtain. 3. As in other bioassays, the route of administration of the hormone is important. Generally intraperitoneal injections result in a smaller response than do those given subcutaneously (Bernstorf, 1957). The dif- ference may be explainable by inactivation of the hormone after intraperitoneal ad- ministration (Bernstorf, 1957). When the hormone is applied locally on the comb, special care should be taken to use the same volume, for Jaap and Robertson (1953) showed that the concentration of the hormone is more important tlian the total amount. 4. Light has been reported to inhibit the response of the capon or the immature cockerel comb to a given dose of androgen (Womack, Koch, Domm and Juhn, 1931; Koch and Gallagher, 1934; Caridroit and Regnier, 1944; Wong and Hawthorne, 1954 ». However, in the experiments of Womack, Koch, Domm and Juhn (1931) the results ascribed to differences in light can also be explained on the basis of differ- ences in temperature, whereas in Koch and Gallagher's experiment, there was no dif- ference in comb response of capons kept in the dark or under light. Inhibition of response occurred only if birds were first kept in the dark and then subjected to light. As Lamoreux (1943) pointed out, birds under these conditions show increased activity, which, as was indicated above, may inhibit the response of the comb to exogenous androgen. Caridroit and Regnier (1944) used only 3 capons and no controls; it was assumed, however, that comb size had equilibrated during the previous sev- eral months of treatment. Their hypothesis that thyroid activity increased in the dark, and in turn caused a greater response to exogenous androgen, was not substantiated by measurements of thyroid activity before or after exposure to darkness. In the experi- ment of W^ong and Hawthorne (1954), in- vestigation showed that light inhibited re- sponse at only 1 of 4 levels of androgen tested. An analysis of variance for the total experiment, instead of the reported series of individual "t" tests for each level of androgen, reveals that light did not affect the assay; neither the main effect of light nor the androgen-light interaction was sig- nificant ip > 0.05). On the basis of the evidence cited, the claim for an effect of light on the androgen bioassay should be given the Scottish verdict "not proven." Lamoreux (1943) showed in a carefully conducted experiment in which temperature and light were controlled variables, that neither visible nor ultraviolet light affected comb size significantly, but that increased temperature caused an increase of the comb size. In contrast Leroy (1956, 1958) found that combs of birds raised in darkness REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1091 were very much enlarged, but Leroy (1958) was also able to obtain these large combs by enclosing the comb in a cloth cover which would interfere with heat dissipation from the comb surface; this suggests that tem- perature may have been an important fac- tor in his experiments. Attempts have been made to use the size of the comb or the rate of comb growth within a breed of chickens as an aid in se- lection of more fertile or of genetically superior males. Although the lines produc- ing fertile males earlier can be selected successfully by choosing the cockerels with the larger combs, no selection can be made in this manner for superior subsequent fer- tility (Parker, 1956). Goodwin, Cole, Hutt and Rasmusen (1955) found that their strain with larger combs, larger testes, and earlier spermatogenic activity was also the strain with the lower fertility. The lower fertility was not caused by the interference of the large comb with mating activity, for all males were dubbed at 9 weeks of age. Pasvogel (1952) tested the hypothesis that comb growth of cockerels might aid in predicting the egg-laying performance of their offspring. In 3 out of 4 trials a selection of males on the basis of their comb growth resulted in higher egg produc- tion of the next generation. Unfortunately, this hypothesis has not been confirmed by more extensive tests. As might be expected, androgens play an important role in the regulation of the ac- tivity and size of the secondary sex organs ; the seminal vesicles in a number of pas- serine birds (Wolfson, 1954a), the vas deferens in the starling ff ..f/. ^K>¥iJlli . //^^_^^K ' :^ -6 "^ ^ - ^ h}dm. tMrim. 0qQ ■^^ ^.^,V;.^«V^■^^»?■ FIG. 18.2. The siMTiiiMlo^cllotir cycle n\ I lie duck according to ClciiiKUit (1958). (N = Scrloli cell; G — spei-matoguniuin ; GM ~ spermatogonium in mitosis; / = primary spermatocyte in interphase; L = spermatocyte in leptotene stage; Z — sper- matocyte in zygotene stage; P = spermatocyte in pachytene stage; Sim = primary spermatocyte in metaphase; SII = secondary spermatocyte; Slim — secondary spermatocyte in metaphase. The numbers indicate the stages of the cycle. (From Y. Clermont, Arch. Anut. microscop. et Morphol. exper., 47, 47-66, 1958.) book). Thus the secretion of progesterone by the testicular tubules may have evolved in this group of birds, whereas in species in which the male does not incubate the eggs or in which progesterone does not induce incubation behavior, this adaptation may be absent. One is reminded of this by the fact that, contrary to Lofts and Marshall 1094 SUBMAMMALIAN VERTEBRATES TABLE 18.1 The cellular composition during the different stages of the spermatogenic cycle of the drake (From Y. Clermont, Arch. Anat. microscop. et Morphol. exper., 47, 47-66, 1958.) Spermatogonia, G Spermatogonia in mitosis, M Primary spermatocytes Interi)hase, I Leptotene, L Zygotene, Z Pachytene, P Diakenesis, D Metaphase, Sim Secondary spermatocj^tes, SII Dividing spermatocj^tes, Slim Spermitid. Spermiogene.sis steps, 1-10 L, Z Stage of Cycle L, Z III IV V V P, D D, S, Im SII Slim 1 A B c c s 1 s 1 S Fig. 18.3. Schematic presentation of renewal of spermatogonia in the drake (Clermont, 1958). A, A' = stem cells, B, C — differentiated spermato- gonia, S rrr spermatocytes. (From Y. Clermont, Arch. Anat. microscop. et Morphol. exper., 47, 47-66, 1958.) (1959j, Fraps, Hooker and Forbes (1949) detected progesterone in the blood of intact roosters but not in the blood of capons, in- dicating secretion of progesterone by testes with presumably normal spermatogenetic activity. Progesterone bioassays of the blood of hypophysectomized roosters treated with either avian luteinizing hor- mone (LH) or follicle-stimulating hormone (FSH) and with a combination of these two hormones should reveal whether or not tul)ules are the source of progesterone in the rooster. After the sperm are released into the lumina of the seminiferous tubules they have to pass through a duct system. The latter has been described in detail by Lake (1957). On leaving the seminiferous tubules the sperm pass first through the tubuli recti, structures characterized by the absence of germinal epithelium and the presence of Sertoli cells. These Sertoli cells apparently secrete a lipoidal material into the lumen. After passage through the tubuli recti the sperm reach the rete testis which are lined by a cuboidal or squamous epithelium. The rete testis converges into the efferent ducts which are convoluted and which form cone- like structures comprising the head of the epididymis. The ducts are lined by alter- nating groups of tall and low cells with in- tense holocrine secretion. The basement membrane is surrounded by a circular smooth muscle. The ductus epididymis, which connects the efferent tubules to the vas deferens, has a tall, pseudostratified, columnar epithelium with nonmotile stereo- cilia. The relationship between these duct systems is illustrated in Figure 18.4. In the epididymis, which is quite small compared with the epididymides of mam- mals, sperm undergo a maturation process which increases their fertilizing ability REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1095 (Munro, 1938a) . Munro found that sperm obtained from the testes fertilized 3 of 69 liens, epididymal sperm fertilized 5 of 39 hens, and sperm from the vas deferens 57 of 77 hens. The differences between testicular and epididymal sperm are statistically not significant, but, in view of the lack of secre- tions in vas deferens, it seems probable that sperm maturation starts in the epididymis. It is not known whether epididymal sperm of chickens show a lower endogenous res- piration rate and a greater Pasteur effect than do ejaculated sperm, as is the case for epididymal and ejaculated bull sperm (Mann, 1954). Munro (1938b) also showed that the change in the fertilization capacity is not affected by androgen. Different parts of the vas deferens were tied off, the birds caponized, and sperm collected at various intervals and tested for fertilizing capacity. Androgen treatment of these capons was without effect on the fertilizing capacity of sperm from different parts of the genital tract. This lack of effect of androgen in the caponized rooster contrasts with the effect of castration and replacement therapy in the rat (see chapter by Bishop). The vasa deferentia, which become highly convoluted with approaching sexual matu- rity, are lined with columnal pseudostratified epithelium and have three muscle layers, an internal longitudinal, an intermediate circular, and an exterior longitudinal layer. At the distal end of the vas deferens in the fowl, at the junction with the ejaculatory duct, there is a small storage space, whei'eas in many passerine birds large "seminal vesicles" are present (Wolfson, 1954a). These seminal vesicles may be organs for storing sperm at lower-than-body tempera- tures (Wolfson, 1954b). 4. Vasa Deferentia The vasa deferentia end in the ejacula- tory ducts which have numerous subepi- thelial sinuses and tortuous arterioles and venules in the submucosa. These structures make the ejaculatory ducts erectile organs; together with lymph folds, the vascular body and the phallus, they form the copu- latory organ (see Fig. 18.5). The phal- lus is formed by a combination of two roimd folds and the "white body." Dur- Testicular artery Vas deferens (exterior) Fig. 18.4. The connections between seminiferous tubules and vas deferens according to Lake (1947). (From P. E. Lake, J. Anat., 91, 116-129, 1957.) Fig. 18.5. Diagram of semen ejaculation in the rooster (according to Nishiyama, 1955). g = longi- tudinal groove of erected phaUus; / = swelled lymphfold; p = erected phallus; c — papillary process of vas deferens; // = 2nd fold of cloaca; /// = 3rd fold of cloaca, i.e., anus; i ejection of semen from vas deferens (y) and outflow of trans- parent fluid from I, as well as ejaculation of the semen (mi.xture of vas deferens .semen with trans- jiarent fluid along g) to the outside of the anus. (From H. Nishivama, J. Fac. Agric. Kvushu Univ., 10, 277-305, 1955.) ing erection the phallus becomes engorged with lymph from the vascular body, whereas the posterior retractor penis muscle relaxes and allows the phallus to protrude. After mating, the lymph can drain back into the vascular bodies. The copulatory organ of the turkey is distinguished from that of the rooster by its lack of a midventral white body and by the presence of a separate white body on the tip of each round fold. In colored varieties of turkeys, these white bodies are highly pigmented (Lorenz, 1959). An intromittent organ, a so-called penis. 1090 SUBMAMMALIAX VERTEBRATES Fic. 18.6. Penis of duck according to EUenberger and Baum (1932). a = cloaca; /) = penis; c - seminal groove ; d — ridge of seminal groove ; e = opening of glandular tube; / = opening of vas dejerens; g = opening of ureter. (From W. EUen- berger and H. Baum, in Handbuch der Verglei- chenden Anatomie der Havstiere, 17th ed., Julius Springer Verlag, 1932.) is found in Anseres, Cracidae, Crypturi and Ratitae (Domm, 1939). Figure 18.6 illus- trates the "penis" of a drake. The semen is transported along the seminal grooves. Stimulation of the nerves originating from the sympathetic plexus and going to the lymph folds has resulted in erection of the penis of drakes (Domm, 1939). The time sperm require to traverse the duct system from testis to phallus has been estimated as 24 hours in an active rooster and 2 to 3 days in a sexually inactive one (Munro, 1938a). The enormous variation of the morjihol- ogy of sperm from different species is il- lustrated in Figure 18.7. The various factors which may affect sperm morphology after ejaculation will be discussed later. The finer structure of the fowl sperma- tozoon has been investigated with the aid of the electron microscope (Grigg and Hodge, 1949; Bonadonna, 1954). The results can be summarized as follows: At the anterior tip of the head there is a small acrosomal spine which is embedded in the head proper. This spine is 1.5 X 0.1 ix m size and is covered by an acrosome cap. The head of the sperm has dimensions of 14 X 0.5 /x, and is slightly curved. After eosin-nigrosin staining a crescent-shaped proximal cen- triole can be seen at the posterior end of the head (Lake, 1954). This centriole does not stain in fresh semen regardless of the mor- phology of the sperm, but after storage of sperm at 5''C. all abnormally shaped sperm show a deeply stained proximal centriole even when the head has not taken up the stain. Occasionally a normally shaped sperm will also show a stained proximal centriole (El Zayat, 1960). The midpiece, between the head and tail, measures 4 X 0.5 IJL and is bounded anteriorly by the anterior distal centriole, posteriorly by the posterior distal centriole. Fibrils of the tail filament arise from the anterior distal centriole, pass medially through the midpiece, and pass through the posterior distal centriole. The tail, which starts at the midpiece tail junc- FiG. 18.7. Spermatozoa of different sjiecies of birds (according to Romanoff, 1959). 1 — Chicken (Gallus gallus); 2 — pigeon {Columba livia) ; 3 = turkey (Meleagris gallopavo) ; 4 — duck {Anus platyrhynchos) ; 5 = sea duck {Aythinae); 6 — ring-necked parrot {Psittacus torcuatus) ; 7 = black-headed gull (Lams fuscus) ; 8 — red-backed sandpiper (Calidris alpina) ; 9 = European wood- cock {Scolopax rusticula) ; 10 — European coot {Fulica atra); 11 — European ruff {Philomachus pugnax); 12 — sparrow (Passer domesticus) ; 13 — greenfinch (Chloris chloris) ; 14 = .songthrush (Turdus philomelos); 15 — chaffinch {FringiUn coelcbs). Magnification 600 X. (From A. L. Ro- manoff, The Avian Embryo, Macmillan Company-, 1960.) REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 109/ tion where the j)osterior distal centriole is located, is over 100 /x long. The tail has a smooth appearance except for the last two fji. It consists of an axial filament surrounded by a thin sheath, which, however, seems absent for the last 2 fi, and shows the fibrils caused by the absence of the thin sheath which surrounds the rest of the axial fila- ment. The axial filament itself consists of 11 fibrils. Nine of these fibrils, the L fibrils, are about 450 A in diameter and are not easily destroyed by distilled water. Two M fibrils are easily destroyed so that no estimate could be made of their diameter. Grigg and Hodge (1949) have speculated that the L fibrils might be the motor elements for the sperm whereas the M fibrils might act as controls for the L fibrils. An extensive re- view of the problem of sperm motility is contained in the chapter by Bishop. B. ENDOCRINE REGULATION OF TESTICULAR ACTIVITY 1. The Pituitary Gland The general concepts of the morphology of the pituitary gland with respect to func- tion and of the physiology of the pituitary gland have been discussed in detail in the chapters by Purves and Greep. It seems thus desirable to discuss here only those aspects which substantiate general principles found to be true also for the avian pituitary and to mention in what respects the avian pi- tuitary differs from the mammalian pitui- tary. A large part of the description of mor- phology has been taken from the excellent comparative account of avian pituitaries by Wingstrand (1951). The avian pituitary (Fig. 18.8) lacks an intermediate lobe in all species investigated. The epithelial stalk, a vestige of the connec- tion between adenohypophysis and its point of origin in the oral epithelium, is more prominent in some species than in others, but it lacks glandular activity in all the species. Within the pars distalis a distinc- tion can be made between caudal and ce- phalic lobes. The Ai cells or dark-staining acidophils (dark red with azocarmine) are restricted to the caudal lobe (Rahn and Painter, 1941; Wingstrand, 1951; Matsuo, 1954; Mikami, 1954), whereas the Ao cells are restricted to the cephalic lobe (Rahn and Painter, 1941; Wingstrand, 1951; IMat- suo, 1954). According to Wingstrand (1951), Ai cells and chromophobes are sometimes difficult to distinguish. Mikami (1955) states that the thyrotrophs, which he distinguishes by their positive periodic acid-Schiff (PAS) reaction are also restricted to the cephalic lobe. Wing- strand ( 1951 ) concluded that the following avian-mammalian homologies exist: the Fig. 18.8. Pituitary of a goose (Anser anser) according to Wingstrand (1951). (From K. G. Wingstrand. The Structure and Development of the Avian Pituitary. CWIv Gleerup, 1951.) 1098 SUBMAMMALIAN VERTEBRATES TABLE 18.2 The relationship between anterior pituitary cells and their hormonal association in the fowl CeH Type Hormone Reference 1 Prolactin Prolactin TSH TSH TSH TSH Gonadotrophin Gonadotrophin Gonadotrophin Gonadotrophin Gonadotrophin Luteinizing hormone? Schooley, 1937; Saeki, 1955 Payne, 1943 Payne, 1944 Perek, Eckstein and Sobel, Brown and Knigge, 1958; gait and Legait, 1955 Legait and Legait, 1955 Payne, 1940, 1944, 1947; rick and Finerty, 1940 Schooley, 1937 Perek, Eckstein and Sobel, Brown and Knigge, 1958; gait and Legait, 1955 Legait and Legait, 1955. Payne, 1955. 2. Small acidophil with large nu- 3. Basophils a. Basophilic cytoplasm cleus like in acidophil b Small PAS + cell nu- 1957 c. ^-Cell:PAS+; AF+ d. /3-Cell blue with Kresazan. . . Le- Her- f Basophil g. Large basophil PAS+. . h. 5 -Cell PAS+; AF- . .. . i Violet with Kresazan 1957 Le- 4 Acidophilic granules in baso- philic gonadotrophs caudal lobe of the avian pituitary corre- sponds with the entire epithelial gland of the mammalian pituitary with exception of the pars tuberalis. The pars tuberalis of the avian and mammalian pituitary are homol- ogous. A very restricted vestigial portion of the mammalian epithelial gland lying near the rostral end of the pituitary corresponds with the cephalic lobe of the avian pituitary. Attempts have been made to establish the relationships of some of the cells of the avian pituitary with specific secretions. The tech- niques used are generally the same as those used for mammals (see chapter by Purves). The differences in terminology used by the different authors sometimes make it difficult to summarize the data, but an attempt to do so has been made in Table 18.2. In studies of these relationships castration of mammals has resulted in the formation of "signet-ring" cells. In the pigeon (Schooley, 1937), and the fowl (Payne, 1940) this does not occur. However, signet-ring cells were observed in young control male and female chicks although never in older chicks. The function of these avian signet-ring cells is not known. Some apparent discrepancies between cy- tologic evidence and hormone assays on pi- tuitaries in other experiments should be mentioned. Nakajo and Tanaka (1956) found that the bioassay of caudal and ce- phalic lobes of chicken pituitaries showed the presence of prolactin in both lobes. The caudal lobe showed a change in potency when broodiness of the hens w^as interrupted by electric shock or by a strong illumination, whereas the potency of the cephalic lobe re- mained unchanged. If the prolactin is pro- duced exclusively by the acidophils or Ai cells, and if these are limited to the caudal lobe, then one would not expect to find pro- lactin in the cephalic lobe. Nevertheless, the potency of the cephalic lobe was higher than that of the caudal lobe in all bioassays re- ported by Nakajo and Tanaka (1956). To prevent confusion in the discussion of the relationship between the nervous sys- tem and the pituitary, a brief description of the vascularization and innervation of the avian pituitary is given here. Branches of the internal carotid artery supply the pitui- tary. After the internal carotid artery has given off the sphenomaxillaris artery and the vidiana artery, the carotids bend dorso- medially and enter the posterior end of the sella turcica where an intercarotic anastomo- ses occurs (Wingstrand, 1951; Green, 1951). Near this point the inferior hypophyseal ar- tery is given off to the neural lobe. After the carotid arteries have separated again, sev- eral arteries branch off, but only the infun- REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1099 dibular arteries supply the i)ituitary. They supply a very dense plexus of capillaries which covers the median eminence and sur- face of the infundibular stem. The capil- laries of this primary plexus fuse in the central zone of the median eminence to form larger vessels, the so-called portal vessels. These vessels are often embedded in the epithelium of the portal zone of the pars tuberalis, but some vessels run directly into the pars distalis. No other arteries supply the pars distalis. In the pars distalis the por- tal vessels send their blood through the sinusoids and then drain into the sinus cav- ernosus or flattened veins connecting with it. In his extensive material Wingstrand found no case in which an artery or its branches penetrated the hypophyseal cap- sule, and in only one pigeon, did he find an artery going from the neural lobe to the pars distalis. The walls of the sinus caverno- sus are formed by the periosteum of the sella, turcica, the capsule of the pituitary, and the connective tissue septa and adventitia of the carotid veins. Blood from the sinus drains into the carotid veins. The neural lobe of Pica, Corvus, Columba, Apus, and others is supplied by blood from the inferior hypophyseal arteries, whereas in passerine birds [Regidus, Panis, Ember- iza, Riparia, Phylloscopiis) and the fowl the blood supply comes from the infundibular arteries (Wingstrand, 1951). The blood drains into the sinus cavernosus from the capillaries of the neural lobe tissue. In addition to this main blood supply of the pituitary, small blood vessel connections sometimes occur between the primary plexus of the median eminence and the capillary bed of the neural lobe, and occasionally single blood vessels are found between the hypothalamic vascular bed and the primary capillary bed of the median eminence. The rarity of such connections makes functional connection between these various capillary beds unlikely. This condition is different from the considerable anastomosis between the vascular beds of posterior lobe and pars distalis in the rat (Landsmeer, 1951). The lack of vascular connections between pars distalis and neurohypophysis plus the presence of a layer of connective tissue be- tween anterior and posterior lobes of the pituitary (Legait, 1959) makes the transport of hormones from one lobe to the other ex- tremely improbable. On the basis of the blood supply of the avian pituitary, Wing- strand (1951) concluded that the blood flows from median eminence to portal vessels. This conclusion has been confirmed by di- rect observation of the blood flow in the duck (Assenmacher, 1958). Studies of the pars distalis of the avian pituitary by Drager (1945) and Green (1951) showed that the nerve fibers never penetrate the glandular part of the pars dis- talis. Metuzals (1955) claimed that a few nerve fibers penetrate into the pars distalis of ducks, but the origin of the fibers could not be established. Wingstrand (1951) wrote, "The pars distalis also contains a few scattered fibres in most preparations of pi- geons and also in the geese and the ducks. The fibres are, however, so rare that large areas in a section must be examined before a single fibre is found." He continued, "It has been seen several times that fibres in the pars distalis are continuous with the autonomic fibre bundles in the capsule of the organ, and the nervous character of the fibres can therefore hardly be doubted. The fibres in the pars distalis are, however, so few that they cannot be able to influence the function of the gland." The posterior pituitary is innervated by the tractiis hypophyseus anterior, the trac- tiis supra opticohypophyseus, the tractus tu- berohypophyseus, and the tractus hypophy- seus posterior, which originate in the lateral and inferior hypothalamic nuclei, the nu- cleus tuberis (Kuhlenbeck, 1937), and the nucleus subdecussationis (Wingstrand, 1951). According to Green (1951), the me- dial forebrain bundle may contribute fibers to the tractus hypophyseus, but Wingstrand ( 1951 ) is not as definite on this. The tubero- hypophyseal tract shows some special adap- tations, in that fibers form "loops" into the glandular layer of the median eminence where the blood supply is most prominent. In this manner the blood vessels can trans- port neurosecretory material to the pars dis- talis and thus affect its function (Wing- strand, 1951). A similar mechanism was projiosed for some fine fibers of the tractus hypophyseus anterior. Because the nucleus 1100 SUBMAMMALIAN VERTEBRATES tuberis is connected with a large number of nonmedullatecl fibers from the preoptic re- gion, the possibility exists that areas of the preoptic region influence the anterior pitui- tary by way of the nucleus tuberis. Experi- mental evidence indicating that the nervous system can influence the avian anterior pi- tuitary will be discussed later. The evidence summarized in Table 18.2 makes it seem logical that the avian pitui- tary exercises the same control over the general reproductive processes that the rep- tilian, amphibian, and mammalian pitui- taries do. Only a few of the numerous ex- periments offering proof of this assumi^tion will be mentioned here. The endocrine function of the avian an- terior pituitary has been investigated by the classical methods such as ablation of the gland, replacement therapy, bioassays of the gland, and purification of the hormones. The aspects which concern the male will be dis- cussed here whereas those [pertaining to the female will be discussed in another section of this chapter. Removal of the anterior pituitary leads to a sharp decrease in testicular size (Benoit and Aron, 1934a; Chu, 1940; Chu and You, 1946; Nalbandov, Meyer and McShan, 1946, 1951; Coombs and Marshall, 1956; Assen- macher, 1958; Lofts and Marshall, 1959), to a decrease in tubule diameter (Chu, 1940; Chu and You, 1946; Nalbandov, Meyer and McShan, 1946, 1951 ; Coombs and Marshall, 1956; Assenmacher, 1958; Lofts and Mar- shall, 1959) , to a decrease in tubule diameter (Chu, 1940; Chu and You, 1946; Nalbandov, Meyer and McShan, 1946; Coombs and Marshall, 1956; Lofts and Marshall, 1956), to steatogenesis of the tubules (Coombs and Marshall, 1956, Lofts and Marshall, 1959), and, as a consequence of decreased Leydig cell activity, to a decrease in comb size (Nalbandov, Meyer and McShan, 1946, 1951), and to atrophy of the vas deferens (Chu, 1940). The effect of hypophysectomy on the his- tology of the Leydig cells is somewhat con- troversial. Chu (1940) remarked that the Leydig cells of hypophysectomized pigeons were healthy in appearance but poor in staining reaction, whereas Coombs and Marshall (1956) stated that the interstitium of hy]5ophysectomized roosters became ex- hausted but that a new generation of Leydig cells with a sudanophilic and a cholesterol- positive staining reaction arose in the ab- sence of the pituitary. This observation seems jmrticularly puzzling in view of the experiments (Nalbandov, Meyer and Mc- Shan, 1951) in which it was found that the combs of hyi)ophysectomized roosters could only be maintained for a limited time with mammalian gonadotrophins. This was not due to antihormone production. The comb of such roosters could respond, however, for an indefinitely long time to avian pituitary extracts. Histologic examination of the in- terstitium during these experimental treat- ments revealed two types of Leydig cells, a nonsecretory type which could be converted into the secretory type by avian gonado- trophin, and a secretory type which could be stimulated to secrete androgen by avian or mammalian LH. These experiments led Nalbandov, Meyer and McShan (1951) to the conclusion that a "third gonadotrophic hormone" may be secreted by the avian pi- tuitary or that avian and mammalian LH are qualitatively different. In any event the experiments support the hypothesis that re- placement therapy stimulates the Leydig cells. The observation of cyclic phenomena in the Leydig cells by Coombs and Marshall (1956) does not seem reconcilable with the histologic studies made by Nalbandov, :\Ieyer and McShan (1951). 'The following observation by Coombs and Marshall (1956) may offer an explanation. "... the hypophy- sectomized bird was killed after only 17 days and before there was any reduction in comb size, so as to examine an early stage of metamorphosis" (italics mine). According to Nalbandov and Card (1943b), the reduc- tion in comb size is obvious 6 days after hypophysectomy. The cyclic phenomena of the Leydig cells observed by Coombs and ^larshall (1956) may thus be the result of traces of anterior pituitary left during the operation. Various gonadotroi:)hic hormone prej^a ra- tions, all containing FSH, have proven to be effective in causing an increase in diameter of the seminiferous tubules, re-initiation of spermatogenesis, loss of cholesterol-positive material from the lumen of the tubules, and REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1101 an increase of testicular size in hypophysec- toraized birds (Chu, 1940; Nalbandov, Meyer and McShan, 1946; Lofts and Mar- shall, 1956j. Bioassays of avian pituitaries have shown that the different gonadotrophic hormones found in mammals are also present in avian pituitaries. FSH and LH were shown to be present by Witschi, Stanley and Riley (1937) and by Leonard (1937), and Bur- rows and Byerly (1936) demonstrated the presence of prolactin in the pituitaries of the fowl, particularly in the pituitary of broody hens. The experiments which proved that the avian pituitary contains these three gonadotrophic hormones have been con- firmed many times. The response of the avian gonads to the purified hormones has already been discussed in connection with replacement therapy experiments. The ap- plication of this response in bioassay meth- ods will be discussed in a separate section. The LH portion of the gonadotrophic complex seems to be the only hormone which can restore the function of the Leydig cells. The restoration or the resumption of sper- matogenesis in an inactive testis can be in- duced in hypophysectomized and in intact individuals of certain avian species by hor- mones other than gonadotrophins. Chu (1940) demonstrated that the germinal epi- thelium of the hypophysectomized pigeon can be maintained by testosterone injec- tions, whereas Chu and You (1944) showed that spermatogenesis can be induced in such pigeons after the germinal epithelium has degenerated and the tubules contain sper- matogonia and Sertoli cells only. Pfeiffer (1947) subsequently observed that testos- terone will enhance further spermatogenesis in sparrows. Passer domesticus, provided spermatocytes were present. Wolf son and Harris (1959) could maintain gonadal ac- tivity of slate-colored j uncos, J unco hye- inalis, and white-throated sparrows, Zono- trichia albicollis, when illumination was reduced from 16 to 8 hours per day. Li the controls gonadal activity decreased. Cler- mont and Benoit (1955) could not increase the size of the testes of either juvenile or adult drakes during the sexual rest period, nor could testicular size be maintained when the drakes were subjected to a sharp de- crease in daily illumination. The species dif- ferences which seem to exist in intact birds do not prove that such differences will exist in hypophysectomized birds. Chu and You (1944), for instance, demonstrated that tes- tosterone does not stimulate the testes of immature or of adult but sexually quiescent male pigeons. The hypothesis that androgen stimulates the testes of hypophysectomized ducks needs to be tested. Doses of androgen capable of inducing spermatogenesis in hypophysectomized male pigeons cause degeneration of the germinal epithelium and a decrease in testicular size when given to intact males (Chu, 1940). Breneman and Mason (1951) found that physiologic doses of androgen are followed by a reduction of gonadotrophic potency. This inhibition was implied in the statement by Kumaran and Turner (1949c) that the same dose of androgen acts as an inhibitor of spermatogenesis at one age but stimulates it at a later age. They suggested that, in the fowl, androgen stimulates only the trans- formation of secondary spermatocytes; higher doses of androgen inhibit the FSH required for the transformation of spermato- gonia to spermatocytes, and no spermato- cytes are available when androgen is given at too early an age. Pfeiffer 's data (1947) for the sparrow and Chu's data (1940) for intact young and adult pigeons also support this concept, but the concept is not consistent with the action of androgen in the hypophy- sectomized pigeon in which only spermato- gonia are present. Further investigation as to what distinguishes the immature testis from the testis of the hypophysectomized pigeon is needed to resolve this question. The testis is also under control of the third hormone found in the avian pituitary, pro- lactin. This hormone reaches its highest con- centration during incubation of the eggs by the parents. It has been found in the pitui- taries of the domestic fowl (Burrows and Byerly, 1936; Saeki and Tanabe, 1955), pi- geons (Schooley and Riddle, 1938) , the Cali- fornia gull, Larus californicus (Bailey, 1952), and pheasants, Phasianus colchicus (Breitenbach and Meyer, 1959). Its physi- ologic effects are many. In the male it causes a decrease in comb size, a decrease in tes- ticular size, a decrease in tubule diameter, 1102 SUBMAMMALIAN VERTEBRATES and steatogenesis of the tubules (Riddle and Bates, 1939; Breneman, 1942; Nalbandov, 1945; Lofts and Marshall, 1956). It also prevents the response of the testes to light in the white-crowned sparrow, Zonotrichia leucophrys pugetensis (Bailey, 1950). Pro- lactin does not affect testicular activity in mammals (Riddle and Bates, 1939; Lofts and Marshall, 1956) . The action of prolactin on avian testes is probably caused by an inhibition of pituitary gonadotrophin se- cretion because small doses of FSH, given simultaneously with prolactin, prevent the decrease in testicular activity (Bates, Rid- dle and Lahr, 1937; Nalbandov, 1945). Breneman (1942) also observed that pro- lactin causes a decrease in the number of basophil cells which are implicated in the secretion of the gonadotrophic hormone. Prolactin also is apparently required for the formation of the incubation patch, be- cause in hypophysectomized passerines the incubation patch can only be formed after combined estrogen-prolactin treatment (Bailey, 1952). Further, the prolactin con- tent of the California gull pituitary was cor- related with the presence of the incubation patch. The occurrence of the incubation patch in both sexes is well correlated with the incubation behavior of the two sexes. If it is present in a species, it occurs in the sex or sexes which incubate (Bailey, 1952). Prolactin injection causes molting in the fowl. This is probably the result of a direct effect on the feather follicle, because the hormone is equally as effective in roosters as in capons ( Juhn and Harris, 1958) . Finally, prolactin is probably concerned with incubation behavior in the fowl (Saeki and Tanabe, 1955), although it seems that progesterone regulates incubation behavior in the ringdove, Streptopelia risoria (Lehr- man, 1958, and his chapter in this book). The fact that hypophysectomy of the pigeon may lead to progesterone secretion by the testes (Lofts and Marshall, 1959) makes such an animal an excellent tool for estab- blishing whether or not progesterone acts directly or by way of prolactin secretion in the pigeon. The following hypothetical series of events may take place in the regulation of the male's breeding cycle of birds in which both sexes incubate the eggs. In the spring under the influence of increasing daylight and provided that other ecologic factors are favorable, the testes of the male reach full activity before the ovaries of the females are fully developed (Benoit, 1956). The court- ship activities and vocalizations may stimu- late the females' ovaries by way of the nervous-pituitary system so that the fol- licles mature and ovulation and oviposition can occur. At the time when about half of the clutch has been laid, estrogen secretion is at its peak and the first signs of vascu- larization of the incubation patch become visible (Bailey, 1952). Visual stimuli from his incubating mate may cause secretion of prolactin by the pituitary of the male, in pigeons at least (Patel, 1936). This release of prolactin could serve a triple purpose of inducing the formation of the incubation patch (Bailey, 1952), precipitating incuba- tion behavior, and causing the functional collapse of the testes. This collapse through withdrawal of androgen might then end sex- ual activities which would divert the male from incubating. Evidence exists that the mechanisms pro- posed above operate in birds as a group, but, as far as the author is aware, this sequence of events has not been proven for any single species. One problem in need of investiga- tion in the male is the source of the estrogen required for the formation of the incubation patch. This sequence of events would also account for the progesterone secretion by the testes of the pigeon. (The assumption is made that the changes in the testes are the same after prolactin secretion as after hy- pophysectomy. Such an assumption seems to be warranted on the basis of the similari- ties in the histology of the testes after these treatments.) The progesterone, in turn, would cause incubation behavior. More re- search is required to determine which secre- tion starts first in the pigeon, prolactin or progesterone. Seeing his mate incubate and finish incubation may release prolactin and so explain the eclipse molt of the mallard drake, which occurs about 3 weeks sooner in mated than in unmated drakes (Hochbaum, 1944). In species in which the male is polygamous and not concerned with incubation and care REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1103 of tlu' younu;, testicular collapse docs not seem to occur until the end of the breeding season. An example of this is found in the pheasant (Greeley and Meyer, 1953). The effects of other anterior pituitary hor- mones on avian testes are difficult to eval- uate because possible contamination by gonadotrophins has not always been ex- cluded. For example, adrenocorticotrophic hormone (ACTH) injections to roosters and capons were observed to be followed by an increase in comb size of roosters but not of capons. No atrophy similar to that in mam- malian testes after ACTH administration (Dulin, 1953) was observed. Dulin (1953) assayed the ACTH preparation for gonado- trophin but used an assay method (chick testicular size as end point) which is not par- ticularly sensitive for LH, the hormone which stimulates androgen secretion. Dulin explained the effect of ACTH by assuming increased endogenous gonadotrophin secre- tion by the ACTH-treated roosters. This as- siunption was based on the increased pitui- tary weights of the treated roosters. In view of assay methods used, we feel that LH con- tamination has not been excluded and that further experimentation is required before the described increase in comb size is as- signed to ACTH per se. Discussion of the effects of nongonado- trophic anterior pituitary hormones on gon- ads does not seem fruitful in view of the above mentioned possibility of gonado- trophin contamination. £. The Pineal Body Of the several endocrine organs which may affect testicular activity, the pineal body has been a most controversial one. It is fortunate that one of the experiments, in which the classical endocrine approach of al)lation and replacement therapy M'as used, was carried out in the fowl. Shellabarger (1953) demonstrated that ablation of the pineal body at an early age caused an increase in testes and comb weight, and a simultaneous increase in gon- adotrophic potency of the pituitary. On the other hand, injection of a pineal-body ex- tract caused a decrease in testicular weight, although a brain extract did not have this effect. Pituitary potency was not affected by either treatment. Pineal extract injec- tions into pinealectomized cockerels reduced testicular size to that of nonoperated or sham-operated controls. Miller (1955) ob- served that the onset of sexual maturity in two strains of chickens, differing widely in the age at which sexual maturity is reached, coincided with the change in the pineal body from the follicular (active) to the lobular (inactive) stage. In vitro studies by Mosz- kowska (1958) established that the gonado- trophic potency of chick pituitaries cultured in vitro was higher w4ien the pituitaries were cultured alone than wdien they were cultured in the presence of pineal body. This evi- dence strongly suggests that the pineal body may act as an inhibitor of pituitary activity. Moszkowska's experiments indicate that this inhibition is probably direct. 3. The Adrenal Gland The role of the adrenal in reproduction is more difficult to evaluate in birds than in mammals. Part of this difficulty is the result of the intermingling of cortical and chromaf- fin cells so that the effect of cortex and me- dulla cannot be separated. The greater dif- ficulty lies in the problem of complete surgical removal of the adrenals and in the high mortality after adrenalectomy (Parkes and Selye, 1936; Biilbring, 1937, 1940; Ta- ber, Salley and Knight, 1956). Leroy and Benoit (1956) observed no mortality after adrenalectomy of drakes, Anas platyrhyn- chos, observations which do not agree with those of other workers. Biilbring (1940) em- phasized that in her work with drakes every case of good survival was correlated with the presence of adrenal tissue. Efforts to sus- tain life of the adrenalectomized drakes, A?ias platyrhynchosl , with desoxycorticos- terone acetate (DCA) were successful only as long as injections were given. A correla- tion was found between the size of the testes at the time of surgery and the dose of DCA reciuired to keep adrenalectomized drakes alive. This correlation was not a result of higher androgen secretion by the larger tes- tes, because testosterone injections did not affect the amount of cortical extract re- quired to keep castrated-adrenalectomized clrakes alive (Biilbring, 1940). After adrenalectomy, roosters maintained 1104 SUBMAMMALIAN VERTEBRATES by salt therapy show atrophy of the testes (Herrick and Torstveit, 1938; Herrick and Finerty, 1940) . The atrophy may be caused by the general metabolic disturbances re- sulting from the adrenalectomy. Taber, Sal- ley and Knight (1956) prevented develop- ment of the rudimentary gonad of sinistrally ovariectomized hens (poulards) by pair feeding them with adrenalectomized pou- lards. Both groups showed similar rudimen- tary gonad development. Leroy and Benoit (1954), who maintained their adrenalecto- mized drakes without special measures, found no testicular atrophy. This observa- tion supports the view that testicular atrophy is correlated with the general meta- bolic disturbances resulting from adrenalec- tomy. Chester Jones (1957) has reviewed the literature on the interrelation between testes and testicular hormones and size of the ad- renal. The data suggest that long-term cas- tration effects (8 to 11 months after opera- tion) may result in a decrease in adrenal size, whereas short term effects (42 days after operation) result in an increase in adrenal size, which can be jircvented by testosterone injections. Administration of adrenal hormones has resulted in contradictory results. Desoxycor- ticosterone acetate (Link and Nalbandov, 1955; Boas, 1958) caused atrophy of the germinal epithelium, edema, and decrease in comb size of the fowl. Corticosterone in- creased testicular size and stimulated sper- matogenesis in domestic mallard drakes (Le- roy, 1952) and roosters (Conner, 1959) and an increase in comb size in the latter (Leroy, 1952) . Dulin (1955 ) , on the other hand, used three different doses of corticosterone and failed to find stimulation of testes or comb, but he observed decreased testes and comb size at the highest dose used. Cortisone caused a slight but significant increase in comb size of capons. The dose used by Leroy (1952) and the highest dose used by Dulin (1955) are essentially the same, but the length of treatment was shorter in Leroy 's experiment. The most important difference between the two sets of experiments was probably in the age of the birds used. In Leroy's experiment the cockerels were 4 months old, whereas Conner (1959) used 6-week-old cockerels, and Dulin's cockerels were from 20 to 40 days of age. One is re- minded of the similarity between these re- sults and those of the experiments of Kuma- ran and Turner (1949c) in which androgen did not stimulate spermatogenesis at the younger age, but did stimulate it after the formation of spermatocytes had occurred. It seems possible, therefore, that cortisone stimulates the transformation of one type of germ cells (spermatocytes?) to another (spermatids?). This interpretation is not consistent with Leroy's conclusion (1953) that cortisone promotes testicular matura- tion in young cockerels. Unfortunately, Leroy gave no evidence to support his state- ment. Cortisone (Leroy, 1953), like andro- gen (Kumaran and Turner, 1949b), fails to prevent estrogen-induced inhibition of the testes and of comb size in the fowl. An observation by Chester Jones (1957) on the relationship between testes and ad- renals should be noted. He observed that the survival of adrenalectomized drakes was inversely related to testicular size at the time of operation. Castrated drakes re- quired lower doses of corticosteroids for sur- vival than did intact drakes. The explana- tion given was that testosterone secretions, by increasing the metabolism of the birds, might increase the requirement for the cor- ticosteroids. The effect of epinephrine on the gon- ads has not been studied in great detail. Wheeler, Searcy and Andrews (1942) ob- served that the injection of epinephrine into sexually mature fowl interfered with spermatogenesis and caused damage to the nuclei of the germ cells. In English spar- rows adrenaline caused regression of the testes and disappearance of the black pig- ment from the beak. Adrenaline also pre- vented the gonad stimulation by gonado- trophins (Perry, 1941) but Wolfson (1945) could not confirm this finding for j uncos, Junco oreganus, nor could Lyman (1942) for pigeons. 4. The Thyroid Gland The thyroid gland is required for' normal development of the testes and for the nor- mal response of secondary sex organs and secondary sex characteristics to androgen. REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1105 After thyroidectomy sexual maturity is de- layed temporarily and in some cases in- definitely (Benoit and Aron, 1934b; Benoit, 1936, 1937a; Greenwood and Chu, 1939; Payne, 1944; Blivaiss, 1947). The develop- ment of the penis of the Pekin drake (Be- noit, 1937b) and of the comb of the rooster (Greenwood and Chu, 1939; Payne, 1944; Blivaiss, 1947) is inhibited after thyroid- ectomy. The lack of development of these organs, which are under androgen control, may be the result either of a lack of andro- gen secretion by the testes of thyroid- ectomized birds or of a diminished tissue responsiveness to androgen in the absence of thyroid hormone. Evidence that the last hypothesis may explain a large part of the smaller combs in thyroidectomized fowl was found in experiments conducted by Morris (1951). In these experiments with intact and thyroidectomized capons, andro- gen elicited a smaller response in the ab- sence of thyroid hormone than in its pres- ence. Administration of thyroid hormone caused l)rccocious sexual maturity of the drake (Jaap, 1933; Aron and Benoit, 1934 » and chicken (Kumaran and Turner, 1949d) with an accompanying increase in comb size. Vaugien (1954) reported that administra- tion of thyroxine to house sparrows during the sexual rest period resulted in recru- descence of the testes and spermatogenesis. Thus all the evidence seems to indicate that severe hypothyroidism interferes with tes- ticular development whereas mild hyper- thyroidism promotes spermatogenesis. The work of Woitkewitch, cited by Hohn ( 1950) , showing that thyroidectomy of starlings in the summer prevents the degeneration of the testes in the fall, seems rather sur- prising. Such work should be confirmed be- fore it is accepted, because it is contrary to observations made in all other species of birds on which data are available. The effect of mild hypothyroidism in- duced by feeding thiouracil indicated that, at certain stages of development of the tes- tes, hypothyroidism promotes earlier sper- matogenesis (Kumaran and Turner, 1949d). However, the fact that the experimental groups sometimes contained only two birds and the well known great variability of testicular size during the stage of rapid testicular development makes it doubtful that the differences were significant. This great variability in testicular size should always be carefully considered in the design of experiments, because it means that large numbers of birds are required to demon- strate significant differences. An illustration of the relative variability of testicular size and stage of spermatogenesis can be found in the following coefficients of variability obtained in two experiments with 6-week- old White Leghorn roosters kept on experi- ment for 10 weeks. Groups were killed at 2-week intervals and body and endocrine organ weights recorded. Weights were ex- pressed as milligrams per 100 grams and stage of spermatogenesis as scores 1 through 7. The following average coefficients of variability were found for 2 experiments: testicular weight, 80 per cent; comb weight, 38 per cent; thyroid weight, 24 per cent; adrenal weight, 31 per cent; body weight, 13 per cent; spermatogenesis, 40 per cent (van Tienhoven, Thomas and Dreesen, 1956). These coefficients are in excellent agreement with the figures published by Fox (1956) from experiments in which the testes had not yet undergone their great increase in size (the average testes weight was about 10 mg. in one experiment and 2.8 mg. in the other). Fox (1956) found that the coefficient of variability for body weight ranged between 4.8 to 14.1 per cent, for comb index between 14.0 to 40.1 per cent, and for testes weight between 27.6 to 160.0 per cent. The results obtained by feeding of iodi- nated casein to different breeds of chickens at a variety of ages are somewhat contra- dictory. A review of these results has been published recently (Turner, 1959) and its repetition seems unnecessary. 5. Progesterone Progesterone administration to cockerels I'educes testicular size (Fox, 1955; Herrick and Adams, 1955), inhibits spermatogenesis (Herrick and Adams, 1956), and reduces comb size (Libby, Schaible, Meites and Reineke, 1953; Fox, 1955; Herrick and Adams, 1956). These effects are comparable with the inhibition of egg laying observed 1106 SUBMAMMALIAN VERTEBRATES when progesterone is given in large doses to laying hens. Apparently there exists a species difference with respect to the effect of progesterone for Kar (1949) reported an increase of more than 75 per cent in testicu- lar size when he administered 0.5 mg. progesterone per day for 30 days. Histologic examination showed that both tubules and Leydig cells were stimulated by the hor- mone. In this case, we might have an ex- ample of a hormone which stimulates the organ which secretes it, for Lofts and Mar- shall (1959) showed that progesterone is present in the tubules of hypophysectomized pigeons. Whether or not progesterone has a role in the regulation of the male pigeon's breeding cycle needs to be determined. If progesterone can stimulate the testes in the absence of the pituitary, as is the case for androgens (Chu and You, 1946) , one might observe "cycles" in the absence of the an- terior pituitary. 6. Estrogen Two lines of indirect evidence suggest that estrogen is secreted by the avian male. (1) In some birds, as for instance in Colymbi- formes, Piciformes, Phalaropodidae, and Jacamidae in which the male incubates the eggs, an incubation patch is formed just before incubation starts. Experimentally this incubation patch will only form under the combined effect of estrogen and pro- lactin (Bailey, 1952). (2) Estrogens have been isolated from the feces of roosters (Hurst, Kuksis and Bendell, 1957). As far as the author is aware no evidence has been published in which the presence of estrogen in the blood was demonstrated. The indirect evidence, although presump- tive, may have to be accepted to explain some phenomenon in the reproduction of birds. The effects of estrogen administration have been studied extensively, both from an endocrinologic point of view and from the angle of practical applications in improving the quality and efficiency of poultry pro- duction. This subject has been reviewed ex- pertly by Lorenz (1954) . Therefore, only the aspects which involve the reproductive sys- tem will be mentioned here. In young cockerels physiologic doses of estrogen will inhibit comb size, testicular size, and gonadotrophin secretion (Brene- man, 1953). The same author demonstrated that comb growth is more sensitive to estro- gen inhibition than testicular size. The latter in turn is more sensitive than gon- adotrophin content of the pituitary. The difference in sensitivity between comb and testes has been confirmed (Bird, Pugsley and Klotz, 1947; Nalbandov and Baum, 1948; Boas and Ludwig, 1950). Lorenz (1959) mentions that the pituitary becomes less sensitive to estrogen-inhibition with in- creasing age. Other effects of estrogen, e.g., lipemia, still manifest themselves at doses of estrogen which do not inhibit the pitui- tary. The inhibition of the comb by estro- gen may be mediated also by an additional mechanism not involving the jiituitary. Martin, Graves and Dohan (1955) surgi- cally divided combs of capons in halves. All half combs received 2 fxg. testosterone propionate and the other half of each comb received, in addition, 0, 80, 100, 160, or 200 yttg. estrone. The results showed that the 100- and 200-fjig. levels inhibited comb growth significantly. The lack of effect of the IQO-fig. dose is puzzling, but the local effect of estrogen seems a real one. These experiments may mean that in adult hens, in which the pituitary apparently has be- come relatively insensitive to estrogen in- hibition, large amounts of estrogen are re- quired to inhibit the comb by local effect. Apparently, the estro^en-androgen ratio in the laying hen is not high enough to cause such a regression. The question has been raised whether a temporary estrogen inhibition of the pitui- tary at a young age would have a ''carry- over" effect at older age, manifesting itself in delayed maturity, damage to testes, small comlD, and decreased fertility. Reports in the literature (Akpinar and Shaffner, 1953; Eaton, Carson and Beall, 1955; Traps, Sohn and Olsen, 1956) seem to indicate that estro- gen inhibition may have a carry-over effect. However, in these cases estrogen pellets were implanted and the possibility exists that the pellets were not completely ab- sorbed before sexual maturity. This sug- gestion receives support from the observa- tion that estrogen administered as a paste REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1107 did not show any carry-over effects (Eaton, Carson and Beall, 1955). Whether or not estrogen i)lays a role in the regulation of some of the reproductive functions of males cannot be stated definitely until evidence of estrogen secretion during various i)arts of the !'ci)i'()(hi('ti\'<' cycle has been o})tained. 7. Xiitritiun 'i'lie importance of various nutrients for the normal development of the rei)roductive tract is hardly in doubt. Unfortunately, as Lutwak-lViann (1958j pointed out, few ex- periments have been carried out on the effects of nutritional deficiencies on repro- duction of male birds, and in these the data presented were not statistically analyzed and do not lend themselves to such analy- sis. The conclusions reported here, therefore, may have to be accepted with reservations. The lack of statistical analysis leads to a criticism of the design of some of the ex- periments, in which it was impossible to distinguish the effect of a specific de- ficiency from general inanition. The latter has been shown to be a cause of atrophy of the testes (Portier, 1920; Parker and Mc- Spadden, 1943; Mason, 1949; chapter by Leathern ) . Some of the studies on the effect of nu- trition on reproduction in male birds have been directed to the problem of vitamin de- ficiencies. Of the fat-soluble vitamins, only A, D, and E have been studied in relation to their influence on male reproductive per- formance. Vitamin A deficiency leads to atrophy of the testes of roosters although body weight is not affected (Lowe, Morton, Cunningham and Vernon, 1957). This effect, therefore, is probably a specific deficiency symptom rather than a symptom of inani- tion. Feeding a vitamin A-deficient ration to mature cockerels resulted in a sharp de- crease in the number of spermatozoa per ejaculate, whereas the incidence of morpho- logically abnormal and of nonmotile sperm increased. These symptoms all proved to be reversible when vitamin A was fed (Paredes and Garcia, 1959) . Vitamin E deficiency resulted in abnor- mal spermatozoa (Adamstone and Card, 1935) and atrophy of the testes (Adamstone and Card, 1934; Herrick, Eide and Snow, 1952). This atrophy may have been the re- sult of a decrease of gonadotrophin secretion by the anterior pituitary (Herrick, Eide and Snow, 1952). Unfortunately, no data were given on body weights, so that it is not certain whether or not the observed effect was the result of starvation. In our laboratory, vitamin E deficiency has often resulted in a decrease in body weight, and thus it seems possible that, in the experi- ments cited, inanition may have been a contributing factor in causing testicular atrophy. Feeding of vitamin D-deficient diets from 6 weeks of age caused a decreased testicular weight in cockerels 14 and 16 weeks of age. (Buckner, Insko, Henry and Wachs, 1951). Body weights of the vitamin-deficient birds were less than half those of the controls, thus indicating that inanition was a con- tributing factor. Investigations of vitamin B deficiencies have been carried out by Haque, Lillie, Shaffner and Briggs (1949). Unfortunately, no statistical analysis of the data was re- ported nor do the published data lend them- selves to analysis. An increase or decrease in testicular weight even at 50 per cent may not be significant, for under normal con- ditions testicular weight shows great varia- bility. A vitamin deficiency may increase this variability even more, because some birds may have a genetically lower re- quirement than others. The latter statement finds support in the observations by Howes and Hutt (1952) and Laraoreux and Hutt (1939, 1948) that breeds differ in their nu- tritional requirements. On the basis of data available in the literature and after careful consideration of the possible effects of inani- tion Mason (1939) concluded that thiamine deficiency per se caused testicular dysfunc- tion. Haque, Lillie, Shaffner and Briggs (1949) determined the response of the comb to a standard dose of androgen in birds fed diets deficient in various vitamins. With the reservation that statistical analysis might prove them incorrect, the tentative con- clusions may be drawn that deficiency of vitamin E, nicotinic acid, or riboflavin re- sults in a greater than normal response of the comb. The explanation for this effect 1108 SUBMAMMALIAN VERTEBRATES of nicotinic acid and of riboflavin may be that testosterone is not inactivated in the liver of these deficient birds. Evidence that these two vitamins are involved in the inactivation of estrogen (Singher, Kensler, Taylor, Rhoads and Unna, 1944; DeMeio, Rakoff, Cantarow and Paschkis, 1948) sug- gests that they may also be involved in the inactivation of testosterone. Such impair- ment of inactivation would result in high circulating testosterone levels. Evidence that would indicate whether or not vitamin E de- ficiency impairs liver function of chickens is not available, as far as I have been able to find, nor is evidence that vitamin E is involved in steroid hormone inactivation. Thus, the increased comb response to andro- gen in vitamin E deficiency may not with certainty be ascribed to effects of the vita- min on liver enzyme systems. The effect of folic acid deficiency on testicular development and on the comb re- sponse to androgen has been investigated in experiments in which inanition effects were separated from specific folic acid ef- fects by the paired feeding technique (Zar- row, Koretsky and Zarrow, 1951). The con- clusion was that folic acid deficiency did not affect testes size of cockerels but that it did increase comb response to a standard dose of androgen. The latter was postulated to be the result of impaired inactivation of the testosterone by the liver. 8. Drugs Brief mention has to be made of the effect of different drugs on testicular de- velopment. Some of these effects were first noted when the drugs were incorporated in poultry feeds to combat various diseases. Enheptin (2-amino,5-nitrothiazole) in- hibits testicular and comb development, probably by inhibiting pituitary gonado- trophin secretion (Pino, Rosenblatt and Hudson, 1954). This conclusion is based on the lowered gonadotrophin content found in the pituitary of Enheptin-treated cockerels and also on the normal testicular develop- ment obtained after gonadotrophin treat- ment of Enheptin-fed cockerels. The re- lated drug, 2-acetylamino-5-nitrothiazole, caused only slight, localized areas of at- rophy of the seminiferous tubules and an accompanying decrease in semen volume without an effect on fertilitv (Cooper and Skulski, 1957). Sulfamethazine, a coccidiostat, causes precocious testicular and comb development of cockerels (Asplin and Boyland, 1947) probably by way of an effect on the thyroid. Increased thyroid size with normal histology and normal P^^ uptake per mg. thyroid led to the conclusion that sulfamethazine feed- ing caused slight hyperthyroidism (van Ti- enhoven, Thomas and Dreesen, 1956). Nicarbazin, another coccidiostat which in- hibits egg production (Baker, Hill, van Tienhoven and Bruckner, 1957) , has no ap- parent effect on testicular size, semen char- acteristics, or fertility (van Tienhoven, Crawford and Duchaine, 1957). Furazolidone .V- (5-nitro-2-furfurylidene) -3-amino-2-oxazolidone, fed at a level of 0.011 per cent of the feed to combat Sal- monella and Histomonas infections, delayed sexual maturity of turkey males. Apparently there is great variability in the response to this drug, for 4 of 30 males had not yielded any semen at 296 days of age, whereas the average age for the first ejaculate for the other 26 toms was 208 days, only one week later than the controls (Redman and Smyth, 1957). Cooper and Skulski (1955) noted that feeding of this drug (0.022 per cent of the feed) caused a marked decrease in tes- ticular size at 12 weeks of age. The lack of an effect noted when either 0.011 or 0.022 i)er cent furazolidone was fed to 0- to 4-week-old cockerels (Francis and Shaff- ner, 1956) may have been a result of the younger age. If this drug interferes with the later stages of spermatogenesis, no difference would be noticeable at 4 weeks of age. The mode of action, whereby the drug interferes with male reproduction, has not been established. In general, our knowledge of the effect of nutrition and of pharmacologic agents on avian male reproduction is fragmentary compared with our knowledge of the same subject in mammals, although the rooster should be a good experimental animal for studying the interaction between metabolic factors and the functioning of the testis. REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1109 C. FERTILIZATION AND SPERM PHYSIOLOGY 1. In ]'ivo Unlike most mammalian sperm, avian spermatoza retain their fertilizing capacity for a long time in vivo. Exceptions are the sperm of bats and of Armadillum vulgare (Mann, 19541. The long functional survival of the sperm is correlated with an interest- ing anatomic adaptation of the infundibu- lum of the oviduct. Van Drimmelen (1951) found "sperm nests," small crypts, in the oviduct with groups of sperm in them. The heads of the sperm WTre oriented towards the oviducal wall and the tails towards the lumen (Fig. 18.9). Their location corre- sponds to the site of fertilization experi- mentally established by Olsen and Neher (1948».\\ccording to Grigg (1957), who passed a cellophane bag filled with Ringer's solution through the oviduct and found many sperm in the oviducal lumen, the sperm are apparently released into the lu- men of the oviduct by the passage of the egg. The sperm nest may serve three purposes : (1) to ensure the presence of large numbers of sperm when required; (2) to supply nu- trients to the sperm; (3) to remove the waste products of sperm metabolism. Un- fortunately, too little is known about avian sperm metabolism to permit a reasonable estimate of the importance of these nests. After their release into the oviducal lu- men, the sperm can penetrate the vitelline membrane and effect fertilization. Penetra- tion of the vitelline membrane by more than one sperm was noted in Aves by Olsen (1942). These extra sperm are called the supernumerary sperm, and special signifi- cance has been given to them by Kushner (1954). When the sperm, which fuses with the female pronucleus, and the supernumer- ary sperm are from different males, the re- sulting chicks are alleged to have increased vigor, to have increased hemoglobin con- tent, and, in some cases, to have characters from both sires (Kushner, 1954). The sug- gestion of bipaternity in birds had been made earlier by Hollander (1949) to ac- count for some mosaics in which sex-linked color patterns were involved in pigeons. The ■T&'yT- Fifi. 18.9. Sperm nests in the infundibulum of the oviduct of the fowl (van Drimmelen, 1951). (From 0. C. van Drimmelen, J. Vet. Res., Siippl. 1,1951.) hyiwthesis of bipaternity was tested by Al- terkirch, Hoffmann and Schaaf (1955) for fowl. Male breeds with definite genetic markers were used. In 57 offspring obtained from hens mated in short succession to such males, no evidence of bipaternity was found. Nalbandov and Card (1943a) pointed out that sperm, during their sojourn in the ovi- duct, may undergo changes which cause ab- normal development of the embryo. The ag- ing of spermatozoa in the oviduct not only decreased fertilizing capacity, but also in- creased embryonic mortality. Dharmarajan (1950) confirmed these results and estab- lished that most of the abnormalities were in the nervous and vascular system. Mc- Cartney (1951) and Hale (1955) made sim- ilar observations of increased embryonic mortality in turkeys. Lorenz (1959) , who re- viewed the literature w^hich appeared before as well as after Nalbandov's and Card's publication, concluded that the available data were consistent with this concept of increased embryonic mortality as a result of fertilization by ''stale" sperm. The ob- served deleterious effect of aging of gametes has also been observed in mammals. In them the aging of ova, but not of sperm, has also led to abnormalities in embryos. This interesting phenomenon is discussed in the chaj^ter by Blandau and in a review by Young (1953). It seems logical to assume that abnormal embryonic development after fertilization by stale sperm is the result of nuclear dam- 1110 SUBMAMMALIAN VERTEBRATES age. Experiments with sperm subjected to x-ray irradiation have yielded results par- alleling those obtained with stale sperm. Kosin (1944) established that embryonic mortality after fertilization with irradiated sperm occurred mostly during the first 4 or 5 days of incubation. This time of maximal mortality is the same as found by Nalban- dov and Card (1943a) for aged sperm. Not enough data are available in the papers of Kosin (1944) and Dharmarajan (1950) to compare the kinds of abnormalities found. The effect of fertilization by sperm stored in vitro on embryonic development has not been studied extensively, largely because of the lack of fertility obtained with stored semen. Wilcox and Shorb (1958) were able to obtain good fertility with semen stored 26.5 hours. No effect on embryonic mor- tality was observed. Lake, Schindler and Wilcox (1959), in experiments which in- volved storing rooster semen for 37 to 38 hours, found a decrease in hatchability of fertile eggs and a statistical analysis of their TABLE 18.3 Comparison of the composition of Imll (ttid rooster seminal plasma All values expressed as mg. per 100 ml. Na. K.. Ca. Mg. B . Cu Zn CI Glucose Fructose Lactic acid Total phosphorus. Glutamate Nonprotein N . . . . Freezing point . . . Bull Ref. 289 5 155 5 39 5 11.6 4 1.48 4 1.36 4 0.02 4 154 5 0 2 540 2 35 2 55 2 7.75 1 .53 2 Rooster Ref. 378-428 39-49 6.9-9.3 8.4 0.145 0.18 197-212 41 4 0 890-1340 142.8 0.64 References: 1. Sarkar, Luecke and Duncan, 1947. 2. Mann, 1954. 3. Schindler, Weinstein, Moses and Gabriel, 1955. 4. Cragle, Salisbury and Muntz, 1958. 5. Cragle, Salisbury and VanDemark, 1958. 6. Lake, Butler, McCallum and Maclntyre, 1958. 7. Lake and Mclndoe, 1959. 8. Personal observations, 1959. data by me showed that storage of tlie semen resulted in increased embryonic mor- tality {p < 0.01). (In the analysis all the data were pooled for stored semen and for fresh semen.) Moravec, Mussehl and Pace ( 1954) observed a sharp increase in embry- onic mortality after insemination of turkeys with semen stored 24, 48, and 72 hours. The data from these two experiments are not conclusive, but there is an indication that aging of fowl semen in vitro may have the same effects as aging in vivo. Nalbandov ( 1958) has stated, without presenting the evidence, that such was the case. £. In Vitro The long functional survival of the avian sperm in vivo stands in sharp contrast to the low fertility obtained after storage of fowl and turkey spermatozoa in vitro. jMammalian sperm, on the other hand, have been stored in vitro with relatively little loss in fertilizing capacity. It seems, there- fore, fruitful to compare the various meta- bolic and physiologic characteristics of avian and mammalian sperm. A detailed review of the composition of cock semen and the metabolic behavior of fowl and turkey sperm in vitro has been published recently (Lorenz, 1959). Some observations made in our laboratory will be added here and a comparison of some aspects of the l^hysiology of avian and mammalian sperm will be made. Bovine semen and rooster semen have been selected as representa- tives of the mammalian and avian classes. The selection was largely based on the amount of data available on the sperm physiology of these species. From Table 18.3 a comparison can be made of the dif- ferences in composition of the seminal plasma. One of the most striking differences is in the glutamate content. In vitro studies in our laboratory showed that dilution of semen with seminal plasma or Tyrode solu- tion, 1 part semen to 4 parts diluent, did not affect respiration rate (van Tienhoven, 1960). However, it was noted that storage of avian semen in Tyrode solution decreased respiration rate and rate of fructolysis, and increased abnormal sperm (El Zayat and van Tienhoven, 1959). These effects were found to be caused by chloride ions, for the REPRODUCTIVE ENDOCRINOLOGY IN BIRDS nil TABLE 18.4 Comparison of the in vitro metabolism of bull and rooster spermatozoa under various experimental conditions Factor Investigated Bull Semen Rooster Semen Glucose Metabolized to lactic acid (2) As bull semen (9) Conversion to fructose (7) Fructose Metabolized to lactic acid (2) As bull semen (9) Glucose metabolized preferentially to As bull semen (9) fructose (7) Metabolized at same rate as glucose Metabolized slower than glucose (9) Phosphate Depresses respiration (5) Depresses respiration (9) Increase fructolysis (5) Depresses fructolysis (9) Glycine Increases respiration rate (6) Decreases respiration rate (9) Decreases lactate gain (6) Same as bull semen (9) Reduces fructose loss in synthetic media (6) Metabolized to CO. (4) Same as bull semen (9) Not metabolized to CO2 (8) Dihition Increases respiration (3) Decreases respiration (9) References, 1. van Tienhoven, Salisbury, VanDemark and 5. Blackshaw, Salisbury and VanDemark, 1957. Hansen, 1952. 6. Flipse and Almquist", 1958. 2. Mann, 1954. 7. Lorenz, 1959. 3. Bishop and Salisbury. 1955. 8. van Tienhoven, unpublished, 1959. 4. Flipse, 1956. 9. van Tienhoven, 1960. incidence of abnormalities was linearly pro- portional to the concentration of chloride ions. Replacement of chloride ions by phos- phate or glutamate prevented the sperm ab- normalities and partly prevented the de- crease in metabolic rates (El Zayat, 1960). On further investigation, it was found that l)hosphate decreased the initial metabolic rates, whereas glutamate did not. Gluta- mate was found to "spare" the utilization of fructose ; apparently, glutamate itself was metabolized to yield COo . In many of the experiments in which efforts were made to store semen in vitro, the diluents contained chloride ions which from our observations clearly seem to damage the sperm. Replac- ing chloride with glutamate in a diluent might prevent the loss of fertilizing capac- ity during storage. Lake (1958) has shown that a diluent high in glutamate can support the maintenance of the fertilizing capacity of sperm for at least 24 hours. Thus, one of the main differences between bull and rooster semen may lie in a difference in sensitivity to chloride ions. Foote (1950) has shown that a Tyrode solution supported bull sperm viability better than any other synthetic diluent. It remains to be deter- mined whether glutamate would improve sperm viability in a bovine semen diluent. Some other differences between bull and rooster sperm are tabulated in Table 18.4. At the moment the significance of these dif- ferences is not known, partly because not enough data are available for rooster sperm to correlate nr vitro findings with fertilizing capacity. III. The Female A. THE GONADS 1. The Right (Rudimentary) Gonad With few exceptions such as Accipitrinae, Falconinae, Buteoninae, Cathartidae, birds normally have only one functional (left) ovary, the right gonad is either absent or very small (Domm, 1939; Stanley and Witschi, 1940) . This asymmetry of gonadal development is already noticeable during embryonic development. In the duck (Anas platyrhynchos) primordial germ cells mi- grate to the left and right gonad primordium in equal numbers until the 28 to 37-somite stage is reached (75 to 85 hours of incuba- tion) . After this, more primordial germ cells migrate to the left, and after about 125 hours of incubation a difference in the asymmetry between different embryos led van Limborgh (1957) to deduce that an 1112 SUBMAMMALIAN VERTEBRATES embryo was female if less than 41 per cent of the total number of primordial germ cells was present in the right gonad, whereas it was male if more than 45 per cent were pres- ent in the right gonad. Simon (1960) on the basis of an extensive study concluded that van Limborgh's conclusions were erroneous and that the ratio of primordial germ cells in left and right gonad of the 25 to 32-so- mite stage chick embryo cannot be used to determine the sex of the embryo. As both authors base their conclusions on statistical considerations of normal and abnormal sex ratios further work using cytologic tech- niques needs to be performed. Kosin and Ishizaki (1959) established that the pres- ence or absence of sex chromatin in the nucleus permits sex identification of chicks. The sex chromatin is found in birds in the cells of females as it is in mammals, in spite of the fact that in birds the female is the heterogametic sex. Ohno, Kaplan and Kin- osita (I960) have proposed that the sex chromatin of chickens represents a single Z chromosome in positive hcteropycnosis. Stanley and Witschi (1940) compared the asymmetric gonads of embryonic chicks with the symmetric gonads in Accipiter cooperii, Buteo amiansis horealis, and Cir- cus cyaneus hudsonius. They found that the distribution of primordial germ cells be- tween the left and right gonads in these latter species was still essentially sym- metric at a stage of development when asymmetry already had developed in the chick. They offered the explanation that, in the chick, primordial germ cells migrate from the right to left, thus disturl)ing the initial symmetric arrangement. Van Lim- borgh (1957) tested this hypothesis by dividing the duck embryo medially and thus destroying all vascular and other con- nections between the left and right side. In the surviving embryos the asymmetry was not different from untreated controls. On the basis of other experiments van Limborgh came to the conclusion that the asymmetric distribution of primordial germ cells could not be explained by the fact ^■i-'at the embryo lies with its left side to- wards the yolk so that the left gonad may be better A^ascularized (an explanation pro- posed by Dantschakoff and Guelin-Sche- drina (1933) nor could it be explained by the secretion of hormones by the gonads which inhibit or stimulate the asymmetric migration of the primordial germ cells. The riddle of the asymmetrical distribu- tion of the primordial germ cells still re- mains unsolved and no real explanation can be given for the difference between the hawks and other birds. The right ovary of the hawks is apparently functional, for no histologic differences between it and the left ovary have been found (von Faber, 1958). Domm (1939) states that yolks can be ovulated from these right ovaries; he postulated that such yolks might be trans- ported through the left oviduct, for the right oviduct is either missing or vestigial (Stanley and Witschi, 1940; von Faber, 1958). Domm based his hypothesis on the absence of yolk material in the body cavity. It has since been shown that yolk can be ab- sorl)ed from the body cavity in less than 24 hours (Sturkie, 1955a). An additional rea- son for believing that Domm's hypothesis is probably incorrect is that the dorsal mes- entery makes it impossible for a yolk to move from the right to the left side of the body cavity. This anatomic arrangement occurs in chickens and it probably is the same in hawks. It is rather difficult to un- derstand the evolutionary significance of the presence of two ovaries with only one ovi- duct and the occurrence of this arrange- ment in only one order. The postembryonic development of the fowl's right gonad after sinistral ovariectomy has been investigated in different labora- tories. Domm (1939) reviewed the litera- ture which had been published to that time. Only the salient features pointed out by Domm and new evidence obtained since then will be presented here. Ovariectomy before 30 days of age results in the development of the right gonad into a testis or an ovotestis, either of which may exhibit active spermatogenesis (Domm, 1939; Kornfeld and Nalbandov, 1954; Kornfeld, 1957) sometimes even at an ear- lier age than in cockerels of the same breed (Taber, Claytor, Knight, Flowers, Gambrell and Ayers, 1958). Later ovariectomy re- sults in a greater incidence of ovotestes and ovaries than does ovariectomy before 30 REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1113 days of uge. Some of these right ovaries resulting from sinistral ovariectomy have follicles which can be ovulated (Nal))andov, 1959a». A number of observations indicate that the apparent effect of the age at which ovariectomy is performed on the type of de- velopment of the rudiment has an endocri- nologic basis. (1) The 20-day-old chick secretes detectable amounts of estrogen as determined by bioassay of the blood ( Korn- feld and Nalbandov, 1954). (2) Small doses of estrogen (2 ^g. per 100 gm.) inhibit the develoi)ment of the rudimentary gonad of ovariectomized pullets (Kornfeld and Nal- bandov, 1957; Kornfeld, 1958). These doses are too small to affect oviduct weight signif- icantly and may, thus, be in the range of the amounts secreted by the immature ovary. (3) If the rudiment develops in spite of es- trogen treatment, the incidence of ovaries and ovotestes is greater than that of testes (Taber and Salley, 1954; Kornfeld and Nal- bandov, 1954). Histologic examinations show also that estrogen inhibits medullary tissue more than cortical tissue (Taber, Clay tor. Knight, Flowers, Gambrell and Ayers, 1958). The following explanation of the age ef- fect is thus in agreement with the experi- mental evidence. Estrogen secretion by the left ovary inhibits the development of the medullary tissue of the right gonad. If this inhibition is removed before 30 days, the medulla can still proliferate. By 30 days of age the estrogen inhibition has apparently destroyed the potential of the medulla to develop and thus no proliferation occurs even when the inhibition is removed. It is understood, of course, that this critical point, may vary between individuals within a strain and even more so from strain to strain. The possible stimulation of cortical tissue development by estrogen is not as clear-cut. Taber and Salley's (1954) and Kornfeld and Nalbandov's (1954) experiments indicate that estrogen favors the development of cortical tissue. However, in a series of ex- periments by Taber, Claytor, Knight, Flowers, Gambrell and Ayers (1958) in- volving large numbers of birds, no evidence was obtained that would support such a conclusion. The development of the rudimentary gonad seems to be under the control of the anterior pituitary. Hypophysectomy (Korn- feld and Nalbandov, 1954) , prolactin in- jections (Kornfeld and Nalbandov, 1954), and estrogen administration (Taber and Salley, 1954; Kornfeld and Nalbandov, 1954; Kornfeld, 1958; Taber, Claytor, Knight, Flowers, Gambrell and Ayers, 1958) inhibit its development. However, replacement therapy in the case of hypophy- sectomized birds (Kornfeld and Nalbandov, 1954) or in the case of estrogen-inhibited birds (Kornfeld and Nalbandov, 1954; Kornfeld, 1958; Taber, Claytor, Knight, Flowers, Gambrell and Ayers, 1958) does not stimulate the rudiment, nor do mam- malian or avian gonadotrophins in poulards (sinistrally ovariectomized hens) (Korn- feld and Nalbandov, 1954; Kornfeld, 1958; Taber, Claytor, Knight, Flowers, Gambrell and Ayers, 1958). The lack of development in the rudiment cannot be explained by the lack of the "third gonadotrophin," because, in some of these investigations, chicken pituitary preparations were used (Kornfeld, 1958; Taber, Claytor, Knight, Flowers, Gambrell and Ayers, 1958) . It seems that the estrogen inhibits the rudiment by a di- rect action, as proposed by Kornfeld (1958) . Recently, Kornfeld (1960) obtained ad- ditional evidence that estrogen is the main agent preventing development of the rudi- ment. Injections of 17a-etliyl-19-nortestos- terone, an anti-estrogen, resulted in its de- velopment to four times the control size in 100-day-old chickens. The lack of develop- ment after hypophysectomy in the poulard may be the result of deficiency of thyroid or adrenal hormones or of generalized meta- bolic disturbances caused by the hypophy- sectomy. It is known that adrenalectomy (Hewitt, 1947) or inanition (Taber, Salley and Knight, 1956) inhibits rudimentary gonad development, and hypophysectomy causes a sharp decrease in thyroid weight (Baum and JMeyer, 1956). As has already been shown in this chapter, reduced thyroid activity severely restricts gonadal develop- ment. The inhibitory effect of prolactin on rudiment development may be explained by a direct inhibitory effect of prolactin on the rudiment or by possible side effects of pro- 1114 SUBMAMMALIAN VERTEBRATES lactin which cause metabolic disturbances that interfere with development of the rudi- ment. Miller (1938) utilized the occurrence of spermatogenesis in the rudimentary gonad in an ingenious procedure for identifying the sex chromosome. Study of actively di- viding spermatocytes in a normal testes and in a rudimentary gonad revealed that the 5th largest chromosome, a V-shaped one, was not paired in the rudiments, but was paired in the testes from a normal male, thereby proving that this chromosome is the sex chromosome. Development of the rudimentary gonad after ovariectomy occurs in ducks and song- birds (Nalbandov, 1958) but not in turkeys (Domm, 1939). In addition to those that can be attributed to the activity of the rudi- ment, other sex abnormalities sometimes occur. Crew (1923) documented a case in which a bird laid eggs and subsequently "sired" offspring. The bird had two testes with vasa deferentia and a diseased ovary and thus was a true hermaphrodite. Hutt (1937) reported a case in which a chicken was first more like a pullet, later showed male characteristics and produced sperma- tozoa (incapable of fertilization) , and later looked more like a female. The bird on au- topsy had a testis and vas deferens on the right and an ovary and oviduct on the left side. This bird was a gynandromorph, as was evident from the feather pattern which was light on the left side (one sex chromo- some) , dark on the right (two sex chromo- somes), and from the fact that the right side exceeded the left in size. The cases re- ported by Crew (1923) and Hutt (1937) show that "sex reversals" can occur with- out involvement of the rudimentary female gonad. Benoit (1950a) hsted other instances in which the same phenomenon occurred. 2. The Ovary The ovary is attached to the body wall by a short mesovarium. In the quiescent state the ovary is a small, flat, yellow organ with small (< 1 mm.) follicles. In the active state it is a large organ composed of 5 to 6 large follicles filled with yellow yolk and a larger number of smaller follicles filled with white yolk. The large follicles are graded in size whereas the smaller follicles are more uniform. In addition to these follicles one may find atretic follicles ; in the early stages of atresia they look somewhat like a shriv- eled balloon but later may become small, dark yellow, flabby masses. After ovulations the ovary also contains ruptured follicles. These ruptured follicles disappear rapidly in the fowl and the rook, Corvus /. frugilegus L. (Marshall and Coombs, 1957), but they persist until the end of the breeding season in pheasants. Counts have been made of these follicles to determine the egg produc- tion of pheasants (Kabat, Buss and Meyer, 1948; Buss, Meyer and Kabat, 1951). The ovary is innervated by the nerves from the abdominal and pelvic plexuses and from the posterior continuation of the sym- pathetic trunk (Bradley, 1950). It receives its blood from the ovarian artery which is usually a branch of the left renolumbar artery but occasionally is a branch of the dorsal aorta (Nalbandov and James, 1949). The ovarian artery divides and sends 2 to 4 branches to each follicular stalk. Spiral arteries provide the main blood supply in the wall of the follicle ; these spiral arteries constrict when the ruptured follicle col- lapses and thus little if any bleeding occurs at ovulation. The ovarian venous system is much more extensively developed than tlie arterial system. Nalbandov and James (1949) classified the venous system of the follicles in 3 layers: (1) a capillary network in the theca of the follicle that drains by venules into (2) a complex network periph- eral to the first layer that drains into (3) a third venous layer consisting mainly of a few large veins that drain into the follicular stalk. The large veins from different follicles anastomose and drain into either the ante- rior or the posterior ovarian vein both of which empty into the vena cava. Histologic examination of the ovary re- veals the presence of the left adrenal and the epoophoron embedded in the stroma (Biswal, 1954). The large follicles (Fig. 18.10) consist of the very vascular theca folliculi, the basement membrane, the gran- ulosa, and the vitelline membrane which surrounds the yolk. An area which macro- scopically seems free of blood vessels stands out sharply in the follicular wall. This area. REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1115 the stigma, is the place where the follicle ruptures. On microscopic examination, small blood vessels are found to cross the stigma (Xalbandov and James, 1949 ». In the theca in tern (I of the rook Marshall and Coombs (1957) found large glandular cells whose cytojilasm contains lipid droplets, similar to those in the Leydig cells of the male. The cells are considered by Marshall and Coombs to be the source of estrogen. In ad- dition to the thecal gland cells, Marshall and Coombs distinguished exfollicular gland cells which may arise from fibroblasts that migrate from the theca into the lumen of atretic follicles. After collapse of such fol- licles till' cells are freed into the stroma. Although they resemble the Leydig cells histologically these cells are not homologous with them, because they arise from follicles and not from stromal tissue. The female Leydig cells, which may be considered ho- mologous with the male Leydig cells, arise from connective tissue cells and may be the source of androgen in the female. After rup- ture of the mature follicle the thecal gland cells disappear and the empty follicles are invaded by erythrocytes, lymphocytes, and a very large number of fibroblasts. In birds tliere is no structure that may be regarded as homologous with the mammalian corpus luteum. The references to the ruptured fol- licles as an avian corpus luteum by Pearl and Boring (1918), Novak and Duschak (1923), and Bradley (1950j are erroneous. 3. Function of the Ovary The avian ovary produces gametes and hormones which play an integral role in the production of the egg. Gametogenesis. Gametogenesis starts in the embryo, so primary oocytes with chro- mosomes in the bivalent state (Hutt, 1949) at the time of hatching are present in the ovary. During the long interval between the time of hatching and about 4 to 5 hours before ovulation, little activity takes place in the nucleus. In sharp contrast, large amounts of yolk are deposited in the follicle in the 8 to 9 days before ovulation. About 24 hours before ovulation, the breakdown of the germinal vesicle starts (Olsen, 1942), but nuclear changes are not vet noticeal)le. The reduction division is lar layer of stalk Fig. 18.10. Hi.stulugy of ovanan folLcle of the chicken according to Nalbandov and James (1949). (From A. V. Nalbandov and M. F. James, Am. J. Anat., 85, 347-378, 1949.) comjileted about 2 hours before ovulation in both the fowl and the turkey (Olsen and Fraps, 1944, 1950). Thus the sex of the fu- ture embryo is determined at ovulation and not at fertilization, as it is in mammals. Investigations to determine the primary sex ratio on fowl, possible only in hatches with 100 per cent fertility and no embryonic death before sex difTerentiation, have shown it to be unity ( Hays, 1945 ; Landauer, 1957) . The nuclear changes in the primary oocyte leading to extrusion of the first polar body and formation of the secondary oocyte are apparently under the control of LH, be- cause LH injections produce such changes prematurely (Olsen and Fraps, 1950). The secondary oocyte extrudes the second polar body after sperm penetration of the vitelline membrane (Olsen, 1942; Olsen and Fraps, 1944). During gametogenesis aberrations can oc- cur which lead to parthenogenetic embryos. In certain strains of turkeys the incidence of parthenogenesis may be as high as 100 per cent ( Poole and Olsen, 1958 ) . The occur- rence of parthenogenesis in birds may have been discovered as early as 1872, as was pointed out by Fraps (1955). Partheno- genesis occurs in turkeys (Olsen and Mars- den, 1953, 1954, 1956; Olsen, 1956) and chickens (Olsen, 1956; Poole and Olsen, 1958). Only a small fraction of the eggs showing parthenogenetic development de- ^'eloped into normal embryos and only a few 1116 SUBMAMMALIAN VERTEBRATES poults have ever been hatched from these. Such poults have always been males and so have the embryos that died but could be sexed (Poole and Olsen, 1957) . The embryos have the diploid chromosome number (Yao and Olsen, 1955; Poole, 1959). Poole (1959) has classified the possibilities whereby par- thenogenetic turkeys with 2N chromosomes could be formed as: (1) suppression of mei- osis I or reentry of the first polar body fol- lowed by reduction division; (2) suppres- sion of meiosis II or reentry of the second polar body; (3) nuclear but not cytoplasmic division of the mature haploid ovum. The observation that all parthenogenetic embryos from turkeys have been males makes it probable that the explanation (1) is not correct, because it would lead one to expect some female offspring. In birds the second polar body is not extruded until the sperm penetrates the egg, so the second mechanism is probably the correct one be- cause the possibility for the two nuclei of the ovum and the second polar body to fuse would exist in this case. Linkage studies with males obtained from parthenogenetic development are needed to provide the answer whether possibly 2 or 3 is the correct one (Poole, 1959) . Attempts to increase the incidence of parthenogenesis have been made in order to find the possible cause of parthenogenesis, and the following factors have been mentioned as causes of parthenogenesis. 1. (31sen and Marsden ( 1953) suggested on the basis of experiments in which turkey hens were housed such that they could see and hear or not see and hear turkey toms, that sound and sight of other turkeys would increase the incidence of parthenogenetic development. The explanation was suggested on the assumption that a neural mechanism would cause the release of a pituitary hor- mone involved in triggering parthenogenetic changes in the oocyte. The hypothesis that such a hormone might exist was founded on evidence that LH injections and also "spon- taneous" LH release cause maturation changes in the nucleus of the ovum and cause extrusion of the first polar body (Ol- sen and Fraps, 1950) . Unfortunately, in the experiments of Olsen and Marsden cited above, the effect of sound and sight of other turkeys cannot be separated from the effect of confinement in cages versus confinement in larger pens and from the effect of artifi- cial light versus natural daylight. In other words, the effect assigned to sound and sight of other turkeys might have been caused also by differences in confinement or differences in light. In a second experiment by Olsen and Marsden (1956) the results were reversed: birds that could hear and see other males laid fewer eggs showing more parthenogenetic development than those hens that could not see, but might have been able to hear the males. From these experiments one might conclude that environment may play a role in increasing the incidence of parthenogenesis; however, to determine which part of the environment plays this role will require a better design such as factorial designs. 2. Olsen (1956) made the interesting ob- servation that vaccination with fowl pox vaccine increased the incidence of parthe- nogenesis in a strain of turkeys in which this trait already occurred. The mechanism whereby this vaccine might act has not been determined. During the period between hatching of the chick and ovulation, many changes take place in the follicle. The major change is the deposition of yolk which occurs usually in three phases. 1. The phase of slow yolk deposition which starts in the embryo and continues for several months or years depending on the species. During this period of yolk dep- osition, the follicle is formed around the vitelline membrane. 2. The intermediate phase of yolk forma- tion during which transparent vacuoles ap- pear and during which yolk is formed in- side the vacuoles (Marza and Marza, 1935) . This phase lasts about 60 days. 3. The phase of rapid yolk formation. During this phase initially white yolk and later yellow yolk is formed and the latebra becomes distinguishable. This period starts 10 to 14 days before ovulation and ends at ovulation. This phase is very rapid, and yolk formation accounts almost entirely for the enormous gain in weight which the ovary undergoes during the 10 to 12 days before ovulation starts. Data of Nalbandov REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1117 and James (1949) show that the chicken ovary may weigh 2 gm. at 150 days of age and 20 gm. at 180 days. A single follicle may increase in weight from 1 to 16 gm. in 9 days. The yellow yolk deposited during this phase is rich in lipids (50 per cent of the dry matter) ; it contains large amounts of cholesterol and is rich in vitellin. The mechanism of yolk deposition is poorly understood, especially the apparent change in the selective permeability of the follicular membranes during the three phases of follicular growth and yolk forma- tion. Although changes in the blood chemis- try under the influence of estrogen in all probability play a role in mobilizing the yolk precursors, these changes do not guar- antee the deposition of yolk in the follicle. Undoubtedly, the anterior pituitary hor- mones influence the permeability of the fol- licular membranes, but how they do so is not known. Endocrine activity of the ovary and THE EFFECTS OF HORMONES. Adequate evi- dence exists to show that the avian ovary secretes three hormones, estrogen, androgen, and progesterone. Evidence for the secretion of each of these hormones will be presented together with the physiologic effects of the hormones as they pertain to reproduction. 1. Estrogen. Estrogen secretion by the ovary was demonstrated by Mario w and Riehert ( 1940) by extraction procedures followed by bioassay of the extract. Sub- sequent chemical analyses demonstrated that the ovary of the laying hen contains estradiol, estrone, and estriol (Layne, Com- mon, Maw and Fraps, 1958). Estrone is present in the blood in the conjugated frac- tion. Kornfeld and Nalbandov (1954) had previously demonstrated the presence of a biologically active estrogen in the blood of 16- to 20-day-old pullets. The evidence strongly favors the concept that estrogen is secreted by the ovary and that secretion starts during embryonic development (see chapter by Burns). The thecal gland cells are considered to be the source of estrogen (Marshall and Coombs, 1957). Estrogens have many effects on the physi- ologic processes of birds. Extensive reviews have been published on various aspects of the effects of estrogens (Nall)andov, 1953; Lorenz, 1954; Sturkie, 1954; Stammler. Katz, Pick and Rodbard, 1955; Urist, 1959) , so only the most salient features will be mentioned here. Estrogen administration causes an en- largement of the oviducts and its ligaments, an effect which will be discussed more ex- tensively later in this chapter. In addition to this effect on a secondary sex organ, estrogens cause marked changes in the composition of the blood which are sum- marized in Table 18.5. The substantial agreement with respect to their nature makes it unnecessary to include all the pub- lications on this subject. Extensive further documentation can be found in the reviews. Investigations have been conducted in order to determine the mechanisms involved in the production of some of the changes in blood composition. The lipid and phos- pholipid responses can be obtained in chick- ens on a fat-free diet and in hypophysecto- mized chickens (Baum and Meyer, 1956). The responses require the normal function- ing of the liver (Ranney and Chaikoff, 1951 ; Vanstone, Dale, Oliver and Common, 1957). The increases in plasma protein and phosphoprotein are also dependent on nor- mal functioning of the liver (Vanstone, Dale, Oliver and Common, 1957). Thyroid hormone administration together with estro- gen abolishes the estrogen-induced lipemia (Fleischmann and Fried, 1945; Hertz, Schricker and Tullner, 1951), proteinemia (Sturkie, 1951), increase in serum vitellin (Hosoda, Kaneko, Mogi and Abe, 1954), increase in biotin (Hertz, Dhyse and Tull- ner, 1949), and calcemia (Fleischmann and Fried, 1945). The mechanism of this inhibi- tion is not clear, but it seems to involve a different mechanism of action of estrogen from that which produces oviducal growth, for estrogen-induced oviducal development is not affected by simultaneous thyroxine treatment (Fleischmann and Fried, 1945; Hertz, Schricker and Tullner, 1949; Hosoda, Kaneko, Mogi and Abe, 1954) . Intensive studies have been made of the effect of estrogen on calcium metabolism; Urist (1959) has given a detailed account. He established that administration of 100 mg. estrone jier week to either roosters or laying hens caused the deposition of large 1118 SUBMAMMALIAN VERTEBRATES TABLE 18.5 Changes in blood composition after estrogen administration Component Sex Control Estrogen Reference Total lipids, mg. per 100 ml M 1100 14210 Urist, 1959 Phospolipids, mg. per cent plasma M 162 934 Ranney, Entenman and Chai- koff,"l949 Sphingomyelin, mg. per cent plasma M 22 54 Rannev, Entenman and Chai- koff,"l949 Cephalin, mg. per cent plasma. . . . M 34 214 Rannev, Entenman and Chai- koff, 1949 Cholesterol, mg. per cent plasma. . . M 235 1136 Stammler, Katz, Pick .md Rod- bard, 1955 Total protein, gm. per ICO ml. serum M 3.90 7.40 Urist, 1959 Albumen, gm. per 100 ml. serum . . . M 1.00 0.60 Urist, 1959 Globulin, gm. per 100 ml. serum . M 2.90 6.80 Urist, 1959 Vitellin (dilution detected) F 0 40 Hosoda, Kaneko, Mogi and Al)e, 1955 Hemoglobin gm. per ICO ml F 9 5.6 Ramsay and Campbell, 1956 Total vitamin A, /ig. per 100 ml.. . F 5.1 46.8 Gardiner, Phillips, Maw and Com- mon, 1952 Vitamin A ester, Mg. l)ei' ICO ml F 0.9 42.8 Gardiner, Philli])-^, Maw and Com mon, 1952 Vitamin A alcohol, /ug. per 100 ml.. . F 4.2 4.0 Gardiner, Phillips, Maw anil Com- mon, 1952 Riboflavin, p.p.m F Trace? 1.22 Common and Bolton, 1946 Biotin (water sol.), nijug. per ml F 1.3 ± 0.22 8.3 ± 1.8 Hertz, Dhyse and Tullner, 1949 Ca, mg. per 100 ml M 10 97 Urist, 1959 U.F. Ca, mg. per 100 ml M 6.50 8.00 Urist, 1959 Mn, jug. per 100 ml F None* 13.8 Bolton, 1955 Inorganic phosphate, mg. per 100 ml M 6.20 20.00 Urist, 1959 Total sulfate, mg. per 100 ml M 5.80 1.70 Urist, 1959 Iron, (Jig. per 100 ml. plasma F 100 700 Ramsay and Campbell, 1956 Controls were nonlaying hens, treated were 11-week-old pullets. amounts of intramedullary bone whether the birds were on a diet with enough calcium or on one completely deficient in calcium. The Ca-deficient diet was also deficient in vitamin D in order to further reduce the intestinal absorption of Ca. Birds fed the Ca-deficient diet deposited somewhat more medullary bone after estrogen treatment than did the estrogenized birds fed a Ca- adequate diet. The bone cortex of the Ca- deficient estrogen-treated birds showed re- sorption cavities and osteoporosis (Urist, 1959). Apparently, the intramedullary bone deposition occurs at the expense of the cal- cium from the flat bones. Estrogen treatment results in an increase in blood calcium, mainly in nondiffusible Ca (Polin and Stur- kie, 1958; Urist, 1959). Discussion of this increase in blood calcium levels requires consideration of the role of the parathy- roids on Ca metabolism. Estrogen adminis- tration causes an increase in size of the parathyroids (Landauer, 1954) and of ac- tivity, as measured histologically (Benoit, 1950b; von Faber, 1954). Is this increase coincidental or physiologic? The following observations may allow the formulation of a tentative explanation for the interaction between estrogen and the parathyroid hor- mone. a. Parathyroid hormone administration results in greater increase in total blood calcium in hens than in roosters (Polin, Sturkic and Hunsaker, 1957). 1). Parathyroidectomy results in a de- crease in total calciimi (Clavert, 1948; Polin and Sturkie, 1957) diffusible and non- diffusible; the decrease in nondiffusible REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1119 Ca is probably partly, if iiot completely, the result of postoperative starvation (Po- lin and Sturkie, 1957). 0. Parathyroidectomy of estrogen- treated capons or roosters reduces the estrogen-induced increase of diffusible cal- cium to about 58 per cent of the pre-opera- tive level, whereas the nondiffusible Ca is only slightly affected. The diffusible cal- cium levels obtained after this treatment were similar to those obtained in parathy- roidectomized birds without estrogen treat- ment (Polin and Sturkie, 1958). d. Estrogen treatment after parathy- roidectomy results in an increase of the nondiffusible Ca in cocks and capons, de- {lending on the level of diffusible Ca pres- ent. The higher the latter the greater the in- crease in nondiffusible Ca after estrogen administration. These data indicate that es- trogen can cause an increase in blood cal- cium when the parathyroids are functional or when enough diffusible Ca is present in the blood. Polin and Sturkie (1957) pro- posed that the level of diffusible Ca in the blood regulates the activity of the parathy- roids. The hormone from this gland must maintain a certain level of diffusible Ca in order to make the estrogen-induced in- crease in nondiffusible Ca possible (Polin and Sturkie, 1958). Thus estrogen-induced high nondiffusible Ca levels coincide with deposition of the resorbed substance. The discontinuation of estrogen administration produces resorption of the intramedullary bone (Urist, 1959). This resorbate may be transported as diffusible Ca, because, after estrogen treatment, the nondiffusible Ca as well as the phosphoprotein levels decrease. Nondiffusible Ca seems to be closely asso- ciated with the phosphoprotein. According to Urist, Schjeide and McLean (1958), the phosphoprotein deposited in the yolk is de- posited with the full complement of Ca that it carried in the serum. In none of the hy- potheses made has an explanation been given for the increased parathyroid size and activity observed after estrogen ad- ministration. This may mean that the in- creased parathyroid activity is coincidental or that the parathyroid is involved in a manner not yet accounted for. The mechanisms bv wliicli estrogen mobi- lizes the various components which are de- posited in yolk are still largely unknown, although the site of action seems to be the liver. The importance of mobilizing the yolk precursors will be discussed under the endocrine regulation of ovarian activity. Increased appetite has been observed after the administration of "artificiar' estro- gens (Lorenz, 1954; Baum and Meyer, 1956; Hill, Carew and van Tienhoven, 1958). Such stimulation of appetite may be an important adaptive mechanism in birds to provide for the deposition of large amounts of high energy materials in the yolk. It has, however, not been determined whether or not natural estrogens have the same appetite-stimulating effect in birds as the artificial estrogens. In experiments which the author was able to find in the literature, in which naturally occurring es- trogens were used in birds, pair feeding was practiced. Thus, the possible appetite stim- ulating effect could not be evaluated. The possibility of a difference in effect on appe- tite regulation by naturally occurring and artificial estrogens was revealed in experi- ments with rats by Meites (1949). This author found that diethylstilbestrol (DES) inhibited food consumption in rats whereas estrone had no effect. AVhether the appetite-stimulating effect of artificial estrogens in chickens is the re- sult of a direct stimulation of appetite cen- ters in the hypothalamus or an indirect ef- fect mediated by a change in the blood or in fat deposition has not been established. The influence of estrogen on feather de- velopment has been extensively reviewed by Domm (1939) and by Benoit (1950a); little needs to be added to their conclusions. In most species in which sexual dimorphism occurs and in which the male has the more ornamental plumage, estrogens are responsi- ble for the female type of feathering (so- called hen feathering). It is apparently true for the chicken, turkey, mallard, ostrich, pheasant iPhasianus colchicus and Phnsia- nus colchicus X gennaeus nyethemerus, Pha- sianus colchicus torguatus) and bobwhite quail (Colinus virginianus) that the male or cock feathering is due to the absence of es- trogen. In all these birds (sinistral) ovari- ectomy results in male type feathering, the 1120 SUBMAMMALIAN VERTEBRATES feather type also in those cases in which no right rudimentary gonad develops (Domm, 1939). Parkes (1952) has pointed out that the feather development of the Brown Leghorn capon may be used suc- cessfully to measure the duration of action of different estrogens. Witschi (1955 for review) has analyzed a peculiar phenomenon of determination of feather coloring which exists in certain species. During the nonbreeding season males and females of the genera Euplectes, Steganura and Quelea have the hen plum- age. Just before the breeding season the male passes through a partial molt, and the new plumage is the brightly colored cock plumage. Castration of the male does not affect the changing of the plumage from eclipse to nuptial plumage, whereas ovari- ectomy of the female causes her to go through the same phases as the male or the castrated male. These experiments proved that plumage color was independent of androgens but, nevertheless, showed cy- clic changes. Experimentally, Witschi dem- onstrated that LH injections will cause color changes of newly formed feathers in these species. The response is specific and sensi- tive enough to be used for bioassays of Lli (see addendum). In intact Euplectes, color changes in plumage and bill occur together under normal conditions. The feather color is a reflection of the LH directly, whereas the bill color is a reflection of androgen se- cretion by the testes under influence of LH. In wydahs (Steganura paradise a) , LH de- termines bill color directly. The lack of cock plumage in normal females is the re- sult of estrogen inhibition of male plumage as Witschi (1937) demonstrated experimen- tally. 2. Androgen. Evidence for the secretion of androgen by the avian ovary can be found in the red vascular com!) of the chicken before and during egg production, in the yellow bill and the stimulated vasa deferentia of the female starling {Sturnus V. vulgaris) during her reproductive season (Witschi and Fugo, 1940). These characters of the starling are stimulated by androgens only and not by estrogen or progesterone (Fugo and Witschi, 1940). Benoit (1950a), Taber (1951), and Mar- shall and Coombs (1957) have suggested that the interstitial cells of the ovary (Be- noit, 1950a; Taber, 1951), or, more specifi- cally, the ovarian interstitial cells arising from the connective tissue cells of the ovar- ian stroma (Marshall and Coombs, 1957) are the source of androgen in female birds. Some of the effects that androgens can have on blood composition have been dis- cussed in a previous section of this chapter. Their main function in female birds may be to act synergistically with estrogen in the stimulation of the oviduct. In chickens an- drogen acts synergistically with estrogen to increase calcium retention (Common, Maw and Jowsey, 1953.) and to increase endosteal bone formation (Jowsey, Oliver, Maw and Common, 1953) ; the interaction may be caused by increased Ca absorption from the gut caused by androgen combined with the increased formation of endosteal bone caused by estrogen (Jowsey, Oliver, Maw and Common, 1953). .3. Progesterone. Evidence that the avian ovary secretes progesterone is provided by the detection of a biologically active pro- gestin in the blood of laying and nonlaying hens (Fraps, Hooker and Forbes, 1948, 1949 1 and by the chemical identification of progesterone in extracts from ovaries of laying hens (Layne, Common, Maw and Fraps, 1957). Progesterone (5 fig. per 100 ml.) as such was detected in the blood of laying hens when loss of progesterone in the peripheral tissue was circumvented (Lytle and Lorenz, 1958). The source of jn-ogesterone in the ovary has not been established. In all probability it is not the ruptured follicle, because the amount of progesterone in it is small. Fraps (1955) proposed that the maturing follicle is a pos- sible, but not the exclusive, source of pro- gesterone, whereas Marshall and Coombs (1957) favored certain amorphous noncel- lular aggregations of cells of cholesterol- positive lipid in atretic follicles. Neither of these tissues has been proven (or disproven) to be the source of progesterone. Whatever the source of progesterone may be, it seems to function largely in regulat- ing the ovulatory cycle, at least in the chicken (a topic discussed under endocrine regulation of ovarian activity) ; progester- REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1121 one acts synergistically with estrogen to stimulate ovidiical development and secre- tory activity (discussed under oviduct de- velopment), and it may play a role in regulating incubation behavior in the ring dove {Streptopelia risoria) (Lehrman, 1958, and his chapter in this book) . Progesterone, in doses which also cause ovarian atresia, causes molting of chickens and seems to stimulate the feather papilla (Shaffner, 1954; Juhn and Harris, 1956, 1958). Juhn and Harris (1955) defeathered birds, then gave progesterone intradermally. No stimu- lating effect of progesterone was observed, although thyroxine treatment under similar conditions was effective in stimulating the feather papilla. Juhn and Harris (1955) used the structure of the new feathers as an ''internal assay" to detect possible stimu- lation of the thyroid by the injected pro- gesterone. No evidence of thyroid stimula- tion was found and the hypothesis that progesterone causes molt by stimulating the thyroid was rejected. Himeno and Tanabe (1957) arrived at a similar conclusion after determination of thyroid activity with I'^^. These authors suggested that the molt is precipitated when the ovary becomes atretic under the influence of progesterone, the con- sequent reduction in circulating estrogens would then allow the feather follicles to become active. In addition, progesterone would stimulate the feather papillae to form new feathers. This hypothesis does not ac- count for the fact that testosterone causes cessation of laying (and thus atresia?) but does not induce molting, nor does it account for the precipitation of molting after preg- nant mare's serum (PMS) or FSH injections (Juhn and Harris, 1956) which cause atresia but do not reduce estrogen secretion (Bates, Lahr and Riddle, 1935) . The endocrine regu- lation of molting may well be different de- pending on the species. Harris and Shaffner (1956) noted that progesterone fails to in- duce molting in pigeons whereas similar doses induce molting in chickens. Kobaj^a- shi (1958) subsequently investigated the effect of 17 a-oxyprogesterone-7-caproate (PC) on molting of 19 avian species. He found that birds which breed all year can be induced to molt by PC injections, but in seasonal breeders the molt fails to occur. In birds of the former type complete thyroid- ectomy prevented the molt response to PC, but gonadectomy had no effect. Kobayashi proposed that the following mechanisms miij;lit ])v involved in the PC-induced molt: (1) inn-eased sensitivity of the feather pa- pillae to thyroid hormone, which can induce molt alone; (2) a synergistic action between PC and thyroid hormone; (3) a combination of 1 and 2. In view of the results of Juhn and Harris (1958) implicating prolactin as a liormone involved in molting, it seems that factorial experiments with hypophy- sectomized-gonadectomized birds should be carried out as a means of establishing the relationships between prolactin, progester- one, thyroid hormone, and estrogen on molt- ing. An excellent review on the endocrine factors involved in molting has been pub- lished by Assenmacher (1958). 4. Huptured follicle hormone? Rothchild and Fraps (1944a, b) noted that removal of the recently ruptured follicle caused re- tention of the egg in the oviduct from 9 hours to 3 days longer than normal. Re- moval of the largest mature follicle caused only a slight delay in oviposition, but when both the recently ruptured and the largest follicle were removed the egg was retained for from 1 to 7 days. Subsequent investiga- tions by Conner and Fraps (1954) demon- strated a rather curious quantitative rela- tionship between the ruptured follicle and oviposition. When half of the ruptured fol- licle is removed some birds show no effect, others retain the egg, and a small number lay the eggs prematurely. The smaller the portion removed, the greater the incidence of premature ovipositions. The time of re- moval, apparently, also played a part, for a maximal incidence of premature oviposi- tions occurred when the operation was per- formed about 9 hours after ovulation. Although the endocrine function of the rup- tured follicle has not been demonstrated by replacement therapy, the evidence strongly suggests that the recently ruptured follicle has a rather short-lived endocrine activity. The short duration of its function is indi- cated by its rapid degeneration and by the lack of effect when the next to last ruptured follicle is removed (Rothchild and Fraps, 1944a). 1122 SUBMAMMALIAX VERTEBRATES 4. The Oviduct A discussion of the effects of the ovarian hormones on the development and function of the avian oviduct requires a brief ana- tomic description of this organ. The second- ary sex organ of female birds usually con- sists of a single fully developed, left oviduct which has developed from the Miillerian ducts. Even in species in which the inci- dence of two ovaries is high, only one ovi- duct is developed (Witschi and Fugo, 1940; Nelson and Stabler, 1940; von Faber, 1958 j. In chickens the incidence of right oviducts varies between different strains, but, in a large number (80 to 85 per cent), some evidence of a right oviduct is encountered (Winter, 1958). Morgan and Kohlmeyer (1957) reported that the incidence of fully developed right oviducts was quite high in one inbred strain of chickens. Usually, how- ever, the right oviducts are thin mem- branous cysts or short tubes (Winter, 1958). Development of right oviducts can be in- duced by estrogen treatment of the embryo, a topic discussed in the chapter by Burns. It is, thus, possible that the higher incidence of fully developed right oviducts in certain strains is the result of a higher estrogen deposition in the yolk and the consequent presence of abnormal quantities of estrogen in the developing embryo. This explanation is speculative, but may be worth further investigation. The left o\'iduct is suspended from the body wall by the dorsal and ventral liga- ments. The ventral ligament, at its caudal end, consists of muscle fibers radiating to- wards the vagina. The left dorsal oviducal ligament, the left abdominal air sac, and the body wall together form the "ovarian pocket" (Surface, 1912) into which the yolk falls after ovulation. The oviduct subsequently engulfs the yolk and trans- ports it to the cloaca. The oviduct can be divided into 5 ana- tomically distinct regions which will be de- scribed from an anatomic and functional point of view. 1. The injundibuhim is about 7 cm. long and consists of a thin funnel. Its lips are continuous with the ventral and dorsal ligaments (Surface, 1912) ; the chalaziferous region if^ tubular in shape (Richardson, 1935; Winter, 1958j. The infundibular walls are comjjosed of the peritoneum, a thin layer of longitudinal muscles, and a nonciliated columnar epithelium. In the funnel the epi- thelium consists of nonciliated and ciliated cells; in the chalaziferous region goblet cells filled with mucin are also present (Richard- son, 1935; Winter, 1958). The mucin cells stain with mucicarmine, thionin, hematoxy- lin, and Bismarck brown. Although the main function of the funnel is to engulf the egg, some mucin is deposited around the yolk while it descends the oviduct. Van Drim- melen (1951) identified the "sperm nests" in the infundibulum. Although a part of the infundibulum has been named the chalazi- ferous region, the chalazae are not formed there nor is the material from which they are formed secreted there. Burmester and Card (1939) resected this region of the oviduct and found no significant decrease in chalazae weight as a result of the re- section. 2. The infundibulum gradually changes into the iiKigninn which is about 34 cm. long in an active oviduct. It has thicker walls than the infundibulum, mainly because of enormously developed tubular glands which secrete albumen. The magnum is character- ized by nuicosal ridges with secondary and tertiary folds, and by an epithelium consist- ing of ciliated cells and of nonciliated goblet cells filled with mucin. The staining af- finities of the goblet cells are similar to those of the infundibulum. The function of the magnum is mainly the secretion of the thick albumen around the yolk. This proc- ess is completed in the relatively short time of 3 to 4 hours (Warren and Scott, 1935). 3. The transition of magnum to isthmus is marked by a sharp band visible to the naked eye. This band is free of tubular glands and is covered by a cuboidal epi- thelium. The isthmus is about 8 cm. long and has a thin tubular gland layer result- ing in a thinner wall than the magnum. These glands of the isthmus secrete ovokera- tin for the formation of the shell mem- branes, a process which occurs during the 1-hour sojourn of the egg in the oviduct (Warren and Scott, 1935). Histologic ex- amination reveals a circular muscle layer REPRODUCTR'E ENDOCRINOLOGY IN BIRDS 1123 which is hctUT dcvclopt'd than that in the magnum. The epithelium consists of ciliated and nonciliated cells and of goblet cells with little or no affinity for mucin-staining dyes (Richardson, 1935). The transition from isthmus to shell gland is gradual. The area is characterized histologically by the presence of special glandular cells which show a distinct vacuolization, a pale cyto- plasm, and a scarcity of granules. In this area tubular glands of the isthmus and of the shell gland api:)arently do not mix (Richardson, 1935). The change in epi- thelium is gradual. 4. The shell gland is about 8 cm. long and has a larger diameter than the isthmus or magnum. The longitudinal muscle layer is well developed. The mucosal folds have diagonal and transverse secondary folds. The tubular gland cells of the shell gland are smaller than those of the isthmus. The epi- thelium consists of a single layer of cells with apical and basal nuclei (Richardson, 1935) and of some goblet cells which lack affinity for mucin stains. In the shell gland the egg receives the thin albumen in 4 to 8 hours. The shell is deposited around the membranes, and indications are that shell formation continues as long as the egg stays in the shell gland which is about 20 hours on the average (Warren and Scott, 1935) . 5. A sphincter separates the shell gland from the vagina. In birds which are not secreting sufficient estrogen to have caused its breakdown (Greenwood, 1935; Kar, 1947a) the occluding plate which is proba- bly homologous with the mammalian hy- men can also be found. The vagina is characterized by its highly developed, circu- lar muscle layer and a mucosa with flat, longitudinal folds. The vaginal epithelium consists of nonciliated and ciliated cells, and of tall goblet cells. The latter stain with the same dyes as do the goblet cells of the magnum. The principal function of the vagina is its j)articipation in the ex- Dulsion of the egg. The infundibulum of the oviduct of the fowl is innervated by nerve fibers originat- ing in the ovarian plexus; these nerves transverse the dorsal ligament before reach- ing the oviduct. The more posterior parts of the oviduct receive nerve fibers from various autonomic jilexuses along the abdominal aorta (Alauger, 1941). The pigeon's oviduct receives blood from the genital artery and from the pelvic artery, a branch of the iliac artery (Bha- duri, Biswas and Das, 1957). The anterior portion of the oviduct of the fowl receives blood from a branch of the renal artery. The magnum is supplied by a branch of the left sciatic artery and the shell gland by a branch of the arteria pundendal communes. The blood from the oviduct drains into the common iliac vein and from this into the vena cava. In the adult bird, the ovarian hormones mainly control structural changes and the secretory activity of the oviduct. The ad- ministration of estrogen to immature pul- lets dramatically increases the size of the oviduct (Juhn and Gustavson, 1930; Kar, 1947a; Brant and Nalbandov, 1956) and of the dorsal and ventral ligaments (Kar, 1947a). It also initiates the breakdown of the occluding plate (Kar, 1947b). Although 1.00 mg. of estradiol benzoate per day may induce a 20-fold increase in oviduct size, it fails to induce development of the tubu- lar glands of the oviduct or to induce al- bumen secretion by the magnum. The de- velopment of the tubular glands and the secretion of albumen can be induced by a combination of estrogen and either pro- gesterone or androgen, whereas neither of these hormones alone produces this effect (Brant and Nalbandov, 1956). Estrogen ad- ministration induces riboflavin secretion in the magnum, but the secretion increases 18 to 35 per cent if the estrogen is given in combination with progesterone or testos- terone or with both (Bolton, 1953). The secretion of avidin by the oviduct is also a synergistic response to combinations of estrogen and progesterone, and to estrogen- desoxycorticosterone acetate (DOCA) com- l)inations. However, DOCA, progesterone, or testosterone when given alone can in- duce avidin secretion by the oviduct (Hertz, Fraps and Sebrell, 1943, 1944). A curious phenomenon is that estrogen and progester- one act synergistically to cause avidin secre- tion, but, at the same time, progesterone inhibits the estrogen-induced increase in oviduct size (Hertz, Dhvse and Tullner, 1124 SUBMAMMALIAN VERTEBRATES TABLE 18.6 Effect uf progesterone on estrogen-induced increase in oviduct weight of immature Jowl Body Weight Estrogen Dose per Day Progesterone Dose per Day Oviduct Weight as Percentage of Oviduct with Author Es- trogen Pro- gester- one gm. 580* Estradiol benzoate 2.0 mg. 1.0 mg. 91.5 Bolton, 1953 150-200t Stilhpstrol 20 Mg. 50 Mg. 108.4 Mason, 1952 20 Mg 500 Mg. 176.1 508.9 200 Mg 50 Mg. 143.2 200 Mg 500 Mg. 61.4 2029 Estradiol benzoate 20 Mg 500 Mg. 360.2 1246 200 Mg 500 Mg. 82.1 3394 100-150t Diethyl stilbestrol 250 Mg 50 Mg. 104.6 Hertz, Larsen and Tull- 250 Mg 100 Mg. 85.1 ner, 1947 250 Mg 150 Mg. 62.6 250 Mg 200 Mg. 47.3 250 Mg 250 Mg. 54.4 250 Mg 300 Mg. 43.0 lOOOt Diethyl stilbestrol 13 mg. pellet 500 Mg. 128.6 1516 Brant and Nalbandov, 1000 Mg. 281.8 2284 1956 2000 Mg. 314.4 2835 4000 Mg. 240.7 2474 180* Diethyl stilbestrol 15 mg. pellet 0.57 mg. 169.0 1127 Adams and Herrick, 1955 550* Diethyl stilbestrol 25 Mg. 50 Mg. 125.8 van Tienhoven, iinpub- 25 Mg 500 Mg. 158.1 lished 250 Mg 50 Mg. 148.2 250 Mg 500 Mg. 188.2 500t Estradiol 25 Mg 5 Mg. 599 373 Breneman, 1956 25 Mg. 70 463 * Average body weight of groups. t Weight estimated from age of birds from Table 9, Nutritional Requirements of Poultry, Nat. Res. Council, Publ. 301, 1954. 1949a). The observation that j)rogesterone alone can inhibit this response to estrogen has been confirmed by some workers, but others have noted a definite synergistic ac- tion. Some of these experiments have been summarized in Table 18.6. The list of ex- periments is not complete, for in some cases no quantitative data were published (Gard- iner, Phillips, Maw and Common, 1952). The difi"erent results are difficult to evalu- ate because of differences in body weight and the uncertainty concerning the amount of estrogen absorbed from implanted pellets. It is possible, however, that an antagonism occurs at the higher doses of estrogen. This is somewhat similar to the inhibition ob- served between some combinations of es- tradiol, estriol, and estrone in immature rats. At lower doses, estrone + estradiol and estradiol + estriol acted synergistically in stimulating uterine weight, but estradiol inhibited the eftect of estriol when estradiol + estriol were given in higher doses (Grauer, Saier, Strickler and Cutuly, 1958). More detailed studies on the dose relation- ship and the ratio of estrogens to progester- one are required in order to determine whether the present variations in results can be explained by competitive inhibition. It is notew^orthy that Brant and Nalban- dov (1956) observed no antagonism between estrogen and progesterone W'hen oviduct weight, tubular gland development, and al- bumen secretion were measured. When es- trogen plus 2 to 3 mg. of testosterone were administered, an optical stimulation oc- curred, but W'hen a larger amount of testos- terone (4 mg. per day) was given with estro- gen an antagonism was indicated by the low^er oviduct weight, the lesser develop- REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1125 ment of the tubular glands and the smaller amount of albumen secreted. Oviducts were still larger than those of birds treated with estrogen only. However, this inhibitory ef- fect of the higher dose of testosterone is surprising in view of the oviduct stimula- tion obtained with even higher doses (per unit of body weight) of androgen in the black-crowned night heron, Nycticorax nyc- ticorax (Noble and Wurm, 1940) , the starl- ing, Sturnus v. vulgaris (Witschi and Fugo, 1940) , the sparrow hawk, Falco s. sparvenus (Nelson and Stabler, 1940), the house spar- row, Passer domesticus (Ringoen, 1943), and the fowl (Kline and Dorfman, 1951). Ringoen (1943) made particular note of the full development of the tubular glands of the oviduct and stated that this might be due to the secretion of estrogen by the ovary, inasmuch as large follicles had developed under the influence of the exogenous testos- terone. The question as to which hormones, an- drogen or progesterone, synergize with estro- gen to give full oviduct development in birds may be answerable only if specified for species, and even then the possibility exists that all three hormones act together. In the fowl, ovary, oviduct, and comb de- velopment occur about the same time; the comb growth is evidence that androgen is secreted in considerable quantities. Simi- larly, female starlings secrete large amounts of androgen which stimulate the vasa def- erentia and cause the yellow coloring of the bill (Witschi and Fugo, 1940). Lehrman and Brody (1957) proposed that progester- one, which acts synergistically with estrogen to cause oviduct development of ringdoves {Streptopelia risoria) , may be the hormone which is secreted and is responsible for the synergism. This hypothesis was sug- gested by the onset of incubation behav- ior after progesterone administration (Lehr- man, 1958). In this case, progesterone might stimulate the physiologic development of the oviduct and simultaneously induce pa- rental behavior. In none of the birds in- vestigated is there any evidence against an hypothesis which assumes that estrogen interacts with both, androgens and pro- gesterone, to stimulate the oviduct. Nalbandov ( 1959a » presented evidence that carbonic anhydrase activity in the shell gland of the fowl may involve the I)ituitary, but the manner in which the pituitary is involved awaits clarification. A brief summary of various other factors involved in the normal development of the oviduct and in the response of the oviduct to exogenous gonadal hormones concludes the discussion of the oviduct. 1. Campos and Shaffner (1952) demon- strated that different sire and dam families show differences in the magnitude of re- sponse of the oviduct to a standard dose of estrogen and androgen. 2. The exposure to infectious bronchitis when the birds are 1 to 14 days old causes development of incomplete oviducts in which a great decrease in size occurs in the magnum and shell gland (Broadfoot, Pomeroy and Smith, 1956). The older the birds at the time of exposure, the less was the incidence of incomplete oviducts. 3. The presence of a well developed right oviduct, induced by estrogen treatment of the embryo, was highly correlated with a decrease in length of the left oviduct in sexually mature hens. The hypothesis pro- posed to explain this phenomenon was that not enough estrogen is secreted by the ovary of the adult hen to stimulate both oviducts (van Tienhoven, 1957) . It may explain also the presence of two completely developed oviducts in certain inbred lines (Morgan and Kohlmeyer, 1957). If the presence of the right oviducts were the result of larger than normal amounts of estrogen deposited in the yolk, one might expect larger than normal estrogen secretion by the hens of these strains, so that sufficient estrogen would be present to stimulate both oviducts. 4. It lias been established that nutritional deficiencies can affect the response of the oviduct to exogenous estrogens. From these investigations it seems that a paradoxical situation exists in that certain deficiencies such as the thiamine (Kline and Dorfman, 1951), nicotinic acid (Haque, Lillie, Shaff- ner and Briggs, 1949; Kline and Dorfman, 1951), riboflavin, pantothenic acid, choline, and vitamin D deficiency (Haque, Lillie, Shaffner and Briggs, 1949) result in a greater than normal response, whereas folic acid deficiency (Hertz and Sebrell, 1944; 1126 SUBMAMMALIAN VERTEBRATES Hertz, 1945, 1948a, b; Haqiie, Lillie, Shaff- ner and Briggs, 1949; Kline, 1955; Kline and Dorfman, 1951b) or vitamin B12 deficiency reduces the oviduct response, with folic acid deficiency resulting in the greatest reduc- tion. The increased response of the oviduct after nicotinic acid or thiamine deficiency may be the result of a decreased inactivation of es- trogen by the liver, which would in effect increase the levels of estrogen reaching the oviduct. Nicotinic acid is part of the coen- zyme involved in estrogen inactivation by the liver (DeMeio, Rakoff, Cantarow and Paschkis, 1948). Whether or not the de- ficiencies of the other vitamins mentioned causes an increased oviduct response by af- fecting liver function has not been estab- lished. Riboflavin according to Singher, Kensler, Taylor, Rhoads and Unna ( 1944) is involved in estrogen inactivation by the liver; however, Kline and Dorfman (1951b) could not confirm the effect of increased ovi- duct response observed by Haciue, Lillie, Shaffner and Briggs (1949). The failure of weight to increase after es- trogen administration to birds in which folic acid is deficient is probably the result of the lack of nucleic acid synthesis. Brown (1953) found that feeding of desoxy[)entose nucleic acid (DNA) to folic acid-deficient chicks partially restored the oviduct response to estrogen. The synthesis of DNA requires in turn the synthesis of considerable amounts of purines. Folic acid is required for synthe- sis of purines (Stokstad, 1954), whereas vi- tamin B12 is implicated in the metabolism of 1 -carbon fragments. On the other hand, the precise role that vitamin B12 plays in the oviduct response to estrogen is not known. Folic acid is also required for the increase in size of the oviduct in response to large doses of testosterone (Kline and Dorfman, 1951). The observation that folic acid is not required for the comb response (Zarrow, Koretsky and Zarrow, 1951 ) to testosterone may be explained by the differences in the nature of the two organs. The oviduct re- sponse involves synthesis of proteins and purines, whereas the comb response involves the deposition of substantial amounts of hyaluronic acid (Boas, 1949; Boas and Lud- wig, 1950). B. ENDOCRIXE REGULATION OF OVARIAN ACTIVITY 1. A)iterior Pituitary After hypophysectomy the avian ovary shows extensive atresia of the follicles and regression of the medullary tissue, especially of the interstitium (Hill and Parkes, 1934; Schooley, Riddle and Bates, 1941; Nal- bandov, 1953, 1959b, c ; Opel and Nalbandov, 1958). Replacement therapy with mamma- lian gonadotrophins is apparently success- ful in the pigeon, Columha livia (Chu and You, 1946), but only partially successful in chickens (Nalbandov, 1953). It is not clear from the i)ublished papers whether or not avian gonadotrophins are completely suc- cessful. According to Opel and Nalbandov (1958), some ovulations were induced (and thus some follicles were maintained?) a few days after hypophysectomy, but some atre- sia of follicles occurred. It appears that for a few hours after the withdrawal of endoge- nous gonadotrophins by hypophysectomy the large ovarian follicles are more suscepti- ble to exogenous LH, because a dose of LH, which does not cause multiple ovulations in an intact laying hen, will cause their occur- rence if injected into a hypophysectomized hen between 6 to 12 hours after the opera- tion. Within this time range, the ovary be- comes more and more sensitive as the time after surgery increases. In intact hens mul- tiple ovulations can be obtained with ex- ogenous LH provided that the hens have been pretreated for about 10 days with PAIS (Fraps, Riley and Olsen, 1942) or FSH (Nalbandov and Card, 1946). Progressive changes take place in the hypophysecto- mized and the PAIS- or FSH-treated bird, which also rc^sult in atresia of the follicles (Fraps, Riley and Olsen, 1942; Phillips, 1943). It seems, thus, that atretic changes in the follicle wall will predispose the follicle to ovulate (Nalbandov, 1959b), but that after atresia has caused breakdown of the vitelline membrane, ovulation can no longer be induced (van Tienhoven, 1955). Accord- ing to Nalbandov (1958), ovulation is pre- ceded by local ischemia of the follicular wall, particularly in the region of the stigma, which causes local necrosis. Whether or not this necrosis is the sole local precipitating REPRODUCTIVE EXDOCRIXOLOC.Y IX BIRDS 1127 factor in ovulation remains to be determined. The occurrence of ovulation in vitro (Neher, Olsen and Traps, 1950), provided the fol- licle is not removed from the ovary until about 2 hours before expected ovulation, suggests that changes other than ischemia also must occur. Removal of the ovary ear- lier than the designated time might be ex- pected to cause ischemia of the stigma as soon as the blood vessels are cut; neverthe- less, no ovulation occurs when this is done. Replacement therapy in hypophysecto- mizccl chickens (Opel and Nalbanclov, 1958; Nalbandov, 1959c) or the injection of gon- adotrophin into intact laying hens (Traps, Riley and Olsen, 1942; Phillips, 1943) does not result in maintenance or formation of the follicles of graded size observed in the normal ovary. Rather, the effect of injected hormones has been akin to an all-or-none effect: either many follicles are stimulated to grow to about the same size, or no stimu- lation occurs (Opel and Nalbandov, 1958; Nalbandov, 1959c). The mechanisms in- volved in the gradation of the follicles in the normal ovary awaits further elucidation. Gonadotrophin administration to intact chickens has different effects depending on the maturity of the birds. Until about 120 days of age, the response of the chicken ovary to mammalian gonadotrophin con- sists mainly of increases in estrogen and an- drogen secretions and of hypertrophy of the ovarian medulla. Evidence for the increase m estrogen production was hypertrophy of the oviduct (Domm, 1937; Domm and Van Dyke, 1930; Asmundson and Wolfe, 1935; Asmundson, Gunn and Klose, 1937; Lorenz, 1939; Nalbandov and Card, 1946 », and in- creased blood lipids (Lorenz, 1939). In- creased androgen secretion was indicated by growth of the comb (Domm and Van Dyke, 1930; Domm, 1937; Asmundson, Gunn and Klose, 1937; Nalbandov and Card, 1946; Taber, 1948; and Das and Nalbandov, 1955). The hypertrophy of the medulla ac- counts almost entirely for the increase in ovarian weight. None of the workers who in- jected mammalian gonadotrophins found normal development of the follicles. This lack of response of the follicles was not en- tirely due to the absence of the "third gon- adotrophic hormone," for even imi)lants of avian pituitaries (Domm, 1931) or daily in- jections of chicken anterior pituitary pow- der (CAP) did not result in large follicles until the birds were about 100 to 110 days old (Das and Nalbandov, 1955; Taber, Claytor, Knight, Gambrell, Tlowers and Ayers, 1958). There are, however, differ- ences in the response of the immature ovary to mammalian and avian gonadotrophins. Taber, Claytor, Knight, Gambrell, Tlowers and Ayers (1958) noted the following: (1) mammalian gonadotrophins fail to induce precocious follicular development, whereas CAP can induce such development; (2) mannnalian gonadotrophins cause medullary distension and consequently increase ovar- ian weight by about 400 per cent, whereas CAP causes no medullary distension and only a small (27 per cent) increase in ovar- ian weight; (3) after 12 days of PMS treat- ment the combs of immature pullets, which were stimulated by the FMS, start to re- gress, whereas with CAP treatment the combs continue to grow, a result similar to that obtained when the combs of hypophy- sectomized and of estrogen-treated roosters were being studied (Nalbandov, Meyer and McShan, 1951). Both mammalian and avian gonadotroph- ins increase the incidence of polyovular fol- licles (Taber, 1948; Taber, Claytor, Knight, Gambrell, Tlowers and Ayers, 1958) , indi- cating that there is some response of the cor- tex of the ovary to mammalian hormones. The changes which make the ovary more re- siionsive to avian gonadotrophins with in- creasing age are not known. It is known that the immature ovary responds to TSH by in- creased respiration (Nalbandov and Nal- bandov, 1949), but whether this response changes with age has not been established. The lack of response of the follicles in the immature ovary stands in striking contrast to the enormous development of ovarian fol- licles in the mature hen after either mam- malian or avian gonadotrophin administra- tion (Traps, Riley and Olsen, 1942; Phillips, 1943). The response, however, requires the presence of the "third gonadotrophin," be- cause in hypophysectomized hens mamma- lian gonadotrophins fail to elicit the re- sponse, whereas avian gonadotrophins can elicit it at least temporarily (Nalbandov, 1128 SUBMAMMALIAN VERTEBRATES 1953; Das and Nalbandov, 1955; Opel and Nalbandov, 1958j . Whether or not the avian pituitary is required for this "maturation" of the ovary has not been determined and not enough data are available for a com- parison of birds hypophysectomized at dif- ferent ages and given similar treatments. In all cases in which ovarian development has been obtained in chickens, large amounts of gonadotrophins, equivalent to 10 to 20 pitui- taries from 12- 14-week-old "broilers," had to be used. If one assumes that the pitui- taries came half from males and half from females, and that female pituitaries have half the potency of male pituitaries (com- parison of data of Breneman and Mason, 1951; and Breneman, 1955), then the 18 to 20 avian pituitaries per day are equivalent to 270 to 300 I.U. gonadotrophin per day be- cause according to Phillips (1959), 1 pitui- tary of a "broiler" rooster = 1 mg. dried powder = 20 I.U. PMS. Mammalian gon- adotrophin injections, equivalent to 500 rat units of FSH (Nalbandov and Card, 1946) or to 100 to 200 rat units PMS (Fraps, Riley and Olsen, 1942) were needed to stimulate follicles in adult intact hens. In contrast to the rather low sensitivity of the ovary with respect to follicular growth, stands the ex- treme sensitivity to LH for induction of ovulation. Fraps, Fevold and Neher (1947) showed that 1 fjig. LH prepared from chicken pituitaries was capable of inducing prema- ture ovulations in 50 per cent of the hens. The effect of gonadotrophin injections into intact female birds other than chickens seems to depend on the species used. Red- billed weavers (Witschi, 1935), European gold finches, Carduelis elegans (Vaugien, 1956), green finches, Chloris chloris, bunt- ings and canaries, Serinus canaria (Vaugien, 1957), and house sparrows. Passer domesti- cus (Riley and Witschi, 1938; Witschi and Riley, 1940; Vaugien, 1954 j can be stimu- lated to lay eggs during the nonbreeding season by injections of about 100 to 150 I.U. PMS every 3 days for 3 weeks. In con- trast to those of chickens, the follicles of these birds show normal gradations in size (Riley and Witschi, 1938; Witschi and Riley, 1940), and no separate LH injections are required for ovulation (Witschi, 1935; Vaugien, 1954, 1957). The lack of ovarian response in the robin, Erithacus r. rubecula, observed by Schildmacher (1939), may be explained by the low dosage used. If one ex- cludes possible differences between species with respect to the dosage required to obtain ovarian stimulation, the generalization can be made that intact songbirds differ from chickens in their response in the following ways. 1. Regular gradation of follicular size is obtained even with rather massive doses of PMS. 2. Ovulations occur "spontaneously" and do not require separate LH injections. It seems, thus, that a comparative approach to the problem of follicle-size gradation might prove to be profitable, as might an investiga- tion of the endocrine regulation of ovulation in song birds. Administration of the third gonadotrophic hormone, prolactin (the luteotrophic hor- mone of mammals), inhibits FSH secretion and results in cessation of laying and in atresia of the follicles (Bates, Lahr and Riddle, 1935; Bates, Riddle and Lahr, 1937) . Juhn and Harris (1956) reported, however, that prolactin counteracts the inhibition of laying by exogenous progesterone. This ef- fect is rather surprising for it would assign to prolactin a true gonadotrophic function which is contrary to its effects in roosters and female pigeons (Bates, Lahr and Riddle, 1935; Bates, Riddle and Lahr, 1937). Fur- ther investigations are needed to establish whether prolactin affects male and female chickens differently. Assays of the pituitaries from chickens (Burrows and Byerly, 1936; Saeki and Ta- nabe, 1956; Nakajo and Tanaka, 1956), pheasants, Phasianus colchicus Breitenbach and Meyer, 1959), and California gulls (Bailey, 1952) reveal that the prolactin con- tent is maximal when the eggs are being in- cubated. Prolactin content decreases when the chicks are hatched, although the hen is still caring for the young. Strong lights or electric shocks to the head interrupted broodiness and decreased the prolactin con- tent of the anterior pituitary, especially of the caudal lobe (Nakajo and Tanaka, 1956) . As we noted in the section on the male, the data from a limited number of species sug- gest that prolactin may be required for in- REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1129 cubation in species which have an incubation patch, whereas in ring doves {Streptopelia risoria), which do not have an incubation patch, incubation is not correlated with an increase in prolactin secretion (Lehrman, 1958, and his chapter in this book) . Gonadotrophin assays of pituitaries from the time of hatching into adulthood when reproductive activity is cyclic seem to have been made only on chickens. The results ob- tained by two groups of workers are sum- marized in Table 18.7. Their results are expressed in the common standard of chick units as defined by Breneman (1955). The results obtained by Riley and Fraps (1942a, b) are in essential agreement with those in the table with respect to the ratio of gonadotrophic potency of pituitaries from roosters and laying and nonlaying hens. They were not included because of the diffi- culty in converting mouse uterine units into chick units. As a part of the survey of the data con- tained in Table 18.7, it should be noted that the amount of gonadotrophin in the pitui- taries of young birds is closely correlated with ovarian weight (r — 0.898). This correlation provides an argument for the concept that the secretion of gonadotrophic hormone can be estimated from assays of the pituitary, in immature pullets as well as in adult hens. Just before ovulation begins in young hens pituitary gonadotrophin po- tency (and secretion?) reaches its peak. It is at a much lower level in older laying hens. This decreased gonadotrophic potency (and decreased gonadotrophin secretion?) is probably due to estrogens which are se- creted in large amounts by the ovary. The lower gonadotrophin secretion would be suf- ficient to maintain follicles already present and to stimulate new ones to grow to ovula- tory size. This concept that less gonado- trophin is required for maintenance and stimulation of follicles already present than is required for stimulation of an immature or an inactive ovary finds support in the following experimental evidence: 1. Vaugien (1957) stated on the basis of a rather small number of experiments that in song birds the ovary is more sensitive to exogenous gonadotrophin wdien one or more medium-sized follicles are present than TABLE 18.7 Gonadotrophic potency of pullet pituitaries in the domestic and hen fowl Age Ovarian Weight of Donor AP Assay Authority mean ± S.D. mg. chick units* 20 days 39.4 d= 11.3 0.4 Breneman, 1955 40 days 99.1 ± 15.3 1.1 Breneman, 1955 60 days . . . 167.9 ± 32.1 3.1 Breneman, 1955 80 days 292.1 ±62.7 4.6 Breneman, 1955 100 days .... 466.3 ± 198.6 6.4 Breneman, 1955 110 days. .. . 401.5 ± 68.1 6.4 Breneman, 1955 126 days .... 5781 ± 2445.0 14.4 Breneman, 1955 Aduh 39.1 gm. i.ot Saeki, Hi- meno, Tanabe and Katsu- ragi, 1956 Adult 3.1 gm. 2.3 Saeki, Hi- meno, Tanabe and Katsu- ragi, 1956 Aduh cock . 7.6 gm. (testes) 4.10 Saeki, Hi- meno, Tanabe and Katsu- ragi , 1956 * Chick unit is eciuivalent to 35 per cent in- crease over control assay. t Calculated (AvT) from data of Saeki et al., 1956. when the ovary contains only small folli- cles. 2. In order to obtain follicles of about 17-mm. diameter Taber, Clay tor. Knight, Gambrell, Flowers and Ayers (1958) had to inject 18 to 20 broiler pituitaries (equiva- lent to 270 to 300 I.U.) per day, whereas when laying hens were injected with 80 to 160 rat units of PMS enormous stimulation of the follicles was obtained in a few days (Fraps, 1955b). to be sure, the ratios be- tween these levels of exogenous gonado- 1130 SUBMAMMALIAX VERTEBRATES trophins are not as great as the ratio be- tween gonadotrophic potency of pullets of about 126 days and laying hens, but, on the other hand, the extents of stimulation ob- tained with the exogenous gonadotrophins in the chickens of the two ages were not directly comparable (the laying hens were overstimulated, the immature pullets not completely stimulated I . 3. On exogenous gonadotrophin adminis- tration the ovary is more sensitive when estrogen is administered before gonado- trophin injection or when administered si- multaneously with gonadotrophins (Phil- lips, 1959) . A more complete understanding of the regulation of follicular growth during the reproductive cycle will require more quantitative data on the gonadotrophic po- tency of pituitaries, especially during the period between the end of laying and the emergence of the new crop of follicles, more data on the sensitivity of the follicles to ex- ogenous gonadotrophins, and pure gonado- trophic hormones (see chapters by Greep and by Young on the ovary). 2. Estrogen Breneman (1955, 1956) investigated the effects of injection of 0.5, 1.0, 5.0 and 25.0 /Ag. estradiol per day for 10 days on the ovary of 30-day-old pullets and found no significant difference from control ovarian weight. Histochemically, there was evidence that 1 /Ag. estradiol caused increased choles- terol deposition in the follicle. Phillips (1959) injected 12.5 mg. DES per week into 6-week-old pullets and obtained a 32 per cent increase in ovarian weight (p < 0.01). Similarly, 10 mg. DES per day, given to adult, nonbreeding black ducks {Anas pla- tyrkynchos) , resulted in a 95 per cent in- crease in ovarian weight (p < 0.01 ) . In neither of the experiments was there any yellow yolk deposition. Chu and You (1946) also failed to induce maturation of follicles by estrogen injections in hypophysectomized pigeons. It is not clear from their paper whether any stimulation of ovarian weight occurred. Schonberg and Ghoneim (1946) reported that feeding of stilbene to pullets 100 days of age resulted in egg production at 114 days of age, whereas egg production in DES-fed pullets did not start until 162 days and egg production in control pullets did not start until 146 days of age. These results suggest that there may be differences in effect between estrogens and it also sug- gests that stilbene feeding just before pul- lets reach sexual maturity may cause earlier egg production. The difference between the two estrogens may be caused, for instance, by differences in inhibition of the pituitary gonadotrophin secretion. Experimental evi- dence that estrogens may synergize with exogenous gonadotrophins suggests that an estrogen which does not inhibit gonado- trophin secretion but mobilizes yolk pre- cursors could cause a somewhat earlier egg production. Clavert (1958) has, largely on theoretical grounds, defended the proposi- tion that estrogens should augment the ac- tion of exogenous gonadotrophins with re- spect to stimulation of follicular growth. Clavert's hypothesis was that estrogen would mobilize yolk precursors immediately and thus facilitate yolk deposition in the follicles under the influence of gonado- trophin. If gonadotrophins were given alone, estrogen would have to be secreted under the influence of gonadotrophin and yolk mobilization could occur subsequently. Tliis hypothesis was tested with nonbreeding black ducks by Phillips (1959). In one ex- periment 4 out of 6 birds treated with the combination of CAP and DES had large follicles with yellow yolk, whereas none of the birds treated with either hormone alone had yellow yolk. In the second experiment 3 out of 5 birds on the combined treatment had yellow yolk in the follicles, whereas none of the CAP-treated birds contained yellow yolk (there were no DES-treated birds in this experiment) . It should be noted that similar experiments wdth 6-week-old pullets failed to show any large follicle for- mation with either CAP, DES, or the com- bination. This may be the result of the un- responsiveness of the immature ovary, a factor which was discussed previously. The administration of estrogen causes de- lay of the next ovulation by suppressing LH release (Fraps, 1954). Progesterone admin- istration under identical conditions results in LH release and premature ovulation. The significance of these findings will be dis- cussed under regulation of the laying and REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1131 ovulation cycle of the fowl. In the meantime the indirect evidence suggesting that estro- gen has its depressing effect on gonado- trophin release by way of a neural mechanism will be cited: (1) estrogen ad- ministration to chickens causes changes in the neurosecretory cells of the paraventric- ular nucleus of the hyopthalamus (Legait, 1959) ; (2) estrogen causes gonadotrophin inhibition by way of the hypothalamus in mammals (Flerko, 1957) ; this is discussed in detail in the chapter by Everett; (3) progesterone, another steroid hormone, causes release of gonadotrophins from the anterior pituitary by way of a neural mech- anism. This possibility is discussed in the present chapter and, for mammals, in the chapter by Everett. 3. Androgen Androgens, apparently, have an effect on the ovaries of hypophysectomized pigeons which corresponds to that on the testes. Chu and You (1946) obtained 4 to 6 mm. fol- licles filled with yolk in androgen-treated hypophysectomized pigeons. A stimulating effect on the ovary was found also in intact immature pigeons, although the testes of immature males failed to respond (Chu and You, 1946). A similar situation exists in sparrows in which injections of testosterone (0.5 to 1.0 mg. per day) stimulated the ovarian follicles to such a degree that their diameters were approximately 75 per cent larger than the average in the controls (Ringoen, 1943). Androgen administration also resulted in a modification of the follicu- lar epithelium from simple to stratified (Ringoen, 1943). Breneman (1955, 1956) studied the effect of androgen on the ovaries and follicular de- velopment in intact, 30-day-old chickens. Five fjLg. TP given daily increased ovarian weight, but administration of either 1.0 or 25 ^g. did not have this effect (Breneman, 1956). Doses of 0.1, 1.0, 10.0, 50, and 100 fig. TP increased follicle area of the ovary significantly, but after a maximum w^as reached with the 0.1- and 10-/>ig. levels, the response tended to decrease. After the ad- ministration of 100 fjig., the follicular area was less than when the smaller amounts were given, but it was still larger than in the controls. The increase in follicle area may well be the result of inhibition of the interstitium combined with stimulation of the follicles. Breneman (1955) emphasized that androgen administration increases the height of the follicular epithelium. Analysis of variance of Breneman's data (by me) showed that only the comparison of control versus all androgen levels combined was significant (p < 0.05) . The lack of a dose- response relationship over such a wide range of doses suggests that the difference between controls and androgen-treated pul- lets was the result of a sampling error, un- less the assumption of an all-or-none re- sponse is made with respect to increases of follicular epithelial height. Androgen, ap- parently, did not affect pituitary gonadotro- phin assays. Breneman's (1955) statement that low doses of estrachol and testosterone facilitate the ovarian response to PMS is not supported by evidence, because no data are given on the effect of PMS alone. Nelson and Stabler (1940» injected large doses of TP (140 mg. in 30 days) into young female sparrow hawks (Falco s. sparverius) and found no effect on either left or right ovary. The limited data available on different spe- cies suggest the following provisional gen- eralization: if androgens stimulate the testes of a species (sparrows, pigeons), then an- drogens will, under similar conditions, also stimulate the ovaries of the females. Testosterone can cause the anterior pitui- tary to release LH, which results in ovula- tion. The incidence of premature ovulations after testosterone injection is about 41 per cent compared with 95 per cent after jiro- gesterone injection. Testosterone-induced LH release is mediated by a neural mecha- nism which will be discussed more fully under progesterone effects in the regulation of ovarian activity. Testosterone, thus, seems to be capable of affecting ovarian activity in two ways. One is by a direct effect on the ovary, as in hy- pophysectomized pigeons, and the other is by an effect on the neural components which regulate anterior pituitary activity. 4- Progesterone Progesterone probably plays a very im- l)ortant role in the regulation of ovarian ac- 1132 SUBMAMMALIAX VERTEBRATES tivity, especially during the period of full reproductive activity, but before discussing this phase of the subject, the experimental evidence that the hormone affects ovarian activity will be reviewed. Nalbandov (1956), in a preliminary note, presented evidence that progesterone pellets implanted into immature pullets hastened maturation of the ovarian follicles with a consequent precocious egg production. This result is consistent with the data obtained by Fraps (1950) with turkeys showing that PMS-stimulated follicles could be main- tained by progesterone injections. It also agrees with the results obtained by van Tienhoven (1958) suggesting that after a broody period, egg production is somewhat enhanced by progesterone pellet implants. On the other hand, Duchaine, Driggers and Warnick (1957) observed that injections of 6 mg. progesterone every other day delayed rather than enhanced the onset of sexual maturity in 16-week-old pullets. Broodiness of turkeys was inhibited by progesterone given in a readily absorbable form (van Tienhoven, 1958; Haller and Cherms, 1959) , but no effect on subsequent egg production was observed. It seems, therefore, that the level of progesterone al- ready in the blood may determine whether the action of exogenous hormone will be stimulating or inhibitory. This may explain the difference between the results of Nal- bandov (1956) and those of Duchaine, Driggers and Warnick (1957). Apparently, no experiments were carried out in wiiich the effect of combined treatments of gonado- trophins and progesterone on the inactive ovary were compared with the effect of single treatments. Large doses of progesterone, administered to laying hens in paste or pellet form, inter- rupt egg production (Adams, 1955, 1956; Juhn and Harris, 1956; Shaffner, 1954, 1955; Harris and Shaffner, 1956), presumably by causing follicular atresia. These results ob- tained with forms of progesterone which are relatively long acting can be explained in terms of the timing of the high progesterone levels with respect to the ovulation cycle of the chicken. Rothchild and Fraps (1949b) demonstrated that progesterone injections about 36 to 38 hours before expected ovula- tion result in atresia of the ovarian follicles. Atresia can be the result of an inhibition of all gonadotrophin secretion or of the release of too small an amount of LH to cause ovu- lation. Which of these two occurs after the progesterone administration is a matter of speculation. If one accepts the evidence that FSH and LH are secreted as one gonado- trophic complex, then the two interpreta- tions are essentially the same and differ only (luantitatively. Recently, van Tienhoven (1959) and Nalbandov (1959a) have de- fended the position that the FSH and LH are released as one complex. If their idea is correct, the question whether progesterone causes a partial or complete inhibition of gonadotrophins could be answered by deter- mining the total gonadotrophic potencies of the pituitaries of the progesterone-treated hens. On the other hand, if FSH and LH are secreted as separate entities, the two inter- pretations for the atresia are qualitatively different, and separate assays for FSH and LH in the pituitaries of progesterone-in- hibited birds should be made. In contrast to the atresia which occurs when progesterone is given 36 to 38 hours before ovulation, pre- mature ovulations result when progesterone is given 2 to 24 hours before the expected ovulation (Fraps, 1955b, for review). Considerable evidence has accumulated indicating that progesterone acts through a neural mechanism to cause the release from the pituitary of the gonadotrophin which in- duces ovulation. This evidence can be sum- marized as follows: 1. Progesterone-induced ovulation in the hen can be prevented by the simultaneous or previous administration of such adrenei'- gic blocking agents as SKF 501 (Zarrow and Bastian, 1953), Dibenamine (van Tien- hoven, Nalbandov and Norton, 1954), Di- benzyline (van Tienhoven, 1955), and the anticholinergic agent, atropine (Zarrow and Bastian, 1953; van Tienhoven, 1955). Re- cently, Moore (1958) questioned the va- lidity of the argument that large amounts of such agents block ovulation by blocking a neural mechanism. As Everett has pointed out in his chapter, "blocking" agents must be given between 2:00 and 4:00 p.m. on the day of the proestrum if they are to block ovulation in rats. Moore (1958) adminis- REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1133 tered Dibenamine or Dibenzyline to rats for 12 days after 5:00 p.m. After this period regular cycles were resumed. Injections of eitiier drug at the "critical period" during the proestrum failed to block ovulation in 80 per cent of the cases. ]\Ioore interpreted the blockade of ovulation observed in rats not pretreated with Dibenamine or Diben- zyline as the result of a shift in the pituitary from gonadotrophin to adrenocorticotrophin production and not to a "neural blockade." This interpretation would cast doubt on the hypothesis that progesterone acts by way of a neural mechanism were it not for rather abundant supportive evidence that this is indeed the case. Fraps and Case (1953) found that diallyl barbituric acid (Dial), Nembutal, and cal- cium ethylisopropylbarbiturate (Ipral) cause premature ovulation of the follicle when given 12 to 16 hours before the ex- pected time of ovulation. The incidence of premature ovulations was 15 to 30 per cent compared with 95 per cent when progester- one was given. Dial and Nembutal acted synergistically with subovulatory doses of progesterone to cause an incidence of 57 per cent and 30 per cent premature ovula- tions, respectively; the same doses of pro- gesterone alone were followed by 5.5 per cent premature ovulations. These findings can be interpreted (Fraps, 1955b) by assum- ing that, after the period of depression, a period of excitation follows which lowers the threshold for the stimuli which cause the release of gonadotrophin from the pituitary. Fraps (1955b) demonstrated that pheno- barbital administration blocks progester- one-induced ovulations. No explanation can be given for this opposite effect of pheno- barbital unless it is that it is longer acting than Dial or Nembutal. 2. Lesions placed in the ventromedian re- gion of the preoptic hypothalamus within about 2 hours after the injection of proges- terone prevent premature ovulation (Ralph and Fraps, 1959) . 3. Injections of small amounts of proges- terone (5 to 10 fig.) into the diencephalon result in premature ovulations only when they are placed in the preoptic region of the hypothalamus (Ralph and Fraps, 1960). In- jections into the caudal extensions of the forebrain are also effective (Ralph and Fraps, 1960). Systemic injections of 10 fxg. progesterone were ineffective as were injec- tions of 10 ^g. progesterone into the anterior pituitary. Taken together, these observa- tions indicate that progesterone causes premature ovulation by way of a neural mechanism, but the possibility that other mechanisms are involved has not been ex- cluded. 5. Corticosteroids Studies on the effects of corticosteroid administration on the avian ovary have been rather limited. Fraps (1955b) reported that DOCA was as effective as progesterone in inducing premature ovulations in chick- ens, and the effect of either can be blocked effectively by phenobarbital. Daily injec- tions of 5 mg. of DOCA caused inhibition of egg production in chickens (Hohn, 1960). Cortisone acetate (2.0 mg. per day) had no effect on egg production. 6. Epinephrine Perry (1941) found that injections of epinephrine daily for 15 to 20 days after sparrows had been exposed to 15 hours of light for 30 days caused regression of the slightly stimulated ovaries and oviduct. The mechanism whereby this occurred was not established. 7. Thyroid Hormone Early thyroidectomy (Blivaiss, 1947) or destruction of the thyroid by large doses of P-^^ (Winchester, Comar and Davis, 1949) results in total lack of ovarian development. Replacement therapy with thyroxine of such chickens results in follicular matura- tion and egg laying (Winchester, Comar and Davis, 1949). Greenwood and Chu (1939) thyroidectomized 5 pullets and found that 2 started to lay eggs at the same average age as the control flock. Winchester (1939) ob- served that thyroidectomy decreased egg l^roduction of 7 hens from 3.77 to 0.42 eggs per week. Whether this difference in results between the experiments of Blivaiss (1947) and Winchester, Comar and Davis (1949) on the one hand, and of Greenwood and Chu (1939) and Winchester (1939) on the 1134 SUBMAMMALIAN VERTEBRATES other hand, are the result of the difference in age at which the thyroid was removed or the result of incomplete removal of all thyroid tissue (especially because ectopic thyroid tissue seems to occur sometimes) cannot be judged from the few data now available. It is interesting to note that, in certain families of White Leghorn chickens, birds without any apparent thyroid tissue or with thyroids consisting of one abnormal follicle occur. In the most severe cases of hypothyroidism ovaries are immature at an age when normal hens of the same strain are in full production. Thyroxine injections will bring such hypothyroid birds into produc- tion in 7 to 21 days (the information on these hypothyroid hens was communicated to the author by Dr. R. K. Cole) . The bal- ance between gonadotrophin and thyroid hormone is apparently rather important. Clavert (1958) noted, for instance, that the ovarian response to P]MS was reduced by simultaneous thyroxine injections of pi- geons. Such an inhibition seems understand- able in view of the inhibition of estrogen- induced lipemia and proteinemia. In effect, thyroxine decreased the concentration of yolk precursors and thus lowered the re- sponse to PMS. In view of this effect of thyroid hormone, it is not surprising that thyroid hormone feeding (mainly as iodi- nated casein) has given opposite results in different experiments. These results have been reviewed recently (Turner, 1959; van Tienhoven, 1959) and do not need to be discussed here. 8. XutritioJi The specific effect of nutrition on ovarian activity has not been studied in great de- tail; this is in contrast to the many studies on the effect of nutrition of the hen on the hatchability and embryonic development (Cravens, 1949; Landauer, 1951). Restric- tion of energy intake delays sexual matu- rity (Bruckner and Hill, 1959). Pullets, after being reared on a restricted diet, when fed ad libitum at the approach of sexual maturity, produced more eggs during the rest of the year than pullets reared and maintained on an unrestricted diet. The mechanisms involved in these relationships have not been studied. It seems probable tliat restricted energy intake results in later gonadotroi^liin secretion, thus delaying sex- ual maturity. ^Yithdrawal of feed from laying hens re- sults in atresia of the follicles and a simul- taneous decrease in serum vitellin (Hosoda, Kaneko, Mogi and Abe, 1955b). These ef- fects can be prevented by injections of FSH or PAIS (Hosoda, Kaneko, Mogi and Abe, 1955a), and follicles so maintained can be ovulated (Hosoda, Kaneko, JMogi and Abe, 1956). The results suggest that starvation prevents production of gonadotrophin, a suggestion supported by bioassays of the pituitaries. Phillips (1959) found that the testes of chicks injected with pituitaries from starved hens with atretic follicles weighed 7.6 mg. compared with 10.11 mg. for the testes from chicks injected with pi- tuitaries from well fed, laying hens. The difference was not statistically significant, but shows a trend in the expected direction. No data seem to have been published on the specific effect of the separate nutrients on ovarian activity. In most studies egg pro- duction was measured and no efforts seem to have been made to separate the effects of inanition from the specific nutrient effect. C. REGULATION OF BREEDING CYCLES OF SEASONALLY REPRODUCING BIRDS Aristotle's observation that the testes are small during the nonbreeding season and large during the breeding season bears wit- ness to the accurate observations that have been made throughout history. It does not require much imagination to visualize that man must have observed the effect of en- vironment on reproduction. The regular flight north of flocks of geese every year must have impressed the hunting tribes. But only recently some understanding has been obtained of the pathways by which the en- vironment can affect reproduction. 1. Hypothalamic-Pituitary System In order to understand the explanations proposed here, a review of the control of the anterior pituitary is required. The dis- cussion is i^urposely limited to birds, because the relationships for other vertebrate classes are described in the chapters by Greep and Purves. It seems that the only manner in which environmental stimuli, especially those such REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1135 as light, could influence the activity of the pituitary is by way of the nervous system. Considerable evidence from different kinds of experiments implicates the hypothalamus as the structure that is specifically involved in the transmission of the stimuli to the anterior pituitary. The hypothesis has been proposed (Scharrer and Scharrer, 1954; Benoit and Assenmacher, 1955, 1959; Assen- macher and Benoit, 1958) that environ- mental factors cause changes in the activity 01 specialized hypothalamic cells, the so- called neurosecretory cells. These cells can be identified by a variety of stains (Assen- macher, 1958; Legait, 1959). They may be considered, on one hand, as nerve cells, on the other, as endocrine cells. Material pro- duced by these cells is transported along their axons to the posterior pituitary. How- ever, loops of these axons in the median eminence come into close contact with the capillary bed of the portal vessels of the hypothalamus where some of the neuro- secretory material (NSM) is picked up by portal vessels which transport it to the anterior pituitary whose cells it stimulates. The evidence in support of this hypothesis will be presented together with the counter argument by Zuckerman (1955), who ques- tioned the validity of this hypothesis. For convenience, the available evidence will be divided into somewhat arbitrary categories. Inasmuch as many details of findings have been published recently in the review papers and chapters of this book cited above, refer- ences will be limited largely to these re- views. Discussion will be limited to the re- sults obtained with birds. Anatomic evidence shows that few, if any, nerves reach the glandular tissue of the anterior pituitary. Even the few fibers found l)y Metuzals (1955) do not seem to have significance, because their origin could not be established. On the other hand, NSAI has been observed in the hypothalamico-hy- l)oi)hyseal tract of ducks (Assenmacher, 1958; Legait, 1959; Benoit and Assen- macher, 19591, chickens, although rarely ( Legait, 1959) , and the white-crowned spar- row, Zonotrichia leucophrys gamhellii (Oks- che, Laws, Kamemoto and Farner, 1959). The axons of these neurosecretory cells form ''loops" which are in close contact with the portal vessels in the strafion (lUinduUire of the "special zone" of the median eminence (Assenmacher, 1958; Oksche, Laws, Kame- moto and Farner, 1959) . The median emi- nence can be divided into three layers: (a) stratum ependymale, (b) stratum fibrosuni, (c) stratum glandulare. The tracts from the hypothalamus to the neurohypophysis are part of the stratum ftbrosimi, and NSM can l3e found here, often in such amounts that the individual fibers of the tracts can be dis- tinguished because of the content of NSM (Oksche, Laws, Kamemoto and Farner, 1959). The stratum gkindidare also con- tains large amounts of NSM arranged in arcades (Wingstrand, 1951; Legait, 1959). In this area the hypophyseal portal vessels make contact with the loops of NSM. The demonstration that the blood flow is frojn the median eminence to the anterior pituitary in ducks (Assenmacher, 1958) , together with all the other anatomic evidence cited above, is certainly in accord with the hypothesis that the neurosecretory material from the hypothalamus is the link between the ner- vous system and the anterior pituitary. Zuckerman (1955) has stressed that nerve fibers such as those found by Metuzals (1955) may form the functional connection between the hypothalamus and the pitui- tary. However, it seems to this author that Zuckerman's argument cannot be accepted for birds until it has been established that the fibers come from the hypothalamus. Further evidence is provided by interrup- tions of the connections between hypothala- mus and anterior pituitary. 1. Lesions in a medial region of the ven- tral portion of the paraventricular nucleus in chickens caused a long lasting interrup- tion of ovulation (Ralph, 1959). Lesions in the same area also prevented progesterone- induced ovulations (Ralph and Fraps, 1959). In other parts of the hypothalamus lesions did not consistently interrupt either "spontaneous" or progesterone-induced ovu- lations, and when they did, the interruption of "spontaneous" ovulations was temporary rather than long lasting. In drakes {Anas platijrhynchos) , fairly large lesions of the anterior hypothalamus prevented the noi-- mal light-induced increase in testicular ac- tivity (Assenmacher, 1958). 2. Complete interruption of the hypo- l)hyseal portal system of laying hens re- 1136 SUBMAMMALIAN VERTEBRATES suited in complete atrophy of the ovaries without any apparent effect on ACTH or thyroid-stimulating hormone (TSH) secre- tion as judged from adrenal and thyroid weights and histology (Shirley and Nal- bandov, 1956b) . Thus the ovaries resembled those of hypophysectomized hens. The thy- roids and adrenals were not affected by in- terruption of the portal vessels, whereas their weights were drastically reduced in hypophysectomized birds. Assenmacher (1958) reported that sec- tioning the portal vessels caused testicular atrophy and prevented compensatory hyper- trophy after hemicastration, light-induced increase in testicular size, and cyclic activ- ity such as that found in drakes even when kept in total darkness. His data do not in- dicate that thyroid weight or histology were affected, but the adrenal weights were slightly lower than in the controls. The evi- dence from the experiments with these two species can be interpreted in two ways. One is that destruction of the portal vessels pre- vents the transmission of the NSM to the anterior pituitary. The second interpretation is that sectioning the portal vessels inter- rupts the blood supply to the anterior pitu- itary, and thus causes ischemia. This latter interpretation deserves emphasis in view of Wingstrand's (1951) statement that the pars distalis has no blood supply other than the portal vessels. Indeed, sectioning of the hypophyseal stalk resulted in "a profound increase in fibrotic tissue as well as a de- crease in the number of the usual cell types" (Shirley and Nalbondov, 1956b). Assen- macher (1958) stated that sectioning portal vessels caused atrophy of the central part of the caudal lobe but did not affect the ce- phalic lobe (the cephalic lobe may still have received some blood from the few anterior portal vessels that are indicated in Assen- macher's drawings). Zuckerman (1955) has emphasized the importance of the second in- terpretation in view of the observed infarcts in the pituitary. Benoit and Assenmacher (1959) have presented evidence that the in- farct per se is not the factor causing testicu- lar atrophy, but that sectioning the portal vessels causes a qualitative difference in the vascularity of the anterior pituitary. First, similar infarcts, obtained when the portal vessels regenerated or were incom- pletely cut, did not impede testicular re- sponse to light nor did they cause atrophy of the testes. Second, the lack of infarcts in the cephalic lobe should allow production of enough gonadotrophin to stimulate the testes, inasmuch as in intact drakes the cephalic and caudal lobes have equal gonadotrophic potencies. Third, the infarcts observed after sectioning of the portal ves- sels leave more than 20 per cent of the gland intact. Previous experiments had shown that, even when 80 per cent of the pituitary was removed during attempted hypophysec- tomies, testicular degeneration did not oc- cur, therefore 20 per cent of the gland was sufficient to maintain the testes. 3. Sectioning the hypothalamico-hypo- physeal tract in the median eminence with- out damag(> to the portal system results in genital atrophy and lack of gonadal stim- ulation by light (Assenmacher, 1958; As- senmacher and Benoit, 1958; Benoit and Assenmacher, 1959). Another line of evidence stems from the correlations between activity of the neuro- secretory cells and the experimentally in- duced gonadal activity observed by Oksche, Laws, Kamemoto and Farner (1959). An increase in daily illumination from 8 to 20 hours increased body and testicular weight of white crowned sparrows. Simultaneously, the amount of NSM in the hypothalamic nuclei and the median eminence decreased. During the dark hours of the day, NSM reaccumulates in these areas. This evidence seems rather convincing, but it should be pointed out that a variety of treatments af- fect the activity of the neurosecretory cells and the accumulation of NSM (Legait, 1959). The findings obtained with the white crowned sparrows are suggestive, but they cannot be regarded as proof. Finally, anticholinergic and antiad- renergic drugs can block "spontaneous" (Zarrow and Bastain, 1953; van Tienhoven, Nalbandov and Norton, 1954) as well as progesterone-induced ovulations (Zarrow and Bastian, 1953; van Tienhoven, Nal- bandov and Norton, 1954; van Tienhoven, 1955), although Zuckerman (1955) and Moore (1958) have questioned the inter- pretation that these drugs act specifically by REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1137 blocking adrenergic or cholinergic stimuli. One might also interpret the different effects of different barbitm^ates on "spontaneous" and progesterone-induced ovulation (Fraps and Case, 1953; Fraps, 1955b) as not pro- viding very direct evidence for the neural control of ovulation. Zuckerman (1955) mentioned especially the unpredictability of the effects of drugs on pituitary activity as an argument against the neurohumoral con- trol of the pituitary. In evaluating all the evidence one has to concede that for each line of evidence mar- shaled in support of the hypothesis of neurohumoral pituitary control, another hy- pothesis can be offered to explain the same phenomenon. It also has to be conceded that, so far, no extract has been obtained which counteracts the effects of lesions of the hypothalamic nuclei or the effects of sectioning of the portal vessels; in other words, there is no evidence available demon- strating that replacement therapy is effec- tive in birds. However, no experiments done with birds have disproved the neurohumoral pituitary control hypothesis. As all the evi- dence seems to support the hypothesis and as no evidence is categorically contra- dictory, it seems to be the most acceptable as a working hypothesis with birds. For the sake of convenience, the various stimuli which have been shown experi- mentally to affect avian reproduction will be discussed separately. 2. Light For centuries the Japanese and Dutch have made use of additional illumination to induce out-of-season singing by song birds (Damste, 1947; Hendricks, 1956). The Jap- anese, presumably because they enjoyed the singing, the Dutch because they wanted to use the singing birds as decoys (Damste, 1947) . The first experimental evidence that light stimulated the gonads and induced the urge for migration was obtained by Rowan (1925). Many research papers have since been published on this phenomenon, and ex- tensive documentation can be found in the reviews by Hammond (1954), Yeates (1954), Benoit and Assenmacher (1955, 19591, Fraps (1955b, 1959), and AVolfson < 1959a, l)L Benoit and Assenmacher ( 1955) and Farner (1959) list the species in which reproductive activity has been induced suc- cessfully by additional illumination. Male birds of the temperate zones can generally be brought into a reproductive state by in- creased day length. The light stimulus can be broken down into several components which may effect the response. The effect of intensity was studied in starlings (Bissonnette, 1931), house spar- rows (Bartholomew, 1949), and bobwhite ciuail (Kirkpatrick, 1955). It is apparent from these studies that a trend exists for greater stimulation as intensity increases. However, the numbers of birds used were small as were the observed differences, con- secjuently the differences may have been sampling errors rather than experimentally induced effects. In Farner's equation log Wt = log Wo + kt 1 in which Wt = testes weight at time t, Wo = testes weight at time 0, /c = rate constant, t = time, the rate constant k was higher at an intensity of 3.0 ft.-candles than at 1.0 ft.-candles for white crowned sparrows (Farner, 1959). No further change in the rate constant was observed between 3 ft.- candles and 37.5 ft.-candles. The time of ap- pearance of nuptial plumage in response to light of intensities between 3.67 ft.-candles and 21.6 ft.-candles showed a graded re- sponse to increasing intensities for Euplectes pijromelana (Rollo and Domm, 1943). Egg production of chickens is not affected by light intensity between 0.5 and 38.0 ft.- candles (Nicholas, Callenbach and Mur- phy, 1944) or between 1.0 and 35.0 ft.- candles (Dobie, Carver and Roberts, 1946) . From these limited data it seems that gonadal response can be obtained as long as the threshold of the stimulus is reached and that an intensity-response relationship exists over a limited range only. The re- lationship approaches that of an all-or-none response. Only light with a wavelength between 4000 and 8000 A causes a testicular response in drakes (Benoit and Assenmacher, 1959), starlings (Bissonnette, 1932; Burger, 1943), chickens (Carson, Junila and Bacon, 1958), turkeys (Scott and Payne, 1937), and spar- rows (Ringocn, 1942). In a series of ingen- 1138 SUBMAMMALIAN VERTEBRATES ions experiments Benoit and Ott (1944) investigated the relationship between wave- length and response. They established that red and orange light, which were more ef- fective in intact birds than was blue, pene- trated deeper into the tissues of the brain than the blue. These observations have been confirmed by more refined techniques (Ben- oit and Assenmacher, 1959). Subsequently, a quartz rod was used to shine the light di- rectly on the hypophyseal-hypothalamus area; under these conditions, blue light was more effective than red (Benoit and Ott, 1944) . Benoit and his co-workers have made a careful analysis of the receptors for the light stimulus. In drakes, sectioning the optic nerve reduced, but did not abolish, the gonadal response to increased light (Benoit and Assenmacher, 1955, 1959) . However, when the drake heads were covered with black cloth no stimulation occurred ex- cept when the eyes were left uncovered, in- dicating that two sets of receptors might exist which allow photostimulation to stim- ulate the pituitary. Further investigations showed that light applied directly on the hypothalamus or the rhinencephalon, by means of a quartz rod was effective in gon- adal stimulation (Benoit and Assenmacher, 1959). Therefore, a set of receptors con- nected with the optic nerve and a set of deep receptors may be involved in the stim- ulation of the hypothalamus. In mammals, Knoche (1956, 1957) has demonstrated un- myelinated nerve fibers which originate in the optic chiasma and, coursing through the lamina terminalis, reach the ependyma of the third ventricle as well as the para ven- tricular nucleus and the nucleus tuberis in- fundibularis. These fibers would provide a connection between the retina and hypo- thalamus. Whether or not similar nerve fibers are present in the avian brain is un- known. The concept presented by Rowan (1938a, b), that light causes gonadal stimulation by inducing wakefulness, which, in turn, af- fects the physiology of the entire body, seems erroneous in view of the evidence now available. Bissonnette (1930) was unable to induce gonadal development by forced exercise. As a matter of fact, when starlings were exposed to light, forced exercise had a slight inhibitory effect, which may, however, not have been statistically significant in view of the variability in testes size and the small number of birds used. Benoit (1935) approached the problem by immobilizing drakes and exposing them to light. No dif- ference in response was obtained between free-roaming and immobilized drakes to in- creased photoperiods. The present concept of the manner in which light induces gon- adal stimulation is that it causes an in- crease in the secretory activity of the neuro- secretory cells; the NSM is subsequently transported down the axons and is picked up by the portal vessels in the special zone of the median eminence and transported to the anterior pituitary where it can have its effect. The duration of the jihotoperiod required to obtain a gonadal response has been studied in detail by Marshall (1959) and by Wolf son (1959a, b). The regulation of the gonadal cycles of l)irds in the Northern zone seems to be largely, but not entirely, regulated by the photoperiod. A short summary of the events in the natural breeding cycle will clarify the experimental ai^proach used in studies of regulation of the breeding cycle. In the spring the testes and ovaries mature, and, under good con- ditions, breeding starts. At the end of spring or in early summer, the gonads re- gress. The testes show steatogenesis of the tubules, the tunica albuginea is renewed, and a new generation of Leydig cells is formed. During this period, increases in ])hotoperiod will not cause recrudescence of the testes. Marshall (1959) believed, on the basis of the histologic appearance of the testes, that the lack of response was the result of unresponsiveness of the testes. However, earlier experiments (Riley and Witschi, 1938; Miller, 1949) had demon- strated that the gonads can respond to gon- adotrophin administration. Lofts and Mar- shall (1958) confirmed this response with small doses of gonadotrophins and aban- doned the idea that the refractoriness to light was caused by unresponsiveness of the testes. In any event, the testes will not respond to light for a time interval after regression which depends on the species REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1139 (Marshall, 1959). The light-induced gon- adal response can be obtained again only after the end of this so called "refractory period." The refractory period plays an im- portant role in the regulation of the gonadal, migratory, and fat deposition cycles of migratory birds (Wolfson, 1959a, b). Wolf- son studied the effect of dift'erent photo- l)eriods on the gonadal and fat deposition cycles of Junco hyemalis and Zonotrichia albicollis. In these birds, in the fall and spring, large amounts of fat are deposited in the subcutaneous and interperitoneal depots. These fat dei)ositions are closely associated with the migratoiy drive (Zugunruhe), and account largely for the increase in body weight at those seasons. Kobayashi ( 1954, 1957) proposed on the basis of light-induced molt and gonadal cycles in male and female canaries that the refractory period reflects an increased secre- tion of TSH at the expense of gonado- troi)hin secretion. This hypothesis needs further verification, however, for gonado- trophins have been found in the anterior pituitaries of drakes during the refractory period. Fall migration will not be discussed be- cause too little is known about the regula- tion and physiologic conditions associated with it. AVolfson (1959a, b) investigated the abil- ity of various photoperiods to induce gon- adal and fat de])osition in birds caught in the fall and spring. These birds w^ere ex- posed to light schedules of 9 L(ight) + 15 D(ark) hours; 12 L + 12 D; 15.5 L + 8.5 D ; 20 L + 4 D ; and 24 L and 9 L + 15 D ; 12 L -h 12 D ; 20 L + 4 D and 24 L, respec- tively. The experiments showed: (1) the rate of the gonadal and fat responses is a reflection of photoperiod, the rate being greater with longer photoperiods; (2) the degree of response is greater with longer photoperiods; (3) even under short photo- periods (9 L ) , a response can be obtained ; (4) the time interval between gonadal stim- ulation and regression is smaller for the longer photoperiods. Wolfson (1959a, b) formulated his sum- mation hy})othesis on the basis of these re- sults. The hypothesis is that the sum of the photoperiods and not the changes in day- light to which the birds are subjected de- termines the response. Subsequent experiments were designed to test the importance of the dark period by interrupting the dark periods by short light l)eriods. These experiments tested the hy- l)othesis of Jenner and Engels (1952) and Kirkpatrick and Leopold (1952) that the dark period has a positive effect. Wolfson compared the effect of 8 L + 7.25 D to 1.5 L + 7.25 D with 8 L + 8 D and found no difference between the treatments. Thus, 8 + 1.5 L in 24 hours was as effective as 16 L per 24 hours, whereas previous experi- ments had shown that 9.5 L in one dose per 24 hours was relatively ineffective. This evidence made a positive effect of dark peri- ods seem unlikely. Experiments were then designed to test the effectiveness of different dark periods in breaking up the refractory period. Ex- posure to darkness had been used for centuries by the Dutch to interrupt the refractory period (Rowan, 1938b; Damste, 1947). The experiments by Wolfson demon- strated that 12 hours of uninterrupted dark- ness per 24 hours were required to abolish the refractoriness to light. However, 12 hours darkness alone does not seem to be sufficient to abolish refractoriness, because on 16L + 16D the refractory period is not broken. This suggests that the photoperiod may also have an effect. On the basis of these results, the regula- tion of gonadal and migratory cycles can be tentatively explained for birds of the temperate zone. For the present dis- cussion the gonadal and migratory (fat deposition) cycles will be regarded as one, although there are some quantitative dif- ferences with respect to the rate of response. Nonmigratory species and races do not show the fat deposition cycles shown by migra- tory races of the same species; the discus- sion here, therefore, is concerned only with the gonadal cycles. In late summer and early fall the birds enter what Wolfson calls a preparatory phase (similar to Marshall's (1959) regeneration phase). Birds need ex- posure to at least 12 hours of darkness per 24 hours to enter this physiologic state. After exposure to such dark periods for a certain length of time, depending on the 1140 SUBMAMMALIAN VERTEBRATES species, the birds will go into the progressive phase. During this phase the rate of gonadal response depends on the summation of photoperiods. In the spring the accumulated effects of the photoperiods become effective in stimulating the gonads, and, if ecologic conditions are satisfactory, breeding can start. For birds migrating to the equatorial zone or remaining in the temperate zone, the same current of events occurs except that a cycle of fat deposition is added to the gonadal cycle. According to Wolfson's hypothesis, the rate of response is determined by the summation of photoperiods so that the response can occur near the equator even though no change in photoperiod occurs. For transequatorial migrants, the exposure to daylight would increase in October and November and decrease after December. Again summation of photoperiod would be the deciding factor, provided that the ex- posure to enough short days has occurred to break up the refractory period. Marshall (1959) has criticized AVolf son's hypothesis mainly on the basis of field studies showing that adult rooks, mallards, and starlings show sexual displays in the fall at the same time that in starlings the bills become yellow and 13.6 per cent of the rooks show spermatogenesis. This, accord- ing to Marshall, argues against a need for an exposure to short days to obtain a response, because the response is obtained before the shortest day. As Farner (1959) stated, these phenomena can be explained by assuming that the refractory period ends relatively early in the late summer, when the photo- period is still long enough to cause stimula- tion. Not all individuals show these cycles because certain other ecologic factors in- terfere with the response. Evidence that a refractory period may be short or even ab- sent is found in the experiments of Kirk- patrick ( 1959) with bobwhite quail. Marshall and Disney (1956) subjected the "summation of photoperiod" hypothesis to an experimental test with the tropical nonmigratory Quelea quelea and observed no response when the daily photoperiod was increased 5 minutes over the natural photo- period, although the summation of the pho- toperiods was an amount which the birds under natural conditions would have ex- perienced only after a period of 27 years. The test proves that the summation of photoperiods does not hold true for this species, but it does not eliminate the possi- bility that it may hold true for temperate zone birds of the Northern hemisphere. In any controversy of this kind it would seem desirable that experiments be undertaken with the same species. Any comparison be- tween Quelea quelea and Zonotrichia al- bicollis should take into account the dif- ferent ecologic factors which may play a role in the determination of gonadal and migratory cycles. For some species living in the temperate zone of the Northern hemi- sphere, light may be the most important single stimulating factor; for another spe- cies, for instance Melopsittaciis undulatus in a different but temperate zone environ- ment, light may not be a factor (Vaugien, 1951,1953). Experiments in which short })hoto])eriods of one to several minutes interrupt long dark periods should be mentioned here. Farner (1959), in a well conducted series of experiments, determined the rate constant of Equation 1 for various lengths of photo- periods with light given in different doses. He established that 6 hours of light in equally spaced 50-minute doses resulted in k value similar to that for 12 hours of light given in one dose. The effectiveness of the short photoperiod was dependent on the intervening dark periods. Farner proposed the following hypothesis to explain this effect: a substance generated during the photoperiod decays gradually during the dark period, but remains able to stimulate the hypophysis for a certain length of time. It has been estimated that it takes about 1 minute to generate the substance in equi- librium amounts, whereas it has been esti- mated that the decay of the substance once generated, takes at least a few hours (Far- ner, 1959). This hypothesis does not assign any i^ositive function to the dark period as suggested by Jenner and Engels (1952) and Kirkpatrick and Leopold (1952). Although light plays a powerful role in inducing spermatogenesis in drakes, cycles of testicular activity can occur in the ab- sence of light. Benoit, Assenmacher and REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1141 Brard (1956) kept young Pekin drakes in total darkness and found that testicular size showed definite cycles which were un- related to temperature or changes in light outside the pens ; however, the cycles of the individual birds parallelled each other. Un- fortunately, no data are available on sper- matogenesis under these conditions. Recently, Benoit, Assenmacher and Brard (1959) reported that drakes kept under continuous light after the age of 3 weeks showed maximal testicular size later than birds kept under natural daylight. After maximal size was obtained the testes showed regular cycles that were apparently unre- lated to outside conditions and again the cycles of the individual males paralleled each other. Vaugien (1951) showed that ovulation and egg laying occur in budgerigars, Melop- sittacus undulatus, kept in darkness, and Vaugien (1953) contended that males reached full spermatogenesis sooner in dark- ness than when kept under light. Marshall and Serventy (1958) criticized this interpre- tation and stated that spermatogenesis had occurred faster under light. They believed that Vaugien (1953) had misinterpreted the histologic data, which, according to Marshall and Serventy, showed that post nuptial degeneration of the tubules had oc- curred. Marshall and Disney (1957) con- firmed, however, that spermatogenesis would occur in total darkness in Zebra finches, Peophila castanotus. These experiments show that gametogenesis does not require light although light may regulate the cycle. An example of the "breaking through" of the inherent rhythm in spite of i)hotoperiods may be found in Australian silver gulls, Larus novne-hollandiae, kept in the Wash- ington Zoo. For two seasons the gulls nested in November, then adapted to the northern spring and summer but later reverted back to nesting during the Australian spring (Davis, 1945). In short-tailed shearwaters, Puffimis tenuirostris, the internal rhythm seems to regulate the onset of the breeding season independentlv of the photoperiod (Marshall and Serveiity, 1959). The differ- ent examples show that in some species, e.g., ducks, light is the main regulatory fac- tor in the initiation of gametogenesis, al- though cycles can occur in the absence of light. In other species, e.g., short-tailed shearwaters, the inherent rhythm seems to regulate the onset of gametogenesis, and in species in the tropics, e.g., Quelea quelea, light can affect the initiation of gametogene- sis (Marshall and Disney, 1956) but in their natural habitat, rainfall and the availability of long green grass initiate gametogenesis and determine breeding success (Marshall and Serventy, 1957). 3. Temperature Considerable observational data from field studies (Marshall, 1959) indicate that tem- perature may be an important factor in the regulation of the breeding season. No ex- perimental data seem to be available to demonstrate clearly that temperature is the factor per se and is not affecting the breed- ing season by making the required food available, but investigations have been made to determine the effect of temperature on the light-induced gonadal response. Burger (1949) mentions that the testes of starlings kept at 98 to 100°F. w^ere larger than those kept at 60 to 70°F., but that the number of eggs laid by house wrens was lower when the birds were kept at 77°F. than at 67°F. Farner and Wilson (1957) determined the effect of temperature on the rate constant k in Equation 1 and found that K,/K„ = 1 + C(T, - Tj 2 in which C = constant, Ka = I'ate constant at temperature A, Kjj — rate constant at temperature B, Ta =' temperature A, T^ = temperature B. The results showed that C = 0.009 for white crowned sparrows; C = 0.02 for j un- cos (data of Jenner and Engels, 1956) ; C = 0.02 for starlings (Burger's data). The con- clusion is that temperature affects the light- induced gonadal response but the effect is rather slight. Kosin and his co-workers carried out ex- tensive investigations on the effect of tem- perature on the reproduction of turkeys which, although domesticated, have sea- sonal breeding cycles. Their work showed that pretreating the toms with a tempera- ture of 65°F. during the period January to March, when outside temperatures may be 1142 SUBMAMMALIAX VERTEBRATES as low as — 20°F., resulted in earlier produc- tion of sperm with high fertilizing capacity (Burrows and Kosin, 1953; Kosin, Mitchell and St. Pierre, 1955a I. On the other hand, "cooling" the toms to 65°r. in the period May to July, when outside temperatures may range between 60° and 100°F., pre- vented the drop in sjiermatogenesis and fer- tility experienced by toms kept outside (Kosin, Mitchell and St. Pierre, 1955b; Law and Kosin, 1958). Kosin (1958) established that the respiration rate of turkey semen, especially during the second hour of incuba- tion, was affected by the environment in which the donors were kept. The respiration rate was higher for semen from toms kept at 65°F., especially during the summer, sug- gesting that high temperatures may be more harmful than low temperatures. In view of the fact that body temperatures rose in the toms kept outside during the summer (Ko- sin, Mitchell and St. Pierre, 1955b), it seems that the higher body temperature may have affected spermatogenesis, as discussed in the beginning of the present chapter. Constant temperature 50° ± 5°F. depressed over-all egg production by turkey hens (Mitchell and Kosin, 1954). Preheated (50° ± 5°F.) hens laid initially at a somewhat higher rate than the hens kept outside, where temi)era- tures were as low as 10°F., but the birds kept in the constant environment had a greater tendency to become broody with an accom- panying decrease in the rate of lay (INIitchell and Kosin, 1954; Kosin, Mitchell and St. Pierre, 1955a). Eggs from turkey hens in the constant environment were significantly smaller (84.5 versus 96.7 gm.) than those from hens kept outside. 4. Rainfall Marshall (1959) reviewed the evidence that rainfall may be involved in regulation of the breeding cycles. Serventy and Mar- shall (1957) observed that unseasonal pre- cipitation in Australia was followed by the appearance of spermatogenesis in a large number of terrestrial as well as aquatic spe- cies. Marshall and Disney (1957) analyzed experimentally in what manner the rain or increased humidity had its effect. Rainfall induced adult nonbreeding Quelea quelea to molt from one breeding plumage dress to the other without the normally interven- ing neutral dress. The urge to build nests was also stimulated by rainfall or humidity, but for the construction to be successful long green grass was reciuired. This grass normally becomes available after the rain. After the nests are built, breeding can pro- ceed; at the same time the seed heads, the staple food of the nestlings, normally ap- pear in the grass. Whether the relationship between the initiation of ovulations and ap- pearance of seed heads is coincidental was not determined. These results suggest that rainfall affects the breeding cycle directly and also indirectly by making suitable nest- ing material (and food?) available so that reproduction can occur. 5. Food The a\-ailability of food may affect breed- ing success, but no evidence is available that demonstrates that a specific food supply reg- ulates the ))r('C(ling cvcle of birds (Marshall, 1959). 6'. \'ocalizatio»s Vaugien (1951) found that female bud- gerigars, Melop.slftacits inuhilatus, would lay in complete darkness, provided they could hear the vocalizations of courting pairs of budgerigars in the aviary. Ficken, van Tienhoven, Ficken and Sibley (1960) investigated the effects of vocalizations by the birds' own mates on gametogenesis. The gonads of pairs isolated from hearing other pairs remained inactive, w^hereas pairs which could hear others showed full spermatogene- sis and ovulations, whether or not they could see the other pairs. No quantitative investi- gations were made to determine how many pairs were reciuired to start tlie chain of events. 7. Xesting Site Marshall (1952) reported that arctic birds of a variety of species seem to be adversely affected by lack of nesting sites. A rather detailed investigation of nonbreeding, or rather decreased laying, has been made by Barry (1960) for the brant, Branta berniela hrota, and blue and snow geese, CheJi caeru- lescens. The data from the two species agree closely enough to be treated here as one REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1143 group. Barry (1960) found a correlation be- tween snow cover and the date of first egg, whereas the correlation between date of first egg and clutch size was 0.79 to 0.85. From his data it appears that the ovaries are in the beginning stages of development when the birds arrive at their breeding grounds in the arctic and that full ovarian development oc- curs during the prenesting interval. Ovula- tion seems to be dependent on the availabil- ity of nesting sites, although lack of food was not completely excluded as a factor. A sharp decrease in average clutch size takes place when the interval between arrival on the breeding grounds and laying of the first egg exceeds 7 to 10 days. Barry (1960» counted the number of atretic and ruptured follicles and compared these with the average clutch sizes. In this manner he could show that each egg not laid because of unfavorable conditions was rep- resented by an atretic follicle. For the males of these species the situa- tion seems somewhat different. Testicular weights decrease and lipoidal infiltration of the tubules starts, apparently immediately after the males arrive at the breeding grounds. Corroborating evidence for the hy- pothesis that spermatogenesis may already be past its peak on arrival at the breeding grounds is found in the fact that neither Barry (1960) nor the Eskimos in this area have ever seen brant or snow geese copu- late. Also, sperm were found in the oviduct of a brant killed on arrival at the breeding grounds. It was not possible to establish whether the rate of testicular collapse would proceed at different rates in the presence or absence of nesting sites and plentiful food. Marshall and Roberts (1959) studied the fish-eating cormorants, Phalacrocora.v carba and P. afncanus, which breed in the northern Lake Victoria region. These species apjiarently can breed the year around, but within the species different segments of the jioi^ulation are in different phases of the reproductive cycle, so that no pair breeds twice without a pause long enough for an- other pair to breed at the nest they have just abandoned. It seems from the observa- tions that the availability of nests and nest- ing sites determines the breeding behavior for each segment of the population. Vaugien (1948), with very few birds, showed that canaries would not lay when the female was deprived of cotton to line the nest bowl. The condition of the ovary was not mentioned. However, when the nest bowl was warmed by an electric coil, ovipo- sitions occurred in the absence of the cotton lining. Unfortunately, no information con- cerning the ovaries was published and so few birds were used that any inter])retation is very tentative. 8. Psychic Factors A variety of psychic factors have been shown to affect the breeding of birds. Craig ( 1913) noticed that a dove which had failed to lay started to do so 9 days after Craig started to stroke her daily. Matthews (1939) later established that an isolated female pigeon could be stimulated to lay a normal clutch by the sight of another pigeon or even the sight of herself in the mirror. Vaugien (1948) noted that the canary needs a part- ner, male or female, in order to lay. House sparrows, in which the male and female are dimorphic, are apparently more discriminat- ing than pigeons, in which male and female look alike. Female sparrows will not show any oviduct response (as a reflection of es- trogen secretion) if caged with other fe- males, but will show enlarged oviducts when caged with males (Folikarpova, 1940, cited l)y Lehrman, 1959). This response may be partly the result of the nest building which is largely done by the male. It may thus have been the nest built by the male rather than the male per se to which the female re- sponded. Burger (1953) observed that cag- ing female starlings with males increased the response of testes to light. Captivity prevents ovarian development in pintail ducks. Anas acuta, captured from migrating flocks while spermatogenesis in the males is unimpaired (Phillips, 1959) . The inhibition is mediated by way of the pitui- tary, which, in the captive birds, contains no detectable amounts of gonadotrophins. Vaugien (1954a) found that wing clipping of house sparrows, so that they could not perch, prevented their testes from being stimulated by light, as measured by sper- matogenesis and l)ill color. The birds did respond to PMS injections, suggesting that 1144 SUBMAMMALIAN VERTEBRATES the inability to perch prevented the secre- tion of gonadotrophins. Ficken, van Tien- hoven, Ficken and Sibley (1960) found that mirrors in cages of pairs of budgerigars de- layed ovulations but did not affect spermat- ogenesis. In this respect the budgerigars may differ from pigeons in which a mirror was stimulatory when one female was in a cage. We were not able to find evidence about the effect of mirrors in cages with pairs of pi- geons. The observations and experiments re- viewed indicate that light may be a very important factor in determining the onset of gametogenesis, of migration, and of fat de- position in certain migratory birds. How- ever, other factors (e.g., rain) may modify the onset of breeding, and in some species these factors rather than light seem to be most important. Such species can then breed at more irregular intervals when conditions are favorable, e.g., after rainfall. Some trop- ical species such as the sooty terns, Sterna fuscata, breed every 9.6 months (Chapin, 1954) , apparently stimulated, not by outside factors, but by an inherent rhythm (Mar- shall, 1959). The multiplicity of factors de- termining breeding cycles of avian species makes it unlikely that any hypothesis will be useful when it takes into account only one of these factors such as light. The discussion of the initiation of the breeding cycle should be followed by a dis- cussion of the factors which terminate the cycle, but relatively little is known. In those species in which the male helps in incubation or in building the nest or stays with the fe- male while she is incubating, the release of prolactin in response to visual and emotional stimuli may cause the regression of the tes- tes. Some evidence for this is found in the experiments by Patel (1936) in which the sight of an incubating mate caused crop gland development in the male. Experi- mentally, crop gland development can be induced by prolactin, and prolactin is known to cause regression of the gonads. In polyg- amous species the regression of the testes does not seem to occur until rather late in the summer when decreasing daylight may be a causative factor. For the females a distinction should be made between deterininate and indetermi- nate layers. Tlie former lay a definite num- ber of eggs per clutch whether or not eggs are removed. Examples are brant, snow geese (Barry, 1960), budgerigars, Agapornis roseicollis, A. taranta, A. fischeri. From the rather scanty data available, it seems that two mechanisms are involved in making these birds determinate layers. In brant and snow geese only a limited number of follicles (5 to 6) reach ovulatory size, and these are ovulated when conditions are appropriate (Barry, I960). Ovulation, or atresia of the largest follicle, permits the next follicle to reach maturity so that it can be ovulated if conditions are right. In Agapornis and in budgerigars more follicles mature than are necessary for the clutch, but after the nor- mal number of eggs (5 to 6) are laid, a gen- eral atresia of the other follicles occurs. This information on Agapornis and budgerigars was kindly given to me by Dr. W. C. Dilger. The factors causing this atresia are un- known. Indeterminate egg laying has been ob- served in many species (Lehrman 1959, and his chapter in this book). In brief, the evi- dence suggests that the female starts to in- cubate after a certain number of eggs are present in the nest, whether laid by herself or placed in the nest by others. After this in- cubation starts (and prolactin is released?) degeneration of the follicles takes place and no further ovulations occur. Lehrman (1959) cites Poulsen's work in which laying could be repressed in pigeons if 2 eggs were placed in the empty nest. As pigeons are determi- nate layers, he could not obtain more than 2 eggs by removal of 1 or 2 eggs as they were laid. This whole field of investigations on the physiologic mechanisms involved in deterininate and indeterminate layers is vir- tually unexplored, in spite of many field ob- servations which suggest what mechanisms may be involved. The following may serve to summarize the present concepts of the regulation of the breeding cycle of seasonally reproduc- ing birds. Various stimuli or combinations of stim- uli, such as light, rainfall, availability of food and nesting material, and vocalization by other birds in the flock, may initiate gametogenesis. In many species the males REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1145 reach the peak of gonadal activity before the females (Benoit, 1956) ; an exception is the turkey in which the female responds to increased photoperiods before the male does (jMargolf, Harper and Callenbach, 1947). The sexual behavior of the male, his sing- ing, and the initiation of nest building, all of which may be dependent on temperature, availability of nest sites, etc., stimulate the females (feenoit, 1956; Lehrman, 1959). Under the influence of this stimulation the female will copulate, and ovulate. She may now lay a more or less predetermined num- ber of eggs (determinate layer) or she may lay eggs until the presence of a sufficient number stimulates incubation behavior and the degeneration of follicles still present in the ovary. Destruction of the nest may restart a new cycle; or, in some species, a new cycle may be started after the first hatch has fled the nest. At the end of the summer, in tem- perate zone birds, the gonads regress, and, in migratory species, fat is deposited in intraperitoneal and subcutaneous depots. ]\ligration southward starts under the in- fluence of yet unknown factors. The re- fractory period, which varies in length for different species, is terminated under the influence of short days. In the spring gonadal activity is reinitiated, fat deposition takes place, and migration north starts again, either because of an "inherent rhythm," or under the influence of photostimulation. For many species w^e know nothing about the regulatory factors involved in their migra- tions; therefore, the above generalizations should be taken as tentative even for the best studied species such as those of the genera Junco and Zonotrichia. D. REGULATIOX OF THE REPRODUCTIVE CYCLE OF THE FOWL For convenience of discussion the repro- ductive cycle of the fowl will be divided into an annual cycle and the laying cycle which encompasses only part of the annual cycle. The annual cycle of females resembles the cycle of wdld birds, especially in breeds and strains in which broodiness still occurs. The cycle of the male is virtually absent and males produce sperm all year. Seasonal variations in fertilitv mav occur, but these fluctuations may be a reflection of high tem- perature, and they are probably not a re- flection of changes in photoperiodicity. Roosters have been kept for years without molting, although hens normally molt. The hens which show broodiness may become broody several times when not allowed to incubate, and they produce eggs between the broody periods. Legait (1959) studied the annual cycle of Rhode Island Red hens which were allow^ed to incubate the eggs. Her study encompasses all phases of the annual cycle with special attention to the incubation period. Unfortunately, the re- sults seem to be based on 1 or 2 birds for each phase of the cycle, but, if allowance is made for the small numbers, several con- clusions may be deduced from the data. 1. The diameters of the nuclei of the para- ventricular cells are about 6.24 fx during the annual rest and during the molt; they are somewhat larger, 6.4 to 6.6 /x, during laying, and considerably larger during incubation, 7 to 12 /x. 2. During molting many granvdes of NSM are present in the paraventricular cells, and the posterior lobe also contains NSM in abundance. During incubation, the amount of NSM in the paraventricular cells and posterior pituitary lobe is small. 3. During laying the percentage of Ao cells in the cephalic lobe of the anterior pituitary is at its maximum and /3-cells are at a minimum. The /?-cells increase during incubation. 4. In the caudal lobe of the anterior pi- tuitary, S-cells increase during incubation and decrease sharply during the molt. 5. Adrenal weight is low during the molt and annual rest, but is high during incuba- tion. Legait (1959) show'ed that the diame- ter of the nuclei of the neurosecretory cells and adrenal weights parallel each other. 6. During incubation and molt, the ovar- ian weight is ciuite small (2 to 3 gm.). The annual cycle seems to be regulated largely by light. Recent investigations all indicate, directly or indirectly, that the daily increment in the photoperiod may be more important than the length of photoperiod (Sykes, 1956; Morris and Fox, 1958). The experiments carried out by Morris and Fox (1958) showed particularly clearly the im- 1146 SUBMAMMALIAN VERTEBRATES l^ortance of daily increments in photoperiod. The correlation between the summation of daily light changes and the age of sexual maturity was 0.96, an unusually high cor- relation in biologic experimentation. A question which has been raised in con- nection with the practice of providing arti- ficial light for growing chickens is that of refractoriness. Tomhave (1954) and Shutze, Jensen and IMatson ( 1959 ) found that arti- ficial light provided during the growing pe- riod causes lowered egg production even when the artificial light is supplied only during the period from hatching to 8 weeks of age. Hutt, Goodwin and Urban (1956 » suggested that the failure of ovaries to de- velop in some birds may be the result of artificially long photoperiods during the growing period. Experiments reported for the domestic Pekin drake indicate also that raising young drakes under continuous light delays the onset of maximal testicular size in the first breeding season (Benoit, Assen- macher and Brard, 1959). Further experi- ments are needed to determine whether the observed effect is essentially the same as the refractoriness in seasonally reproducing birds and whether it can be interrui)ted by exposure to short days. After egg laying has started, 13 to 14 hours of light per 24 hours seem to be opti- mum, provided the photoperiod is given in one dose (Dobie, Carver and Roberts, 1946). AVith interrupted photoperiods the results are similar to those described for seasonally reproducing birds (Fraps, 1959). When hens are exposed to continuous light, egg produc- tion is higher initially than in birds ex- posed to 13- to 14-hour photoperiods, but egg production decreases sooner (Penquite and Thompson, 1933; Greenwood, 1958). This decrease may be the result of refrac- toriness of the hypothalamus or of the pi- tuitary to light (Byerly and Moore, 1941). Greenwood (1958) observed that chickens kept in a constant environment (continuous light?) showed no molting, but there was a steadily decreasing rate of egg production and of hatchability of fertile eggs. After the birds were 3.5 years old, 94 per cent died, all of adenocarcinomas. Although carried out with a limited number of birds, the ex- periment suggests that molting observed in l)irds undei- continuous light is precipitated by factors other than light. The high mor- tality may have been the result of the con- tinuous light, although in Greenwood's ex- periment other environmental factors cannot be separated from the light effect. However, data obtained by Benoit, Assenmacher and Brard (1959) with ducks suggest that light may be the most important factor. These in- vestigators found poorer survival by Pekin drakes raised and kept under continuous light than by ducks kept either under day- light conditions or in total darkness. Greenwood's data (1958) also suggest that a constant environment is not very de- sirable for optimal reproduction, an observa- tion confirmed in the experiments with tur- keys, mentioned before. Byerly and Moore (1941) demonstrated that an unnatural photoperiod of 14 L + 12 D resulted in con- siderably increased egg production and clutch (number of eggs laid on consecutive days) length. They offered the following ex- l)lanation for this phenomenon. 1. The long dark period prevents the on- set of refractoriness to light stimulation. 2. Tli(> limiting effect of the onset of dark- ness is partially removed. As will l)e dis- cussed later, the onset of darkness may de- termine the length of the clutch (Warren and Scott, 1936). No further experimental work to test the hypothesis of Byerly and ]\Ioore (1941) seems to have been reported. The effects of intensivity, wavelength, and interrupted photoperiods have been dis- cussed previously for seasonally reproducing birds. The experimental evidence indicates no fundamental differences between such birds and chickens; thus no further dis- cussion seems to be required. Photoperiod is the main regulator of gonadal activity, but other factors such as nutrition and tem- perature may modify the response to light; some of these factors are discussed later when consideration is given to the several hypotheses offered to explain the timing of the ovulation cycle. An understanding of the regulation of the laying cycle requires a short introduc- tion to the events which occur in the forma- tion of the egg after ovulation. Excellent reviews on this subject are available for more details (Romanoff and Romanoff, REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1147 1949; Fraps, 1955b). After ovulation, the yolk (secondary oocyte) either falls into the ovarian pocket and is subsequently en- gulfed by the funnel of the oviduct, or it may immediately fall into the funnel when the latter is partly covering the mature fol- licle. The yolk is moved down the oviduct by peristaltic movements, and, during this passage, albumen is deposited around it. The albumen has been accumulated in the glands of the oviduct during the period between successive yolks coming down the oviduct (Conrad and Scott, 1942). On pas- sage of the yolk, the albumen is hydrated and transferred to the lumen of the oviduct (Smith, Hoover, Nordstrom and Winget, 1957). Part of the albumen accumulation in the magnum of the oviduct seems to occur continuously whereas another part is se- creted in larger quantities when ovulation is imminent (Smith, Court and Martin, 1959) . The egg is transferred from magnum to isthmus where the membranes are formed around the albumen. After this stage is com- pleted, the egg is moved into the shell gland where the thin albumen is added through the egg membranes to give the egg its plumped appearance. This process takes 3 to 5 hours, and during this process shell deposition also starts (Bradfield, 1951). Of the egg-shell calcium, 60 to 75 per cent comes directly from the food (Driggers and Comar, 1949). These workers found that an egg laid within 10 minutes after an oral dose of Ca^^ con- tained radioactive calcium, evidence for the very rapid transfer as w'ell as for the con- tinuous deposition of calcium while the egg is in the oviduct. In some breeds of chickens porphyrin is deposited on the shell to give it a brown color. According to recent in vitro studies, the porphyrin is synthesized from 8-amino-levulinic acid by the shell gland tissue (Polin, 1957). In vitro no dif- ference was found between the amount of porphyrin synthesized by tissue from breeds laying white-shelled eggs and breeds laying brown-shelled eggs. Polin (1957) suggested that white shells are white because of a lack of 8-amino-levulinic acid in the tissues, and not because of a lack of enzyme systems for the synthesis of porphyrin from S-amino- levulinic acid. During the sojourn in the shell gland, the egg is rotated on its longi- tudinal axis (Conrad and Phillips, 1938), which causes some of the mucin material in the albumen to form strands. The formation of these twisted strands squeezes out some of the water to form the inner thin albumen (Conrad and Phillips, 1938). Shell forma- tion continues until complete oviposition occurs. The physiologic mechanisms which ini- tiate oviposition are not well understood. A variety of treatments, including injection of posterior pituitary hormone, vasopressin, and oxytocin can cause premature oviposi- tion. Soft-shelled eggs can be obtained in this manner (Burrows and Byerly, 1942; Burrows and Fraps, 1942). It is known that the posterior pituitary glands of chickens contain vasopressin and oxytocin (de Lawder, Tarr and Gelling, 1934; Heller, 1950) ; however, posterior pituitary removal does not interfere with either ovulation or oviposition, although it does result in dia- betes insipidus, thus indicating that the an- tidiuretic hormone is absent (Shirley and Nalbandov, 1956a). An analogous situation is observed in rats from which the posterior pituitary is removed. Such rats are unable to nurse their young because the milk ejec- tion cannot occur in the absence of the pos- terior pituitary hormones. After a subse- quent pregnancy these rats will deliver the young normally indicating normal release of oxytocin. The dams can nurse their young normally in spite of diabetes insipidus (Ben- son and Cowie, 1956). These data indicate that oxytocin can be released from the re- generated stalk, where the NSM from the hypothalamic nuclei has accumulated. This release occurs in response to a reflex stimu- lus, but for some reason, the antidiuretic hormone is not released. It is entirely pos- sible, therefore, that the posterior pituitary hormones, or rather the NSM from the hypo- thalamus, are involved in oviposition, but this is a matter of speculation. In addition to posterior pituitary hor- mones, acetylcholine injections cause pre- mature expulsion of the egg, and ephedrine delays oviposition (Weiss and Sturkie, 1952). As mentioned, the ruptured follicle and the largest maturing follicle may secrete a hormone involved in expulsion of the egg, 1148 SUBMAMMALIAN VERTEBRATES but no evidence is available as to the nature of this hormone. Fraps (1942) showed that premature induction of ovulation with LH also resulted in premature expulsion of the oviducal egg. This means that either LH per se caused the expulsion or that the ovula- tion liberated a hormone which caused ex- pulsion of the egg. "Spontaneous" ovulation is preceded by increased activity of the fun- nel of the oviduct, as was shown in a film by Warren and Scott. It is possible that such activity also causes contractions in the shell gland that would result in ovi- position. That this is not the only mecha- nism involved in oviposition is evident from the terminal oviposition in a clutch which is not accompanied by ovulation. Be- fore oviposition takes place, the egg is turned 180° on its short axis so that it is laid blunt end first (Bradfield, 1951). The find- ings by Olsen and Byerly (1932) that about 80 per cent of the eggs are laid small end first, were criticized by Bradfield ( 1951 ) . He states that normally the bird gets uji, turns the egg in the shell gland, settles again, and lays the egg about one hour later. Ac- cording to Bradfield (1951), Olsen and By- erly (1932) disturbed the hens when they picked them up to determine the orientation of the egg at oviposition and thus prevented the egg from being turned. Bradfield used radiographic examination which did not dis- turb the hens. Fraps (1955b) has presented a detailed study of the time relationships between ovu- lations, between ovipositions, and between ovulations and ovipositions. His termi- nology will be used here. This discussion will be largely devoted to the relationship between events in the egg laying cycle of chickens kept under 14 hours of light, from 6 a.m. to 8 p.m. The oviposition cycle is the number of consecutive days on which ovi- position occurs plus the number of days on which oviposition fails to occur before its resumption. In the following equation some of the re- lationships are presented : / = n/in + z) 3 in which / = oviposition frequency within a cycle, n = number of days on which ovi- position occurs (singly or consecutively), z = number of days intervening before ovi- position is resumed. The discussion will be mainly concerned with those cycles in which z = 1, the so-called closed cycles. Using Fraps' terminology, the consecutive ovi- positions of a cycle may be called Ci , ... €„ . The difference in time of day when successive ovipositions occur has been called "lag"; total lag is the difference in time of day between first and last oviposition of a clutch ; mean lag is total lag divided by {n — 1). Fraps calcu- lated the mean lag from a large number of observations in which birds were exposed to artificial light from 6 a.m. to 8 p.m. From these calculations the following became ap- parent: (1) the lag of the terminal oviposi- tion is greater than at preceding places in a clutch; (2) this lag decreases as n increases; (3) lag for positions between the initial and terminal ovipositions decreases as n in- creases and this lag may approach zero for large n. These facts for the oviposition clutch have a bearing on the ovulatory cycle. Warren and Scott (1935) established that ovulation occurs within 14 to 75 minutes after ovi- position of an egg, except in case of the last oviposition of a clutch. Ovulation, however, is not caused by oviposition. When the egg in the oviduct is broken so that it is ex- pelled prematurely ovulation does not oc- cur prematurely (Warren and Scott, 1935). Premature expulsion of an egg caused by in- jection of posterior pituitary extracts is not followed by premature ovulation. On the other hand, oviposition can be induced pre- maturely by inducing premature ovulation with LH (Fraps, 1942). Fraps (1955b) calculated the regression of interval between oviposition and ovula- tion on mean lag between successive eggs. The regression showed that for each in- crease in mean lag of 1 hour the mean inter- val between oviposition and ovulation in- creased about 10 minutes. By considering these characteristics (Fraps, 1955b for de- tails) for ovulation and oviposition clutches, Fraps observed that the lag between the first and second ovulations of a clutch was of the same order of magnitude as the lag between penultimate and ultimate oviposi- tions in the clutch. A consideration of lags REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1149 in the ovulation sequence compared to lag in the oviposition secjuence showed that the lag for the second place in the ovulation sequence remained relatively large, whereas in the oviposition sequence it became rapidly smaller as the number of eggs per clutch increased. Thus, the time that the second egg remains in the oviduct decreases more rapidly as clutch length increases. Another important characteristic of the ovulation cycle is the interval between terminal ovula- tion of one clutch, C,, , and ovulation of the first follicle, Ci', of the next clutch, an in- terval w^hich is of the order of 44 hours when }i = 2 and 40 hours when n = 6. The difference between this value and the mean interval between ovulations in a clutch gives an approximation of the "period of lapse." This period of lapse approximates the ad- ditional time elapsing between C„ and Ci follicles over what might be expected, had ovulation occurred on the day of the missed ovulation. In order to explain these conclusions of Fraps, consideration will now be given to the effect of environmental factors on the ovulation sequence. A fundamental study by Warren and Scott (1936) demonstrated: (1) under normal daylight, the onset of darkness seems to determine the termination of the clutch and more than 90 per cent of the eggs are laid during the day; (2) when lighting is continuous and entirely artificial, time of laying is equally distributed over the 24 hours; (3) when light and dark periods were reversed, the hens would lay in the dark for a few days, demonstrating that ovi- position can occur in the dark; the hens would shift gradually to laying in the light period; (4) hens laid 90 per cent of the eggs during the day when daylight was supple- mented with continuous artificial light; this was attributed to a psychologic factor. McNally (1947) found that the period of feeding determined the time of oviposition for chickens kept under continuous light, and Fraps, Neher and Rothchild (1947) investi- gated both the effect of feeding and time of light with respect to time of lay. Birds were first exposed to 14 hours light; they laid dur- ing the light period. After the ovijiosition pattern was established, lights were turned on continuously, and eggs were still laid dur- ing the daytime. By changing the period at which the birds were fed and tended, tlie investigators were able to shift the time of lay to the period in which feed was availa- ble. An incidental observation was that the time of minimal body temperature coincided wiih the time of release of gonadotrophin to cause ovulation. We have tried to estab- lish experimentally whether lowering of body temperature would result in prema- ture ovulations. Hens were placed in i-e- frigerators (with good ventilation), with and without lights. This was done during the period of the cycle (day of lay of the last egg of the clutch) when progesterone injections cause premature ovulations. Body temperature dropped 1° to 3°F., but in no case were we able to induce premature ovu- lation. This suggests that the observation made by Fraps, Neher and Rothchild ( 1947) may be the result of coincidence. Bastian and Zarrow (1955) recorded ac- tivity of hens during light and dark periods and found that practically all activity oc- curred during the light hours. As release of the gonadotrophin for ovulation occurs dur- ing the dark period of a 14 L + 10 D day, the investigators determined the effect of light and enforced activity on ovulation of the Ci follicle. Enforced activity on the evening (9:00 p.m.) of oviposition of the terminal egg of a clutch delayed ovula- tion 26 minutes. A combination of light and enforced activity caused a delay of 2.16 to 4.5 hours. Light plus Nembutal anesthesia (to prevent increased activity) did not af- fect the time of ovulation. They concluded that the daily fluctuations in light and ac- tivity of the hen prevented the release of the hormone for ovulation on the day of lay of the last egg of the clutch. A brief discussion of the hormonal re- lationships in the induction of ovulation is required before the hypotheses offered to explain the laying cycle of chickens can be considered. For the present it will be assumed that LH and the ovulation-inducing hormone are the same, a point not proven but made very acceptable by the observation that as little as 1 /xg. LH from chicken pituitaries can induce ovulation in 50 per cent of expei'i- mental bird^ (Fra'^s, Fevold and Neher. 1150 SUBMAMMALIAN VERTEBRATES 1947). Ovulation occurs 6 to 7 hours after intravenous LH injection (Fraps, Riley and Olsen, 1942). After intravenous progesterone injection which induces gonadotrophin re- lease from the pituitary, ovulation occurs in 7 to 8 hours (Fraps, 1955b). The ques- tion of how long the pituitary would have to remain active in order to secrete enough gonadotrophin for ovulation was investi- gated by Rothchild and Fraps ( 1949a j. Hypophysectomy was performed at various intervals before "spontaneous" ovulation. The results showed that ovulation would still occur in 60 per cent of the cases when the interval was 5 hours, but only 20 per cent would ovulate when the interval was 7.2 hours. Progesterone-induced ovulation provided, of course, a better estimate how long it takes to secrete sufficient gonado- trophin because the time of stimulation of the pituitary can be timed better. As the intervals between LH or progesterone in- jection and ovulation agreed rather closely, it was assumed that the stimulation for gonadotrophin secretion after progesterone was immediate. Rothchild and Fraps (1949b I established that the pituitary has to remain in situ 2 to 4 hours after the in- jection of progesterone in order to obtain ovulation. The question as to the duration of stimulation of the pituitary was investi- gated by "blocking" the stimulus with atropine (van Tienhoven, 1955), and by destroying the hypothalamic centers (Ralph and Fraps, 1959) at various time intervals after progesterone injection. In van Tien- hoven's experiments the estimate was that 26 minutes was the minimum and about 2.5 hours the maximum, whereas Ralph and Fraps found that the lesion had to be made within 2 hours after the injection. In a typical closed cycle which has been discussed so far, the Ci follicle is ovulated about 6:00 a.m. on day 1 and laid the next day about 8:00 a.m. The C2 follicle ovulates about 8:30 a.m. of day 2 and is laid about 10:00 a.m. of day 3. The C„ follicle is ovulated about noon and laid in the after- noon of the nth + 1 day of the cycle. On this day no ovulation occurs in spite of: (1) greater sensitivity of the C'l follicle (first follicle in the second clutch) at this time to LH than any subsequent follicles of the same clutch (Fraps, 1946; Bastian and Zarrow, 1953); and (2) competence of the ])ituitary to secrete ovulation-inducing amounts of gonadotrophin when stimulated by progesterone. Neher and Fraps (1950> were, for example, able to add as many as 13 eggs to the clutch by injecting proges- terone at times calculated to stimulate ovu- lation at a time corresponding to the nth + 1 follicle of the clutch. In this manner, the C'l follicle became the /^th + 1 follicle of the first clutch. By injecting progesterone from then on at 26-hour intervals more eggs were added to the clutch. Thus, the situa- tion is, in a typical closed cycle, that ovula- tion fails to occur in spite of the presence of an ovulable follicle and in spite of the abil- ity of the pituitary to secrete enough gon- adotrophin. Various hyi)otheses have been proposed to exjilain this jihenomenon. Bastian and Zar- row (1953) and Fraps (1955b) agree that light and activity are the regulatory mecha- nisms which impose the rhythm of ovula- tions. Bastian and Zarrow (1953) proposed that two independent rhythms, a 24-hour daily rhythm and rhythmic maturation of follicles, interact to produce an asynchro- nous ovulation cycle. The inference would l)e, if this is true, that succeeding follicles in a clutch would be more and more imma- ture as the ovulations of the clutch pro- gressed. Some evidence was obtained to show that this occurred: the yolks of the second and third eggs of 2- and 3-egg clutches were indeed smaller than the yolk of the first egg of the clutch (Bastian and Zarrow, 1953) ; furthermore, the follicles became less and less sensitive to LH. As stated above, the first follicle of a clutch is more sensitive to LH than the succeeding ones. Thus, the fail- ure of the C/ follicle to ovulate as the nth + 1 follicle would be due to its low sensitivity to LH. This is not in agreement with Fraps' data (1955b) showing that the sensitivity to gonadotrophins for the follicles of a clutch is about the same at the same time interval after the preceding ovulation. Thus, the fol- licle is equally sensitive to LH at the same interval after previous ovulation as all folli- cles of the clutch are after that interval. In- asmuch as no ovulation occurs, sensitivity of the Ci follicle increases, so that at the same REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 151 10 p.m. 6 a.m. 10 p.m. Fig. 18.11. Relationship between diurnal rhythm of neural thre.shold (E) and excitation hormone concentration (C) 7-day cycle. Shaded areas are times when release of ovulation- inducing hormone occurs according to Fraps (1954). (From R. M. Fraps, Froc. Nat. Acad. Sc, 40, 348-356, 1954.) interval before expected ovulation the Ci follicle is more sensitive than the other fol- licles. Fraps' hypothesis can be explained best by reference to Figure 18.11. According to him, diurnal variations in threshold oc- cur, as represented by the curve Ei , E2 . . . Eg . Ci , C2 . . . Ce , C'l indicate the excita- tion hormone levels. Only when these hor- mone levels reach the threshold values, do ovulations take place. Follicle C'l would not be ovulated as follicle C- of the previous clutch because the threshold value of E7 is too high and cannot be reached. When the threshold values go down, the hormone level reaches its threshold value and C'l ovulates. This hypothesis needs experimental testing to demonstrate the fluctuations in the ex- citation hormone and the diurnal rhythm in thresholds. Nalbandov (1959b) proposed a mecha- nism based on a phenomenon discovered by Huston and Nalbandov (1953) . When a loop of thread was placed lengthwise in the ovi- duct of laying hens, ovulation was inter- rupted and the ovary remained fully func- tional, but no follicular maturation occurred. Evidence was obtained that this effect was probably neural in nature. Secondly, not more than one ovulation per hen was ob- tained. However, daily progesterone in- jections induced ovulations in the same hen fit different days. The experiments thus showed: (1) the thread inhibited gonado- trophin release, but enough gonadotrophin was secreted to prevent atresia of the fol- licles and regression of the comb (Huston and Nalbandov 1953) ; (2) according to van Tienhoven (1959) and Nalbandov (1959c), the results of this experiment indicate that gonadotrophin is secreted or released as a complex, for when progesterone causes re- lease of hormone for ovulation it also causes maturation of the next largest follicle. Further investigations showed that a thread placed in the isthmus prevented ovu- lation for a longer time and in a greater per- centage of birds than did a thread placed in the funnel (infundibulum) or in the upper, middle, or lower magnum (van Tienhoven, 1953). Sykes (1953) subsequently showed that a thread in the shell gland caused pre- mature oviposition but no change in ovula- tions. Nalbandov (1959c) proposed that, after ovulation, the egg in the oviduct in- hibits gonadotrophic secretion until the egg passes from isthmus to shell gland. The pi- tuitary then requires time to recover and produce enough gonadotrophin to induce the next ovulation. Clutch length thus would be- come a function of recovery rate of the pi- tuitary. This recovery rate would be af- fected by light only insofar as light causes production of gonadotrophins. The hypothe- sis is attractive because it provides for an 1152 SUBMAMMALIAN VERTEBRATES internal mechanism for the reguhition of ovulation. It fails to account for the shift in ovulation time by 12 hours when the time of feeding is shifted 12 hours. A more serious objection to the hyj^othesis is that release of gonadotrophin from the pituitary, and subsequent ovulation, can be induced by progesterone injection, during the "re- covery" period. In Nalbandov's hypothesis one of the crucial points in the regulation of timing is the slow recovery by the pitui- tary, but the fact that the release of enough gonadotrophin can be induced to stimulate ovulation indicates that the pituitary is competent, but that a stimulus is lacking or that the threshold is too high for the stimu- lus to be effective. In each of the hypotiieses, fluctuations in threshold of the ovary to LH (Bastian and Zarrow, 1953), neural threshold (Fraps, 1955b j, or pituitary recovery (Nalbandov, 1959c) are an essential part of the hyi)othe- sis. Whether these changes actually occur remains to be proven in each case, and, there- fore, it seems that judgment as to the cor- rectness of each hypothesis must be with- held. Indirect evidence from other species can often be marshalled, but applying data obtained with one species to another si)ecies is fraught with danger, especially in the case of a species which has been as strongly influenced by artificial selection as the chicken. 1. l^se of Birds in Bioassays Birds can be used in a variety of l)io- assays that w^ill be described briefly in this chapter. In several cases reference will be made to reviews because they contain the essential information on sensitivity and de- tails of procedures. Gonadotrophin as.says. Total gonado- trophins. 1. One-day-old cockerels are kept in shipping boxes with food and water. In- jections are made subcutaneously at 12- hour intervals and the chicks are killed 12 hours after the last injection. Usually a total of 5 injections is made (Breneman and Mason, 1951). At autopsy the testes are re- moved and weighed to the nearest 0.1 mg. With this assay a minimal total dosage of 5.0 I.U. PMS, 5.0 ^ig. rSH, or 50 /.g. LH give responses significantly different from water-injected controls. The index of pre- cision for each of the three gonadotrophins in this assay was 0.5734, 0.5766, and 0.4459, respectively (Breneman, Zeller and Beek- man, 1959). Each of the gonadotroj)hins gave a linear log dose-response relationship. No response was obtained with human chorionic gonadotrophin nor was there evi- dence of a FSH-LH synergism. Ten to fif- teen chicks should be used for each point on the regression. In this assay no reduc- tion of error mean square was obtained by adjusting testes weights for differences in body weight of the chicks by the use of a covariance analysis (Phillips, 1959). By measuring P^- uptake by chick testes Flor- sheim, VelcofT and Bodfish (1959) were able to detect 0.1 I.U. of human chorionic gon- adotrophin, 0.05 I.U. of PMS, and 0.5 to 1.0 |ug. of LH and FSH. 2. Nall)andov and Baum (1948) proposed the use of estrogen-inhibited roosters. Ac- cording to their data, FSH would cause an increase in testicular size and an increase in tubule diameter, but no increase in comb size, whereas LH would cause an apprecia- ble increase in comb size, but only a small increase in testicular size. By this method an estimate of FSH and LH content could be made in unpurified gonadotrojihin ex- tracts. As far as the author is aware, no use has been made of this method to make such estimates. LH assay (Weaver-Finch test). In this assay male, female, castrate, or noncastrate Euplectes, Steganura, or Quelea are partially deplumed and given a rest for 5 days. The feather follicles reorganize and the tips of new feathers are formed. The material is injected subcutaneously in 1 or 2 doses. In a positive reaction a colored bar is formed on the new feather. The test is specific for LH and human chorionic gonadotrophin (Wits- chi, 1955; Segal, 1957). The response is not affected l)y the presence of FSH, jirolactin, ACTH, or TSH in the material to be tested, and as little as 5 /*g. LH can be detected (Witschi, 1955; Segal, 1957). Prolactin assay. Several methods for the assay of prolactin on pigeons have been de- scribed in a review by Meites and Turner (1950). In each of the assays the stimula- tion of the pigeon crop gland is measured. A REPRODUCTIVE ENDOCRINOLOGY IN BIRDS 1153 very sensitive method has recently been described by Grosvenor and Turner (1958). Adult pigeons, Columba livia, between 240 and 360 gni. were kept in a room with a tem- perature between 78 and 80°F., artificial light-supplemented daylight during daylight hours. Two solutions to be tested were in- jected intradermally on opposite sides of each crop sac. The volume injected was 0.1 ml. Four injections were made at 24-hovu- intervals in the same area marked with a nontoxic dye. The birds were killed 24 hours after the last injection, and the crop sac dis- sected and stretched over a light source. The area stimulated was estimated with the aid of plastic discs with diameters ranging from 0.5 to 4.0 cm. With 15 pigeons per dose level and a linear log dose-response rela- tionshii^ was found for a range of 0.00072 to 0.02240 mg. per bird (1 mg. = 20 I.U.). The index of precision was A = 0.11. ACTH. Bates, Lahr and Miller (1940) used 2-day-old chicks for the assay of ACTH. White Leghorn cockerels were in- jected 3 times per day for 5 days. The adrenals were weighed. Ten mg. of a par- tially purified ACTH preparation resulted in a 25 per cent increase in adrenal weight. The assay, however, is relatively insensi- tive. TSH. In order to compare different meth- ods it was necessary to convert U.S. P. to Junkmann-Schoeller (J.S.I units. In these calculations 1 I.U. = 1 U.S.P. unit = 0.1 J.S. unit (Brown, 1959j. The use of chicks for bioassay of TSH has been reviewed by Turner (1950) and by Brown (1959). 1. Gravimetric methods are too insensi- tive to be of much value. 2. Histometric methods. One-day-old cockerels are injected five times at 12-hour intervals and the chicks killed 12 hours after the last injection. The thyroids are removed, fixed in Bouin's solution, sectioned at 4 to 6 /x, stained with hematoxylin and eosin, or Mason's triple chrome stain. The height of one cell in each of 100 acini is measured, the cells to be measured being selected at random or the height of the highest and lowest cell in each of 50 acini is measured. If the latter method is used the acini are selected at random. In our labora- tory the highest and lowest cell in each of 20 acini is measured and with this method 0.003 U.S.P. units can be detected (van Tienhoven, unpublished data). The assay can be made more sensitive by adapting Uotila and Kannas' (1952) technique of l)rojecting the thyroid section on a screen and estimating the percentage of epithelium. Using this method, 0.0001 U.S.P. units can be detected (Saatman and van Tienhoven, unpublished data). 3. Intracellular droplet method. In this method 3-day-old chicks are injected with the preparation to be assayed and the chicks killed 2 hours later. The thyroids are re- moved and fixed in Carnoy's fluid for 1 hour at room temperature, embedded in paraffin, sectioned at 4 /a, and stained with Heiden- hain's azan. The droplets in 25 successive cross sections of follicles are counted. The assay is about 70 times as sensitive as the gravimetric method and 5 times as sensitive as the cell height measurement method. 4. P'" depletion method. Bates and Corn- field (1957) used 1-day-old chicks and in- jected them with 0.2 ml. P=*i solution (2 to 3 fxc). After 24 hours counts were made in vivo with the aid of a scintilation counter. The assay solution was injected and simul- taneously 0.2 cc. of a solution containing 8 /i.g. thyroxine and either 5.0 or 0.5 mg. pro- pylthiouracil (PTU) was injected. The PTU was injected to prevent re-utilization of I''^\ and the thyroxine was used to inhibit secretion of TSH from the chick's anterior l)ituitary. This procedure was repeated daily for 2 to 3 days and daily counts were made. A linear relationship between log dose and response was obtained. The index of ac- curacy was A = 0.20. 5. I^^^ accumulation. One-day-old cock- erels are kept without food or water and receive 5 injections at 12-hour intervals. Twelve hours after the last injection 0.1 to 10 /AC I^^^ are given and the chicks are killed 5 hours later. Thyroids are removed and ra- dioactivity counted. This test can detect 0.001 and 0.005 U.S.P. units, whereas the histometric methods (cell height) could not detect these levels (Shellabarger, 1954). Oxytocic principle. Adult chickens (1.8 to 2.2 kg.) are anesthetized with sodium phenobarbital and blood pressure is taken witli tlie aid of a mercurv manometer with 1154 SUBMAMMALIAN VERTEBRATES a recording pen. Blood pressure is taken from the ischiatic vein. Injections of the assay material are made in the crural vein. Material can be injected at 3- to 5-minute intervals and materials or doses can be used in any desired sequence. A drop in blood pressure of 20 to 40 mm. Hg (Thorp, 1950) is caused by 0.2 units of oxytocin. Androgen. Assays for androgen activity can be made with capons and with baby chicks. Dorfman (1950) has given a de- tailed account of the methods used and their sensitivity. Also part of the problems in- volved in the bioassay have been discussed previously in this chapter. A very sensitive method which does not seem to have been used very extensively is the sparrow bill test. Either adult females or adult castrated males can be used. Local ap- plications of as little as 1.0 fig. testosterone in 16 divided doses will evoke a response in 4 of 6 castrated males. The response con- sists of blackening of the bill. Acknowledgements. I would like to ac- knowledge the great help received from Dr. R. E. Phillips. He has discussed many points of avian biology with me and has greatly improved the manuscript by suggesting changes in grammar, as well as by pointing out ambiguities in the original draft. Thanks are also due to Dr. Richard iM. Fraps, who in spite of sickness, showed interest in this paper while it was being written and who encouraged me to undertake this assignment. Finally, my thanks to my wife for encour- agement and much help in completing a large, but short-time assignment. rV. References Adams, J. L. 1955. 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Bisexual nature of the hen and experimental hermaphroditism in in hens. Tr. Lab. Exper. Biol., Zoopark. Mos- cow, 2, 164-179. Zimbardo, p. G. 1958. The effects of early avoid- ance training and rearing conditions upon the sexual behavior of the male rat. J. Comp. & Physiol. Psychol., 51, 764-769. ZiTRiN, A. 1942. Induction of male copulatory behavior in a hen following administration of male hormone. Endocrinology, 31, 690. Zuckerm.w, S. 1952. Chairman's closing re- marks. Ciba Foundation Collocjuia Endocri- nol., 3, 239-246. Zuckerm.\n, S., and Parkes, a. S. 1939. Observa- tions on secondary sexual characters in mon- keys. J. Endocrinol., 1, 430-439. 20 GONADAL HORMONES AND SOCIAL BEHAVIOR IN INFRAHUMAN VERTEBRATES A. M. Guhl, Ph.D. PROFESSOR OF ZOOLOGY, KANSAS STATE UNIVERSITY, MANHATTAN, KANSAS I. Introduction 1240 theniian, 1939 ; Noble, 1939a, Yerkes, 1939 ) . II. Social Organization 1240 ^-^^^ ^^ ^j^j^ j^^^^^, relationship has been III. Historical Background 1242 , , ., , . x i tx i i r i IV Methods 1242 abundantly demonstrated. It has been loimd y. Elements Comprising THE Substrate 1244 also that social relationships sometimes fa- A. Heredity and Levels of Aggressive cilitate and sometimes inhibit the display Behavior 1244 ^f gg^ual behavior. It is the object of this c: Z:n::S^^^. :::::::::: Im f l-Pter to discuss the mAuence of gonadal D. Social Inertia and the Development hormones on certam social behavior pat- of Social Behavior 1247 terns and, reciprocally, the influence that E. Interaction of Drives 1249 ^ome social factors have on the display of VI. Gonadal Hormones and Social Be- sexual behavior HAVIOR 1250 A. Social Behavior and the Reproduc- tive Cycle 1250 II. Social Organization B. Androgens and Aggressiveness... 1254 C. Estrogens and SubmLssiveness . ... 1255 The term social IS used here in a broad D. Are Aggressiveness and Submissive- sense, which is generally acceptable to zoo- ness Separate Behavior Patterns? 1257 Jogists working in the field of sociobiologv. E. Gonadal Hormones and the Devel- ^^^-^^ behavior is any behavior caused by opment 01 Social Behavior l^o/ n- ,- ^i ■ i n ^ ±^ VII. Releasers and Other Mechanisms ^v affecting another animal, usually of the IN Social Behavior 1258 same species. Although sexual and parental VIII. Social Stress and the Endocrine behavior are also social, this chapter is con- ,r ^ System 1261 cerned essentially with agonistic behavior, IX. Concluding Remarks 12b2 i • i ■ i j • i i r • X. References 1262 ^^'^"^^^ includes aggressive and defensive ac- tions and escape and submissive behavior. I. Introduction Agonistic behavior is especially important Most investigations of the influence of during the etstablishment of any degree of gonadal hormones on behavior have been intraspecific organization. Because it is so focused on reproductive behavior, probably conspicuous at such times, there is a ten- because the various patterns are readily ob- dency to report the behavior of animals servable and follow a sequence or reaction which are organized on the basis of such chain which facilitates analysis. Social be- behavior. However, there are groups which havior patterns, on the other hand, are often might be integrated as a social unit through less obvious; indeed, it was not until 1939 other behavior patterns; e.g., reciprocal in- that their manifestation w^as related to teractions such as grooming, play, parent- gonadal hormones (Allee, Collias and Lu- young relations (Carpenter, 1942, 1952; 1240 GONADAL HORMONES AND SOCIAL BEHAVIOR 1241 Scott, 1945), or what Schneirla (1946j in- cludes under trophallactic relationships. Social organizations can be classified into two general categories: social hierarchies and territories. Some species show either one or the other seasonally or throughout the year, whereas others may have elements of both concurrently. Still others have ter- ritories during the reproductive phase and hierarchy during the rest of the year. Once established, or even during the incipient stages, a pattern of social organization is typical for a given species. The variation is considerable from species to species, al- though comparable types of organization are found throughout the vertebrates. Allee (1952) recognized two major kinds of hierarchies: one based on unidirectional (despotic) domination and the other based on relative despotism in which pecking be- tween any two individuals is bidirectional. The former is often referred to as a "peck- right" system, in which the individuals are ranked in an order according to the number of individuals each can dominate without any attack or threat in return. Such a domi- nance order is usually quite stable, and species so organized are suitable for ex- perimentation insofar as controlled situa- tions can be maintained. A hierarchy based on bidirectional pecking is more fluid be- cause there is an exchange of aggressive acts, and the individual delivering the most "pecks" is considered the dominant mem- ber of the pair. In species so organized there is an overlap with territoriality, inasmuch as each individual becomes more dominant as it approaches the center of its territory. Credit for the development of the con- cept of territoriality is usually given to Howard (1920), although Lack (1953) and Carpenter (1958) cite even older reports in which some aspects of this behavior were recognized. Evidences of territoriality are often reported in studies on reproductive be- havior and have been found in ^-arious classes of vertebrates. Territorial organization has many forms, depending on how it functions for a par- ticular species. It is often defined as "a defended area." However, there is no evi- dence that it is the area per se that is de- fended. According to Emlen (1957), "the term territory is generally applied to an area or space in which a particular animal is aggressive and largely if not supremely dominant with respect to certain categories of intruders." The biologic significance of territory for birds has been discussed by Hinde (1956a), and for all vertebrates by Carpenter (1958). The latter concluded that territoriality apparently, when once established, reduces stress, pugnacity, and nonadaptive energy expenditure. Aggressive behavior has the tendency to disperse the individuals, as manifested in territorialism. Such behavior is mediated by hormones. Some behavior patterns operate in the opposite direction and result in ag- gregations by members of the species (Allee, 1931). No gonadal hormones have been discovered which influence gregariousness (other than sexual and parental bonds). Emlen (1952) discussed the social forces which cause the centripetal and centrifugal actions in flocks of birds, and concluded that flocking responses have their physiologic basis in stereotyped neural patterns and are influenced by hormonal factors only so far as these incite disruptive responses as- sociated with sexual or parental activity. Tendencies to aggregate or to disperse may be seasonal or diurnal (Emlen, 1952), and physical factors such as temperature and light may exert an action (Allee, Em- erson, Park, Park and Schmidt, 1949, p. 393). Species of vertebrates vary in the relative distance at which one individual will tolerate the presence of another. Hcdi- ger (1950, p. Ill) calls this "individual distance" and distinguishes betw^een "dis- tance animals" and "contact animals." The relative proximity at which chaffinches tol- erate each other varies with dominance rank and with sex (Marler, 1956). Dominant fe- males allowed subordinate females to come closer than other females, and males per- mitted females to come closer than males. Submissive behavior promotes toleration. The forces of mutual attraction may de- velop early in life (Collias, 1952, 1956) by mechanisms that suggest imprinting (Lo- rcnz, 1935). In many instances changes in agonistic behavior appear to precede sexual behavior and thereby to prepare the proper social en- vironment for sexual and parental behavior. IVIoynihan (1958) described the behavior of 1242 HORMONAL REGULATION OF BEHAVIOR several species of North American gulls in the process of pair formation. The hostility which precedes pairing is associated with the establishment of their territories. Pair- formation activities seem to be followed by a reduction of intersexual hostility and the gradual emergence of sexual behavior. According to Tinbergen (1953), the pre- nuptial behavior of the female appeases the male, suppresses her escape behavior and, together with the courting by the male, fa- cilitates the synchronization of sexual be- havior patterns. The selective value of aggressiveness has been discussed by Collias ( 1944) and Car- penter (1958). Selection may operate on the level of the individual, with the more ag- gressive ones usually having precedence to food, mates, and cover. On the group level the more socially stable units conserve en- ergy and may leave more progeny. III. Historical Background The discovery of a social organization based on agonistic behavior was made by Schjelderup-Ebbe (1913) during a study of calls or sounds made by chickens. Later he summarized observations of the domestic fowl and other birds (Schjelderup-Ebbe, 1935). Many of these were repeated by Sanctuary (1932) and Masure and Allee (1934a), working with chickens. Masure and Allee (1934b) also observed common pigeons and shell parakeets and discovered, contrary to Schjelderup-Ebbe, that some birds do not show absolute dominance, but rather bidirectional pecking (called peck- dominance in the earlier reports). Allee (1936) summarized these initial observa- tions and suggested a plan for analytical studies, which included alteration of the physiologic state by hormonal treatment. Noble and his associates (Noble, 1939a, b; Noble and Curtis, 1939; Noble and Borne, 1940; Noble and Wurm, 1940; Noble and Greenberg, 1941) made extensive studies of behavior which stimulated a general in- terest in the relationship between the endo- crines and social behavior in fishes, am- phibians, reptiles, and birds. The social organization in baboons was described by Zuckerman (1932), and in monkeys by Maslow (1934). Carpenter (1934) reported the virtual absence of a dominance order in the howler monkey. Yerkes (1939) observed dominance relations between uni- sexual and heterosexual pairs of chimpan- zees and related changes in dominance- submission to the sexual status of the female. Since this early period, many investi- gations have been directed at a clarification of the relationship between the gonadal hormones and social, but particularly ago- nistic, behavior. Something of the progress that has been made will be apparent from what follows. However, many reviews al- ready exist^ and in this place a particular effort will be made to present a contempo- rary cross-sectional view of the subject that will contain an indication of the many problems that are in need of study. IV. Methods Many experimental techniques have been used in studies of agonistic behavior. As would be expected, they vary greatly, de- pending on the species, the question it is hoped the experiments will answer, and the connotation of aggressiveness that is ac- cepted. Potter and Allee (1953), Kislack and Beach (1955), Scott and Fredericson (1951), and Scott (1958b) considered ag- gressiveness as a tendency to start fights. According to this view, the levels of aggres- siveness would be measured by latency, i.e., the time between the meeting of two indi- viduals in a test situation and the first overt display of agonistic behavior. However, the term as it is used in this chapter refers to the ability to be self-assertive or to display independence of action (Collias, 1944) . Ag- gressiveness in this sense is a tendency whereas aggression is an activity. Factors ^ Comprehensive reviews of aggressive behavior among vertebrates, including a discussion of hor- monal factors, have been prepared by Collias (1944, 1950). Social organization and related phenomena in vertebrates were considered bv Allee, Emerson, Park, Park and Schmidt (1949), Allee (1952), Tin- bergen (1953) and Scott (1956, 1958a). General in- formation bearing on the territorial behavior of vertebrates has been brought together by Bourliere (1952) and Carpenter (1958). Other reviews deal with the social behavior of fishes (Aronson, 1957; Baerends, 1957), birds (Hinde, 1956a), ungulates (Darling, 1952), other lower mammals (Hediger. 1952), and subhuman primates (Carpenter, 1952) GONADAL HORMONES AND SOCIAL BEHAVIOR 1243 which evoke aggression seem to vary among species and may inchide such as close prox- imity, training, sex hormone, and pain re- sulting from attack. The pattern of response may depend on the stimulus situation, the strength of the stimulation received, and the physiologic state or level of response thresh- old. In general, in studies of aggressiveness, conditions are best controlled when the ani- mals are observed in pairs or in small groups and this has long been done by many in- vestigators. Paired encounters were used by Maslow (1936) to determine dominance re- lationships among subhuman primates. If the pairings were made between unac- quainted individuals, they were called ini- tial encounters or initial contacts. These were used by Collias (1943) to determine tlie factors which make for success in estab- lishing dominance in chickens, and by Brad- dock and Braddock (1955) in their work on the fish, Betta splendens. Pairings of mice were used to analyze the effects of thiamine deficiency on fighting success (Beeman and Allee, 1945) and to condition individuals to win or to lose encounters (Ginsburg and Allee, 1942) . Pairings of chimpanzees were made in order to ascertain the effects of the female sexual cycle in female pairs (Craw- ford, 1940) and between mates (Yerkes, 1940; Young and Orbison, 1944). Corres- ponding techniques served to test the effects of gonadal hormones in chickens (Allee, Collias and Lutherman, 1939; Allee and Collias, 1940) and of androgen in mice (Beeman, 1947). Support for the opinion that staged initial pair contests in neural areas, or cages, give better estimates of levels of aggressiveness than rank in a social order or the frequency and intensity of aggression comes from a recounting of what has been found during work with chickens. In a flock of chickens the frequency of pecks delivered by an in- dividual on others is not a measure of the individual's native aggressiveness. In the determination of a peck-order the tabula- tion of pecks delivered by each bird usually shows no apparent correlation with rank in the social order. Those in the top rank have more individuals to peck and therefore the highest rate of pecking may be expected. However, the highest rates of pecking by one bird on another may occur between birds at any level above the lowest ranks. These interindividual interactions have been called "antipathies." Unexpected toleration, i.e., low rates of pecking, also may be found at any level. Antipathies may develop when the flock is assembled as a result of a hard fight, or later when a revolt is unsuccessful. Toleration may follow a passive submission in the initial meeting of unacquainted birds. Furthermore, the rate at which one bird pecks another may vary from week to week according to incidents which arise. The peck-order is learned and the laws of re- inforcement and extinction apply. Rank in the hierarchy, or the number of individuals dominated, may be used as a measure of aggressiveness, and may agree with the results of paired encounters (Guhl, 1953). However, the reliability of esti- mates based on rank in the hierarchy or the number of individuals dominated may de- pend on the conditions under which the flock was first assembled. At the first meeting the birds engage in initial encounters by pairs which meet at random. Fatigue may set in early for those engaged in lengthy fights, and such individuals may refrain from fighting, or lose subsequent encoun- ters. If new individuals are added after the peck-order is formed, they usually assume low rank. A significant correlation was found by Guhl and Allee (1944) between seniority and social position. These results lead to the conclusion that stimulus situa- tions and other factors (Allee, Collias and Lutherman, 1939) which make for winning encounters need to be controlled and that this can best be done in initial paired en- counters. When groups of animals were employed in the study of aggressive behavior the de- sirability of certain practices became ap- parent. Sanctuary (1932) found, when strange hens were added to organized flocks, that the fewer the newcomers in relation to the residents, the greater was the dis- advantage to the introduced hens. Flocks of equal size could be combined with the least disparity. In another study social organization was kept in a state of flux by regularly shifting hens from isolation to a 1244 HORMONAL REGULATION OF BEHAVIOR flock or between flocks (Giihl and Allee, 1944). The degree of domination or sub- ordination of a hen may be measured by the number of individuals it pecks or avoids. The top-ranking hen habitually pecks all and submits to none, whereas the one lowest in rank has a strong habit for avoid- ance and does no pecking. Those in inter- mediate ranks show varying degrees of both habits commensurate with social status. Therefore, the frequency with which individuals display dominance or submis- siveness may be altered by subflocking without disrupting the dominance relations among the birds in the smaller groups (Guhl, 1950). Of extreme importance in the method- ology for studying aggressive behavior is the control of what we will refer to as the elements composing the substrate. The prob- lem can be explained, Init it will l)e at some length. V. Elements Comprising Substrate The study of animal behavior is beset with problems of multifactorial relation- ships. This produces either a perplexing situation or an intriguing one, depending on the viewpoint. It is virtually impossible to control all of the known factors in a single experiment, and some apparently minor ones may gain in their influence when others are controlled. There is a continual interaction between the environment and the organism. The experimenter is one of the factors. The importance of this fact, which is also recog- nized clinically (Matarozzo, Saslow, Mata- rozzo and Phillips, 1958), has not always been appreciated. The presence of the in- vestigator, his mannerisms, and the methods of handling the animals may alter behavior. It must be recognized, too, that behavior is the expression of an effort to adapt or to adjust, and different conditions may lead to different results. Animals are often main- tained in one location and moved to an- other for experimentation. Many animals can adapt readily to such technicjues, but the time required for adjustments should be considered. In general, domestic species adjust to laboratory conditions with less diflficulty than do wild animals; Hediger (1950) suggests that the confined environ- ment of wild animals requires certain fea- tures of their natural environment. Other parts of the substrate are the genie back- ground, meteorologic conditions, and the interaction of drives. All these factors will be considered in the discussion of the in- fluence of the endocrines on social behavior which follows. A. HEREDITY AND LEVELS OF AGGRESSIVE BEHAVIOR The wealth of information from com- parative studies provides abundant evidence that the genie background influences the character of an animal's behavior. We will mention only a few: phylogenetic studies of social behavior patterns have been made for orders of insects (Michener, 1953), ter- mites (Emerson, 1938; Schmidt, 1955), bees (Michener and Michener, 1951 ; Michener, 1953), fishes (Winn, 1958), and birds, e.g., anis (Davis, 1942) and tits (Hinde, 1952). The importance of the genie factor is also apparent from observations of and experi- ments with hybrids {e.g., finches, Hinde, 1956b j, from the existence of species dif- ferences {e.g., fishes, Schlosberg, Duncan and Daitch, 1949; Clark, Aronson and Gordon, 1954) and breed differences (e.g., dogs, Scott and Charles, 1953, 1954; chick- ens. Potter, 1949; Hale, 1954; Allee and Foreman, 1955). Scott (1954) discussed the effects of selection and domestication on various behavior patterns in the dog. In genetically different stocks of male guinea pigs, factors peculiar to the strains affected the behavioral responses to testosterone propionate (Riss, Valenstein, Sinks and Young, 1955). Strain differences were also found in the response of female guinea pigs to estradiol and progesterone (Goy and Young, 1957). Most of the studies cited above were not concerned with agonistic behavior per se, but it must be presumed that strain differences would be as im- portant for the display of agonistic behavior as for all other social behavior including reproductive behavior. Some breeds of domestic animals have been developed by selection for particular behavior patterns. Terriers among dogs, gamecocks among chickens, and Siamese fighting fish have a long, and largely un- CxONADAL HORMONES AND SOCIAL BEHAVIOR 1245 known, history of selection for fighting abil- ity. Scott (1942) found that inbred strains of C57/10 black, C3H agouti, and C (Bagg) mice differed in aggressiveness, activity, and other traits. When C57/10 and C prog- eny were cross-fostered the behavior char- acteristic of the strains remained true to heredity (Fredericson, 1952) . A selective breeding program for levels of aggressiveness in Leghorn chickens was conducted by Guhl, Craig and Mueller ( 1960) . The males and females of the parent generation were of different strains merely because these were available, and there was no information on the relative aggressive- ness of either strain. Selection in each gen- eration was based on the percentage of ini- tial encounters won, as supported by rankings in the peck-order. Individuals ranking highest and lowest were used for breeding. This technicjue limited the num- ber of individuals wdiich could be tested. Some of the results are indicated in Figure 20.1 for four generations of selection. Re- ciprocal crosses were made with the breeders of the F3 generation, and their offspring tended to be intermediate in ag- gressiveness. These results are indicated by four })oints in the F4 . However, due to uncontrollable circumstances, only a few of these chicks lived to sexual maturity and testing. The upper points are means of 7 males and 8 females from "high" dams, and the lower two points are the means of 3 males and 1 female from "low" dams. The number of individuals selected for breeding and their tested progeny is given beneath the figure for all generations ex- cept the crosses. A x" analysis based on the mean percentage of encounters won by in- dividuals from the "high" and "low" lines showed statistically significant differences between the two lines of selection from the F2 through the F4 in both sexes. Calhoun (1956) made an interesting at- tempt to determine the extent to which heredity might modify social behavior. He used physiologically unstable DBA/2 and physiologically stable C57BL/10 inbred mice. Among other differing traits, the MALES FEMALES 100^ 90- ■^ / 100- 90- \ ,^ / y" HIGH LINE /' \HIGH line/ / \ / /■ 80- / / 80- \ \ / / \ / ' 0 70- y' 70- V / $ '' / if) OL UJ =) 0 0 60- 50- BREEDERS ONLY ALL PROGENY 60- 50- / / / BREEDERS ONLY ALL PROGENY ■z. u 40- \ 40- \ L^ \ 0 \ \ 0 30- ^^ \ 30- ^ 0-? ^ \ \,---'""\ \ \ 20- LOW LINE \ / ^^ 20- LOW line\ V^^ 10- \/ ~'^' 10- v^"~-.: P F| F2 F3 F4 P F| F2 F3 F4 BREEDERS 2 6 4 4 3 7 7 4 PROGENY 66 32 25 27 53 45 29 26 Fig. 20.1. Result.? of selective breeding for high and low levels of aggressiveness in White Leghorn chickens. Figures show the percentage of initial encounters won by sires, dams, and their progeny. All encounters were between individuals of high and low hnes. 1246 HORMONAL REGULATION OF BEHAVIOR DBA/2 mice have a high incidence of mammary tumors and marked susceptibility to lethal audiogenic seizures, whereas the C57BL 10 strain has a low incidence of mammary tumors and susceptibility to au- diogenic seizures. Both lines had been through extensive artificial selection and always reared in small cages with a re- stricted social environment. He wished to ascertain whether exposure to a more com- plex physical and social milieu than pre- viously experienced in many generations of rearing would provide a marked taxing of the physiologic homeostatic mechanisms available to the mice. Four colonies, two of each strain, were established in standard- ized pens with 17.5 M.- of floor space. The arrangement of nests, food, and water en- couraged social interactions. The DBA's fought more frequently and intensely than did the C57's, and the latter developed more toleration and made more passive social ad- justments. The C57 mice were more suc- cessful in reproduction and died off at a lower rate than did the more aggressive DBA mice. B. METEOROLOGIC FACTORS Some of the more immediate effects of meteorologic factors on reproductive and parental behavior can be readily observed. Modification of other forms of social be- havior, such as shifts between group living and territoriality, have been reported in some vertebrates to be associated with marked changes in the weather. Petersen (1955 1 noted that the activity of migratory bank swallows on arrival in the spring was influenced by weather conditions. Days of fair weather with temperatures near or above normal appeared to be necessary for taking up territories. Collias and Taber (1951 j found that ring-necked pheasants roosted closer together and in larger groups when the weather was very cold. According to Stoddard (1931), the male of the bob- white quail {Colinus virginianus) tolerates other males until the first warm days of February. Actions preliminary to pairing may be noted, although the coveys do not break up normally until late April, and even then may reassemble to a certain extent when the weather is cold and raw. Apparently, adverse weather conditions ex- ert some suppressive effect on aggressive- ness, and with increased toleration the ter- ritorialism reverts to flocking. Scott and Fredericson (1951) concluded that heat and probably cold tend to reduce the amount of fighting behavior in mice and rats. Combats between mice were more sluggish and shorter at temperatures over 28°C. ; inex- perienced mice did not fight at 27 to 28° C. C. PSYCHOLOGIC FACTORS In a social organization based on domina- tion, each individual forms special habits toward each member of the group. As these habits of domination or subordination be- come well established, the agonistic be- havior patterns are reduced in intensity and become symbolic. This mutual interindi- vidual adaptation promotes toleration and has been called social inertia. Such adjust- ments have been demonstrated in small flocks of chickens (Guhl and Allee, 1944; Guhl, 1958). Evidence that the principles of complex learning apply to agonistic be- havior would seem to have been provided following the training of chickens (Radlow, Hale and Smith, 1958), mice, rats (Gins- burg and Allee, 1942; Scott and Fredericson, 1951) , and rhesus monkeys (Miller, Murphy and Mirsky, 1955; Murphy, Miller and Mirsky, 1955) to be either dominant or subordinate. In connection with all this, it is appropriate to ask what effect the psycho- logic state associated with social inertia has on the behavioral response to hormonal treatment, be it reproductive or social. Taking the former first, Baerends and Baerends-Van Roon (1950) found in the cichlid fish, Hemichromis, that males that were unsuccessful in establishing territories lacked color markings, but when the domi- nant and territorial male was removed one of these showed reproductive markings and set up territory. When the new domi- nant was removed a third male reacted similarly. They concluded that ''although many of the members of the school are physiologically able to assume reproductive markings and to perform reproductive ac- tivities, in a number of them the reproduc- tive motivation was suppressed by the ac- tivities of the territorial fish." In chickens, GONADAL HORMONES AND SOCIAL BEHAVIOR 124: males ranking lowest in the peck-order among the cocks may show varying degrees of suppression of sexual behavior to the point of psychologic castration (Guhl, Col- lias and Allee, 1945; Guhl and Warren, 1946). One male failed to mate with the hens of the flock even when his social superiors were temporarily removed, but he did mate with strange hens from another flock. Similar situations have been reported by Darling (1952j among wild white cattle of Northumberland, England, and water buffalo of southeast Asia. It is pertinent to the point being discussed that the domi- nant bull water buffalo must have two other bulls to dominate before he is potent. The habits associated with the inde- pendence of action at top levels in a peck- order may suppress receptivity in females of some species. Hens at upper ranks in the flock tended to be least receptive and those at the lowest levels the most receptive. The females at the top of the hierarchy were in the habit of dominating, whereas those at the bottom submitted readily. The sub- missive attitude is a component of recep- tivity. When the degree of domination or submissiveness was altered by subflocking, the differences in the rates of displaying the submissive sexual crouch were reversed (Guhl, 1950). With fewer birds to domi- nate there was less reinforcement of the aggressive habit and the sexual crouch was evoked. Similar tests with other species of vertebrates should be made to determine the extent to which this psychologic effect is applicable. Relationships between physio- logic and psychologic aspects of display behavior in birds have been discussed by Armstrong (1947, Ch. 22). He states that the interconnection of internal and en- vironmental factors has the broad effect of bringing the birds into breeding con- dition, and the fine adjustments which achieve the final sexual synchronization are given by psychologic factors. D. SOCIAL INERTIA AND THE DEVELOPMENT OF SOCIAL BEHAVIOR To answer the second part of the question, social inertia is also important for the de- velopment of social behavior. Chicks reared in groups established certain behavior pat- terns in relation to each other (Guhl, 1958), the social inertia mentioned above, and re- quired 8 to 13 weeks to develop a new peck-order. Similar chicks reared in partial isolation and assembled at 8 to 10 weeks of age were devoid of such habits. They en- gaged in fighting and pecking, and formed peck-orders in a matter of hours. Chicks treated with an androgen while reared to- gether established peck-orders somewhat earlier than normal group-reared chicks. Another experiment was devised to com- pare the effects of androgen with those of social inertia. The specific question was whether chicks treated with androgen dur- ing partial isolation from hatching would form a i^eck-order earlier than treated chicks reared as a group. There were 5 groups of chicks (Fig. 20.2). Two groups of 11 each were pen-reared controls; the in- dividuals of one group received 0.5 mg. tes- tosterone propionate daily, those in the other group were normal controls. Three groups composed of 10 chicks each were reared in partial isolation and treated with the androgen. One was assembled when 31 days old, another at 41 days, and the third when 51 days old. The establishment of dominance relationships (peck-rights) was observed for 56 days in the pen-reared groups and for 7 days after assembly in the case of the 3 isolation-reared groups. The results are shown in Figure 20.2. The ratios at each curve indicate the number of peck-rights established at that point in re- lation to the possible number of unidirec- tional dominance relations in the flock. The group assembled on day 31 nearly com- pleted a peck-order in 1 week, and ex- ceeded the treated males reared as a group. The treated chicks assembled on day 41 failed to establish any dominance relations on the first day and formed only 20 out of a possible 45 pecking relationships by the end of a week. This performance was in- ferior to the normal pen-reared controls. The results with the group assembled on day 31 indicate that in the absence of social inertia, androgen treatment resulted in some precociousness of agonistic behavior. The results obtained from the males as- sembled on day 41 are difficult to explain. In nature behavior j^atterns tend to appear 1248 HORMONAL REGULATION OF BEHAVIOR 4 5- 40 35- 25 a. 20- 5- 14 NORMAL CONTROLS INJECTED CONTROLS 0 0 ASSEMBLED 31st DAY ^ X ASSEMBLED 41st DAY ASSEMBLED 51st DAY 0 42/45 53 /55 I 28 35 42 — I — 49 56 DAYS OF AGE Fig. 20.2. The total number of peck-rights established at various ages among males reared as a group and others reared in partial isolation and assembled at different ages. The isolated chicks and one group, reared together, were injected with an androgen (Guhl, 1958). in a certain sequence; sexual behavior usu- ally follows agonistic behavior. However, the typical behavior of this group was sexual. Attempts to mount were frequent and the birds showed strong avoiding re- actions. The indications were that at this age there was a conflict between aggressive behavior and the newly developed sexual behavior, which had not yet been sub- jected to any adjustment. The group as- sembled on day 51 showed both aggressive and sexual behavior, but with somewhat better adjustment to the conflicting drives. Untreated females (not shown in Fig. 20.2) assembled from isolation did not show this complication, because sexual behavior pat- terns appear much later in the female. The significant point is that hormonal treat- ment may shorten the time between the appearance of sequential behavior patterns to the point where psychologic adjustments cannot be made and an imbalance of drives occurs. Endogenous hormones rise more slowly in concentration and allow more time for experience, or learning the adaptive process. In large groups the variability of individuals in development is also a com- plicating factor. In the same series of experiments an at- tempt was made to demonstrate the in- fluence of social inertia on the development of social behavior bv normal male chicks. GONADAL HORMONES AND SOCIAL BEHAVIOR 1249 Rotation from isolation to group Sporri ng Peckin g X X Fighting 0 ——0 Avoiding Rotation from group to group Rotation from group to group 1 2 3 4 5 6- WEEKS OF AGE Fig. 20.3. Differences in the frequency of some behavior patterns of male chicks rotated between isolation and a group, and of others rotated from group to group. The results of group-to-group rotation for each of these two groups are shown separately, and these show social inertia (Guhl, 1958). Four groups of 10 chicks each were used. The cages were similar except that one was jiartitioned for partial isolation. Two types of rotating group memberships were de- vised. Every other day, from the 3rd day of age, 1 chick was taken from isolation and placed with one group and a group member was placed in isolation. Chicks were also shifted between the other two groups which were maintained as flocks. Rotation fol- lowed a schedule and a chick was in isola- tion (or in one of the three group cages) for 20 days. Those returned to isolation had nearly 3 weeks during which any social habits or individual recognition might be extinguished, whereas those rotated between flocks continued to have social contacts but with different individuals. The results are given in Figure 20.3 and show that the group into which isolates were rotated had the highest frecjuency of agonistic behavior. In addition to the reduced social inertia, the newcomers from isolation acted as a greater stimulus for aggression than did newcomers from the cages containing flocks of chicks. E. INTERACTION OF DRIVES The tendencies toward aggressiveness and submissiveness may be viewed as drives in the sense that they are forces prompting an animal to activity directed toward cer- tain ends. In a flock of goats (Scott, 1948), delayed feeding increased the amount of fighting in dominant animals, and recent feeding decreased the amount of aggression in dominant animals. In subordinate indi- viduals, delaved feeding caused them to take 1250 HORMONAL REGULATION OF BEHAVIOR more punishment but almost never caused aggression. It was concluded that dominance sti-ongly modified the effect of frustration in causing aggression, and that frustration caused aggression in situations in which ani- mals were in the habit of being aggressive. In goats there was no fighting and no domi- nance when they were given plenty of food scattered about to prevent crowding while feeding. In mice hunger and thirst did not affect the tendency to fight (Ginsburg and Allee, 1942), nor did vitamin Bi deficiency cause a loss of social status in high ranking indi- viduals until they were in an advanced stage of avitaminosis (Beeman and Allee, 1945). Aggressive behavior was not affected until the individual was physically weak- ened, and staged pair contacts were strongly influenced by psychologic factors. In the chaffinch (Marler, 1955) starvation, how- ever, reduced the tendency of subordinates to avoid dominant individuals. Inasmuch as females are less aggressive than males, and avoidance reactions are less intense, the toleration which developed toward low ranking individuals was more pronounced in female flocks. In rats Hall (1936) found that nonhungry individuals display greater emotionality than do hungry rats. He con- cluded that ''needs, other than the need to escape, inhibit the display of emotional be- havior by distracting the animal from the fear-provoking aspects of the situation." Among birds there are a number of situa- tions in which two or more drives are in con- flict. The conflict may be between aggressive and sexual activities, or between attacks and escape behavior. What makes the con- flict apparent is that sometimes the re- sultant pattern of behavior is atypical of the drives in conflict. These patterns are called displacement activities and have been vari- ously named and defined. According to Bastock, Morris and Moynihan (1953) "dis- placement activities apparently can occur in two situations. (1) Displacement ac- tivities may be performed by an animal in which two or more incompatible drives are strongly activated; each drive prevents the expression of the other (s). (2) Displace- ment activities may also be performed by an animal in which one drive is, at the same time, both activated and thwarted." A number of such conflicting situations is mentioned by Armstrong (1947, pp. 99- lOlj. An interesting study of the conflict be- tween the tendencies of attacking, fleeing, and courting in the male chaffinch was made by Hinde (1953). The male is dominant over the female in winter, and in the spring the dominance is reversed. The male's dis- play occurs in those situations in which his tendencies to approach (court) and to flee from the female are in approximate balance. A similar analysis can be applied to the female. Attempts to copulate may be un- successful if the sex drives of both indi- viduals are not sufficient to inhibit aggres- sive behavior. The presentation of the material which follows is given with the assumption that the many factors enumerated above have been controlled. VI. Gonadal Hormones and Social Behavior A. SOCIAL BEHAVIOR AND THE REPRODUCTIVE CYCLE The stimulating action of gonadal hor- mones on social behavior might long ago liave been postulated from the close re- lationship between reproductive state and behavior, in wild species in which reproduc- tion is generally seasonal, and in many laboratory and domesticated species in which cyclic activity is continuous without intervening periods of anestrum. Relation- ships of this sort are well known in birds (Armstrong, 1947) and many mammals: the red deer, Cervus elaphus (Darling, 1937), the wapiti, Cervus canadensis, the moose, Alces americana shirasi, the chamois, Rwpi- capra rupicapra, the wild boar, Sus scrofa europ. (Altmann, 1952, 1956), and others. Territorialism may develop during the breeding cycle among frogs (Martof, 1953; Test, 1954) and reptiles (Greenberg and Noble, 1944). In fish, seasonal modification in breeding aggregations has been described by Aronson ( 1957 ) . It is common knowledge that the males of many species show com- bative behavior during the breeding season. Rowan (1931) observed that the male bobo- GONADAL HORMONES AND SOCIAL BEHAVIOR 1251 link attacks other males in the spring, al- though peaceful unisexual flocks are formed when the breeding season wanes. Fighting during the reproductive phase was reported in the gentoo penguin, Pygoscelis papua (Roberts, 1940) and in the herring gull, Lams argentatus (Boss, 1943). Studies have been made which show a cyclic relationship between changes in the size of the gonads and the sequence of social and reproductive behavior patterns occurring before and during the breeding season. Such a relationship is presumed to be universal in seasonal breeders — the ex- ception would be the cause celebre. Changes in the grouping and dominance relations among free ranging ring-necked pheasants {Phasianus colchiciis) are related to sea- sonal increase in the weight of the testes (Collias and Taber, 1951 j. During the gonadal quiescence of winter the birds re- main in marshes as small groups with shift- ing memberships. Evidence of peck-orders among cocks and hens was found. The males pecked the females during competi- tion for food. As the breeding season ad- vanced there was a gradual increase in an- tagonism between members of the same sex and in attraction between individuals of opposite sexes (Fig. 20.4). With an increase in testis weight the male groups broke up, territories were established, and harems were formed. Cocks that ranked high in the winter dominance order established ter- ritories, whereas those of low social status failed to do so. Genelly (1955) reported on PERIOD IN BREEDING CYCLE Transition Spring Dispersal Period period of period of territory harem ^stablishj Lformotionj Fig. 20.4. Expicssions of display or dominance between and within sexes of ring-necked pheasants, from January through April. Broken lines represent infrequent occurrence; solid line, frequent occurrence. The sequence of behavior changes is superimposed upon the in- crease in testis size (Collias and Taber, 1951). 1252 HORMONAL REGULATION OF BEHAVIOR a study of the annual cycle of the Cali- fornia quail {Lophortyx calif ornica) . Of particular interest is the shift from a peck- order type of organization during sexual quiescence to territoriality during the breed- ing season. The establishment of breeding territories by the Anna hummingbird {Ca- lypte anna) has been correlated with testis volume and histologic changes (Williamson, 1956). An extensive study in which behavior was related to the seasonal cycle was made by Petersen (1955) with the migratory bank swallow (Riparia riparia). These birds ar- rived in the spring in flocks which congre- gated at breeding sites on warm days. Fig. 20.5. Summary of behavior elements in the breeding cycle of tlio Ijank .swallow (Peter- sen, 1955). GONADAL HORMONES AND SOCIAL BEHAVIOR 1253 Fig. 20.6. Summary (Petersen, 1955). Unpaired birds, proba])ly males, selected burrowing sites and set up limited territory. Pairing resulted from the persistent return- ing by the female to an area in the face of aggressive attack. After the pair-bond was formed, both mates shared in the attack on trespassers. After the reproductive cycle was comjileted, and after the end of nest- ing, toleration of other individuals was re- stored and flocking ensued. Peterson related the sequence of social, sexual, and ])arental behavior patterns (Fig. 20.5) to physiologic and morphologic changes (Fig. 20.6). Modifications in agonistic behavior occur in continuous breeders and under the nonsea- sonal conditions of the laboratory. The non- 1254 HORMONAL REGULATION OF BEHAVIOR spawning cichlid fish, Aeguidens latifrens, fights the male more often than does the gravid female (Breder, 1934) . Female guinea pigs become submissive to, or tolerant of, the male during estrus (Young, Dempsey and Myers, 1935). According to Pearson (1944) the female short-tailed shrew, Blarina bre- vicauda, fights the male when nonreceptive, but during estrus is quite docile, as is the golden hamster (Kislack and Beach, 1955) . Female chimpanzees become dominant (in the sense of attaining a response priority during food-getting tests) over other fe- males during the period of maximal genital swelling (Crawford, 1940). Not to be overlooked is the probable sig- nificance of the fact that such changes in behavior are seen in females, in which ovar- ian activity is cyclic, rather than in males in which testicular activity is continuous. B. ANDROGENS AND AGGRES.SIVENESS In most subhuman vertebrates which have been observed in sociobiologic studies the males are more aggressive than the fe- males and aggressiveness increases during the sexual development of maturing animals as well as during the breeding season. That early castration of domestic male animals reduces pugnacity has long been known (Rice, 1942). Goodale (1913) noted the lack of combativeness in the capon, and Scott and Payne (1934) found that castration eliminates fighting in the tom of the bronze turkey, Meleagris gallopavo. According to Uhrich (1938), male mice castrated as adults continue to fight, whereas those gon- adeetomized prepubertally rarely fight. Evans (1936) observed that castrated males of the lizard, Anolis carolinensis, continue to fight, and that females rarely fight unless they are ovariectomized. From this, he con- cluded that ovarian hormones inhibit fight- ing. However, Greenberg and Noble (1944) expressed the opinion that both sexes are innately aggressive and that the seasonal increase in testicular hormone in the male transforms mere antagonism into territori- ality. Castration of the male gobiid fish, Bathygobius soporator, results in the disap- pearance of combative behavior, but court- ship is not impaired. Nonspawning males show normal combat, and hypophysectomy is followed by a cessation of both combat and courtship (Tavolga, 1955). A continued display of pugnacity was observed in cas- trated starlings, Sturnus vulgaris (Davis, 1957a), and in castrated male pigeons (Car- penter, 1958) . Geldings ranked among mares in a common dominance order (Montgom- ery, 1957). These observations point to androgen concentration in the blood as in- fluencing the level of combativeness. Ap- parently, however, the extent to which cas- tration affects the level of aggressiveness varies among species, and according to the age at which the animal is gonadectomized. The experimenter's connotation of aggres- sive behavior and the method of its meas- urement may result in variations in the in- terpretation of the effects of castration. The administration of androgen often produces an increase in aggressiveness in normal adults and in castrates, and precoc- ity in immature individuals of either sex. Treatment of fishes has been followed mostly by observations of sexual behavior (Aronson, 1957, p. 286), but Noble and Borne (1940) reported advancement in rank by spayed and intact female swordtails, Xiphophorus helleri, after implanting pel- lets of testosterone. Juvenile and young adult urodeles, Tritunis viridescens, treated with whole pituitary and luteinizing hor- mone (LH) fraction pre-empted first place in their hierarchies (Evans, 1956). Male Anolis treated with testosterone propionate fought more than the controls (Noble and Greenberg, 1941). Subordinate males of Sceloponis grammicus received an androgen (Perandrenj and rose in their respective hierarchies (Evans, 1946). Among birds, androgen treatment caused an increase in aggressiveness in the ringdove, Streptopelia risoria (Bennett, 1940), the herring gull, Larus argentatus (Boss, 1943) , and in pou- lards (Davis and Domm, 1943). Female rats injected with androgen became irritable and pugnacious (Ball, 1940; Huffman, 1941; Beach, 1942). Tollman and King (1956) in- jected an androgen into young gonadecto- mized male and female C57BL/10 mice and found that the males responded more ag- gressively toward each other than did the females. They concluded that either the nervous systems of both sexes responded GONADAL HORMONES AND SOCIAL BEHAVIOR 1255 differentially to the hormone or that females do not provide as adequate a stimulus for aggressive responses as do males. Increase in the social status of androgen- treated individuals has been recorded for chickens (Allee, Collias and Lutherman, 1939; Douglis, 1948), mice (Beeman, 1947), and chimpanzees (Clark and Birch, 1945; Birch and Clark, 1946). However, three males of free-living valley quail which re- ceived pellets of testosterone propionate be- came pugnacious toward other males but failed to alter their positions in the peck- order of the covey (Emlen and Lorenz, 1942). Hens receiving androgen also failed to rise in the social order (Williams and McGibbon, 1956) . In both of these instances the group remained intact, and presumably the social habits withstood the influence of lowered thresholds for aggressive action l^'oduced by the androgen. These results differ from those obtained by Allee, Collias and Lutherman (1939), as did the proce- dure, because in the latter experiment pair contests were conducted between individuals of different flocks containing the treated birds. It is possible that a treated hen, if promptly returned to her flock from an ini- tial encounter (which she usually won) , was still highly stimulated, and if she engaged in fighting with a threatening superior pen- mate, a reversal of dominance would occur. An experiment which is the converse of those described above has recently been performed, but not previously reported, by Robert Buchholz of Kansas State Univer- sity. He attempted to determine whether a low ranking bird could be made the domi- nant individual in the flock by varying the psychologic state rather than by treatment with androgen. The chickens used were males which had been reared together. The lowest ranking bird, which had never been observed to peck a penmate, was selected for experimentation. When 11 weeks old, 7 birds were isolated for 33 days to extinguish the memory of past associations. When ap- proximately 16 weeks old, the lowest rank- ing male was placed into a pen for 1 week to give him the advantage associated with prior residence. When a female was added he dominated her without any difficulty. After 2 days she was removed and a former penmate, which was immediately above him in rank previously, was introduced. The formerly subordinate male gained domi- nance over him and all of his other former penmates which were introduced during the course of 1 day in the reverse of the former peck-order. By this method the original lowest ranking male became the top ranked bird. It is apparent that the chicken as well as the mouse (Ginsburg and Allee, 1942) is a species in which psychologic factors can be instrumental in the attainment of a state that formerly would have been thought of as solely the consequence of hormonal ac- tion. Changes in agonistic behavior after the cessation of hormonal treatment have been investigated. Birch and Clark (1946) noted a reversal of dominance to the pretreatment status in adult ovariectomized chimpanzees, whether they were given methyl testosterone or estradiol. In ringdoves Bennett (1940) also found a tendency of previously treated birds to return to their former social rank. However, Allee, Collias and Lutherman (1939) reported that the social position which treated hens won persisted as long as these flocks were under observation. These contrasting results on the two species of birds may perhaps be explained on the basis of qualitative differences in the social in- ertia of these species. In the peck-right sys- tem found in chickens, repeated pecking re- inforces the unidirectional pecking and maintains dominance relations, whereas the bidirectional pecking characteristic of doves results in a more fluid social order. Thus, the relative permanence of social gains or losses induced by exogenous hormone may be influenced, among other factors, by the type of social behavior patterns peculiar to the species. C. ESTROGENS AND SUBMISSIVENESS The reports that have been made follow- ing observations and experiments on the re- lationship between estrogens and the social order have resulted in a confused picture. There are species such as the red-necked ])halarope, Phalaropus lobatus, in which the female in the reproductive phase is the more colorful, sings, fights, and entices the male (Tinbergen, 1935) . But in the case of many 1256 HORMONAL REGULATION OF BEHAVIOR of the lower mammals that have been stud- ied, the female is submissive when follicular development is at its height, and resistant to the male or even strongly aggressive at other times. This was long ago noted in the female guinea pig (Young, Dempsey and Myers, 1935) and has more recently been recorded for other species. The female opos- sum has been observed after a heat period to attack and kill a male double her weight (Hartman, 1945). Pearson (1944) states that the nonreceptive female short-tailed shrew faces the male whenever he is near and repulses his advances with lunges, loud squeaks, and sometimes a long, shrill chat- ter. Evans' (1937) observation that ovariec- tomy of the lizard, Anolis carolinensis, in- creased the territorial response, may not be unrelated. The change in behavior at estrus, such as that described for the guinea pig, opossum, and shrew, was made the subject of an investigation by Kislack and Beach (1955). Intact, estrous and diestrous fe- males, and estrogen-treated spayed animals were tested in pairs with males. Spayed hamsters were continually aggressive, al- though somewhat less so than intact di- estrous females. The administration of estrogen alone was followed by a slight in- crease in aggressiveness, but progesterone injected after estrogen eliminated fighting and reduced other forms of aggression. Progesterone alone had no influence on the behavior of spayed hamsters. It was con- cluded that there is a negative relationship between sexual receptivity and aggressive- ness, w^iich depends in large measure on ovarian secretions. In the words of the au- thors, the ovarian hormones that render the female receptive also inhibit her tendency to attack the male. In other experiments the results of treat- ment with estrogens were ambivalent or negative. Allee and Collias (1940) injected estradiol into intact hens and discovered only a slight tendency toward reduced ag- gressiveness and lowered status in the peck- order, even w^hen large amounts were given. Both male and female chicks of the domestic fowl injected with an estrogen formed a hierarchy which was determined largely by avoidance reactions (Guhl, 1958). Individ- uals of both sexes gave the submissive sex- ual crouch when a hand was held over them. Pecking was rare and the social order was called an avoidance-order rather than a peck-order. Emlen and Lorenz (1942), on the other hand, did not observe a behavioral response in valley quail given estrogen. Shoemaker (1939) and Noble and Wurm (1940) also obtained negative results with canaries and night herons, respectively. Young rhesus monkeys were tested with an androgen and an estrogen (Mirsky, 1955). Unisexual social groups of 5 were organized on a hierarchial system. Those in ranks 5 and 2 received implanted pellets of the androgen or the estrogen in two dif- ferent periods separated by an interval without treatment. Two pairs of males and two pairs of females of known dominance relations were tested. The subordinate mem- ber of each pair was given either androgen or estrogen. In no case was hormone admin- istration accompanied by a significant de- crease in either dominance or subordinate l)ehavior scores. Except for the report that a prepubertally castrated male showed enhanced dominance status under androgen therapy, and sub- ordinate status as a result of estradiol ad- ministration (Clark and Birch, 1945), in- vestigations on the chimpanzee have yielded results that are not clear. According to Yerkes (1939), the importance of the sexual condition was established. The dominant female of a pair, w^hen in estrus, ordinarily grants privilege to her subordinate com- panion, whereas the subordinate female, when in estrus, may achieve privilege and act as if temporarily in control. The state- ment is not made that the subordinate mem- ber of a pair becomes dominant when she is in estrus. Crawford (1940) provided addi- tional evidence that a response priority in a food competition situation is related to the sexual status of the females. In 9 of 13 pairs in which changes in response priority oc- curred, the subordinate member obtained food more often when in the follicular phase of the cycle. Again, the behavior was de- scribed as a yielding of priority by the dom- inant subject with only the weak suggestion of an increase in aggressiveness on the part of the subordinate animal. However, Birch and Clark (1946, 1950) and Clark and GONADAL HORMONES AND SOCIAL BEHAVIOR 125: Birch (1945) state that estrogens increase the aggressiveness and dominance tendency of female chimpanzees and that the grant- ing-of-privilege concept is superfluous. Certain circumstances should be noted in any consideration of these latter studies. Only three females were used and Yerkes and Crawford had observed much variabil- ity in their experiments. Further, the sub- jects had been ovariectomized postpuber- tally and had been together intermittently for several years. The dominance order was Lia, Nira, May. Nira was paired with Lia in a food competition situation before, dur- ing, and after treatment, and May was tested similarly in pairings with both of her social superiors. Nira obtained higher com- petition scores during treatment, whereas May failed to show any changes. Before ovariectomy. May mated readily with the males when she was in the swelling phase, whereas Nira during 5 years as a mature female had been known to copulate only once despite frequent attempts to breed her. Two questions arise: Did Nira's failure to breed before ovariectomy indicate abnor- mality? Did agonistic experiences before ovariectomy augment or suppress any prob- able effects of estrogenic treatment? D. ARE AGGRESSIVENESS AND SUBMISSIVENESS SEPARATE BEHAVIOR PATTERNS? Before leaving the subjects of estrogens and submissiveness, and androgens and ag- gressiveness, the alternative possibilities must be considered that these antithetical behaviors are (1) independent tendencies or (2) extremes on the same scale of social interactions. As a starting point for the dis- cussion, the reader will be reminded that, except for the claim by Birch and Clark that estrogens increase the aggressiveness of the female chimpanzee, opinion is general that dominance is lowered (or submissive- ness is increased) by these hormones (Col- lias, 1944; Beach, 1948). To the holder of this view who also accepts the conclusion that androgens enhance aggressiveness, it is only a step to the hypothesis that aggres- siveness and submissiveness are independent traits, that aggressiveness is essentially but by no means exclusively masculine and sub- missiveness essentiallv but bv no means ex- clusively feminine. But the alternative con- cept that aggressiveness and submissiveness are merely opposites on the same scale of social interactions must be considered. Sup- {)ort for such an opinion might come from the fact that both types of reaction are shown by the same individual in appropri- ate situations. However, this is also true of sexual behavior, but in the case of sexual behavior, a rather convincing body of evi- dence has accumulated that two mecha- nisms are involved (for a review of the subject see the chapter by Young, and for new data obtained during an investigation of the patterns of inheritance of masculine and feminine behavior in female guinea pigs see Goy and Jakway, 1959). A solution of the problem may come from further work with gamecocks which have been selected for persistence in fighting and may fight to the death without showing avoidance be- havior. It was a strain of guinea pigs (strain 2), in which the females normally display little or no masculine behavior, that provided the genetical evidence for the inde- pendence of masculine and feminine com- ponents of sexual behavior (Goy and Jak- way, loc. cit.). E. GONADAL HORMONES AND THE DEVELOPMENT OF SOCIAL BEHAVIOR A discussion of the development of social behavior should be related to the heredity- environment or instinct-learning contro- versy, which, however, is beyond the scope of this chapter. Should there be a need for references to recent views on this subject, several publications are available (Tin- bergen, 1951; Lehrman, 1953; and Emlen, 1955). Some observational studies on the socialization of young animals have been reported for birds (Collias, 1952), mice (Williams and Scott, 1953), sheep (Scott, 1945), sheep and goats (Collias, 1956), and dogs (Scott and Marston, 1950) . Most of the experimental studies have been focused on the precocious display of sexual behavior and are discussed in the chapter by Young. Collias (1950) began treatment of male and female domestic chicks with testoster- one and estradiol when they were 2 days old. Dosages of 0.1, 0.3, 0.5, and 0.7 mg. of 1258 HORMONAL REGULATION OF BEHAVIOR either hormone were given daily to different chicks for 30 days. The number of aggres- sive pecks observed was proportional to the dosage of androgen given to male chicks. With the lower dosage, pecking was more frequent than attempted matings. Testos- terone was more effective than estradiol in inducing aggressive behavior. Males were much more responsive to the androgen than females. Submissive behavior was not re- ported. The conditions under which these chicks were reared were not given, nor were the dominance relations mentioned. Experiments to determine the approxi- mate age at which chicks develop agonistic behavior and establish a social order, and the effects of gonadal hormones and social inertia on the precocity of such behavior were reported by Guhl (1958). In small groups of chicks the males developed ag- gressive behavior earlier than the females; in mixed flocks there were heterosexual peck-orders, with males pecking more fre- quently than females. There was a gradual trend toward unisexual pecking. Some chicks were reared in partial isolation from the second day after hatching and as- sembled into groups when their respective control flocks of the same sex established a peck-order (8 to 9 weeks for males, 10 weeks for females). These birds formed a peck- order in a matter of hours, indicating that it did not require 8 to 10 weeks of learning to form a dominance order. This difference between group-reared and assembled iso- lation-reared birds may have been related to either the maturation of the endocrine and nervous systems, or it may be that so- cial inertia in the group-reared chicks sup- pressed the influences of developmental processes thereby producing a time lag in the evocation of agonistic behavior. As a part of the same investigation, in- dividuals in small unisexual groups were in- jected with either testosterone or diethyl- stilbestrol. Each treated group was matched with a control group from the same hatch. In all but one group the injections were be- gun the 2nd or 3rd day after hatching. Dom- inance or subordination was established somewhat earlier in the treated groups, de- pending on the hormone used. The andro- gen-treated chicks, irrespective of sex. showed increased aggressiveness, whereas the estrogen-treated chicks, again irrespec- tive of sex, were more submissive and the social order was determined by the consist- ent avoidance by each chick of speciflc pen- mates. However, the differences between the means of experimentals and controls are small and do not suggest any marked pre- cocity in agonistic behavior as a result of treatment with gonadal hormones. In the same study (Guhl, 1958) capons were used to determine the age at which dominance-subordination relations (peck- rights) may be established in the absence of androgen (Table 20.1). For comparison there was one group of normal males and one group of capons receiving large dosages of testosterone propionate daily (0.5 mg. beginning the 10th clay which was increased by 0.5 mg. weekly for 4 weeks) . Caponiza- tion was on the 9th day of age, and therefore the untreated capons had little or no en- dogenous androgen (Breneman and Mason, 1951). The mean age at which the untreated capons established peck-rights was 13.7 weeks, whereas the mean for the normal males was 9.9 weeks, and for the treated capons 7.8 weeks. It is of interest that a so- cial order developed by the 17th week in the apparent absence of androgen, and that the high level of treatment enhanced the formation of a peck-order over the controls by only 1 week, the 12th week compared with the 13th week for the normal males. VII. Releasers and Other Mechanisms in Social Behavior Secondary sex characters such as adorn- ment and color, postures, odors, and certain sounds, have a place in the mediation of social behavior and are thought to function as releasers of specific behavior patterns. Such stimuli may be simple or very com- plex configurations (Tinbergen, 1951). In an excellent review of the behavior of cichlid fishes, Baerends and Baerends-Van Roon (1950) related chromatophores to be- havior patterns. Six types of chromato- phores were discussed showing various methods of development and control. In Tilapia natalensis the black reproductive markings developed under the physiologic conditions typical of the reproductive pe- GONADAL HORMONES AND SOCIAL BEHAVIOR 1259 TABLE 20.1 The effect of androgenic treatment on the development of peck-rights in caponized chicks as compared with normal cockerels and capons of the same age Arrows indicate deviations from a straight-line peck-order. Number Pecked Weeks of Age Normal males 8 GV 1 3 3 1 7 GY 1 3 2 1 6 VY 2 1 1 1 1 5 V 2 2 1 3 ^ ^ 1 2 3 Cvv 1 1 1 3 \ry 1 1 1 1 YY 1 0 BB 36 Total 1 3 7 6 4 6 3 « Androgen-treated capons, 10th day to 63rd day 45 GB GV B ^ R VB, Y BB-^ V GR VY Total 3 2 1 2 1 5 2 1 1 1 2 1 2 2 1 1 1 1 2 1 1 1 1 1 1 1 1 2 1 1 1 10 5 5 » 3 7 4 2 Capons 8 R 2 1 4 1 7 GV 1 1 1 2 2 6 GY 1 1 1 2 1 4 _GR 1 1 1 1 4 \VY 1 1 2 4 1 3 2 GB 1 1 1 GG 1 0 G 36 Total 1 2 0 1 4 3 7 1 6 2 9 riod, but contraction and expansion were controlled by the nervous system. The bright red markings of Hemichromis were also shown only when the fish engaged in courtship. The reproductive colors function in fighting displays and in territoriality dur- ing the breeding season. Nonterritorial fish were pale, and individuals crossing terri- torial boundaries were not molested if their chromatophores were contracted. It was suggested that the development of these chromatophores probably is under the con- trol of the pituitary. Noble and Curtis (1939) found that ripe females of Hemichromis himaculatus se- lected bright red males when given a choice among females and males in different stages of maturity. Tests by Tinbergen (1951, p. r2()0 HORMONAL REGULATION OF BEHAVIOR 27) showed that the red undersides of the three-spined stickleback, Gasterosteus acu- leatur, elicited aggression irrespective of the size or shape of the models. Apart from color, a head-downward posturing was also effective. The hormonal relation to color or posturing was not established. In Gambusia hurtadoi the intensity of yellow markings on the dorsal fin, at the base of the caudal fin, and on the ventral portion of the caudal peduncle were correlated with rank in the social hierarchy (McAlister, 1958). Evans (1955) discussed various types of releasers found among reptiles, which in- cluded postures, colors, and sounds. Many of these occur only in sexually mature in- dividuals. Nocturnal species tend to use auditory cues whereas diurnal species do much posturing, often with pigmented areas drawing attention to these movements. The close relationship between hostile and sexual behavior patterns was shown by Greenberg and Noble (1944) in Anolis carolinensis, because reaction patterns may shift readily from one form to the other in either direc- tion. The crest in this species was stimulated by testosterone propionate. Color in birds was one of the first of the secondary sex characters to receive atten- tion in its relation to agonistic behavior (Huxley, 1934). The songs of birds are gen- erally accepted as indicators of the repro- ductive phase and often are related to terri- toriality (Armstrong, 1947, p. 293). Lorenz (1950, p. 242) , in his discussion of the subject, defined a social releaser as a "device — either a property of color and/or shape, or a special sequence of movements, or, for that matter, of sounds, or a scent — specifically differentiated to the function of eliciting a response in a fellow member of the species. To every releaser, as an organ for sending out sign stimuli, there corre- sponds a perceptual correlate, an 'organ' to receive sign stimuli and to activate the an- swering reaction." The latter he called a releasing mechanism. The point of interest is whether any of the releasers, or the so- called releasing mechanisms, are influenced by gonadal hormones. With respect to the former, when they are clearly defined secondary sex characters, the answer has been given in countless in- vestigations and reviews (see particularly Lipschiitz, 1924, chapter 2; Allen, Danforth and Doisy, 1939, p. 185, 251, 340, 499, 545; Beach, 1948, chapter 10; and the chapters by Forbes and van Tienhoven in this book). Whether the functioning of releasing mecha- nisms, that is, visual, auditory, olfactory, and tactile receptors, and central neural tis- sues, is influenced by gonadal hormones is less certain. The subject is discussed at length by Lehrman and Young in the parts of their chapters dealing with the mechanism of the hormone actions which stimulate pa- rental and mating behavior. Briefly, the opinion has been expressed that gonadal hor- mones act on peripheral receptors (Carter, Cohen and Shorr, 1947 ; Beach and Levinson, 1950) and olfactory sensitivity (LeMagnen, 1952a, b, 1953). Tavolga (1955) castrated males of the gobiid fish, Bathygobius sopo- rator, and noted that they act as though they do not "perceive" the difference between males, gravid females, and nongravid fe- males. He concluded that testicular hor- mones affect the threshold of visual, chemi- cal, and possibly auditory sense organs. Particularly pertinent to the subject is the discussion presented by Birch and Clark (1950) following their investigation of the mechanism of estrogen-induced dominance in chimpanzees. As they wrote, the problem centers around the differential effectiveness of estrogen in producing changes in the dominance status of males and females. Tes- tosterone, whether given to males or females, was assumed to facilitate aggressive be- havior by an action on the central nervous tissues. Estradiol reduced aggressive tend- ency in the two sexes and the effect was as- sumed to be central. But there is a second effect on the female which is peripheral and results in the swelling and increased irrita- tion of the sex skin. The facts that domi- nance-status paralleled the engorgement of the sex skin, and that prevention of the latter by the simultaneous administration of progesterone reduced the dominance- status, were the basis for concluding that the peripheral effectiveness of estrogens ac- counts for their effects on dominance. The case is weakened, unfortunately, by the authors' failure to eliminate the possibility that the progesterone given to inhibit sex- GONADAL HORMONES AND SOCIAL BEHAVIOR 1261 skin tumescence may also have antagonized other actions of the estrogen (for a discus- sion of this action of progesterone see the chapters by Hisaw, Villee and Young on the ovary). If progesterone had this effect in these animals, the reduction in dominance which followed its administration was not necessarily related in any direct way to the prevention of swelling. VIII. Social Stress and the Endocrine System Heretofore, discussion has been limited to the gonadal hormones and aggressiveness. There remains another axis, i.e., the effect of aggressiveness on reproduction. The na- ture of the relationship does not appear to l)e that of a feed-back, as our statement of the problem suggests. Females as well as males are affected, and much of what has been done indicates that the pituitiary-ad- renal axis is involved. Mammals with cyclic populations seem most susceptible. Christian (1950) reviewed the symptoms and conditions associated with the die-offs in mammals having population cycles. These were related to the general-adaptation syn- drome of Selye (1947). A working hypothe- sis was developed for the population crash which terminates the cycle. "Exhaustion of the adrenopituitary system resulting from increased stresses inherent in a high popula- tion, especially in winter, plus the late winter demands of the reproductive system, due to increased light and other factors, precipitate population-wide death with the symptoms of adrenal insufficiency and hy- poglycemic convulsions." Elements of this hypothesis are supported by rather impres- sive evidence. Southwick, (1955a, bl, in a carefully con- trolled experiment with house mice {Mus tnuscuhis) , found that the amount of ag- gressive activity increased as the density of the population increased. Crowcraft and Rowe (1957) in a similar experiment, but with some modifications, found that re- duced fecundity of the females was the most important single factor limiting population growth. They concluded that some factor other than fighting or food shortage appears to inhibit ovarian activity. Christian (1955) showed that mice mantaincd in groups have heavier adrenal glands than mice kept in isolation, and that the adrenals and repro- ductive organs of wild house mice are more responsive to stress than those of laboratory albino mice. In a subsequent study of popu- lations of different sizes and of isolated controls, and with food and water in excess, Christian (1956) analyzed the endocrine re- sponses. The results were indicative of an amount of stress proportional to population density. The secretion of adrenocortico- trophin increased in response to stress and gonadotrophin decreased. Increased adrenal size and low eosinophil counts were taken as evidence for an increase in adrenocortical activity in a dense population of the meadow vole, Microtus pennsylv aniens (Louch, 1958). The results from one study of the chicken (Siegel, 1959) suggests that the response of this species may perhaps be similar to that of the mouse and meadow vole. The adrenals were heavier in birds under crowded conditions than in those with more floor space per bird. With progressive decrease in the density of the flocks, adrenal weights declined. Three urban populations of Norway rats {Rattus norvegicus) with histories of stable high population densities were reduced by trapping to an average of 32 per cent below the maximal density. There was a mean de- cline of 28.3 per cent in the adrenal weights immediately following population reduction, and 7 months later the adrenal weights were 22.4 per cent below the original values (Christian and Davis, 1955). Richter (1954) found that the cortexes of adrenal glands were much smaller in the domesticated labo- ratory rat than in the wild Norway rat. Population cycles of the vole {Microtus arvalis Pallus) in Germany were investi- gated by Frank (1957). He concluded that intraspecific social behavior was of great importance for the events of population dynamics. Crowding favored competition and caused a state of psychologic excitement which was transformed by the pituitary- adrenocortical system into physical stress. This, acutely combined with the stress of food shortage, produces a "readiness" for a crash in the vole population. The ultimate trigger was an additional stress of meteoro- logic events. Davis (1957b) stated that 1262 HORMONAL REGULATION OF BEHAVIOR competition and fighting increase as the population increases and this results in a number of responses including the hyper- trophy of the adrenal cortex. A number of ensuing responses reduces the reproductive rate and increases mortality. Thus reproduc- tion is reduced or, if the behavior patterns and physiologic responses are not precisely adjusted, a population decline may occur. In this manner, behavior acts as a homeo- static mechanism for populations. Conceivably, this important generaliza- tion is premature, at least in this form. The extensive data on the Iowa muskrat, On- datra zibethicus (Errington, 1957), do not indicate that social stress is a major factor in the population cycle of this species. There may be other exceptions. In addition, the application of newer tests of adrenal func- tion would be desirable. IX. Concluding Remarks Much work has been done in an effort to ascertain whether a relationship exists be- tween gonadal hormones and the social be- havior which is displayed so conspicuously at the time of reproduction. There will be disappointment that more exact information has not been obtained. This can be explained in part by the circumstance that endocrino- logic, neurologic, and psychologic processes of the most complex types are involved. Such being the case, any analysis of the many problems requires the utilization of endocrinologic, neuroanatomic, neurophysi- ologic, and psychologic techniques. Unfor- tunately, however, application of the rigor- ous tests which are a part of these techniques has not been possible. The end points thus far available to the investigator of social behavior are not as sharp as those on which the endocrinologist would insist; neural centers which might be inactivated or stim- ulated electrically or chemically are not known to exist, and the psychologist is handicapped by the variables inherent in any study of a behavior involving interac- tion with other animals. On the other hand, it may be expected that as more work is done, the handicaps imposed by these re- strictions will be overcome and a more gratifying progress may be anticipated. X. References Allee, W. C. 193L Animal Aggregations: A Study in General Sociology. Chicago: Univer- sity of Chicago Press. Allee, W. C. 1936. Analytical studies of group behavior in birds. Wilson Bull., 48, 145-151. Allee, W. C. 1952. Dominance and hierarchy in societies of vertebrates. 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Psychol., 1935. Cyclic reproductive behavior in the fe- 37, 107-143. male guinea pig. J. Comp. Psychol., 19, 313- Zuckerman, S. 1932. The Social Behavior of 335. Monkeys and Apes. New York: Harcourt, Young, W. C, and Orbison, W. D. 1944. Changes Brace & Company. 21 HORMONAL REGULATION OF PARENTAL BEHAVIOR IN BIRDS AND INFRAHUMAN MAMMALS Daniel S. Lehrman, Ph.D. PROFESSOR OF PSYCHOLOGY AND DIRECTOR, INSTITUTE OF ANIMAL BEHAVIOR, RUTGERS, THE STATE UNIVERSITY, NEWARK, NEW JERSEY I.' Introduction 1269 11.^ Hormones and Parental Behavior in Birds 1269 A. Nest-building 1269 1. Varieties of nest-building 1269 2. Correlations hplween nest-building and other behavior 1271 3. Nest-l)uilding and gonadal cycles 1272 4. Physiologic induction of nest- building behavior 1274 B. Egg-laying 1276 1. Egg-laying behavior in birds 1276 2. Hormonal relations in ovulation and egg-laying 1277 3. Stimulation of ovulation 1278 C. Incubation 1284 1. Incubation patterns 1284 2. Hormonal regulation of incuba- tion 1284 3. Interaction between internal and external environments in the regulation of incubation behav- ior 1294 4. Some remarks on the onset of incu- bation 1297 D. Care of the Young 1298 1. Types of young and methods of feeding them 1298 2. Hormonal induction of parental be- havior toward .young 1299 3. Induction of parental behavior to- ward young by external stimuli 1300 4. Physiologic nonidentity of incuba- tion behavior and brood}' care of the young ' 1302 III. Hormones and Parental Behavior in Infrahuman Mammals 1304 A. Nest-building 1305 1. Nest-building patterns in mam- mals 1305 2. Hormonal basis of nest-building. 1305 3. Induction of nest-building behav- ior by external stimuli 1309 B. Behavior during Parturition 1310 1268 1. Patterns of parturitive behavior. 1310 2. Physiologic aspects of parturitive behavior 1312 C. Retrieving of the Young 1313 1. Retrieving behavior 1313 2. Physiologic regulation of retriev- ing behavior 1314 ]). Nursing and Suckling Behavior 1321 1. Behavior of the nursing mother. . 1321 2. Milk ejection 1321 3. Mother-young relationships and the regulation of lactation 1325 4. Nursing behavior and the condi- tion of the mammary gland 1330 IV. General Discussion: the Psychobiol- oGY OF Parental Behavior and the Role of Hormones 1332 A. Learning and Hormone-induced Pa- rental Behavior 1332 1. General: formulation of the prob- lems 1332 2. Learning and parental behavior . 1332 B. Hormone Secretion as a "Behavioral" Response 1341 1. Neural conti'ol of hormone secre- tion 1341 2. Hormone secretion as a reflex. . . . 1342 3. Hormone secretion as a condi- tioned response 1343 4. Parental behavior and reflexly in- duced hormone secretion 1343 C. Mechanisms of Hormonal Action on Behavior 1344 1. Formulation of the problem 1344 2. Examples of peripheral contribu- tions to hormonal effects on be- havior 1345 3. Central hormonal effects on behav- ior 1348 4. The importance of behavioral anal- ysis 1351 D. Genetic and Evolutionary Aspects of Hormone-induced Parental Behav- ior 1352 PARENTAL BEHAVIOR 1269 1. Taxonomic diftVitMifes in parental behavior and in the mechanisms underlying it 1352 2. Strain differences and genetic fac- tors 1354 E. The Role of Parental Behavior in the Development of the Young 1355 1. In birds 1355 2. In mammals 1357 V. Scientific Names ok Animals Men- tioned IN Text 1359 A. Birds 1359 B. Mammals 1360 VI. References 1360 I. Introduction Almost all species of birds and mammals exhibit special behavior patterns the func- tion of which is to warm, feed, protect, or otherwise foster the development of their eggs and/or young. Although these behavior patterns vary widely in form, physiologic organization, ontogenetic origin, and degree of psychologic complexity, it is nevertheless sometimes convenient to group them to- gether under the rubric of "parental be- havior." Such behavior is of course appro- priate to, and ordinarily occurs only at, the stage of the reproductive cycle when there are eggs or young present or impending. Its regulation is, therefore, in part, a function of the reproductive cycle, and it is the pur- pose of this chapter to discuss the relation- ships between the (endocrine) reproductive cycle and the occurrence and organization of parental behavior. Decisions about what should be included in such a discussion are not easy to make, because what we call ''parental behavior" is not always as clearly differentiated within the animal's repertoire of behavior patterns as it is in our own conceptions. For example, mice of almost any age, either sex, and any physiologic condition may do a certain amount of nest-building, which is, to the observer, only quantitatively different from the more intense nest-building of the pre- parturient animal. Should a detailed dis- cussion of such behavior necessarily be in- cluded in a chapter on "parental behavior"? The nest-building of most species of birds is associated with the period of maximal sex- ual activity, and in some cases is incorpo- rated into the courtship pattern. How do we, for purposes of a book like this, draw a shari) line between "sexual behavior" and "parental behavior"? It is obvious that such decisions must sometimes be more or less arbitrary. For the purpose of this discussion, "pa- rental behavior" will include nest-building and behavior toward eggs and young, ending with the time at which the young are able to obtain food independently of their par- ents. It is perhaps inevitable that detailed physiologic experimentation should be for the most part limited to a few species of animals which are cheap and easy to breed in the laboratory, like rats, mice, and guinea pigs; or economically important, like do- mestic chickens ; or similar to human beings, like monkeys and chimpanzees. This con- centration of analytic work on a relatively few species has the effect of partially con- cealing from view the enormous diversity of behavioral and physiologic patterns that characterize the adaptation of animals to their environment. The diversity of behav- ior in different species, and of the physio- logic mechanisms underlying the behavior, may be just as great as is their diversity of form. This is not the appropriate place for an exhaustive survey of the varieties of parental behavior found in nature, but it may help to put the available work on hor- monal regulation of such behavior into per- spective if we from time to time briefly indicate the types which can be found, and point out that they are by no means limited to those characteristic of domestic and labo- ratory animals. II. Hormones and Parental Behavior in Birds A. NEST-BUILDING 1. Varieties of Nest-biiilding Structure and location. Each species of bird has its characteristic method of pro- viding a place for the deposition and incu- bation of the eggs, and the variability of nest construction within species is quite small. Between species, however, there are very wide variations. Some species build only above the ground (American robin, Herrick, 1911) or over water (tricolored red-winged blackbird, Emlen, 1941), some build only on the ground (herring gull, 1270 HORMONAL REGULATION OF BEHAVIOR Tinbergen, 1953) , in burrows that they make in the ground (bank swallow, Petersen, 1955) , or in natural cavities (purple martin, Allen and Nice, 1952). The pied-billed grebe builds a semifloating nest on the water (Glover, 1953) . The Baltimore oriole builds an elaborate covered woven nest of grasses (Herrick, 1911), the Florida jay a woven open cup (Amadon, 1944b), the storm petrel a simple scrape with a few scraps of material laid in it (Davis, 1957) , the black guillemot no nest at all, merely holding the egg on top of the webs of its feet (Storer, 1952). Swifts use their own saliva as cementing material, or in some species as almost the sole build- ing material (Lack, 1956a). Various species of megapodes, instead of building nests, build large mounds of vegetable material which creates the incubation temperature when it rots (Fleay, 1937; Frith, 1956b). Most birds build individual nests, but some species build communal nests in which sev- eral birds lay (smooth-billed ani, Davis, 1940a) , or massive woven communal struc- tures within which each pair has a separate chamber (sociable weaverbird, Friedmann, 1930). Share of the sexes in building. Male and female may share in nest-building, either approximately equally, as in house sparrows (Daanje, 1941), Florida jays (Amadon, 1944b), and great crested grebes (Simmons, 1955a), or in a A-ariety of special ways. In a number of species, the male is more active in nest-building at first, with the female doing more of it later on (e.g., red-backed shrike, Kramer, 1950; herring gull, Paludan, 1951; cliff swallows, Emlen, 1954; black- headed gull, Ytreberg, 1956). A frequently occurring special case is one in which the male builds the nest, and the female merely adds the lining (house wren, Kendeigh, 1941 ; graceful warbler, Simmons, 1954; coot, Kor- nowski, 1957) . In the green heron, the male at first selects the nest site, and does all of the gathering, carrying, and weaving of twigs into the nest. He does not permit the female to enter the nest until some time after he has taken up his territory. Once he has allowed her to enter the nest, however, he does most of the gathering and carrying of twigs, and the female does most of the building (Meyerriecks, 1960) . The male may build the nest with little or no help from the female, as in the red- shank (Grosskopf, 1958), the rook (Mar- shall and Coombs, 1957) , and many species of weaver finches (Friedmann, 1949). In the zebra finch (Morris, 1954), and the bronze mannikin (both of them weaver finches) (Morris, 1957), the male builds the covered nest, and the female shapes the in- side by sitting in it and making appropriate turning movements. In most species of meg- apodes, it is the males alone that construct the large mounds in which the eggs are to be laid (Coles, 1937, Frith, 1956b). An interesting variation of male nest- building is one in which the male builds several nests and the female selects one of them as the repository of the eggs. This oc- curs in several species of wrens, in which the nests are built by the male before the arrival of the female in the spring, and in which the female may line the nest she se- lects (long-billed marsh wren, Weller, 1935; house wren, Kendeigh, 1941 ; European wren, Armstrong, 1955). The male Carolina wren builds several such nests, but when the female arrives, both may build a new nest for the eggs (Kendeigh, 1941 ; Nice and Thomas, 1948). A similar pattern is found in many waders, in which the male makes several nests (mere scrapes in the sand) in the presence of the female, during courtship, and the female lays eggs in one of them (ringed plover, Laven, 1940a; lapwing, Laven, 1941). Building by the unassisted female is far more common than building by the male alone. When the nest is built entirely by the female, the role of the male may vary greatly. In some species, the male and fe- male associate only for the purposes of courtship and copulation, and nest-building and rearing of the young are done elsewhere entirely by the female (Gould's manakin. Chapman, 1935; boat-tailed grackle, Mc- Ilhenny, 1937; bower birds, Marshall, 1954; blackcock and rufi", Selous, quoted by Arm- strong, 1947) . When the male and female as- sociate on a territory during the breeding season, the role of the male may vary from complete indifference to the nest-building activities of the female (ovenbird, Hann, 1937), through merely accompanying her PARENTAL BEHAVIOR 1271 on her trips when she collects material (great tit, Hinde, 1952; bullfinch, Nicolai, 1956) to collecting material, but not build- ing (pied-billed grebe, Glover, 1953; Clark nutcracker, Mewaldt, 1956). Observers of some species have noted that the males show all the elements of the nest-building behav- ior in their courtship or other behavior, without ever integrating them into effective building (great reed warbler, Kluyver, 1955). Schantz (1937) observed a nest built by a male song sparrow, a species in which the nest is ordinarily built entirely by the female (Nice, 1943). In some species of birds, the male may play an important part in the selection of the site, even when he does not participate in nest-building (Hinde, 1952; Haartman, 1957). 2. Correlations between Nest-building and Other Behavior xA.s a first step in gaining some insight into the relationship between nest-building be- havior and the reproductive cycle, let us consider how the occurrence of nest-build- ing is related to other behavioral aspects of the cycle. Copulation and nest-building. In a num- ber of species, it has been reported that nest- building begins at about the time when the female becomes sexually receptive. The fe- male snow bunting, after being vigorously courted by the male over a 2 to 3 week period, permits copulation for the first time on the same day on which she first picks up and carries nesting material (Tinbergen, 1939b). The female ruffed grouse, too, be- comes sexually receptive the day she begins to build a nest (Allen, 1934) . In both species, the nest is built entirely by the female. Nest- building and copulation may also begin at the same time in species in which both sexes build (house sparrow, Daanje, 1941; cedar waxwing, Putnam, 1949; gulls, Paludan, 1951; Brewer's blackbird, Williams, 1952). Many observers have noted that, in vari- ous species of birds, copulation is limited to the nest-building period, which comes to an end before the eggs are laid (tricolored red- winged blackbird, Emlen, 1941; purple martin, Allen and Nice, 1952) . This implies that the eggs, some of which may be ovu- lated 8 or 10 days after the last copulation, must be fertilized by spermatozoa held in the oviduct for at least that time. Elder and Weller (1954) found that domestic mallard ducks could lay fertile eggs up to 17 days after being isolated from drakes, and Riddle and Behre (1921) report female ring doves laying fertile eggs after up to 8 days of iso- lation. Domestic hens may lay fertile eggs after 20 or more days of isolation (Hart- man, 1939) . It will be recalled that the male house wren builds several nests before the arrival of the female in spring, and that the female finishes one of them some time after her arrival. In this species, copulation is limited to the period of female nest-building (Ken- deigh, 1941). In the cliff swallow, another species in which the male does a substantial amount of nest-building before the pair is established in spring, copulation between members of the pair is not seen until the nest is well under way (the mechanics of copulation in this species are such that it cannot be performed at the nest-site vmless there is a partially built nest there) . How- ever, promiscuous copulations not involving the members of the forming pair may be seen earlier, at the places where mud is be- ing gathered for the nests (Emlen, 1954). Female white-crowned sparrows seem to start building a few days before the first copulations are observed (Blanchard, 1941). In domestic canaries, the peak of copulatory activity is normally slightly later than that of nest-building activity. However, if the partially built nest is removed each day, so that no nest accumulates in the nest- bowl, the peaks of copulation and of nest- building activity occur at the same time. This indicates that the peak of copulation usually occurs later than that of nest-build- ing behavior only because the presence of the nest inhibits nest-building activity (Hinde, 1958). Various elements of the nest-building be- havior, such as the billing or carrying of nesting material, are sometimes observed as part of courtship activity early in the season (Noble, Wurm and Schmidt, 1938; Armstrong, 1947) . Most of the correlations discussed in the foregoing paragraphs are derived from field 1272 HORMONAL REGULATION OF BEHAVIOR observations, which vary widely with re- spect to the continuity of observation, num- ber of birds observed, distribution of ob- servations during the day and during the cycle, etc. There is nevertheless a strong impression that, in many species of birds, the physiologic conditions encouraging cop- ulation {i.e., sexual receptivity of the fe- male) are the same as those inducing nest- building. Nest-building not correlated with copula- tion. There are some significant exceptions to this general impression. The female rook usually does not permit copulation until after the nest has been built by the male (Marshall and Coombs, 1957) . In the Euro- pean coot, another species in which the nest is built by the male, copulation is also de- layed until after the main shell of the nest is built (Kornowski, 1957) . In these cases it may be suggested that the nest-building activity of the male may play some role in stimulating those physiologic changes in the female which induce sexual receptivity (see below, p. 1275. Nest-building during incubation. Cases in which nest-building continues into the incu- bation period are for the most part of two general types, (a) There are some species in which the main part of the nest is com- pleted before any eggs are laid, and in which the lining of the nest (with a material differ- ent from that used for the main construc- tion) may continue during incubation (Cape weaver, Skead, 1947; graceful warbler, Sim- mons, 1954; bank swallow, Petersen, 1955). This suggests that, in such species, the se- lection of the different materials may have different hormonal bases, a suggestion for which there is some experimental evidence (see below, p. 1274). (b) Many species of gulls, terns, and shorebirds continue to build up the nest during the incubation period by virtue of a tendency to pick up nesting materials and drop or incorporate them in the nest whenever the birds' need to sit on the eggs is frustrated, or in conflict with some other behavioral tendency. Such nest- building has been called "displacement nest- building" (Tinbergen, 1952). It may occur, for example, when the bird is sitting on eggs abnormal in shape, size, or number (Moyni- han, 1955; Baggerman, Baerends, Heikens and ]\Iook, 1956), or when the members of the pair relieve each other at the nest {e.g., Cuthbert, 1954; Ytreberg, 1956). Baerends (1959) found that such displacement nest- building by a sitting bird also occurs when the temperature of the eggs departs too much from an optimal level. 3. Nest-building and Gonadal Cycles So far, we have established a probable temporal relationship between nest-building and copulation, at least in those species in which the female participates in nest-build- ing. As a further step in the analysis of the cyclic basis of nest-building behavior, we may now look into the relationship between the timing of nest-building activity and of the maximal activity of the ovarian follicle. In the absence of very much direct evidence on this point, let us adopt the somewhat roundabout procedure of considering, first, the timing of follicle growth, and then the timing of nest-building activity, in the cycle. Relation of follicle growth to time of egg- laying (see chapter by van Tienhoven). The ovary of a bird at the egg-laying stage looks like a cluster of ova of varying size, the larg- est being the one that is nearest to being ovu- lated. If these ova are measured at the au- topsy of a laying bird, the measurements form a graded series, which can be arranged in order, from most mature to least mature. If the interval between successive eggs is known for the species, and if the time of last ovulation is known for the individual, it is a simple matter to calculate the age (in days before ovulation) of each of the larger ova. The size of the ova can then be plotted as a function of pre-ovulatory age. In addition, the growth rate at each day before ovulation can be calculated by comparing the sizes of successive ova, the growth rate between day a and day b being a function of the in- crease in size of the ovum between day a and day b, in relation to the absolute size on day a (Romanoff, 1943). In a wide variety of species of birds which have been studied, there is a sharp increase in follicle growth rate starting from 4 to 11 days pre-ovulation, depending on the spe- cies (Romanoff and Romanoff, 1949). Ro- manoff (1943) has shown that the actual PARENTAL BEHAVIOR 1273 growth rates, and the changes in growth rates, are identical in a number of species. Riddle (1916) found that the rate of growth of the ovum of the domestic hen in- creased quite suddenly by a factor of about 25, some 5 to 8 days before ovulation (cf. Stieve, 1919; ^Yarren and Conrad, 1939; Marza and Marza, 1935) . In seasonal breed- ing birds (including most wild birds) the picture is basically the same. The ovary re- mains in a regressed state during the off- season; ova increase in size slowly during the early part of the breeding season, then very rapidly during the few days before ovulation. Bissonnette and Zujko (1936) found that the size of the largest ovum of the female starling increases very slowly for about 108 days (from December to March), and then very rapidly for about 26 days. During the 108-day period of slow growth, the growth rate remains stable at about 0.009 mm. per day; it then increases quite sud- denly to 0.285 mm. per day, an increase of about 31.6 times. The 26-day period of rapid growth found by Bissonnette and Zujko is based on the average sizes of ova from many different birds, which may have been at slightly dif- ferent stages of the cycle, although collected on the same days. When the sizes of the various ova in individual ovaries are plotted as a function of serial position, in birds that are already ovulating, it becomes apparent that the period of final rapid growth in any one ovum is about 10 to 11 days. Riddle (1911) and Bartelmez (1912) re- l^orted that the growth rate of ova in the domestic pigeon increases sharply (by 8 to 20 times) starting 5 to 8 days before ovula- tion {cf. Cuthbert, 1945). Stieve (19191 found that the volume of the largest ovum of the female jackdaw, after having re- mained quite constant during the months before the breeding season, increases from 24 cu. mm. to 1600 cu. mm. during the last 5 days before ovulation. Paludan (1951) observed that the ova of two species of gull, observed in the wild, grew most rapidly during the last 9 to 10 days before ovulation. It is clear that the characteristic pattern of growth of the avian follicle is that of a long period of slow, steady growth followed by the sudden onset of a short period of ex- tremely rapid growth which ends only when the ovum is ovulated. This final period of rapid growth lasts from about 4 to about 11 days, depending on the species. Relation of nest-building behavior to time of egg-laying. In most species of wild birds, nest-building appears to be sharply limited to a few days during the cycle. Female ruffed grouse begin to build about 6 days before the first egg (Allen, 1934) . Tricolored red-winged blackbirds (Emlen, 1941) and song sparrows (Nice, 1937) start about 4 to 5 days before the first egg. Similar patterns are found in other species of passerine birds (e.g., Clark nutcracker, Mewaldt, 1956; snow bunting, Tinbergen, 1939b). The usual description of this type of rela- tionship in the literature of field ornithology merely states that nest-building takes place during the few days before the first egg. When more exact quantitative observations are made, however, the situation seems somewhat more complex. Hinde (1958) weighed the amount of nesting material built into the nest on each day of the cycle in a number of female domestic canaries. He found that the intensity of nest-build- ing activity reached a peak some time be- fore the laying of the first egg, and then waned. The timing of the peak was subject to considerable variation, ranging in dif- ferent individuals from 7 to 0 days before the first egg. Field observations on the cedar waxwing by Putnam (1949) show a similar pattern. In this species nest-building ac- tivity reached a peak 2 or 3 days before the laying of the first egg. In some species the first egg appears, not immediately on the completion of the nest, but after an interval of several days follow- ing the last nest-building activity (purple martin, Allen and Nice, 1952; white-crowned sparrow, Blanchard, 1941 ; ovenbird, Hann, 1937; shrike, Miller, 1931). No information is available concerning possible differences in pre-ovulation changes in the ovary be- tween species having the two different pat- terns. Data on nest-building behavior and gon- adal condition. The data so far presented clearly suggest that nest-building behavior is often associated with the period of maxi- 1274 HORMONAL REGULATION OF BEHAVIOR mal follicle growth. In a few cases, observa- tions of follicle growth and of nest-building behavior have been made on the same spe- cies, and have usually led to the conclusion that this association does in fact exist. Em- len (1941, tricolored red-winged blackbird), Paludan (1951, herring gull), Petersen (1955, bank swallow), and Marshall and Coombs (1957, rook) have all noted that the period of nest-building coincides with that of maximal follicle growth. Mr. S. Glucksberg, in an unpublished study, de- stroyed the nests of several pairs of ring doves at the end of each day, and made daily counts of the number of pieces of nesting material built into the nest. He found that the amount of nest-building ac- tivity increased with increasing follicle size, reaching a peak at the time of ovulation. Similar observations have been made by Clausen (1959) on the homing pigeon. Unfortunately, there are not yet any data on nest-building and male gonadal cycles to compare with those available for the re- lationship of this behavior to the female cycle. It is clear that the nest-building ac- tivity of seasonal breeding birds in which the male participates in building usually oc- curs during the part of the year when tes- ticular secretory activity is at its height (Marshall and Coombs, 1957), but no ob- servations have been made on detailed changes in testicular activity, correlated with detailed observations on behavior. 4. Physiologic Induction of Nest-building Behavior The foregoing discussion makes it plain that, in the large number of species in which the female does most or all of the nest- building, nest-building behavior is associ- ated with the final period of maximal follicle activity. We may now examine evidence bearing more or less directly on the prob- lem of the hormonal induction of nest- building behavior. This evidence may be divided into two general categories: the in- duction of nest-building behavior by direct injection of hormones; and the elicitation of nest-building behavior by external stimuli. Hormonal induction of nest-huilding be- havior. The coincidence of nest-building be- havior and the period of rapid follicle growth just preceding egg laying strongly suggests that nest-building behavior is in- duced by ovarian hormones. We have gone into such detail about these and other co- incidences because very little direct experi- mental evidence is available, but what evi- dence there is confirms the impression that ovarian hormones often provide the physio- logic background for nest-building behav- ior. Lehrman ( 1958b) reported that the injec- tion of 0.4 mg. diethylstilbestrol daily over a 7-day period induces nest-building behav- ior in ring doves. Hinde and Warren (1959) indicate that the injection of estrogenic hor- mone into female canaries also induces nest- building behavior, but only in near-lethal doses. Hinde's observations on the nest- building behavior of canaries (1958) indi- cate that these birds change over from the use of grass to the use of feathers (which is the nest lining) shortly before egg-laying is due. This change-over occurs to some extent even though the stimulus situation in the cage remains the same (the nest is re- moved daily so that the birds cannot be stimulated by a completed nest) . This sug- gests that the change from the use of grass to the use of feathers is controlled in part by a change in hormonal condition, although such a change has not yet been induced by means of hormone administration. The suggestion is particularly interesting in view of the facts that, in some species of birds, the building of the main part of the nest stops abruptly with the beginning of egg- laying, but the addition of a lining of dif- ferent material may continue thereafter, and that in still other species, the male may l)uild the main part of the nest, whereas the female merely adds the lining (see above, page 1270. Cole and Hutt (1953) studying a number of nonlaying hens, found on autopsy that some of them had ovulated, failing to lay because of interrupted oviducts, impacted oviducts, etc. Others had not ovulated. The ovulators among the nonlayers were seen to enter nests on about 47 per cent of the observed days (about the same percentage as in the case of normal laying hens) . Non- ovulators entered the nests in only 5 per cent of the cases. This indicates that the be- PARENTAL BEHAVIOR 1275 havior toward the nest is influenced by hor- mones associated with ovidation, even in those birds in which the egg could not be l)roduced because of abnormalities in the oviduct. Progesterone has not yet been shown to induce nest-building behavior in birds. War- ren and Hinde (1959) found that this hor- mone had no effect upon nest-building in the domestic canary, either alone or in com- bination with estrogen. In view of the correlation between nest- l)uilding behavior, on the one hand, and, on the other hand, follicle growth, oviduct growth (Petersen, 1955) , and the readiness of the female to copulate, it is interesting to note that estrogenic hormone induces ovi- duct development in various birds (Brant and Nalbandov, 1956; Lehrman and Brody, 1957; see chapter by van Tienhoven), and female sex behavior in the domestic chicken (Adams and Herrick, 1955). Although it is reasonably certain that estrogenic hormone is the principle physio- logic initiator of nest-building in those typi- cal species in which the female does most or all of the nest-building, the situation is most unclear in those cases in which the male participates. Although we have noted in our laboratory that nest-building can be induced in ring doves by estrogen injection, and not by testosterone (see above), we do not yet have adequate observational evi- dence concerning the specific effects of es- trogen injections on the male and on the female. This evidence, when it is available, will be important and interesting, because in these birds the male typically brings the nesting material to the nest, and the fe- male builds it into the nest (Goodwin, 1955). In the brush turkey, the male builds a large mound by scraping leaves, mold, soil, etc., backwards wdth his feet. The fe- male later lays the eggs in holes burrowed into this mound, and they are incubated by heat generated by decaying leaves and mold. The male's head and neck are almost featherless, and covered with a red skin. This skin becomes brilliant red in each breeding season, a few days before the be- ginning of mound-building. This suggests, of course, that in this case male sex hormone is involved in nest-building activity. How- ever, during the period of most intense mound-building activity, the male does not allow the female near the area, which suggests further problems about the rela- tionship between male sex behavior, mound- building, and the hormonal bases thereof (Fleay, 1937). In the black-crowned night heron both the male and the female nor- mally participate in nest-building, incuba- tion, and the rearing of the young. Noble and Wurm (1940) found that testosterone propionate would induce nest-building be- havior in both males and females, whereas estrogenic hormones had no effect on the nest-building behavior of either sex. Fur- ther, they found that the change in color of the bills (yellowish to black) and legs (yel- lowish to rich pink), which normally oc- curs in both sexes at the beginning of the breeding season in the spring, can be in- duced in the off-season by injection of tes- tosterone propionate, but not by estrogenic hormones. The hormonal background of nest-building behavior in this case is differ- ent from that of the species in which the fe- male alone builds the nest, but it is also dif- ferent from the situation in the ring dove, in which both sexes take part in building. These cases indicate the complexity of patterns and mechanisms involved, and are only a sample of the considerable variety of unsolved problems posed by the nest-build- ing behavior of many species of wild birds, problems which are only hinted at in the work so far done on domesticated birds. Induction of nest-building by external stimnli. In species in which the female builds the nest unassisted (and this includes most of the species for which useful infor- mation is available), it seems that stimuli provided from the environment, including stimuli coming from the behavior of the male, may induce the hormonal changes which lead to the onset of nest-building. Howard (1920) described the typical breed- ing pattern of many species of songbirds, in which the male arrives first on the spring migration, and has his territory established by the time the female arrives some time later. The male appears to be ready to court and eager to copulate as soon as the female arrives, but the female at first does not per- mit copulation. There is a period of some 1276 HORMONAL REGULATION OF BEHAVIOR days or weeks during which the courtship attempts of the male end in "sexual flights" during which the male chases the female through the territory in a characteristically zig-zag flight path, at the end of which con- tact is abruptly broken off. Only after a period of such flights is the female ready to copulate. Tinbergen (1939b) reported that the female snow bunting begins to build a nest (and to copulate) after about three weeks of this type of courtship stimulation by the male. Captive female chaffinches build nests and lay eggs more readily when the}^ are stimulated by males (Marler, 1956). Vaugien (1948), working with serins, and Polikarpova (19401, working with house sparrows, found that females placed in cages without males would build no nests, whereas the presence of a male bird in a cage stimulated the females to build. Male and female herring gulls both build, but the female seems to become stimulated to take a greater part in nest-building by the ac- tivities of the male, who is more active at the beginning (Paludan, 1951). Lehrman (19o8a) reported that, when a jiair of ring doves which have had previous breeding experience are placed together in a breeding cage with an empty nest bowl and a supply of nesting material, nest-build- ing occurs after a 1- to 3-day period of courtship. If a male and female are kept for several days in a cage containing no nest bowl and no nesting material, they will be ready to start building a nest immedi- ately after the subsequent introduction of nesting material into the cage. If both birds have been pretreated with estrogen before being introduced into the cage (see above), the nest-building starts immediately, rather than after a preliminary period of courtship (Lehrman, 1958b). This suggests that par- ticipation in courtship may have brought the birds into nest-building condition be- cause it stimulates the secretion of estrogen. This is confirmed by Lehrman, Brody and Wortis (1961), who found that the oviduct of the female ringdove increases in weight about 5-fold, solely as the result of associa- tion with a male for 7 days. Significant in- creases in oviduct weight can be seen after less than 48 hours of stimulation by the courting male. Warren and Hinde (1960) found that the presence of the male domestic canary speeds up the development of nest-building behavior in the female in spring, when nest- building is presumably induced by endoge- nous estrogen. On the other hand, the l)resence of males has no effect upon nest- building behavior induced by estrogen in- jection in the winter. This undoubtedly means that the stimulation of nest-building behavior by the presence of the male is, at least in part, by way of the stimulation of estrogen secretion. Lack (1956b) noted that the male and female members of a pair of swifts (which keep the same mates year after year) may arrive at the nesting place on different days, but that nest-building does not start until the second member of the pair arrives from the south, even though the two members of the pair collect and use the material inde- pendently of each other. Many field ob- servers have noted that the songs and pos- tures of male birds may stimulate nest-building behavior on the part of the fe- male, but it is not always certain in these cases whether what is at issue is the stimula- tion of a hormonal change by the behavior of the male, or the stimulation of a behavioral response of which the female is capable as a result of hormonal changes which have already taken place. There is no doubt that both of these effects occur (Blanchard, 1941; Armstrong, 1955). Other external factors, such as the occur- rence of rainy seasons for tropical birds (Bullough 1951), the presence of suitable nesting sites (Lack, 1933; Marler, 1956), etc., seem to stimulate the onset of nest- building behavior. B. EGG-LAYING 1. Egg-laying Behavior in Birds The typical egg-laying pattern of the domestic hen, in which eggs are laid on several consecutive days, there is a gap of one or more days, and egg-laying is then resumed, and in which such clutches occur repeatedly during much of the year, is by no means typical of the egg-laying behavior of most species of birds. In fact, there oc- curs among wild birds just as great a vari- PARENTAL BEHAVIOR 1277 cty of egg-laying patterns, and therefore of relevant endocrine situations, as we noted in the case of nest-building. Size of clutch. Birds typically lay a clutch consisting of a definite number of eggs, and then incubate the eggs until they hatch. Some species of birds produce only one such clutch per year; others may breed twice, or even three times a year, but with the breed- ing always restricted to a definite part of the year. In the temperate zones breeding is always in the spring and summer; in the tropics some species may breed during the wet season, others during the dry season, and in a few cases breeding may be all year round. The number of eggs constituting a clutch varies from species to species within wide limits. Some birds, such as the large pen- guins, most auks and murres, petrels, and some others lay only 1 egg. Some birds, such as most species of pigeons and doves, char- acteristically lay 2 eggs. Most gulls lay and incubate 3-egg clutches. Most songbirds lay from 4 to 7 eggs. Large clutches, rather variable in size, are laid by ducks and geese, and gallinaceous birds such as par- tridges, pheasants, etc., may lay up to 20 eggs in a clutch. The domestic chicken is, of course, derived by selection from ances- tral birds of the latter type (Mayaud, 1950). Laying pattern. In those species in which the clutch consists of more than one egg, the interval between eggs is subject to wide in- terspecific variation (Mayaud, 1950). Data on the exact time of egg-laying are not as generally available for wild birds, of course, as they are for domestic birds, but certain wild birds, such as some species of ducks, appear to have a pattern like that of the domestic hen — they lay eggs at intervals of 20 to 24 hours. Most pigeons lay their 2 eggs about 40 hours apart (Whitman, 1919) . Although the most common pattern appears to be for the birds to lay their eggs on suc- cessive days, there are species in which the interval is much longer such as the black- headed gull, in which the interval is about 42 hours (Weidmann, 1956), some boobies, in which the interval may be 6 or 7 days (Mayaud, 1950) , and many others. Brood parasitism. An unusually interest- ing jihenomenon which poses several un- usual endocrinologic problems is the occur- rence of brood jiarasitism in several families of birds. Parasitic birds do not build nests of their own, nor do they incubate their eggs. Instead, the female lays her eggs in the nests of other (''host") species, and the hosts incubate the eggs and rear the young. This type of breeding habit appears to have evolved independently in several different families of birds. Of the 200 species of the order Cuculiformes, some 80 are to some degree jiarasitic (Makatsch, 1937) , includ- ing all 40 of the species of cuckoos living in the old world (Southern, 1954). Parasitism has also evolved among the cowbirds, a sub- family of blackbirds living in the new world (Friedmann, 1929), and in the honey guides of Africa (Friedmann, 1955). In addition, individuals of many other species of several families, especially ducks, quail, and pheas- ants, may breed parasitically more or less frequently (Weller, 1959). i. Hormonal Relations in Ovulation and Egg-laying This is not the place for a detailed dis- cussion of the hormonal basis of ovulation and egg-laying, since these matters are ex- tensively discussed in the chapter by van Tienhoven. However, a brief summary will serve as an introduction to certain prob- lems concerning the regulation of egg-laying behavior. The ovarian follicle grows under the in- fluence of a gonadotrophic hormone from the pituitary gland, which is presumably similar to mammalian follicle-stimulating hormone (FSH). The growing follicle se- cretes estrogenic hormone, which in turn stimulates growth of the oviduct. When the follicle has reached ovulatory size, an ovu- lation-inducing hormone, presumably simi- lar to luteinizing hormone (LH), induces the release of the egg. Traps <1955) sug- gests that progesterone (or a progestin) from the ovary induces the secretion of the ovulation-inducing hormone by the pitui- tary gland (Rothchild and Fraps, 1949). Progesterone has been found in the blood plasma of laying hens (Fraps, Hooker and Forbes, 1948; Layne, Common, Maw and Fraps, 1957; Lytle and Lorenz, 1958), non- 1278 HORMONAL REGULATION OF BEHAVIOR laying hens, and cocks (Fraps, Hooker and Forbes, 1949), but not in that of capons. In addition, progesterone induces the formation of secretion products by the albumen-secret- ing glands in the oviduct, the growth of which has been accomplished under the previous influence of estrogen (Brant and Nalbandov, 1956j. It thus seems probable that there is a short episode of progestin secretion by the ovary, just preceding ovu- lation, and that this follows a period of estrogen secretion. (See chapter by van Tienhoven.) The actual laying of the egg appears to involve some posterior pituitary activity. Injection of posterior pituitary preparation induces the laying, within 2 to 25 minutes, of eggs already ovulated, but which would not normally have been laid for up to 20 hours (Burrows and Byerly, 1940, 1942). When the neurohypophysis is removed no oviposition takes place until after there has been time for the regeneration of the nerve connection between the hypothalamus and the pituitary gland (Shirley and Nalban- dov, 1956a, b). Rothchild and Fraps (1944) removed the ruptured follicles and all the rapidly growing preovulatory follicles in hens, so that an ovulated egg was in the oviduct, but no more eggs could be ovu- lated. They then placed some of these hens in normally lighted rooms, others in rooms on reversed light cycles. The majority of eggs in both groups were laid during the daylight hours. They therefore concluded that a light-sensitive, nonovarian process was involved in the laying process, in addi- tion to those factors controlling ovulation itself. (See chapter by van Tienhoven.) 3. Stimulation of Ovulation Our interest in the nature of the condi- tions stimulating ovulation derives from the fact already pointed out, that nest-building activity is in part based on physiologic con- ditions induced by hormones coming from the developing egg follicle; and from the further fact that the physiologic events associated with ovulation somehow set the stage for the occurrence of incubation be- havior, which normally follows egg-laying. Neural stimulation of ovulation. It has become abundantly clear in recent vears that the activity of the pituitary gland is controlled and influenced in considerable detail by the hypothalamus (Harris, 1955; see chapters by Greep, Everett, and van Tienhoven). The physiologic and anatomic details of the relationship between the hy- pothalamus and the pituitary gland are ade- cjuately discussed in these other chapters, and do not concern us here. We may, how- ever, describe a striking example of the evidence for neural control of pituitary ac- tivity. Huston and Nalbandov (1953) sewed a loop of thread into the magnum of the oviducts of a group of domestic hens, and tied it into place, so that it provided a con- stant mechanical stimulation of the oviduct wall. Domestic hens normally ovulate 30 to 60 minutes after the laying of the previ- ous egg. During the 25 days following the operation, however, 58 to 75 per cent of the operated birds laid no eggs. Among the op- erated birds which did lay eggs, the mean number of eggs laid was 1.5 per bird; in sham-operated birds wdth no loop sewed into the oviducts, the number of eggs laid was 5.5 ])er bird. LH or progesterone injection could induce ovulation at any time in those birds which were not laying because of the ]:)resence of the thread. The ova of the ex- perimental birds did not degenerate, and their oviducts and combs remained normal. These data suggest that the mechanical stimulation of the oviduct wall inhibits LH secretion by the pituitary gland, without substantially interfering with the secretion of FSH. Huston and Nalbandov suggest that the jiresence of an egg in the oviduct acts in this way to prevent the ovulation of the succeeding egg until after the previous egg has been laid. External stimuli and ovulation. Since the secretion of gonadotrophic hormones can be influenced and controlled by the hypothala- mus, and by stimuli arising in the body outside of the central nervous system, it is reasonable to expect that external stimuli representing various environmental situa- tions and events may have an influence, through this neurohypophyseal link, on the activity of the ovary. (a) Light has long been known to influ- ence gonadal activity. In seasonally repro- ducing birds, the increasing length of the PARENTAL BEHAVIOR 1279 day i^^ the most important factor which en- sures that the reproductive system will be active in the spring. In addition, experi- mental work with domesticated birds indi- cates that the timing of ovulation and ovi- position during the day are influenced by the day-night light cycle (Farner, 1955; see chapter by van Tienhoven). In addition, there is considerable evidence that other environmental variables, such as those re- lated to temperature, food supply, and so on, play a significant role as regulators of the breeding season (Thomson, 1950; Mar- shall, 1959). (b) Stimuli provided by the courting male apparently influence the secretion of gonadotrophic hormones by female birds. It will be recalled that, in our discussion of the hormonal basis of nest-building behav- ior, we pointed out that nest-building be- havior is sometimes induced in the female as a result of stimulation by the courting male, and that there is reason to believe that the basis for this effect is that the courtship of the male stimulates the secre- tion of estrogenic hormones in the female. We may now examine some further evi- dence of the effect of stimuli provided by the mate upon the growth and ovulation of the egg. Bartelmez (1912) noted that the ovary of an unmated female domestic pi- geon contains follicles which do not exceed 5.5 mm. in diameter. When such a pigeon is lilaced with a male, she lays an egg after about 8 days (Harper, 1904). The growth of the ovum to the ovulation size of about 20 mm. is clearly caused by stimuli pro- vided by the male. Craig (1911, 1913) kept several pairs of doves so that the males and females could see each other from adjoining cages. The males were allowed in the cages of the females daily, but were prevented from copulating by the experimenter, who separatefl them with a wand at appropriate times. All of these females laid eggs within 9 days of the beginning of contact with the male, although, when these birds w^ere kept in isolation for a year preceding the begin- ning of the experiment, 5 out of the 6 fe- males had laid no eggs at all. Matthews (19391 showed that the short period of tac- tual contact between male and female which Craig had allowed was not necessary for the stimulation of ovulation. He showed that a female domestic pigeon would lay eggs as a result of seeing a male court her through a glass plate. We have found the same result in my laboratory, using ring doves. Both Matthews and Harper noted that, when two females are placed in a cage together, both may be stimulated to lay eggs. However, Collias (1950) found that ring doves in heterosexual groups laid more eggs than those in unisexual groups of the same size, indicating that the behavior of male doves is more stimulating to the secretion of gon- adotrophins in females than is the pseudo- male behavior which some of the female doves will adopt when no males are in the group. Polikarpova (1940) placed 50 female house sparrows in cages in which they re- ceived additional illumination daily, start- ing in the late fall. Twenty-five of them had males in the cages, the other 25 were alone. After about 50 days, none of the isolated females had started to build a nest, and of 17 such birds killed for autopsy, only 3 showed enlarged oviducts. On the other hand, all the females with males in their cages had nests, and 5 out of 8 birds ex- amined had fully developed oviducts. Burger (1942, 1949) kept female starlings in groups of various sizes, with or without males. He found that, when he provided ad- ditional illumination to such females either isolated or in groups, their ova were stimu- lated to grow to about 3 mm. When groups of males and females were caged together, the ova grew rather larger (5 mm.). When a single male and a single female were caged together, the ova of the female grew to about 10 mm. This indicates that the stimu- lus for the growth of the ovum is not merely the presence of a male, but probably also the presence of conditions which facilitate the formation of a pairing relationship nor- mal for the species. When Lack ( 1940, 1941) caged two pairs of robins or chaffinches in one aviary, the dominant pair bred nor- mally, the subordinate pair did not. In such birds, the full expression of normal male courtship behavior toward the female re- quires that the male be the territory holder, which in turn means that he must be the dominant bird, or the only male, in a con- 1280 HORMONAL REGULATION OF BEHAVIOR siderable space. Vaiigicn ( 1948) found that single female serins in individual cages would not lay eggs, but that eggs would be laid within a few days if a male was placed in a cage with the female. In the case of the shell parakeet, Vaugien (1951) showed that the sounds made by other birds influenced the growth of the oviduct. When female parakeets were kept isolated in small dark boxes of various sizes, they laid no eggs. When the box was placed inside an aviary containing a breeding pair (so that the ex- perimental bird could hear, but not see the breeding birds) about half of the experi- mental birds laid eggs within 12 days. When the remaining birds were sacrificed for au- topsy some 3 weeks later, they had enlarged oviducts, with the largest ova averaging 9 mm. in diameter. Controls kept out of hear- ing of breeding birds laid no eggs and, on autopsy, were found to have ova no larger than 1.5 mm. in diameter. Ficken, van Tienhoven, Ficken and Sibley (1959) veri- fied this effect on ovarian activity of sounds made by other individuals in parakeet flocks, and also reported that testis devel- opment was stimulated. Marshall (1952, 1954) described the mating behavior of bower birds, in which the male has a special display ground, where he builds a bower and displays to the female. This display stimulates the female to go off and build her nest and rear her young alone. It is clear from the above data that stim- uli provided by the courting male induce the secretion of gonadotrophic hormones by the female, and that this is possibly a source of the synchronization of the sexual cycles of male and female birds during the breeding season. (c) The presence of an appropriate nest- ing site and the availability of nesting ma- terial seem to be important factors in the conditions stimulating normal gonadotro- phin secretion during the breeding season in birds. Lack (1933) observed three col- onies of arctic terns at three different loca- tions near a lake. The first location was per- manently dry ; the second was water-logged for a short period in the spring, because of melting snows; the third was water-logged for a longer period, until a marshy area dried up. Although birds were present from the beginning of the season in all three of these locations, the birds in the first colony laid their eggs earliest, those in the third colony latest. Similar observations were made by Linsdale (1938), who found that the yellow-headed blackbird, which builds its nest only over water, will abandon the nest in midbuilding if the water dries up while the nest is being built, and will then build a new nest elsewhere, this involving a delay in ovulation. However, if the eggs are laid first, so that incubation is in progress when the water dries up, the birds stay on. It can thus be stated that the presence of ai)propriate nesting conditions facilitates ovulation, and thus presumably the secre- tion of gonadoti'oi)hic hormones by the pi- tuitary gland. The induction of ovulation by the avail- ability of nesting material has been shown experimentally in several species of birds. Like some other tropical species (Roberts, 1937; Bullough, 1951) , the red-billed weaver finch of central Africa breeds at irregular times, always following rainfall. Marshall and Disney (1957) showed that the stimu- lating factor following the rainfall is actu- ally the availability of nesting material. During the dry season, when no reproduc- tion was taking place in free-living birds of the species, Marshall and Disney kept groups of male and female red-billed weav- ers in four outdoor cages variously provided with combinations of the following: insect food, artificial "rain" from a sprinkler, dry grass of the type normally used by the birds as nesting material, and green grass of the same type. Birds having green nesting grass available built nests, regardless of whether "rain" was falling, and regardless of whether insect food was available. Further- more, the only females to lay eggs during the experimental period were those in the cages in which the males were building nests. Clearly, manipulation of nesting ma- terial by the males induced in the females hormonal changes leading to ovulation. Marshall and Disney also noted that the bills of the females kept with such males assumed breeding color earlier than did other females. This change in color is, of course, under hormonal control (Witschi, 1938). PARENTAL BEHAVIOR 1281 Whitman (1919) found that various spe- cies of doves and pigeons would not ovu- hite unless nesting material and nesting lo- cations were provided. Lehrman, Brody and Wortis (1961) found that the presence of nesting material plays a significant role in the stinmlation of ovulation. Female ring doves kept with males in cages not sui)plied with a nest bowl or nesting ma- terial will not ovulate as soon or in as high a percentage of the cases as will such fe- males kept in cages with males and an ade- (luate supply of nesting material (the inci- dence of ovulations after 6 days in the cage is 55 per cent without nesting material, 95 l)er cent with nesting material) . Differences in oviduct weight (and in frequency of ovu- lation) between the groups of birds with and without nesting material in the cage do not become apparent until some 5 or 6 days after the birds are placed in the cages, al- though, as reported above, increases in ovi- duct weight as a result of association or nonassociation with a male are to be seen within less than 48 hours. Since male doves collect most of the nesting material, while the females build most of it into the nest, and since no oviduct development is stimu- lated by nesting material in the absence of the male, it seems likely that the courtship behavior of the male ring dove which is not yet interested in nesting material causes estrogen secretion {i.e., FSH secretion by the pituitary gland of the female), whereas nesting material (or the behavior of the male which has nesting material available) later facilitates the secretion of progesterone (I.e., LH secretion by the female's pituitary gland). We may recall that progesterone induces both the final ovulatory pulse of LH from the hypophysis, and the histologic changes in the oviduct which occur after the albumen-secreting glands have been formed under the influence of estrogen. From the above data, it is clear that in some species in which the male participates in nest-building, the presence of nesting ma- terial and/or the change in behavior of the male which is made possible by the presence of nesting material, helps to stimulate ovu- lation in the female. There is some evidence that, in those cases in which the female does most or all of the building, ovulation may also depend to some extent on stimuli pro- vided by the nesting material and/or by participation in nest-building. Polikarpova (1940) starting on January 1st kept 11 fe- male house sparrows in cages supplied with a nest box and nesting material, while 10 females were kept in cages with neither nest box nor nesting material. On April 28th, when birds caught in the wild had fully de- veloped oviducts and eggs ready to ovulate, 10 of the 11 birds with nesting material had enlarged oviducts with a fully formed shell gland, whereas none of the 10 birds without nesting material had advanced beyond the first stage of oviduct enlargement. Vaugien (1948) removed the nest from the cage of female serins while it was being built or just after it was built, and reported that this prevented the birds from laying eggs. When he later replaced nests in the cage, eggs were laid within a few days. Berry (1943, 1944) found that geese of several different species could be induced to lay eggs by the pro- vision of artificial nests, although some of these birds had been in the park for years without laying. Hinde and Warren (1959) found that the absence of nesting material and a nest bowl delay ovulation in domesti- cated canaries. The presence of a nest and/or nesting ma- terial clearly facilitates ovulation, at least in some species of birds. Further, the effect of the presence of nesting material is, at least in some cases, quantitatively or quali- tatively different from the effects of stimuli provided by the courtship of the male. (d) A special, and most interesting prob- lem is posed by the egg-laying of brood parasites such as the cuckoos and cowbirds. How is the egg-laying behavior of such birds synchronized with the availability of host nests? Although there are some excep- tions (Kabat, Buss and Meyer, 1948; Davis, 1958), most birds do not normally lay eggs unless they have first built a nest. Although, as we shall see later, the laying of eggs in- volves hormonal changes which facilitate the subsequent occurrence of incubation be- havior, the brood parasites lay eggs without having built a nest, and without incubating the eggs afterwards. Hann (1937, 1941) states that the female cowbird first finds the nest by seeing the 1282 HORMONAL REGULATION OF BEHAVIOR host building it. She watches intently and for long periods during the nest-building. She visits the nest regularly in the absence of the owners before laying. Hann suggests that the development of the eggs, and their ovulation, in the cowbird are stimulated by the sight of the potential host building a nest, and that this accounts for the syn- chronization of the laying of the cowbird's and of the host's eggs (note that the para- site's eggs must be laid at a time when a host is prepared to incubate them). Accord- ing to Hann's observations, the cowbird's egg is laid some 4 to 5 days after she first begins watching, so that his hypothesis about stimulation of ovulation is plausible. However, observation of the behavior of cowbirds (Nice 1949), as well as histologic studies of their ovaries (Davis, 1942a I, in- dicate that the cowbird's eggs are laid in clutches of 3 to 5 eggs, with a rest period of some 5 to 8 days between clutches. This sug- gests a possibility that the cowbird, when such a clutch is growing, must find a nest, and that it finds a series of host nests be- cause it is about to lay the eggs, rather than laying the eggs because it has found the host nests. However, a series of studies by Chance (1940) on the European cuckoo in- dicates very strongly that a brood parasite may actually be stimulated to lay eggs by the availability of host nests. Chance in- duced cuckoos to lay abnormally long series of eggs (on the order of 20 to 25) by remov- ing eggs from the nests of foster species {i.e., potential hosts) so that they built new nests and relaid. He thus managed the situ- ation so that potential host nests were avail- able to the cuckoo over a much longer pe- riod of time than that during which the cuckoo normally lays eggs, and during which it normally has hosts available. By this method, he induced cuckoos which nor- mally lay 5 to 7 eggs to lay 20 to 25. The anis are a New AVorld subfamily of cuckoo-like birds, closely related to the parasitic cuckoos. Although not themselves, for the most part, brood parasitic, their breeding habits are peculiar in that some of the species nest in communal nests, several females laying in one nest. In some species only a few of the females will incubate, even though many more have laid the eggs. Davis (1940, 1942b) reported that these birds may lay their eggs on the ground, even quite far from the nest. Such eggs, of course, are not incubated. He also found that ovulation may be stimulated by the presence or ac- tivity of other birds. In several flocks, he noted that no egg-laying might take place for a long time, and that a sudden burst of egg-laying activity would occur after a new female joined the flock. Davis suggested that the breakdown of the normally rigid relationships between nest-building, egg- laying, and incubation, and the ability of these birds to lay eggs in response to visual and/or auditory stimulation by other birds, regardless of whether they have built a nest and regardless of whether they will incu- bate, may be features of their reproductive cycle which encourage the development of brood parasitism. Effect of eggs in the nest on ovulation. In nature each species of bird lays a charac- teristic number of eggs in a clutch, the vari- ation within species sometimes being ex- tremely narrow. In some cases the number of eggs laid is independent of the presence of other eggs in the nest. In other cases the number of eggs laid may be considerably extended by removing eggs as they are laid, the bird continuing to lay until the number of eggs present in the nest is approximately the normal clutch size. The term "determi- nate layer" is commonly used for those spe- cies in which the number of eggs laid is rather rigidly determined by physiologic re- lationships internal to the bird, whereas the term "indeterminate layer" is used for those species in which the size of the clutch may vary according to the situation in the nest (Cole, 1917; Laven, 1940b; Lack, 1947; Davis, 1955) . Among the domestic birds, the pigeon is a familiar example of a determi- nate layer, whereas the domestic hen is, of course, an indeterminate layer. Among wild birds, too, there are variations from species to species. For example, the lapwing (Klomp, 1951), and some songbirds (Davis, 1955) seem to be determinate layers, at- tempts to increase the size of the clutch by removing eggs as laid having been unsuc- cessful. On the other hand, female house sparrows have been reported to lay up to 50 eggs in regular succession when the eggs PARENTAL BEHAVIOR 1283 ^vere removed daily (Pearl, 1912; Witschi, 1935), and a flicker from whose nest an egg was removed daily, starting with the 2nd egg, laid 72 eggs in 73 days (Phillips, 1887). The wryneck has similarly been reported to lay up to 48 eggs under the same conditions (quoted by Pearl, 1912). Goodwin (1948) reported that the golden pheasant may lay clutches of up to 40 eggs if eggs are removed as laid. The best experimental work on indetermi- nate egg-laying has been done with gulls, which normally lay 3 eggs, at intervals of about 2 days (Goethe, 1937; Tinbergen, 1953). In the case of the black-headed gull, the average time between the laying of the 1st and the 3rd eggs is about 84 hours {ZV2 days) (Weidmann, 1956). The experiments of Weidmann on the black-headed gull may be summarized as follows (see also Ytre- berg. 1956): If the 1st egg is removed just after it is laid, the birds will lay a 4th egg, so that they end up with a 3-egg clutch. In these cases the 4th egg is laid at a normal interval after the 3rd. If successive eggs are removed as they are laid, most birds will lay more than 4 eggs, Weidmann having found birds laying up to 7 eggs. If the 1st egg is left in the nest, and subsequent eggs are removed as they are laid, no birds lay more than 3 eggs. If both eggs are removed after the 2nd