THE EGGS OF MAMMALS EXPERIMENTAL BIOLOGY SERIES Editors: Philip Bard, Johns Hopkins University; L. R. Blinks, Stanford University; W. B. Cannon, Harvard University; W. J. Crozier, Harvard University; J. B. Collip, McGill University; Hallowell Davis, Harvard University; S. R. Detwiler, Columbia University; Selig Hecht, Columbia University; Hudson Hoagland, Clark University; J. H. Northrop, Rockefeller Institute for Medical Research; G. H. Parker, Harvard University; Gregory Pincus. Harvard University; L. J. Stadler, The University of Missouri; Sewall Wright, University of Chicago. PACEMAKERS IN RELATION TO ASPECTS OF BEHAVIOR. By Hudson Hoagland NEUROEMBRYOLOGY. By Samuel R. Det- wiler THE EGGS OF MAMMALS. By Gregory Pincus Other volumes to follow ^ 1- THE EGGS OF MAMMALS BY GREGORY PINCUS Assistant Professor of General Physiology Harvard University NEW YORK THE MACMILLAN COMPANY 1936 Copyright, 1936, By the MACMILLAN" COMPANY ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER, EXCEPT BY A REVIEWER WHO WISHES TO QUOTE BRIEF PASSAGES IN CONNECTION WITH A REVIEW WRITTEN FOR INCLUSION IN MAGAZINE OR NEWSPAPER Published, August, 1936 SET UP AND ELECTROTYPED BY T. MOREY * SON PRINTED IN THE UNITED STATES OF AMERICA This Book Is Dedicated to W. E. Castle and W. J. Crazier PREFACE I should like to express my appreciation to Dr. J. B. Collip, Dr. H. Selye, Dr. D. L. Thomson, and Dr. W. J. Crozier for their kindness in reading the manuscript of this book before publication. Their comments have been taken advantage of in a manner for which I, not they, am responsible. I am indebted too to Dr. F. H. A. Marshall and Mr. John Ham- mond of Cambridge University for encouragement and interest which led to the undertaking of this monograph, and to my friend and collaborator Dr. E. V. Enzmann who actively assisted in a number of the investigations herein described. The National Research Council Committee for Problems of Sex and the Josiah Macy Jr. Foundation pro- vided grants making possible most of my own work, and the preparation of the monograph itself is due in no small measure to their assistance. To the editors and publishers of the following journals I am indebted for permission to reprint the various tables and figures indicated in the text: the American Journal of Anatomy, the American Journal of Physiology, the Anatomical Record, Archives de Biologic, the Biological Bulletin, the Carnegie Institution of Wash- ington Publications in Embryology, the Journal of Anatomy, the Journal of Experimental Biology, the Journal of Experi- mental Medicine, the Journal of Experimental Zoology, the Journal of Morphology, the Quarterly Review of Biology, and the Proceedings of the Royal Society. I ask the understanding of the reader if this account of the development of mammalian eggs seems at times to deal in summary fashion with some of the voluminous literature on this subject. The investigative aspects are what interest and intrigue me. I emerge confessedly with the impression that at best a qualitative basis for future work has been estabhshed, and since I am possessed by the belief that viii PREFACE accurate quantitative observatioDs afford the means for elucidating the nature of biological processes, I feel that this is a book of interrogation, not explanation. If it does indeed create curiosity its major objective will be attained. Gregory Pincus Cambridge, Mass. July, 1936. TABLE OF CONTENTS PAGE Preface vu CHAPTER I. Introduction 1 II. The Origin of the Definitive Ova 5 III. The Growth of the Ovum 32 IV. The Development and Atresia of Full-Grown Ova and the Problem of Ovarian Parthenogenesis ... 42 V. Methods Employed in the Experimental ]\Ianipula- tion of ^Mammalian Ova 62 VI. The Tubal History of Unfertilized Eggs .... 68 VIL Fertilization and Cleavage 75 VIII. The Activation of Unfertilized Eggs 98 IX. The Growth and Implantation of the Blastodermic Vesicle 112 X. Summary and Recapitulation 128 Bibliography 131 Author Index 155 Subject Index 159 IX THE EGGS OF MAMMALS CHAPTER I INTRODUCTION The behavior of mammahan eggs from the time of their genesis in the ovary to their implantation in the uterus is the subject matter of this book. The attempt has been made to include experimental investigations of the growth and development of ova rather than morphological descriptions. This is not an easy task, because an acute morphologist may make deductions about the nature of his material which are far more illuminating than those of an eager but inexpert experimenter. Furthermore, except for certain notable investigations of ovarian dynamics, there has been no extensive inquiry into the physiology of living mam- malian ova. It has been tacitly assumed, for example, that the reactions involved in the activation of non-mammalian ova occur also in mammalian eggs. Until quite recently no attempt has been made to test even this assumption. Since the middle of the last century a controversy has raged about the possibility of ovarian parthenogenesis. Almost every observer of mammalian ovaries has contributed an opinion, but no one has tried to see if ovarian eggs can be induced to develop parthenogenetically. Experimentation has lagged presumably because of the difficulty of handling living ova. It is interesting to note that the discovery of the mamima- lian egg by von Baer in 1827 led initially to extensive ob- servations of living ova. At first the exact morphology of the egg and its membranes was a matter of some debate (see Wagner, 1836; Jones, 1837, 1838, 1885; Barry, 1838; 1 2 THE EGGS OF MAMMALS Bischoff, 1842). Following Barry's (1839) initial observation of cytoplasmic cleavage there ensued a long series of ob- servations on the developmental history of fertilized eggs. Attention gradually shifted from living eggs to fixed speci- mens, chiefly employed for the determination of the exact cytology of fertilization and the histological changes occur- ring during differentiation. This resulted in the publication of numerous detailed descriptions of the early embryology in various classes of mammals (Bischoff, 1845, 1852, 1854; Bonnet, 1884, 1891; Caldwell, 1887; Hartman, 1916, 1919; Heape, 1883, 1886; Hensen, 1876; Hill, 1910, 1918; Hill and Tribe, 1924; Huber, 1915; Hubrecht, 1912; Jenkinson, 1900, 1913; Keibel, 1888, 1894, 1899, 1901, 1902; Lams and Doorme, 1908; Lams, 1910, 1913, 1924; Melissinos, 1907; Minot, 1889; van Oordt, 1921; Reichert, 1861; Rein, 1883; Robinson, 1892; Sakurai, 1906; Selenka, 1883, 1884, 1887; Sobotta, 1893, 1895; Tafani, 1889; Van Beneden, 1875, 1880, 1899, 1911, 1912; Van Beneden and Julin, 1880; Weil, 1873; Wilson and Hill, 1907). The hving egg was neglected presumably because no technique was developed for pre- serving it intact in vitro long enough for any extensive experimentation to be performed. Nor did the possibility of experimental manipulation of ova in vivo receive more than passing attention (see Grusdew, 1896; Novak and Eisinger, 1923). Since the pubhcation of Stockard and Papanicolou's (1917) and Long and Evans' (1922) exhaustive accounts of the oestrus cycle of the guinea pig and rat respectively, a new era in the study of sexual physiology has been initiated. Enormous strides have been made in the discovery and purification of the hormones regulating the activities of the genital tracts of mammals. The ovarian control of the various phases of the sex cycles in the female has received exhaustive attention, and the control of gonad function by the anterior pituitary has been investigated in detail. Despite the enor- mous accumulation of data on the endocrine regulation of the ovarian and oviduct environment of ova, the ova them- INTRODUCTION 3 selves have received relatively little attention. The study of the hormonal control of ovarian function has centered upon the relation of hormone activity to the development of follicle and corpus luteum. The ovary has been largely considered as a sort of diphasic machine geared for hormone production by certain specialized follicle components. Its primary function as a producer of gametes has been rela- tively neglected. The endocrine control of the proliferative, secretory and contractile activities of the oviducts them- selves is known in detail, and it is tacitly recognized that all these activities have as their end and aim the nutrition and protection of the developing egg. Yet the exact nature of the dependence of the o\aim upon these activities is still problematical. We are now provided with the sort of knowledge that should certainly make profitable in vivo experimentation with eggs. Brachet (1912, 1913) did indeed take advantage of the development of a tissue culture technique in order to in- vestigate a specific stage of development in rabbit ova. But neither the availability of the technique nor Brachet 's suggestive discourse led to any acti\^e investigation until. 1929 when Lewis and Gregory published their account of the cinematography of rabbit ova developing in culture. Since then a number of workers associated with Lewis (Gregory, 1930; Squier, 1932; Lewis and Hartman, 1933; Lewis and Wright, 1935) have conducted a fairly intensive examination of living ova, chiefly with the object of cul- turing fertilized eggs. In addition to these investigations and similar work undertaken by Nicholas and his coworkers (Nicholas and Rudnick, 1933, 1934; Defrise, 1933), the physiological properties of developing ova have been ex- amined from quite different angles. So there exists a meas- urable body of work of recent origin which is properly experimental. Wherever possible the factual data of this work have been presented in the hope that these, speaking for themselves, may stand side by side with any interpreta- tion herein presented. 4 THE EGGS OF MAMMALS It is the earnest belief of the writer that these experi- mental inquiries represent a small fraction of the work that should and will be done. The enormous variety and richness of mammalian material that is available and un- tapped should provide an extraordinary temptation to ex- ploitation now that a beginning has been made in the de- velopment of technical facilities for the manipulation of this material. I emphasize that only a beginning has been made. This book is a beginning. CHAPTER II THE ORIGIN OF THE DEFINITIVE OVA A long-lived controversy concerns itself with the origin of the definitive germ cells. Do they arise de novo from somatic tissue in the sexually mature adult, or are they segregated as primordial precursors early in embryogeny? Weismann's theoretical considerations (1883, 1904, also Nussbaum, 1880) on the continuity of the germplasm led initially to the active investigation of this problem. In the light of modern the- oretical genetics the strict interpretation of the Weismannian dogmata is probably no longer necessary. For, since the data of genetics indicate that every normal nucleus in the organism contains the full complement of genes and that somatic segregation of genes is a rare and exceptional phe- nomenon, it is no longer necessary to postulate the trans- mission of a special, unimpaired germ tissue. The problem of the origin of the germ cells thus properly becomes one concerned with the dynamics of embryonic differentiation and peculiarly one of regeneration. In fact most of the recent experimental approaches have been concerned with the probability of the regeneration of germ cells from somatic tissues. Able reviews of the general problem are contained in the paper of Heys (1931) and the monograph of Harms (1926). Since we are concerned specifically with the origin of the definitive ova of mammals the question that we may set is concerned less with general theory and more with pertinent fact. We want to know what processes are responsible for the emergence in the ovary of the functional eggs. We may at once distinguish two types of investigation. The first, essentially descriptive, is concerned with the de- velopment of the ovary and its germ cells from early em- 5 6 THE EGGS OF MAMMALS bryonic life through sexual maturity. The second is con- cerned with varying the conditions of ovarian growth by experimental means and deducing from the derived data the nature of the factors concerned in the production of functional eggs. We shall assume that these two types of observations are distinct, and consider them separately as: (1) the morphogenesis of egg cells and (2) the experimental investigation of the growth of egg cells. The Morphogenesis of Egg Cells Thanks to the Weismannian controversy we have avail- able a fairly detailed description of oogenesis in embryonic life. It is unnecessary here to enter into a detailed descrip- tion of the embryogeny of the mammalian ovary (see Jenkinson, 1913, de Winiwarter, 1901, de Winiwarter et Sainmont, 1909, Brambell, 1927 and esp. 1930). Our inter- est lies in the so-called ^'primordial" germ cells of the em- bryo, since it is to these cells that a number of observers trace the origin of the definitive ova. The general opinion seems to be that large wandering cells originate from the entoderm of the gut before or at the time of the formation of the genital ridges (Nussbaum, 1880; Fuss, 1911, 1913). These primordial germ cells migrate to the gonad site and enter the genital ridges. The ridges are first seen as thickenings of the peritoneal epithelium between the base of the mesentery and the Wolffian duct on the ventral side of the developing mesonephros. The thickened peritoneal epithelium becomes the germinal epi- thelium and the primordial germ cells complete their migra- tion when they become arranged beneath this epithelium which then proliferates medullary tissue into the germ cells. The underlying mesenchyme forms connective tissue trabec- ulae in the medulla and also the primitive tunica albuginea which separates the medulla from the germinal epithelium. There are among investigators various opinions about the role of the primordial germ cells. A number maintain that these are the only germ cell precursors. The increase in THE ORIGIN OF THE DEFINITIVE OVA 7 number of these cells is by mitosis only, and no new cells are recruited from somatic tissue. This view is set forth at some length by Hegner (1914, also Vanneman, 1917). It leads naturally to the conclusion long maintained as a biological truism that by the end of embryonic life or shortly thereafter the complete quota of future eggs is attained (c/. Waldeyer, 1870 and 1906; Felix, 1912 and Pearl and Schoppe, 1921). The calculations of Aschner (1914) indi- cating the presence of some 400,000 ova in the human ovary at birth furnishes an apparent statistical substantiation. Furthermore, meiotic phenomena are observable in these primordial germ cells during embryonic and prepubertal life (Cowperthwaite, 1925) but not thereafter, and the as- sumption is made that typical meiosis is necessary for the formation of definitive ova. This conception of a large early store of future ova is scarcely controverted by a second group of investigators who admit the primordial germ cells as precursors of the future ova, but who claim that additional egg cells are supplied by proliferations from the germinal epithelium. Brambell (1927) in a careful study of the developing gonads of the mouse finds that the primordial germ cells persist throughout embryonic life and undergo maturation stages, but declares that additional cells from the germinal epi- thelium must be responsible for the large increase of cortical cells found in the gonad before the formation of the tunica albuginea in ten and twelve day embryos. Perhaps the largest group of observers consists of those who also consider post-pubertal production of new egg cells non-existent or negUgible but who find that the primordial germ cells degenerate and are replaced by secondary pro- liferations during embryonic or prepubertal life. Thus Rubaschkin (1908, 1910, 1912) decided that the large dif- ferentially staining primordial germ cells with their prom- inent attraction spheres degenerate in the early guinea pig embryo and are replaced by two successive proliferations from the germinal epithelium. De Winiwarter and Sainmont 8 THE EGGS OF MAMMALS (1909) describe a degeneration of the primordial germ cells in the cat ovary and their replacement by ingrowths from the germinal epithelium from three and one-half to four months after birth {cf. Kingsbury, 1913 and 1914a; Foulis, 1876 and Balfour, 1878). De Winiwarter (1910) observed the same phenomena in human ovaries. In the rat embryos Firket (1920) observed a secondary proliferation following degeneration of the first generation of germ cells. Kingery (1917) in a detailed study of oogenesis in the mouse found that the definitive oocyte arose from secondary pro- liferation begun at three to four days before birth and last- ing until thirty-five to forty days post partum. He found no evidence for oogenesis after puberty. In the rabbit Buhler (1894) also found only prepubertal ovogenesis. Simkins (1923 and 1928) questions the vahdity of the term primordial germ cells, going so far as to state that in the human embryo they are not large wandering cells at all but large liquefied areas surrounding degenerating nuclei. He attributes complete autonomy to the genital ridge. Kohno (1925) recognizes primordial germ cells in the hu- man embryo but declares their origin is in lateral plates of the mesoderm whence they reach the gonad via the gut epithelium and mesentery. Hargitt (1925) also denies the peritoneal origin of the germ cells in rat embryos declaring that large differentially staining cells are found throughout the embryo in the epithelium, mesoderm, ectoderm, gut entoderm and extra embryonic tissues. The disappearance of these cells he attributes to division, not to migration into the genital ridge. A number of more recent investigators have observed a more or less continuous proliferation of ova from the ger- minal epithelium throughout life. The chief modern protag- onists of this view are Robinson (1918), Arai (1920a and 6), Allen (1923), Papanicolou (1925), Butcher (1927), Swezy (1929a, 1933a and h) and Evans and Swezy (1931). Their histological studies are essentially confirmations of earlier observations on post natal ovaries (Pfluger, 1863 — cat; THE ORIGIN OF THE DEFINITIVE OVA Schron, 1863 — cat and rabbit; Koster, 1868 — man; Slawin- sky, 1873 — man ; Wagener, 1879 — dog; Van Beneden, 1880 — bat ; Harz, 1883 — mouse, guinea pig, cat; Lange, 1896 — mouse; Coert, 1898 — rabbit and cat; Amann, 1899 — man; Palladino, 1894, 1898— man, bear, dog; Lane-Claypon, 1905, 1907— rabbit; Fellner, 1909 — man) save that the work of Allen and those who follow takes advantage of recent discoveries of the nature of the oestrus cycle, and presents observations made upon ovaries taken at def- inite times during the cycle. Since the embryogenesis of the primordial germ cells and the germinal epithelium are separate loo — and distinct it follows from the findings of these observers that the definitive ova of adult hfe do not arise from the primordial germ cells at all. Most of the earlier workers observed evi- dences of growth and thickening Fig. l. The frequency of mi- of the germinal epithelium or r^^rttiaTa'cf AUe"! even extensions of germinal epi- 1923. Open circles indicate com- thelium into the ovarian cortex. ^^,f ^^^^ on semi-spayed mice. Halt circles mdicate normal un- In some cases these signs of operated controls. Abscissae are activity were associated with stages of oestrus cycle; l, early pro the period of heat. Allen (1923) whose investiga- tions are perhaps pioneer to the most recent developments mitoses per mouse. (From the distinguished four stages in the ^^nerican Journal of Anatomy.) behavior of the germinal epithelium of the adult mouse during the oestrus cycle. The first, characterized by ex- tensive mitotic activity occurs just before and during oestrus (see Figure 1). The second is marked by a fairly abrupt decrease in mitosis frequency, and a position of the daughter epithelial cells one cell layer below the germinal epithehum oestrus; 2, late prooestrus; 3, pro- oestrus to oestrus; 4, early oestrus; 5, oestrus; 6, early metoestrus; 7, metoestrus; 8, dioestrus. Or- dinates are average number of 10 THE EGGS OF MAMMALS due to the plane of cell division (Figure 2). In the third stage the daughter cells extend two cell layers below the epithelium. And by the fourth stage, occurring during dioestrus, several hundred young ova surrounded by a few folUcle cells are found just beneath the epithelium (Figure 3). vm~' Fig. 2. A late anaphase in the germinal epithelium of the mouse. The plane of division is nearly parallel to the surface of the ovary. (From the American Journal of Anatomy.) According to Allen the tunica albuginea forms ^'from con- nective tissue ingrowth during the absence of ovogenetic proliferation of the germinal epithelium." Allen notes a relatively intact tunica in animals that have had a long period of dioestrus and also a complete or an almost complete absence of young follicles. Cowperthwaite (1925) has criticized Allen's data On the grounds that he gives no demonstration of the presence of meiosis in these presumable new ova. Typical meiotic phenomena in adult ovaries have, in fact, rarely been ob- served. De Winiwarter (1920) noted oocyte formation in the region of the hilum in ovaries of cats shortly after puberty but no such process in the remaining tissue, and Gerard THE ORIGIN OF THE DEFINITIVE OVA 11 (1920) observed typical meiotic prophases in nests of young oocytes in the adult ovaries of Galago. On the basis of these observations and the presence of typical oocytes in certain undescribed adult ovaries of Loris (material of Prof. J. P. Hill and Dr. A. Subba Ran), Brambell (1930) inclines to the belief that these primate oocytes derive from primitive oogonia, not the germinal epithelium. Fig. 3. A stage 4 ovum (see text) in the mouse. Note complete layer of follicle cells. (From the American Journal of Anato7ny.) In rodents, however, such typical meiotic prophases have never been described. Here the observations of Swezy (1929a) and also of Evans and Swezy (1931), are very much to the point and apparently resolve the mystery. Swezy found the classical meiotic stages in the oocytes of rat embryos and young rats up to five days post partum (Plate I, Figs. 1-5), but she noted definite degeneration of all these ova by the loth day post partum. By the 10th day definitely atypical synizesis and pachytene stages occur (Plate I, Figs. 6-13) and in 15 day old rats (Plate I, Figs. 14- 16) synizesis stages are rare or missing, the pachytene mod- ified to a chromatin aggregation much less sharp than in typical stages, and the diplonema chromosomes also less distinct. On twenty day old rats (Plate II, Figs. 17-22) nuclear growth of oocytes involves essentially similarly mod- ifications, and in the adult the new ova derived from the ger- Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 f-'^fl:^ '#^ fT,« t->"-% ^ Fig. 7 Fig. 8 \ \ \ '^' \ A 1 Fig. 9 ,5 '"^^ ^^ Fig. 10 Fig. 11 Fig. 12 Fig. 13 -.. r 1^ Fig. 14 Fig. 15 Fig. 16 Plate I. (From the Journal of Morphologij) Figs. 1-5. Nuclei of ova from ovary of rat 5 days post partum. 1, Deutobroch nucleus in germinal epithelium. 2, Leptotene nucleus. 3, Synizesis. 4, Pachynema. 5, Diplonema. Figs. 6-9. Nuclei of ova from ovary of rat 8 days post partum. 6, Deutobroch nucleus. 7, Synizesis. 8, Stage following 7, evidently modified pachynema. 9, Dip- lonema. Figs. 10-13. Nuclei of ova from ovary of rat 10 days post partum. 10, Deutobroch nucleus. 11, Synizesis. 12, Modified pachynema. 13, Diplonema. Figs. 14-16. Nuclei of ova from ovary of rat 15 days post partum. 14, Deutobroch nucleus. 15, Modified pachynema. 16, Masses of chromatin changing into loose threads. 12 'f^'-i-yl. ^:, -'^ :-/ ^" , V»' Fig. 17 Fig. 18 Fig. 19 Fig. 20 Fig. 23 Fig. 24 Plate II. (From the Journal of Morphology) Figs. 17-22. Nuclei of ova from ovary of rat 20 days post partum. 17, Deuto- broch nucleus. 18, Beginning of the formation of clumps shown in next figure. 19, Modified pachynema. 20, Later stage showing characters of diplonema. 21, Nu- cleus toward the end of the growth period. 22, Final stage in twenty-day rat. Fig. 23. Nucleus in mature follicle from adult rat. Fig. 24. Nucleus from ripe follicle from adult rat. 13 14 THE EGGS OF MAMMALS minal epithelium contain mature nuclei (Plate II, Figs. 23- 24) in which the modification presaged in the younger animals attains culmination. These definitive ova show then a modified type of meiosis which involves essentially the dis- appearance of leptotene and synizesis, and the formation of an atypical pachynema and diplonema. Evans and Swezy (1931) obtained confirmation of these findings in the guinea pig, cat, dog, monkey and man. They point out that instead of being long-lived, the egg cells of mammals are subject to heavy mortality and exhibit a very short life cycle, correlated apparently with the length of the normal ovarian rhythm. In those animals in which the oestrus and ovarian cycles coincide {e.g., rat, mouse, guinea pig) the length of the oestrus cycle is a measure of the lifetime of the ovum in the ovary. These rather straightforward histological findings seem to indicate, on the whole, that the definitive ova originate from the germinal epithelium. All our recent knowledge of the rhythmic activity of the ovary with its periodic produc- tion of large numbers of young ova (Allen, Kountz and Francis, 1925) militates against the assumption of a single large initial store of ova gradually being exhausted through- out sexual maturity. The Experimental Investigation of the Growth of Egg Cells Any attempt to analyze the experimental data pertinent to the problem of the origin of the definitive ova encounters two difficulties. First of all many of the experiments are concerned with the simple Weismannian problem and ignore certain now obvious endocrinological implications. And secondly, the difficulty of experimental treatment of mam- malian embryos makes for a hiatus in our knowledge that can only be bridged by indirect deduction. The information that we do have at hand is derived from experiments concerned with the effects resulting from (1) bi- lateral ovariectomy, (2) partial ovariectomy, (3) ovarian THE ORIGIN OF THE DEFINITIVE OVA 15 transplantation, (4) the irradiation of ovaries with x-rays, (5) hypophysectomy, (6) the injection of gonad-stimulating hormones and (7) the transplantation of embryonic gonad rudiments. Bilateral ovariectomy has been extensively employed in order to determine whether ovarian tissue and eggs can be derived from somatic cells. It is a common experience that ovariectomized animals apparently regenerate ovarian tissue some time after the operation. Thus Davenport (1925) observed as many as 64 per cent of bilaterally ovariecto- mized mice with apparently functional ovarian tissue ap- pearing within a few weeks to several months after the ovariectomy. Such data may be explained as due either to: (1) regeneration of germinal tissue de novo from somatic cells or (2) the presence of accessory gonadal tissue distinct from the ovary and not removed during the operation or (3) the incomplete removal of ovarian tissue so that frag- ments remaining hypertrophy and attain dimensions suf- ficient to permit the manifestation of ovarian function. If the first alternative is accepted then it follows that neither germinal epithelium nor, presumably, primordial germ cells are necessary for the production of ova. The two latter alternatives exclude the first but scarcely affect the problem of origin via germinal epithelium or primordial germ cell though careful observation of the process of hypertrophy may yield pertinent data. Even if the first alternative is acceptable and may thus very well settle the ghost of germ- plasm continuity, it does not necessarily inform us about the normal process of egg production. In rodents accessory gonadal tissue is rarely, if ever, present. On the other hand, it is known that fragments of ovarian tissue, remaining after incomplete extirpation of the ovaries, will hypertrophy to such a remarkable degree that a completely normal ovary will be reestablished from which fertilizable ova are liberated (c/. Haterius, 1928; and Pincus, 1931). Furthermore it is quite possible to fail to extirpate small fragments of the irregularly lobed encap- 16 THE EGGS OF MAMMALS sulated rat and mouse ovaries, or even after careful excision to drop very small crushed fragments. A number of in- vestigators have therefore repeated Davenport's experiments using extreme operative precautions, in some instances going to the trouble of making serial sections of the extirpated ovaries in order to be certain of the completeness of removal. In practically every instance the per cent of animals showing return of oestrus symptoms or of detectable ovarian ^X_J Fig. 4. Section through ovary of young rat showing small, compact ovary. YF, young foUicle; C, ovarian capsule. LL, line of excision. (From the Quarterly Re- view of Biology.) tissue has been much below that reported by Davenport. Fallot (1928) found return of vaginal cornification in three out of twelve ovariectomized rats within six to six and one- half months after operation, and ovarian tissue was found in two of these. Parkes, Fielding and Brambell (1927) detected oestrus symptoms after operation in eleven out of one hundred and twenty-one mice, identifying ovarian tis- sue in eight of these eleven. Haterius (1928) also found apparent regeneration in 10 per cent of the mice he ovari- ectomized, and attributed the regeneration to incomplete extirpation. Pencharz (1929) reported return of oestrus in only three of 118 ovariectomized rats and mice, and demon- strated by serial sections of the ovarian region that incom- plete removal had been made in the case of these three. Heys (1929 and 1931), in an extremely careful analysis of THE ORIGIN OF THE DEFINITIVE OVA 17 a series of double ovariectomies in the rat, has demonstrated the presumable source of regenerated tissue in animals with apparently completely extirpated ovaries. In an initial series of 105 double ovariectomies she found germ cells at the ovarian site in eight cases, and observed that all eight YF -».FA Fig. 5. Section through the ovary of mature rat showing the iobed condition. YF, young fol- licle; F, follicle; FA, fatty tissue. (From the Quarterly Review of Biology.) occurred in the sixty animals over forty days of age. She noted that in females under forty days of age the ovary is relatively smooth and compact and not very heavily em- bedded in fat (Figure 4), whereas in older animals the ovary is Iobed and surrounded by a la]:ger amount of fat (Figure 5). She accordingly ovariectomized a second set of animals con- sisting of eighty-five females under forty days of age and twenty-three older females. Three of the older animals re- generated germ cells but none of the younger ones did. In several of the positive cases serial sectioning of the removed ovaries gave no detectable indication of lost fragments, but Heys believes that certain narrowly constricted lobes of 18 THE EGGS OF MAMMALS ovarian tissue might very well be lost and the loss not noticed upon serial sectioning (see Figure 5). Heys' results can scarcely be due to chance alone, the difference in regen- eration incidence between the young and older rats being 3.43 times the standard error of the difference, i.e., the odds are over 3000 to 1 against this being a chance difference. It is clear, therefore, that regeneration of ovogenetic tissue from somatic tissue is improbable in mammals. And cer- tainly the definitive ova are normally not recruited from somatic cells. We must turn to other experimental pro- cedures to obtain some insight into the processes that lead to the birth of ova in normal functional ovaries. The simple observation that unilateral ovariectomy or incomplete total ovariectomy leads to a compensatory hyper- trophy of the remaining tissue has led to a long series of researches which, often incidentally, form the basis for our modern knowledge of the elements of ovarian dynamics. The fact that such hypertrophy occurs was originally estab- lished both clinically (Robertson, 1890; Gordon, 1896; Sut- ton, 1896; Morris, 1901; Doran, 1902; Kynoch, 1902; and Meredith, 1904) and experimentally (Kanel, 1901; Bond, 1906; Carmichael and Marshall, 1908). An almost exact doubling of weight in the remaining ovary of unilaterally ovariectomized rats has been reported by Stotsenburg (1913) and Hatai (1913, 1915) and the number of eggs shed is demonstrably equal to the number normally produced by two ovaries (see Lipschiitz, 1924; Hanson and Boone, 1926; Crew, 1927; and Slonaker, 1927). In the opossum Hartman (1925) has reported a tripling of the weight of the remaining ovary and a similar threefold increase in the number of eggs shed. In the rabbit (Asdell, 1924; Hammond, 1925; Lip- schiitz, 1928) and the cat (Lipschiitz and Voss, 1925) a single remaining ovary or even small ovarian fragments produce the typical adult number of ripe follicles and eggs, but an exact compensatory hypertrophy of ovarian tissue is not so evident. Emery (1931) in a large series of uni- laterally ovariectomized rats found not a doubling in weight. THE ORIGIN OF THE DEFINITIVE OVA 19 but a one and one-half times compensatory hypertrophy when careful comparison with a control series was made. It is significant that in Emery's material about 50 per cent of the rats were found at autopsy to have large ovarian cysts. Similar cystic formations were observed in about half of the semi-spayed females in Wang and Guttmacher's (1927) series, and Wilhams (1909) reports that such cysts are commonly found in ovarian fragments left after incom- plete ovariectomy. Arai (19205) found definitely that the compensatory hyper- trophy in the rat is due exclusively to an increase in the number of large follicles and corpora lutea. Semi-spaying before puberty when the formation of corpora lutea does not normally occur led to a 40 per cent increase in ovarian weight, whereas semi-spaying after puberty led to a 100 per cent increase. Furthermore, by careful counts he established that the total number of follicles in the ovary does not in- crease after semi-spaying. In this Arai was confirmed by Alien (1923) who found that in semi-spayed mice the num- ber of ova differentiating from the germinal epithelium during stages 2 and 3 (vide supra) was scarcely larger than, normal whereas the average number of mature ova formed was normal. The implication from these studies is that the germinal epithelium produces a large more or less constant number of young ova, that some extra-gonadal factor is responsible for the ripening and maturation of a Hmited number of follicles, and that the maturing crop of ova are chiefly involved in the compensatory hypertrophy. It is now well established that an enormous atresia of young follicles occurs during the course of a single oestrus cycle. Thus in swine 14 per cent of the visible follicles less than 3 mm. in diameter become mature (Allen, Kountz and Francis, 1925) and in the rat of the ova less than 20 fx in diameter only 0.8 per cent attain a diameter greater than 60 M (Arai, 1920a). This extensive destruction of young ova and follicles is particularly striking in the dog and cat (Evans and Swezy, 1931) where all the new eggs (except 20 THE EGGS OF MAMMALS those ovulated) formed in the metoestrum and anoestrum preceding ovulation are completely degenerated by the time of ovulation. That the germinal epithelium is the source of new ova formed in hypertrophying ovarian tissue is demonstrated by the behavior of transplanted ovarian tissue. Among those who have observed the histological development in such tissue only Marshall and Jolly (1907, 1908) report complete disappearance of germinal epithelium with reten- tion of function. Lipschlitz (1928) notes a decrease in the number of primary oocytes in small fragments of rabbit ovaries in incomplete ovariectomy when comparison is made with similar sized fragments isolated from the ovaries in unovariectomized controls. But it is notable that his proto- cols describe a partially preserved or ''flattened" (degenerat- ing?) germinal epithelium in the experimental group whereas the germinal epithelium in the control fragments is appar- ently much better preserved. Tamura (1926) examining a series of ovarian transplants made onto the kidneys of male mice found the presence of primary follicles and many young ova associated with an actively mitotic germinal epithelium. Where the degree of activity of the germinal epithelium is less and more varied, small and medium sized or various sized folUcles are present. Apparently the activ- ity of the germinal epithelium is largely conditioned by the pressure of overlying connective tissue growths since its activity is greatest at free surfaces. Nonetheless, Tamura claims a rhythmical proliferation of ova from the germinal epithelium, but assigns a length of ten days to the ovogenetic cycle which is twice the length of the normal five-day oestrus cycle. Schultz (1900) and Voss (1925) also observed the persistence of functional germinal epithelium in their series of transplantations, but offer no such detailed an analysis as Tamura. Butcher (1932) has examined the nature of ovo- genesis in ligated ovaries and in autotransplantations of ovarian fragments and observed that the development of young ova is definitely associated with the activity of the THE ORIGIN OF THE DEFINITIVE OVA 21 germinal epithelium. Furthermore, in the hgated ovaries the follicles become necrotic and new ova are proliferated from the germinal epithelium which is relatively unimpaired. Athias (1920) has described proliferation of new ova from the germinal epithelium of transplanted guinea pig ovaries. No attempt has been made to make a quantitative study of the relation between the number of new ova formed and the amount of functional germinal epithelium in trans- planted or fragmented ovarian tissue, but it seems evident that the formation of new ova in such tissue occurs in the germinal epitheUum. Thus in Tamura's material the few cases of degenerated transplants were marked by a complete absence of germinal epithelium. It is possible, however, to preserve an intact germinal epithelium with total disappearance of follicles in x-rayed ovaries (Parkes, 1926, 1927a, b and c; Brambell, Parkes and Fielding, 1927a and h). Parkes and his coworkers have described in some detail the replacement of degenerated folUcular tissue by cellular proliferations from the germinal epithelium in the irradiated ovaries of mice. These pro- liferations never give rise to ova, however, though the ovaries seem to retain their hormone-producing capacities as evi- denced by the continuance of oestrus cycles of normal length in the irradiated animals. In the ferret (Parkes, Rowlands and Brambell, 1932) x-ray sterilization is also marked by an obliteration of the follicles and oestrin secretion, whereas in guinea pig ovaries (Genther, 1931, 1934) a transformation to luteal tissue usually occurs with only occasional follicle formation. Brambell (1930) inclines to the belief that the destruction of primordial ova is responsible for the lack of ovogenesis, but it is equally likely that the x-rays affect differentially the ovogenetic and hormone-producing capac- ities of ovarian tissue. It is notable therefore that the pro- liferation of new tissue from the germinal epithelium in x-rayed mice resembles the production of anovular follicles. Hill and Parkes (1931) have attempted to induce germ cell formation in mice with irradiated ovaries by means of in- 22 THE EGGS OF MAMMALS jections of pituitary and pregnancy urine extracts, but no ova were ever produced in the injected animals. That the early stages of ovogenesis in adult ovaries are scarcely under the control of pituitary hormones is abun- dantly evident from observations made upon the ovaries of hypoph3^sectomized animals. Smith (1930) noted that in completedly hypophysectomized rats no new large folUcles or corpora lutea develop, but the proliferation of young follicles goes on unimpaired for many months after hypoph- ysectomy. Swezy (19336) has presented quantitative meas- ures of the rate of ovogenesis in hypophysectomized rats, and her data indicate that a larger number of young ova may be produced in hypophysectomized females than in normal non-pregnant animals. In Table I is presented a suEMnary of her findings. TABLE I Numbers of Ova, Follicles axd Corpora Lutea in a Single Ovary of THE Rat during the Oestrus Cycle, Pregnancy and Pseudopreg- NANCY, and after Hypophysectomy AND THYROIDECTOMY. (From Swezy, 19336) Day of Average Stage Num- ber OF Rats Cycle (or Days AFTER Oper- ation) Age, Days Number of Ova AND Primary Follicles Average Number of Larger Follicles Average Number OF Corpora Lutea Total Oestrus cycle 5 2nd(l), 4th (4) 206-208 1809 171 27 2007 Pregnant and pseudopreg- nant 10 5 to 22 98-224 3857 311 16 4184 Hypoph3'sec- tomized 8 12 to 90 95-202 4164 — 20* 4184 Thyroidec- tomized 3 36 to 42 403 1371 193 15 1579 * Persisting old corpora. Swezy concluded from these data that there is a basic rate of ovogenesis which is observed in hypophysectomized animals. That the increased number of ova in hypophysec- tomized animals is due to an increased rate of production and not merely to accumulation is proven by the absence of any unusual number of degenerated ova. This rate is THE ORIGIN OF THE DEFINITIVE OVA 23 decreased when the hypophysis is secreting active maturity hormone as in non-pregnant females. The maturity hormone is concerned with the ripening of large follicles, ovulation and corpus luteum formation. During pregnancy and pseu- dopregnancy maturity hormone is secreted only in sub- threshold amount, as evidenced by cyclic ovarian changes OVA OF LARGER SIZES \ \ A -20 4 B -40-f C-OV 0" 400 - \ ER 60 " 200 - iW- /•^. — X- -^ — xB 100 - .....X.— J *^ - NUMBER OF OV^ \ (TOTA L) :f^z-: — ^C ^, V _ ^ • t _ — — 100 200 300 700 800 900 1.000 400 500 600 AGE - DAYS Fig. 6. Showing the total number of ova as well as the number of ova of different sizes in the albino rat at different ages (condensed). (From the American Journal of Anatomy.) in the ovary during pregnancy (Swezy and Evans, 1930), so that the hypophysectomized level is attained. During the normal non-pregnant ovogenetic cycle that portion marked by the presence in the ovary of ripe follicles and fresh corpora lutea is always associated with a minimum of small, newly formed ova. The pituitary secretions, then, are concerned with promotion of o\ailation and luteinization and presumably inhibit ovogenesis to a certain extent. The factor controlling ovogenesis is unknown although the effects of thyroidectomy indicate that the thyroid may promote ovogenesis to a certain extent. It should be pointed out, however, that the thyroidectomized rats were much the eld- est of the lot and Arai (1920a) has demonstrated a small 24 THE EGGS OF MAMMALS decline of ovogenesis with age in adult females (see Fig- ure 6) . The experiments of Engle (1928) demonstrate adequately that pituitary secretions are responsible for the later stages of maturation. He injected anterior lobe tissue into normal and semi-spayed rats and found that the per cent of hyper- trophy due to pituitary stimulation was approximately equal in the two groups of animals. We have already noted that in compensatory hypertrophy the increased ovarian weight is due to the doubling of large follicle and corpus luteum number, the number of primary follicles being the same in a single ovary whether the second ovary is present or not. Swezy (19336) also determined the effect of various pitu- itary hormone preparations upon ovogenesis in adult and immature rats. Her data are collected and summarized in Table II. Immediate verification of the conclusions deduced from Table I is found in the data derived from the injection of rat hypophyses into adult and immature rats (columns [11, [10] and [11]). Rat hypophyses are notably rich in gonad stimulating hormones (Smith and Engle, 1927), and their administration results in a decrease in the rate of ovogenesis, and an increase in total ovarian tissue. The data on the immature rats are particularly striking, for a few days of pituitary administration results in a halving of the total number of ova. Arai (1920a) found that the average total number of ova in prepubertal rats was about 10,000 and approximately 6000 in post-pubertal animals. Beef hypophyses, on the other hand, are relatively poor in maturity hormone and rich in growth hormone. Evans and Simpson (1928) have demonstrated an antagonism be- tween the growth and gonad-stimulating hormones of the anterior pituitary. The increase in follicle number following beef hypophysis administration (column 2) might then be interpreted as a neutralization of the intrinsic maturity hormone effect by the growth hormone of the beef pituitary. TABLE II The Number of Ova, Follicles, Cysts and Corpora Lute a in Sin- gle Ovaries of Rats Subjected to Various Hormone Treat- ments. (From Swezy, 19336) Treatment (1) Rat hypoph- ysis (2) Beef Vsc.c. hypoph- ysis (3) Beef Vs c.c. hypoph- ysis plus rat hypoph- ysis (4) Preg- nancy urine (5) \U c.c. theeUn (6) 21-34 c.c. follicular fluid (7) V4-I c.c. growth hormone (8) V4-I c.c. growth hormone (9) 0.5 c.c. growth hormone (10) Control (11) Rat hy- pophysis No. OF Rats Age OF R.A.TS (Days) Days OF .\D- MINIS- TRA- TION Ova AND Pri- mor- dial Fol- licles Large Fol- licles Corpora Cysts To- tals 1661 5 153-182 9-20 1436 155 58* 12 4 153-168 9 4017 228 20 3 4268 1 154 9 1476 295 4 none 1813 2 172-174 10 3322 216 20 12 3570 6 181-190 18 3574 245 22 none 3841 6 183-254 10-14 2183 203 18 none 2404 6 255 35-97 4996 144 3 5143 2 255-408 60 and 394 2277 190 60 — 2527 2 1 139 and 141 24 9 1952 7225 300 31 — 2283 7225 5 24- 26 2- 8 3664 — present in some 3664 Weight MGMS. 227=* 42 97 59 26 sub- nor- mal (3) and hy- pophy- secto- mized types 76 ma- turity type 35 (mixed type) 9.5 67t * Varied with amount of hypophysis. t Average of three. 25 26 THE EGGS OF MAMMALS Simultaneous injection of beef and rat hypophysis tissue results in inhibition of ovogenesis (column 3). When, however, examination was made of the ovaries of animals receiving injections of growth hormone extracts various results were obtained. In six of the ten animals ob- served (column 7) the expected result was obtained, namely an inhibition of ovarian growth and a rise in the rate of ovogenesis. Two animals (column 8) with normal, good sized ovaries exhibited a normal rate of ovogenesis, and two animals (column 9) with somewhat decreased ovarian weight gave no indication of increased ovogenesis. Two interpretations of these data are possible: (1) the growth hormone preparations may in some instances have con- tained sufficient maturity hormone to overcome the typical growth hormone effect or (2) there may have occurred in some of the injected animals a conversion of growth hormone to maturity hormone (c/. Evans, Meyer and Simpson, 1932; Evans et at., 1933). It should be pointed out that Reiss, Selye and Balint (1931a, h) have obtained from the pituitary extracts free of growth hormone which also inhibit the action of maturity hormone. Swezy's extracts are not made in a manner that would free her preparations of such ma- terials. Obviously the use of highly purified extracts and carefully timed injections should assist in resolving the situation. Pregnancy urine extracts (column 4) seem to increase ovogenesis to some extent. It is known that pregnancy urine is only partially effective as a maturity hormone (Engle, 1929; Evans and Simpson, 1929). Prolonged oestrin injection is known to reduce ovarian growth (Doisy, Curtis and Collier, 1931; Leonard, Meyer and Hisaw, 1931; Spencer, D'Amour and Gustavson, 1932; Pincus and Werthessen, 1933), presumably by inhibiting secretion of maturity hormone from the anterior pituitary (Meyer, Leonard, Hisaw and Martin, 1932). One would expect therefore that the data of columns 5 and 6 should show an enhanced ovogenesis. It is interesting to note THE ORIGIN OF THE DEFINITIVE OVA 27 that this seems to be the case when relatively light oestrin doses are injected (column 5), but not with heavy doses (column 6). The theelin-injected animals received about 6.25 r.u. per day, and while continuous vaginal cornification resulted, an apparently normal cycle of uterine changes occurred and the ovaries appeared relatively unimpaired. It is possible that in the animals receiving light doses the ovogenesis inhibiting capacity of maturity hormones was impaired but not the follicle stimulating capacity. The heavier dosages may have caused the hydropic degeneration of the germinal epitheUum described by Doisy, Curtis and Collier (1931) and so prevented maximum ovogenesis, al- though Swezy makes no note of such degeneration. Swezy, noting that normally during the oestrus cycle there is a drop in the production of new ova at the period just suc- ceeding the period of maximum oestrin production (e.^., ovulation), is inclined to attribute this drop (and therefore the results in her oestrin-injected animals) to a factor other than the ''suppression" of hormone secretion from the pituitary. Recently Hisaw and his collaborators have advanced an explanation of the oestrus rhythm which involves a sep- aration of the maturity principle of the pituitary into two hormones (Fevold, Hisaw and Creep, 1934; Lane and Hisaw, 1934; Hisaw, Fevold, Foster and Hellbaum, 1934; and Lane, 1935). One hormone is follicle stimulating, the other lutein- izing and a chemical separation of the two has been attained (Fevold, Hisaw and Leonard, 1931 ; Fevold and Hisaw, 1934). These investigators report an increase in the total number of follicles in rat ovaries on administration of follicle stim- ulating hormone to prepubertal rats but no increase when luteinizing hormone is administered. Their count of ''total follicles" includes only ova in definitely formed follicles. Swezy (19336) attributes the ovogenesis inhibition to the luteinizing hormone. It is possible, therefore, that in addi- tion to the ovogenetic activity which is independent of the hypophysis {e.g.^ the ovogenesis seen in hypophysectomized 28 THE EGGS OF MAMMALS animals) a stimulation to ovogenesis may be engendered by the follicle stimulating hormone. Hisaw and his collab- orators find that corporin (the hormone of the corpus luteum) exerts effects on the ovary like those of the follicle stimulat- ing hormone while oestrin decreases the secretion of follicle stimulating hormone and stimulates luteinizing hormone production from the hypophysis. Pregnant and pseudo- pregnant animals may therefore exhibit an increase in ovo- genesis due to direct action of corporin from their corpora lutea, whereas animals in oestrus and those receiving oestrin injections show reduced ovogenesis perhaps because of the action of the induced luteinizing hormone secretion. It is obviously not possible to arrive at any final decision concerning the factors governing ovogenesis until additional pertinent data are available. The most concise summary of the evidence indicates that ovogenesis occurs from the germinal epithelium at a typical intrinsic rate which may be reduced by the action of a hormone or hormones from the anterior pituitary. But even this deduction requires further verification in the form of careful quantitative esti- mates of ovogenesis in its relation to atresia, and particularly an inquiry into the nature of the atresia of young ova and folhcles. We are completely unaware of the intimate nature of the intrinsic proliferative capacity of the germinal epi- thelium. How does it compare with the mitotic index of tissues generally? Is it a self-perpetuating phenomenon in the sense that the atresia of its products releases substances stimulating cell division? We shall see for example that the atresia of maturing follicles is often accompanied by the formation of mitotic spindles and it is well known that cytolized cell products (trephones) promote cell division. An extraordinary variety of problems suggest themselves. Patience and the formation of substantiated hypotheses will result in their solution. In summating the evidence relating to the normal ovo- genetic processes in prepubertal and post-pubertal animals little doubt remains that the definitive ova are proliferated THE ORIGIN OF THE DEFINITIVE OVA 29 from the germinal epithelium. What then is the role of the primordial germ cells of the embryo? Are they essential structures or merely incidental? There are practically no illuminating experimental data on the development of em- bryonic gonads. The experimental manipulation of mamma- lian embryos is dependent upon the elaboration of techniques now in the process of initiation. Certain investigations of gonadogenesis in amphibian and chick embryos offer provocative suggestions, but their applicability to mammals has yet to be proven. In the chick a gonad or gonad-like organ may form free of primordial germ cells. This can be demonstrated by removal or destruction in three to nine somite embryos of the anterior crescent in which the primordial germ cells originate. The embryos nonetheless develop small gonad rudiments (Rea- gan, 1916; Benoit, 1930). Willier (1932, 1933a and h) has excised the germ cell crescent and transplanted the entire blastoderm and found a sterile gonad developed in the transplant. In the frog (Kuschakewitsch, 1910) sterile gonads free of germ cells develop from the genital ridge when delayed fertilization prevents germ cell migration, Humphrey (1928), on the other hand, finds that in Ambly- stoma gonads form in grafted tissue only when a sufficient number of primordial germ cells are located beneath the coelomic epithelium which gives rise to the germinal epi- thelium. ' It is notable that in all instances gonads arising free of primordial germ cells are sterile. Thus Domm (1929) found in the fowl that if the large functional left ovary is removed prior to the time of the disappearance of the germ cells from the small rudimentary right gonad the latter develops into a testis which produces sperm. If excision of the left ovary is delayed until the time when the germ cells of the right gonad are no longer present (the germ cells normally dis- appear by the third week after hatching) a sterile testis develops. Willier (1933a and h) has demonstrated by means of 30 THE EGGS OF MAMMALS chorio-allantoic grafts of the gonad-forming areas of chick gonads that germ cells remaining outside the germinal ridge area do not differentiate into oogonia or spermatogonia, whereas those that become situated under the germinal epithelium develop as typical sex cells. On the basis of this and other evidence he agrees with Witschi (1929) that the cortex {e.g., the cortical sex cords) of the gonad acts upon the germ cells as a specific organizer of female sex cells, and the medulla as organizer of spermatogenetic tissue. In the free-martin of cattle, which is a female twin develop- ing in utero under the influence of the hormones of its male partner, a sterile testis-like organ develops. It is notable that while typical male sex cords are present, germ cells are absent (Chapin, 1917; Willier, 1921). Perhaps in the case of the free-martin (as in the frogs with delayed ferti- lization) a spermatogenetic tissue is not formed because primordial germ cells do not reach the gonad. If these data are generally applicable to manamals it would seem that although ovogenesis takes place from the germinal epithelium the formation of a functional ovary is dependent upon the primordial germ cells. We have seen, in the case of x-rayed ovaries, that an ovary with morphologically normal germinal epithelium may be incapable of forming ova. A necessary mechanism is lacking. It may be that the primordial germ cells are the precursors to this mechanism in normally developing ovaries. The evidence from the free-martin and recent data on the transplantation of embryonic gonad rudiments indicates that, as in amphibia and birds, the development of an ovary in embryogeny is dependent upon the formation of a cortex in the developing gonad. Normally in ontogeny the gonads of both sexes are morphologically indistinguishable for some time. The genital ridge, as already noted, consists of ger- minal epithelium overlying primordial germ cells. At about the 10 mm. stage in both the pig (Allen, 1904) and cat (Sainmont, 1905) and at the 12th day post coitum in the mouse (Brambell, 1930) the germinal epithelium begins to THE ORIGIN OF THE DEFINITIVE OVA 31 proliferate the primary sex cords from its inner surface. During the formation of these cords (or nest of medullary cells as in man [Felix, 1912]) the gonad is still morphologi- cally indifferent. Morphological differentiation may be con- sidered as initiated when these primary cords become iso- lated in the medulla by the formation of the primitive tunica albuginea under the germinal epithelium in the male gonad and the proliferation of a second set of cortical sex cords from the germinal epithelium in the female gonad. In the embryonic ovary the medullary cords persist for some time but are rarely found after birth; the cortical cords break up to form primitive follicle cells surrounding the primordial ova. Buyse (1935) has transplanted rat gonads in the morpho- logically indifferent stage onto the kidney of adult rats of both sexes. Over 60 per cent of the transplants developed as testes, 16 per cent as ovaries and the remainder were bisexual gonads or gonads of undetermined sex. A small percentage of the gonads classified as rudimentary testes seemed to be transformed ovaries. It will be seen that if these are included in the group of gonads other than testes the normal sex ratio is approximated. Since the type of gonad developed was not correlated with the sex of the host Buyse concludes that adult sex hormones do not affect sex differentiation. The differentiation was then dependent on the history of the sex cords in the transplanted tissue. Presumably the clear cut segregation of testes was due to the presence of formed primary sex cords, e.g., the testis organizers, whereas various types of zygotic ovaries were obtained dependent on the probability of formation or partial formation of the cortical sex cords. CHAPTER III THE GROWTH OF THE OVUM We have seen that the production of ova from the germinal epithehum may proceed in the absence of the hypophysis. But does the formation of mature ova depend upon hypo- physeal hormones? It is clear that ovulation and particularly the number of follicles that liberate ova is dependent upon hypophyseal hormones. Does this dependence involve merely a maturation of the follicular apparatus or is the actual growth of the ova also concerned? In fixed material cells distinguishable as primary ova are in the mouse a little less than 7 microns in maximum diameter (Pincus, unpublished data), in the rat 8 microns (Aral, 1920a). They eventually attain maximum diameters of 65 to 70 microns. What are the factors governing the growth of these ova to maximum size? While direct measurements are unavailable it seems obvi- ous that in hypophysectomized animals the ovum attains the maximum size. Smith (1930) notes that the primary follicles in hypophysectomized rats ^^continually are under- going development, but invariably undergo atresia not later than the stage of cavity formation." Swezy (1933) notes the presence of a follicle having a diameter of 270 microns in a rat ovary 90 days after hypophysectomy and mentions foUicles with diameters of 200 microns. It is evident from the figure in Selye's (1933) paper that foUicles with antra occur in 43 day old rats hypophysectomized at 18 days of age. In the dwarf mouse the largest follicles are about 200 microns in diameter and contain antra (Pincus, un- published data). Now it has been demonstrated (Brambell, 1928) that in the mouse the diameter of the follicle when the ovum is 32 THE GROWTH OF THE OVUM 33 fully grown is 125 microns and in the rat (Parkes, 1931) the maximum diameter of the ovum is attained when the folhcle is 160 microns in diameter. Full growth of the ovum, then, is attained just before the time of antrum formation which begins in rats and mice in follicles having diameters of about 200 microns. We may therefore deduce that the ova of hypophysectomized animals attain the di- mensions of the mature ova in o\ailating animals, and that the growth of the ova (and early follicular growth) is inde- pendent of the hypophysis. This conclusion is supported by various independent lines of evidence. Aral (1920a) found that ova over 60 ijl in diameter appear in the ovaries of rats between the 15th and 20th days of age. Engle (1931a) found pseudomaturation spindles, which appear only in ova of full size, first evident in 16 day old mice and no follicles more than 180 /z in diameter in 14 day old mice. Smith and Engle (1927) found that 10 day old mice treated with gonad-stimulating pituitary implants had to have daily implants for 5 days in order that full ovarian response should be attained, whereas 17 day old mice showed full response in 36 hours to 3 days. Corey (1928) found practically no ovarian response to pituitary extracts in rats until after the 15th day, and Selye and Collip (1933) found no follicular maturation in 6 to 12 day old rats treated with anterior pituitary-like hormone (see also Zondek, 1931). In rabbits (Hammond and Marshall, 1925) the antrum develops later than the 10-1 1th week of life. Hertz and Hisaw (1934) were able to obtain definite follicular response to follicle-stimulating and luteinizing hor- mones only in juvenile rabbits (12 to 13 weeks old), not in infantile rabbits. Casida (1935) reports that pig ovaries show definite response to pituitary hormones only when antrum- containing follicles are present. Nonetheless, fully potent pituitaries are present in 5 to 8 day old rats (Smith and Engle, 1927; Lipschutz, Kallas and Paez, 1929) as judged by their effects in transplantation to immature recipients. It would seem, then, that the at- 34 THE EGGS OF MAMMALS tainment of a certain degree of follicle maturity and full ovum size is necessary before activation of the pituitary hormones can be attained in developing animals. It is to H O o o p, 50 O 10 X J«i. . X-rX— X- _x__x_^x- X X V r X X X X x — X -/ / ' / 1 1 1 1 1 100 500 600 2U0 300 4U0 DIAMETER OF FOLLICLE Fig. 7. Showing the relation of ovum growth to folhcle growth. Data on the mouse. (From Brambell, 1930, courtesy of The Macmillan Company.) be remembered, however, that the release of substances from the normal gland in vivo and the injection of excised 70- > o o g 40- H < 50 30 20 (b) / •: . OBSERVATIONS X CALCULATED POINTS 50 100 400 450 500 Fig. 8. 150 200 250 300 350 DIAMETER OF FOLLICLE, n. Same as Fig. 7. Data on the rat. (From the Proceedings of the Royal Society.) preparations are not comparable phenomena. Furthermore, dwarf mice pituitaries can stimulate ovarian growth in im- mature recipients (Smith and MacDowell, 1931) yet their THE GROWTH OF THE OVUM 35 follicles do develop to the stage of antrum formation. The absence of eosinophile cells in the pituitaries of dwarf mice may, however, indicate the absence of a necessary link in the chain of steps involved in the hypophysis-gonad relationship. Whatever the effect of ovarian maturation upon the pi- tuitary may be, it is plain that no follicular response to pitui- tary hormones occurs until the time when full sized ova are present. Does this mean that the maturation of the follicle 120 iioH 100 ^.90 ^ 80 ^ 70 O 60 H 50 H S ^ 30 (a) . / (b) . OBSERVATIONS X CALCULATED POINTS 100 200 300 400 500 600 700 800 900 1000 DIAMETER OF FOLLICLE, (jl. Fig. 9. Same as Fig. 8. Data on the ferret. (From the Proceedings of the Royal Society.) is dependent initially upon some influence of the ovum, or is the simultaneous development of the ovum to full size and follicular growth to stimulable size a coincidence only? The ovum may grow to full size without an investiture of follicle cells as attested by the frequent presence of such ova in the ovaries of dwarf mice (Pincus, unpublished data). On the other hand, anovular follicles do occur in mammalian ovaries (League and Hartman, 1925) though those of large size represent follicles with completely resorbed ova (Engle, 19276). It is interesting to note also that frequent produc- tion of anovular follicles from the germinal epithelium takes place in senile rats (Hargitt, 1930). 36 THE EGGS OF MAMMALS That the growth of the folHcle beyond the antrum stage is independent of the growth of the ovum is amply evident from the data presented by Brambell (1928), Parkes (1931) 90 P 70 fe 60 O g 50 H W S 40 < SO 20 (b) • OBSERVATIONS X CALCULATED POINTS 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 DIAMETER OF FOLLICLE, fx. Fig, 10. Same as Fig, 7, Data on the pig, (From the Proceedings of the Royal Society.) and Pincus and Enzmann (19366). In Table III are presented the data collected by Parkes on the relation of ovum size to body weight and follicle size in seven species of mammals. 150 - / D — o ^ 6 7 8 9 100 ~ / / A 50 t ^ A n h 1 1 1 I 1 1 1 1 200 400 600 800 1000 1200 1400 1600 1800 DIAMETER OF FOLLICLE Fig, 11. Same as Fig. 7. Data on the rabbit. The lower curve represents ovum diameter plotted against folhcle diameter for the nine types of follicles (see Plate III) distinguished by Pincus and Enzmann, 19366. Figures 7 to 10 relate the various diameters of ova to the diameters of the enclosing follicles. In the rabbit, Pincus and Enzmann (19366) have identified 9 types of follicles each distinguished by characteristic features of the develop- ing ovum, granulosa and theca (see Plate III). When the k^ '9 Fig. 1 Fig. 2 Fig. 3 Fig. 7 Fig. 8 Fig. 9 Plate III. The development of the folhcle and ovum in mature rabbit does. Fig. 1, Type 1 folhcle to the left, type 2 follicle to the right. Nuclei in late con- densation of prophase. Fig. 2, Follicle type 3. One row of follicle cells. Fig. 3, Fol- hcle type 4. Two rows of follicle cells. Fig. 4, Follicle type 5. Many rows of follicle cells. Nucleus migrating to periphery. Fig. 5, Follicle type 6. Antra forming. Fig. 6, Follicle type 7. Numerous antra. Fig. 7, Follicle type 8. Ovum suspended in "spider web" of follicle cells. Corona formed. Fig. 8, Follicle type 9. Last preovulatory stage. Fig. 9, Showing position of various follicle types beneath the germinal epithelium. 37 38 THE EGGS OF MAMMALS mean ovum diameters are plotted against the mean follicle diameters (see Figure 11) the resulting curves essentially resemble those illustrated in previous figures, the full ovum size being attained in follicles of type 5 which just precede antrum formation. TABLE III Size of the Graafian Follicle at Various Stages of Its Life-History (From Parkes, 1931) Species 1 Approximate Weight of Young Adult Female 2 Diameter OF Ovum 3 Diameter of Follicle WHEN Ovum Is Fully Grown 4 Diameter of Follicle when Antrum Appears 5 Diameter of Follicle at Ovulation gm. M /^ M mm. Mouse 2 X 10 70 125 200 0.55 Rat 1.2 X 102 63 160 200 0.90 Ferret 5 X 102 108 170 230 1.4 Rabbit 2 X 103 84 145 250 1.8 Baboon 1.2 X 10^ 83 180 310 6.0 Pig 5 X 10^ 76 300 400 8.0 Cow 4 X 10^ — — — 15.0 The data plotted in this manner give no indication of the absolute rate of growth of ova though the relative growth rates may be deduced from the rising segment of the curves drawn to these data. These first segments are plotted in Figure 12, wherein it might be deduced that the ferret ovum grows at the most rapid rate, the pig ovum at the slowest rate, if com- parable rates of follicular growth occur in the various species. If it be assumed that the various types of follicles de- scribed by Pincus and Enzmann represent developments occurring at equal time intervals then the lower curve of Figure 11 may be taken as a representation of the growth curve of the ovum. The sigmoid shape of this curve is in fact reminiscent of general growth curves. It cannot be taken as a true growth curve, however, until the time necessary for the development of each type of follicle is accurately known. Such information might very well be obtained from ovaries subjected to x-irradiation and ex- amined at various intervals after exposure. THE GROWTH OF THE OVUM 39 110- 100 90- 60- 50 The data of Aral (1920a) give a slight indication of the rate of growth of ova since his tables show no ova above 20 /z in diameter in 1 day rats, the first appearance of 20 to 40 ijl in ova in 3 day rats, the first appearance of 40 to 60 /jl ova in 10 day rats, and ova over 60 ijl in rats over 15 days of age. Thus it may be inferred that growth to full size is attained in a little over two weeks. Whether this time is taken also in adult animals is not known exactly, but the minimum period is at least ten days since irradiated mice pro- duce fertile eggs up to ten days after irradia- tion (Parkes, 1926- 27). This unplies that there is a sensitive period to x-irradiation in young ova. Mar- shak (1935) has shown that the pachytene stage of meiosis is es- pecially sensitive to x-rays, and young ova enter into a modified pachytene shortly after leaving the ger- gj^^h^of minal epithelium. mammals. Not all ova grow to '^^^"^^^•^ mature size. This is evident at once from Aral's data which show that an average of 0.8 per cent of the ova under 20 /x in diameter attain a diameter greater than 60 fi, and only 2.7 per cent reach 20 to 40 jj. in diameter. The factors con- cerned in the atresia of young ova are as unknown as those determining their growth. The absolute size attained by mature ova varies from species to species, but the limits are rather narrow (espe- o o w H W O 40- 30 20 1 1 1 100 200 300 DIAMETER OF FOLLICLE, n- Same as Fig. 7, showing comparative the ovum in the various species of (From the Proceedings of the Royal 40 THE EGGS OF MAMMALS TABLE IV Estimates of the Diameter of Full-Grown Mammalian Ova (From Hartman, 1929) Animal Monotremata Platypus Echidna Marsupialia Dasyurus Didelphis Edentata Armadillo Cetacea Whales Insectivora Mole (Talpa) Hedgehog (Erinaceus) Rodentia Mouse Rat Guinea pig Lagomorpha Rabbit Carnivora Dog Cat Ferret Ungulata Horse Sheep Goat Pig Cheiroptera Bat Lemurs Tarsius Primates Gibbon M. rhesus Gorilla Man ^losT Pkobable Size OF Egg in Micra 2.5 mm. 3.0 mm. 240 140-160 80 140 125 100 70- 75 70- 75 75- 85 120-130 135-145 120-130 120 135 120 140 120-140 95-105 90 110-120 110-120 130-140 130-140 cially in the placental mammals) when comparison is made with other vertebrates or inveterbrates. Hartman (1929) has extensively reviewed the available data on fixed and living material and has estimated the average size of the Ii\dng OA^m for a number of species making allowance for the degree of shrinkage in fixed preparations. His estimates THE GROWTH OF THE OVUM 41 are given in Table IV. Subsequent measurements on living ova have proved these estimates to be on the whole remark- ably exact. An excellent brief account of the comparative morphology of living mammalian ova in several species is given by Streeter (1931). CHAPTER IV THE DEVELOPMENT AND ATRESIA OF FULI^GROWN OVA AND THE PROBLEM OF OVARIAN PARTHENOGENESIS Even when the ova have attained maximum size a major- ity of them are destined to degenerate. We have already mentioned that Allen, Kountz and Francis (1925) estimated that only 14 per cent of the 70 60 50 30 20 10 / \ ATRETIC ' \ FOLLI \ CLES \ PSEUDO Y S MATURATION / ^^ SPINDLES / \ / \ f \ \ \ N — ""rp.s. medium sized follicles of the pig ovary attain maturity. Engle (19276) finds that in the mouse the percentage of atresia among follicles with antra varies with the stage of the oestrus cycle, the maximum percentage of 86 per cent being recorded at the cornified cell stage. While the percentage of atretic follicles with mature ova was highest at the oestrus stage the maximum number was observed at the beginning of the dioestrus. This is obvious from the data of Table V and Fig- ure 13 which summarize the data on 50 ovaries from non- pregnant mice taken at four stages of the cycle. These data include small atretic follicles as well as antrum-containing folUcles, but the fact that the data for pseudomaturation spindles (which occur only in full sized ova) parallel those 42 CORN L.E.l L E.^ STAGE OF CYCLE Fig. 13. Showing the number of atretic folUcles and pseudomaturation spindles in the median ovary at four stages of the oestrus cycle in the mouse. (From the American Journal of Anatomy.) OVARIAN ATRESIA AND PARTHENOGENESIS 43 for follicles indicates that the total number of atretic mature ova reach their maximum in early dioestrus shortly after ovulation. This is doubtless due to the continued formation TABLE V The Degree of Atresia of Ovarian Follicles of the Mouse at Four Stages of the Oestrus Cycle. (From Engle, 19276) Stage OF Cycle Median OF Spindles Average OF Spindles Range OF Atresia Median OF Total Atresia Average OF Total Atresia Range of Total Atresia Corn 16 18.5 6-40 59 60 36- 85 LEI 26 28.4 7-51 71 77.9 50-129 LE2 11 11.2 5-17 36 42.1 29- 63 13 12 0-26 39 39.8 15- 70 of antrum-containing foUicles at a fairly high rate for a short time after ovulation. We have seen that ovogenesis continues during preg- nancy. Engle's data dem- onstrate that the formation ^. 30 and atresia of full-grown ^^ ova also occurs during preg nancy, for he observed an t.§ appreciable number of c^gio pseudomaturation spindles in ovaries taken during the first 43/2 days of pregnancy. These data are summarized in Table VI and Figure 14. It is notable that both the total amount of atresia and the atresia of mature ova is ATRETIC FOLLICLES PSEUDO-MATURATION >-... SPINDLES I ^--'' •---.i \-^ STAGE OF DEVELOPMENT -Fig. 14. Showing the number of , , , i ii • • 1 atretic folhcles and pseudomaturation less throughout this period spindles in the median ovary at four than during the period of stages in early pregnancy in the mouse. 1 i J i J- • (From the American Journal of Anat- least destruction m non- ^^^^y^ pregnant mice. Unfortu- nately, Engle does not give the percentages of atresia during early pregnancy. The presence of cycles of atresia and growth in animals 44 THE EGGS OF MAMMALS other than the mouse has already been noted (Evans and Swezy, 1931). According to Asami (1920) the rabbit ex- hibits a constant rate of folUcular atresia before and after TABLE VI The Degree of Atresia of Ovarian Follicles of the Mouse Stages of Early Pregnancy. (From Engle, 19276) AT Four Stage of Tubal Ova Median OF Spindles Average OF Spindles Range OF Spindles Median OF Total Number Atresia Average OF Total Number Atresia Range of Total Number Atresia To 2 pronuclei 2 to 4 blastomeres Morula Blastocyst 5 2 3 6 5 2.5 5.6 7.1 0-15 0- 7 2-20 3-14 22 17 13 15 23 18 16.5 17.3 11-41 10-31 7-38 14-28 pregnancy. Pincus and Enzmann (19366) found that the younger folUcles (types 1, 2 and 3 — Plate III) of the rabbit show a much lower percentage of atresia than the larger follicles. The atresia of mature ova can be prevented by pituitary hormones. This is deduced from the phenomenon of super- ovulation observed in animals receiving pituitary implants (Smith and Engle, 1927; Smith, 1932). These authors describe, for example, the presence of 49 ova in the tubes of a mature mouse receiving anterior lobe implantations. An adult mouse produces from six to twelve corpora lutea at an ovulation, the absolute number varying with weight of the mouse, the number of previous pregnancies, and certain genetic factors (MacDowell and Lord, 1925; MacDowell, Allen and MacDowell, 1929). In Smith and Engle's mice the largest number of ova ever found in one tube of a normal mouse was seven, and in an immature mouse showing super- ovulation a maximum of 48 ova was observed in a single tube. Thus the maximum number normally found in one tube is 14.5 per cent of the maximum number super ovulated. Furthermore, if we assume from MacDowell's data that 9 is roughly the number of ova normally ovulated this is 18 per cent of the 49 superovulated in the adult mouse. These percentages agree with the estimations of per cent OVARIAN ATRESIA AND PARTHENOGENESIS 45 of antrum-containing follicles maturing. The paucity of antrum-containing follicles and reduction of atresia is di- rectly noted by Smith and Engle. Finally, the ovulated ova are fertilizable although Engle (19316) found evidences of the degeneration of a number of them in the fallopian tubes. An interpretation of the foregoing data is that normally only a limited amount of pituitary secretion is available to the ovary and consequently only a certain percentage of the ova are able to obtain the amount necessary to prevent their atresia, whereas in animals receiving large amounts of pituitary hormones from implants an abnormal number of ova have available sufficient amounts of atresia-suppressing hormones. It cannot be decided, however, whether the effect on the ova is directly exerted by these hormones, or whether the stimulated follicle tissue produces substances ensuring normal ova, or whether some extraovarian sub- stance released into the circulation by pituitary stimulation reacts upon the ova. Loeb (1917; see also Meyer, 1913) has indeed suggested that the ovum itself is the controlling factor in follicle development citing the frequent presence of mitoses in fol- licle cells adjacent to the ovum as well as certain histological evidence that the cumulus oophorus develops under the influence of the ovum (Walsh, 1917). Allen and his collab- orators (1924) also maintained that the ovum is the dynamic center of the follicle apparently on the assumption that the mitosis-inducing action of oestrin upon vaginal and uterine epithelium is reflected in the higher mitosis rate in cells adjacent to the ovum becausB the ovum either produces oestrin or induces oestrin formation. In the opossum the presence of many atretic ova is correlated with prolongation of the dioestrus interval (Hartman). This supposed oestrin- ogenic action of the ovum has, however, been largely con- troverted (1) by the discovery of oestrin in corpora lutea as long as two weeks after ovulation (see Allen, 1932) and (2) by the observation that oestrin is produced in x-rayed 46 THE EGGS OF MAMMALS ovaries lacking ova (Parkes, 1926-27). This evidence, how- ever, does not prove that normally oestrin-production may not be under the control of the action of pituitary hormones upon the ovum itself. In fact, aside from the presumable atresia-inhibiting in- fluence, there seems to be only one other clearly evident influence of pituitary hormones upon the activities of the »^sfr'*-T^""' ?^ Fig. 15. Ovum removed from a preovulatory follicle of an unmated rabbit showing the vesicular nucleus. (From the Journal of Experimental Medicine.) ovum. That is that the production of the first polar body is dependent upon stimulation by pituitary hormones. Since this phenomenon is of some consequence to any discussion of the activation of mammalian eggs the writer, in collaboration with Dr. E. V. Enzmann (Pincus and Enz- mann, 1935), has undertaken an examination of the mecha- nism of polar body formation in the rabbit ovary. The rabbit was chosen for these experiments because it ovulates only after copulation and the ova are liberated regularly between 93^ and 103^ hours after copulation (see Heape, 1905; Walton and Hammond, 1932; Pincus, 1930; Pincus and Enzmann, 1932). Furthermore, the mature ova form polar bodies only after copulation. According to Heape (1905) OVARIAN ATRESIA AND PARTHENOGENESIS 47 two polar bodies are formed in the ovary by 9 hours after copulation. Our observations indicate that only the first polar body is given off in the ovary and then the metaphase plate of the second polar spindle is formed. Robinson (1918) observed in the ferret, which also ovulates only after copu- lation, that only the first polar body is given off in the ovary some time after copulation. ^ - i m ^M Fig. 16. Ovum removed from a ripe follicle of a rabbit doe at two hours after copulation. Note beginning of chromatin condensation. (From the Journal of Experimental Medicine.) Before copulation occurs the mature ovum contains a single large vesicular nucleus about 30 microns in diameter (Figure 15; see also Plate III, Figs. 4 and 5). At two hours after copulation signs of change are partially evident: some of the ripe ova show the beginnings of tetrad formation in the nucleus but the nuclear membrane is still intact (Fig- ure 16). By four hours after copulation the tetrads of the first polar spindle are formed and the nuclear membrane is ordinarily dissolved (Figure 17). The metaphase plate has a diameter of a little over 10 microns. The first polar 48 THE EGGS OF MAMMALS body is given off and the second polar spindle formed at or shortly after 8 hours post coitum (Figure 18). The follicle enlarges during this period also, the first signs of follicular development being evident at two hours after copulation. An exactly similar sequence of events occurs when prolan (pregnancy urine extract) or anterior pituitary extracts are injected. -# ^mf9 ^^^ Fig. 17. Ovum from follicle of ral)l)it don taken 4 hours after copulation. Formation of metaphase plate and dissolution of nuclear membrane. (From the Journal of Experimental Medicine.) It has been definitely established that prolan and anterior pituitary hormones cause ovulation when injected into the rabbit (Bellerby, 1929; Friedman, 1929). The ovulation occurring after copulation occurs because of the increased level of pituitary hormones secreted into the blood. This level is increased by nervous stimulation of the pituitary consequent on the orgasm. It has been shown by Deansley, Fee and Parkes (1930) that hypophysectomy within one hour of copulation prevents ovulation in the rabbit (see also Smith and White, 1931), and McPhail fl933) has demon- OVARIAN ATRESIA AND PARTHENOGENESIS 49 strated similarly that the critical period of secretion increase in the ferret occurs during the first hour of coitus. It seems evident, therefore, that pituitary secretions are responsible for the activation of the egg resulting in the formation of the first polar body and the second polar spindle. Further- more, certain observations of Hinsey and Markee (1933) indicate that the threshold for activation is lower than the ^ '" ™ i Fig. 18. Ovarian ovum of rabbit doe mated 9 hours previously. First polar body and second polar spindle. (From the Journal of Experimental Medi- cine.) threshold for ovulation. They observed that ovulation does not occur in large sized (2.6 kilograms and over) hypophy- sectomized rabbit does if prolan injection is made more than four hours after hypophysectomy. And in small sized hy- pophysectomized does (less than 2.3 kilograms) prolan ovula- tion never occurs. Nonetheless in all non-ovulating does polar body formation took place. Friedgood and Pincus (1935) found that stimulation of the cervical sympathetic of the rabbit resulted in maturation phenomena in those preovulatory follicles which failed to liberate ova. The sympathetic nerves presumably stimulated in these cases 50 THE EGGS OF MAMMALS the secretion of sub-ovulatory amounts of hormone from the anterior pituitary. Finally, Pincus and Enzmann (1935) found definite ovum maturation with as little as 34 the minimal ovulating dose of maturity hormone. In the ovaries of rabbit does which have copulated and then received pituitary injections within six hours after copulation the writer has observed the accelerated ripening of a new set of follicles and the formation of the first polar body. In these rabbits no accessory ovulation occurred though the pituitary extract dosages were at least two to three times greater than those necessary to cause ovulation in unmated does. The absence of ovulation indicates pre- sumably that the expulsion of ova can occur only from full sized follicles, whereas the activation processes may be in- itiated in ova whenever a sufficiency of pituitary hormones are available. It should be neted, however, that the nuclear activity occurred only in medium sized follicles and never in follicles without antra or with small antra forming. Since the ovum in the rabbit grows to some extent after antrum formation (see Figure 11) it is possible that functional matu- rity is attained at some time after antrum formation. A new crop of follicles begins to mature in the mated rabbit, and may certainly be stimulated to ovulate by the 4th day of pregnancy as Wislocki and Snyder (1931) have demonstrated by producing superfetation at that time with simultaneous pituitary extract and sperm administration. It is evident, therefore, that any attempt to dissociate in vivo the processes involved in polar body formation and those involved in ovulation depends in the mature rabbit upon hormone ad- ministration during the very short interval of time following copulation in the hope that active substances reaching the medium sized follicles will differentially affect foUicular growth and ovum maturation. Since the pituitary secretes a thyroid-stimulating as well as a gonadotropic hormone it is possible that maturation (and ovulation) is due directly to thyroid activity and only indirectly to pituitary stimulation. Pincus and Enzmann OVARIAN ATRESIA AND PARTHENOGENESIS 51 (1935) tested this possibility by injecting crystalline thyroxin and thyroprotein into rabbit does on heat. In no instance did ovulation occur but large doses of thyroxin did initiate follicular atresia and a limited degree of o\aim maturation. Again we see that atresia-inducing conditions also initiate maturation. The common feature of atretic follicles and preo\ailatory follicles is an isolation of the ovum from its connections with the follicular epithelium. It is safe to conclude from the foregoing analysis that the formation of the first polar body in the rabbit ovary (and in the ferret's also) is dependent upon an increase of pituitary hormones in the circulating blood. It happens that in all spontaneously ovulating mammals except the dog the forma- tion of the first polar body occurs in the ovary. Even in the dog (Evans and Cole, 1931) certain signs of nuclear matura- tion are observable in ovarian eggs. It is natural to infer that in spontaneously ovulating animals the pituitary level reached during oestrus is normally sufficient to induce ovula- tion as well as polar body formation. Now it is notable that the atresia of ovarian eggs is often initiated by the formation of a maturation spindle. We have noted that Engle has designated the spindles of ova destined to atrophy as '^pseudomaturation" although there is no evidence that they are in fact typically unlike those observed in normally maturing ova. Such spindles are ob- served only in ova of full size. Measurements of spindle containing ova in mouse ovaries give an average maximum diameter of 70 microns, and mature ova with vesicular nuclei had an average of 69 microns. The writer has also made careful examination of a large number of rabbit ovaries and has never observed typical spindles in immature eggs. What ordinarily occurs is a complex fragmentation of the chromatin (see Figure 19). That the spindles are the indices of impending atresia is indicated by the observation that when they are at a maximum the total follicular atresia is also at a maximum (see Figures 13 and 14). A possible in- terpretation of their presence may be that they occur as a 52 THE EGGS OF MAMMALS result of pituitary hormone action and the subsequent atresia of the ova containing them occurs because these ova are not Uberated and fertihzed. " Pseudomaturation " spindles have not been reported in hypophysectomized animals although atresia has. It has long been the contention of certain observers of ovarian atresia that the apparent parthenogenetic develop- t^ 'Mm: '»!. Fig. 19. Atretic ovum from type 3 follicle in the rabbit. Note fragmentation of cytoplasm and chromatin. ment of ova destined never to be liberated is simply an incident of the process of degeneration and is not in fact true parthenogenesis (Hensen, 1869; Balfour, 1882; Sobotta, 1899; Janosik, 1897; Bonnet, 1899; Rubaschkin, 1906; Ath- ias, 1909; Kingery, 1914; Kirkham, 1916; Stockard and Papanicolou, 1917; Addison, 1917; Long and Evans, 1922; Clark, 1923; Engle, 19276; Kampmeier, 1929). These in- vestigators have observed varied types of fragmentation of OVARIAN ATRESIA AND PARTHENOGENESIS 53 egg nucleus and cytoplasm, most of which cannot be con- sidered the result of true cleavage processes though in some instances a remarkable resemblance to cleaved ova is at- tained (see Plates IV and V). Another group of investigators generally admit that complex pseudoparthenogenetic frag- mentation occurs, but claim that a varying number of ova enter into true parthenogenetic development (Pfluger, 1863; Flemming, 1885; Paladino, 1887; Lowenthal, 1888; Schott- lander, 1891; Henneguy, 1893; Grusdew, 1896; Rabl, 1898; Gurwitsch, 1900; Spuler, 1900; Van der Stricht, 1901; Loeb, 1901, 1905, 1911, a and b, 1912, 1915, 1923, 1932; Newman, 1912, 1913; Sansom, 1920; Haggstrom, 1922; Courrier and Oberling, 1923; Courrier, 1923; Branca, 1925; Bosaeus, 1926; Lelievre, Peyron and Corsy, 1927). The resolution of such alternative points of view depends first of all upon a clear definition of what parthenogenesis is and secondly upon the interpretation of the ovarian structures designated as embryonic. If by parthenogenesis is meant the development of a mature individual from an unfertilized egg then it is at once certain that parthenogenesis does not take place in mamma- lian ovaries. If, on the other hand, a cleavage of the ovum with an equational division of the chromosomes is the cri- terion then there is some evidence (Sansom, 1920; Branca, 1925; Engle, 19276) that occasionally parthenogenesis occurs in ovarian eggs (see Plates IV and V). Certainly it is not permissible to consider as parthenogenesis an exact repro- duction of events taking place in the fertilized egg, since it is well known, for example, that parthenogenetic individuals arise from ova in which second polar body formation is suppressed. It seems appropriate, in seeking an understanding of the physiological processes occurring in developing eggs, to dis- tinguish between parthenogenesis and activation. A definite series of physical and chemical events ensue in eggs treated by agents inducing parthenogenesis. An apparently iden- tical set of changes occurs at fertilization. This process 54 THE EGGS OF MAMMALS which Needham (1932) has designated ''an opening of doors'' in the cell initiates the development of the ovum and makes of a static cell one capable of transformation. What happens subsequent to the activation process is often independent of the process itself. The probability of cleavage and the formation of a complete individual depends in part on the nutritional environment and the chromosome constitution of the activated egg. The activation process in non-mammalian ova has been described in physico-chemical terms (see J. Loeb, 1913; F. Lillie, 1919; Just, 1928; Runnstrom, 1933; Whit aker, 1933; R. Lillie, 1934). There exists no similar information partic- ularly for the ovarian eggs of mammals. The only estab- lished index of an activation of ovarian eggs is the described formation of the first polar body. It is conceivable that this represents the first step in an activation process that would go to completion if conditions were propitious. Perhaps the same pituitary stimulus that induces polar body formation might cause the formation of a cleavage spindle. The first cleavage spindles observed by Branca (1925) may then be considered the result of an activation process carried to completion because adequate pituitary stimulation was avail- able. On this basis the liberation of ova from the ovary results in such a change of environment that the stimulus to completion of activation is ordinarily no longer available. Similarly mature ova retained in the ovary at the time of ovulation ordinarily degenerate either because the proper type of pituitary hormone is not active (c/. Hisaw's concep- tion of the alternative action of follicle stimulating and luteinizing hormones) or because of the partition of the active hormone to other tissues {e.g., corpora lutea). We may consider two further alternative explanations of the activation of ovarian eggs. It is possible that activa- tion occurs in the ova of degenerating follicles because (1) the breakdown of cells near the ovum results in the re- lease of activating substances or (2) the initial stages of atresia in the egg cytoplasm frequently involve structural OVARIAN ATRESIA AND PARTHENOGENESIS 55 changes in the egg cytoplasm which are identical with those changes occurring during normal activation. According to the first of these two alternatives cell divi- sion stimulating substances are released as break-down prod- ucts (Gutherz, 1925). That such substances are actually formed by mammalian cells has been attested by the study of the growth of tissue cultures (Carrel, 1924; Fischer, 1925) where they have been given the name trephones. Further- more, signs of atresia in theca and granulosa cells are cyto- logically evident before signs of ovum breakdown. It has never been conclusively demonstrated, however, that treph- ones can activate ova (but see Haberlandt, 1922). On the other hand, it is conceivable that regardless of trephone action, the degeneration of follicle cells leads to a stimulating concentration of cytolizing substances {e.g., fatty acids which are known to act as activating agents) or even to a sufficient hypertonicity in the region of the ovum. The second of these alternatives implies that ^Hhe open- ing of doors" occurring in normal activation is an aspect of degeneration. Atresia certainly involves changes in the colloidal structure of cells, and we have pointed out {vide supra) that definite changes in cortical structure mark the activation process. It is interesting, therefore, to note that the cytological appearance of the cytoplasm of retained ova with spindles is markedly similar to that of fertilized eggs. Thus the cytoplasm of unfertilized eggs have upon fixation a rough coarsely reticular appearance (see Figure 15 and Plate III, Fig. 4), whereas retained ova with spindles, like normally activated or fertilized eggs, have a uniformly granular cytoplasm (Figure 18). Whether stimuli from degenerating follicle cells or endog- enous structure changes are involved, it is evident that these factors are in turn conditioned by the supply of avail- able hormone. Insufficient pituitary hormone results in the creation of ovum activating conditions. This is on the face of it, in direct contradiction of the first hypothesis which states that a supraliminal supply of hormone may also 56 THE EGGS OF MAMMALS initiate activation. But this contradiction may be resolved if we consider that the same conditions may be created by either active pituitary stimulation or absence of it. It has been shown that pituitary hormones themselves TABLE VII The Development of Ovarian Eggs of the Rabbit in Media Containing Various Hormone Preparations. (From Pincus and Enzmann, 1935) Number Time of OF Medium Results CULTURING Cultures 20 min. 14 Ringer-Locke + 1 drop beef pituitary Vesicular tetrads formed in all cases 2 hrs. 11 Ringer-Locke -|- 2 drops beef pituitary In some cases vesicular tet- rads and some free tetrads were formed. Some formed polar bodies 24 hrs. 9 Ringer-Locke -f- 1 drop maturity hormone Vesicular tetrads in all cases except 3 which had free tetrads 25 hrs. 4 Ringer-Locke -f 2 drops maturity Vesicular tetrads in all cases A. hormone 25 hrs. 7 Ringer-Locke + 3 drops maturity hormone Vesicular tetrads, free tet- rads, structures resembling fusion nuclei 2 hrs. 18 Ringer-Locke Vesicular tetrads and free tetrads 4 hrs. 3 Ringer-Locke Free tetrads 6 hrs. 3 Ringer-Locke Rudiment of first polar spin- dle 20 hrs. IG Ringer-Locke Vesicular tetrads, free tet- rads, fusion nuclei 24 hrs. 6 Plasma + 1 drop thyroxin 22 hrs. 4 Plasma + 3 drops thyroxin 22 hrs. 3 Phisma -|- 4 drops All cultures showed about the B thyroxin same phenomena which in- 24 lirs. 8 Plasma -|- 6 drops thyroxin cluded tetrad formation in all cultured eggs. In some 24 hrs. 4 Plasma -|- 8 drops thyroxin of the cultures polar bodies formed, or the vesicular 20-24 hrs. 22 Plasma -|- 2 drops Ringer-Locke sol. membrane dissolved . 20-24 hrs. 8 Plasma -|- 6 drops Ringer-Locke sol. OVARIAN ATRESIA AND PARTHENOGENESIS 57 do not act directly upon the ova (Pincus and Enzmann, 1935) by experiments in which ovarian ova with vesicular nuclei were cultured in media containing various pituitary extracts. The data of these experiments are summarized in Table VII-A. They show that in both the extract- containing media and the extract-free media maturation proceeds at about the same rate. Furthermore thyroxin which causes a certain degree of maturation when injected in vivo (see page 51 above), causes in vitro no further degree of development than thyroxin-free controls (Table VII-B). The isolation of the ova from the normal follicular environ- ment is sufficient to initiate activation. This implies that in preovulatory follicles maturation is caused by either (1) the mechanical separation of the ovum and its corona or (2) the removal of an inhibiting influence. Mechanical separation undoubtedly occurs (c/. Plate III, Figs. 8 and 9), but one cannot estimate the exact degree of isolation necessary to initiate maturation, for it is certain (Pincus and Enzmann, 1935) that maturation is initiated in ova still having strands connecting them to the follicular epithelium. In certain forms {e.g., man) the ova remain embedded in the cumulus mass till just before ovulation and the corona forms late. It is notable that Allen, Pratt, Newell and Bland (19306) were able to obtain only one maturation stage in some two hundred ova recovered from 3 to 20 mm. fol- licles. The writer (unpublished data) has observed one maturation occurring in a primate ovarian ovum, but when primate ovarian ova are cultured in vitro considerable nu- clear activity occurs. During the first stages of pituitary- induced maturation in the rabbit a secretion of secondary liquor folliculi is observed (Pincus and Enzmann, 1935). This secretion may remove an activation-inhibiting influ- ence. The maturation observed in ova of atretic follicles may be due to a similar sort of secretion rather than to simple isolation of the ovum from its follicular epithelium. On the basis of the foregoing considerations one might conceivably encounter occasional evidences of activation Fig. 1 Fig. 2 ^ \ ^ r:? Fig. 3 Fig. 4 Fig. 5 'X^« Fig. 6 Plate IV. Various stages in the development of the mature oocyte. (From the Archives de Biologie.) Fig. 1, First maturation spindle — guinea pig. Fig. 2, Binucleated ovum, chro- mosomes oriented for the metaphase of a mitosis — guinea pig. Fig. 3, Binucleated ovum with formed maturation spindles — mouse. Fig. 4, Multinucleate cytoplasm — mouse. Figs. 5 and 6, Typical uninucleate cleaved ovocytes. Fig. 6 shows deuto- plasmic extrusions — guinea pig. 58 OVARIAN ATRESIA AND PARTHENOGENESIS 59 where alterations in normal hormone balance occur which are sufficient to cause a preponderating activation stimulus. Such may in fact be the basic cause of certain undoubtedly normal early development in ovarian eggs reported by a number of observers. In Plate IV, Figures 1 to 3 and Fig- ure 1, Plate V, are presented various stages of pre-cleavage development found in ovarian eggs. The multinucleate con- dition of the egg of Figure 4 may be due to chromatin fragmentation, but the cleavages of the eggs of Figures 5 and 6 of Plate IV and Figures 2 and 3 of plate V are com- pletely normal. It seems clear that at any one of these stages definite atresia of the ovum may set in, preventing further development. Similar arrests of development may occur in parthenogenetically activated invertebrate ova if the acti- vating treatment is not carefully controlled (c/. Loeb, 1913; Just, 1928). Entrance into the cleavage process is likewise dependent upon a rather nice balance of developmental events. Furthermore, the processes involved in cleavage may indeed be independent of the activation process. Runnstrom (1933) has shown that sea-urchin eggs poisoned by monoiodoacetic acid can be fertilized but that segmenta- tion soon ceases and ordinarily just before the dissolution of the nuclear membrane of the first cleavage division. In later chapters we shall discuss further the problems involved in parthenogenetic activation. Now it is sufficient to indicate that there is a probability of activation of ovarian eggs but that a complete activation is dependent upon a balance of events which must presumably be rarely attained in the ovary. Even if the activation reaction proper occurs and segmentation ensues the probabilities that post-cleavage stages will be entered are made extremely small not merely because of the physical limitations imposed by the structure of the ovary, but because, as we shall demonstrate later (Chapter IX), the growth stage of the embryo is entered into only as the result of a definite hormonal stimulus during the luteal phase, and conversely is definitely inhibited by oestrin. It is therefore surprising that the blastula and Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Plate V. (Figs. 1 to 3 from the Journal of Anatomy; Figs. 4 to 7 from the Archives de Biologie.) Fig. 1, Line drawing of a section through an atretic foUicle of the mouse. First mitotic anaphase. Cytoplasmic division not completed — mouse. Fig. 2, Typical 4-celled ovarian ovum — water vole. Fig. 3, Typical 2-celled ovarian ovum with intact zona — water vole. Fig. 4, Early blastocyst of ovarian ovum — mouse. Fig. 5, Many-celled blastocyst in ovarian ovum — guinea pig. Fig. 6, Multinucleate blasto- cyst-like ovarian ovum — mouse. Fig. 7, Blastocyst-like ovarian ovum — guinea pig. 60 OVARIAN ATRESIA AND PARTHENOGENESIS 61 neurula-like formations, described by Courrier (1923) (see Figures 4 to 7, Plate V) and Courrier and Oberling (1923) and the atypical ovarian embryos observed by Loeb (1932) should be found. The solution to the controversy concern- ing their exact nature must await evidence as to the pos- sibility of their formation by experimental means. It can be seen that the chance of atretic degeneration continually besets the ovarian egg. The evidence indicates this process can be avoided only if sufficient pituitary hor- mone is available to the ovary. There exists also the possi- bility that atresia is endogenous in the sense that the ovum as a cell attains a certain maximum degree of development and then inevitably goes down hill. Only the sudden inter- vention of ovulation and fertilization prevents this process. Such a conception is scarcely amenable to experimental verification chiefly because of the intimate association of the ovum and its follicle. Furthermore, signs of ovum de- generation are preceded by degenerative phenomena in the granulosa cells. If the granulosa and corona cells act as nurse cells to the ovum it is obvious that their behavior must largely condition the behavior of the ovum. Often the ovum becomes detached from the granulosa and corona radiata and floats practically free in the liquor folliculi. We do not know to what extent diffusion of a sufficiency of nutritive substances through the liquor folliculi is possible. The problem of the viability and senescence of the ovum still awaits experimental attack. CHAPTER V METHODS EMPLOYED IN THE EXPERIMENTAL MANIPULATION OF MAMMALIAN OVA The first investigators of living tubal eggs (Barry, Cruik- shank, Bischoff, Spee, et al.) used rather laborious methods of dissecting the tubes (see Squier, 1932, for an interesting historical discussion). The modern technique of securing eggs from the fallopian tubes of most mammals is a fairly simple one. Nonetheless, certain surprising differences in the behavior of the obtained ova arise when exactly the same methods are applied to two different species. Among the laboratory mammals the rabbit is by far superior, and for one very simple reason, namely, rabbit ova seem to withstand the process of handling better than other ova. Mouse, rat and guinea pig ova, for example, begin to frag- ment very soon after removal from the tubes (Lewis, 1931; Gilchrist and Pincus, 1932; Squier, 1932; and Defrise, 1933) and to date it has been possible to observe at most one or two cleavages in culture, whereas rabbit ova will go through the whole course of cleavage and blastulation in vitro. The long, fairly straight tubes of most mammals can easily be washed through by a Ringer-Locke or similar bal- anced salt solution. The writer has found that a Ringer- Locke solution to which has been added an equal amount of homologous blood serum is most useful. It is necessary only to free the tubes of their mesenteric connections, and if the tubes only are to be employed to cut them away from the uterus. It is ordinarily best to cut off the uterus at about one-half inch from the ampulla so that if washing backward toward the fimbria is desired a certain length of uterine lumen will be available for the guidance of the washing pipette. When ova are to be washed downward 62 METHODS FOR THE MANIPULATION OF OVA 63 from the fimbriated end of the tubes a rather broad bored capillary pipette is used; washing upward from the uterine end requires a very fine pipette. The ova are washed into Syracuse watch glasses and are easily observed under low magnification of a dissecting microscope. In animals like the rat, mouse and guinea pig with coiled tubes a different procedure is followed. Here the coiled tubes are cut into several fairly straight portions and are squeezed with a pair of fine iris forceps or stroked gently with blunt needles. The contents of the tubal lumen are extruded and the ova are found among the cellular debris. Ova from the uterus are obtained simply by flushing the uterine lumen with the washing fluid. Allen, Pratt, Newell and Bland (1930a) describe a method for obtaining human tubal ova without removing the tubes or uterus. ^'The ovaries were examined as soon as possible after the abdominal cavity was opened. In some instances the findings at operation necessitated removal of the most recently ovulating ovary and its tube was not justified, the tube was flushed in situ and the corpus luteum alone re- moved from the ovary. This method consisted of clamping the cervix with a special clamp and injecting isotonic saline solution directly into the uterine cavity from above by hypodermic syringe while first one and then the other uterine tube was gently pinched by the assistant. The injected solution in most cases flowed back freely through the tube and was collected in a series of watch-glasses held beneath the fimbriated end. Apparently the development of valve- Hke folds of mucosa at the tubo-uterine junction as described for several mammals by Lee (1928) is not appreciable in woman. Usually from 10 to 30 c.c. was flushed through each tube. The most recent corpus was carefully excised from the ovary, and since it is a transitory structure, without sacrificing any considerable amount of ovarian tissue. "It is believed that this method of flushing the tubes in situ is harmless to uterus and tubes and opens up new 64 THE EGGS OF IMAMMALS possibilities, not only for the recovery of human ova, but also for checking the patency of tubes at operation. ''The tubes which could be removed were washed by direct injection through either the uterine or the fibriated ends after first trimming the tube carefully along the attachment of the mesosalpinx. The trimming seemed advisable, for otherwise when the tube was distended with injected fluid it would often kink badly. ''A search for human tubal ova is sometimes complicated by the follicle cells of the cumulus still surrounding the specimens which make difficult clear observation and certain identification. Although while fresh such specimens are fairly transparent, it is often difficult to observe or measure them accurately. Since it is probable that ova may remain in the tubes for three or more days, degenerative changes may be expected in a certain number of unfertilized tubal ova. Also small masses or balls of cells are often encountered in the tubes. These may originate in the peritoneal ca\'it.y, be pinched ofT from the fimbria of the tube, or (in cases where injected fluid is forced back through the tubes from the uterine cavity) derived from cast-off endometrium. Sometimes such cell balls contain structures which before sectioning can easily be mistaken for ova. For this reason unless an ovum is free from follicle cells or the cells of cumulus are partly dispersed, it w^ould seem necessary that it be sectioned before certain identification is possible. Further check should also be made by histologic study of the most recent corpus luteum." In obtaining both unfertilized and fertihzed ova for cul- ture in litro the use of a warm washing solution is preferable. This is often practically difficult and rabbit ova at least are not materially affected by handling at room temperature over a period of several hours. The usual methods of tissue culture have been employed in the cultivation of mammalian ova. These include the hanging drop with the ovum held in a plasma clot on a coverslip over a fluid-free cavity; a plasma clot occupying Mirrifons for tiik ma\ipi:latio\ of o\'a go the total area under a raised coverslip; the CJarrf^I flask; and the watch-glass teehnirjue in which the sterile watch glass containing the culture rnediun^i is contained in a nnoist chamber, iilood plasma or serum ordinarily form the basis of the most successful culture media. I'he longest perirjd of rfigular development of normally fertilized rabbit ova has bef*n obtained by IxnvLs and Gregory (1929j who photo- graphed the development of rabbit ova from the initial cleavage stages through late blastocyst stages. They placed the ova in homologous plasma upon glass slides. Pincus (unpublished data) has obtained similar development by this technique and also with ova grown in Carrel flasks. IxwLs and Ilartman (\iy.V4) observed the development of a Macacus rhesus ov^um from the 2-cell to the 8-cell stage using the Ixwis and Gregory technifjue. The ova of the rat, moase, and guinea pig have failed to de\'elop beyond one or two cleavages with the use of a \'ariety of culture media. I'hus Defrise 0933; used the following media for culturing rat ova: (\) Ringer's solution, bufferr.'d or not with sodium bicarbone; (2) Tyrode's solution, (\) Isotonic with NaCl, milimol: fa) 120, (h) VM), ((■) 151, (B) K+ - Ca^+ - Mg"+ equilibrium on the basis of the triangular diagram of Loewe, milimol: Ta; 2.05 KCL, 1.90 CaCU, 2.20 MgCF,, ^b; 5.63 KCl, 3.60 CaCU, 0.52 MgCla: the above solutions were used at pll 6.8, 7.2, 7.6; f3j Tyrode's solution rXaCl: milimol 136 - KCl: 5.6 - CaCU: 2.16 - MgCU: 0.52 - XaHC03: 8.6 - CJI 1206:5.5) with the addition of gelatine 0.5 per cent; (4) Tyrode's solution (as above) with the addition of blood serum: faj 1/1, fb) 3/1; (o) Tyrode's solution (a.s above) with the addition of plasma (heparin): (a) 1/1, (b) 3/1; (6) pure blood serum of (a) pregnant female, (b) male, (c) newborn; (7) plasma (secured from the heart and mixed with heparin): (a) pregnant female, (b) male; (8) spinal fluid (secured by suboccipital puncture) ; (9) foetal hystolymph (secured by Mart.ino\itch's technique); HO) uterine fluid (II oestral period): (a) pure, (h) with the ad- dition of blood serum. 66 THE EGGS OF MAMMALS In a few cases, in some of the above media, and especially inthis:NaClmilimol 130 - KCl 2.65 - CaCU 174 - MgCls 1.18 - NaHCOa 8.6 - at pH 7.2 - drops III = blood- serum drops II, one or two mitoses were obtained. The addi- tion of small quantities of embryonic extract, of rat foUic- uline, of extract of the anterior lobe of the hypophysis to the medium (either solid or liquid, natural or artificial) has not noticeably modified the culture results. Squier (1932) using a less extensive variety of media was similarly unsuccessful with guinea pig ova (see also Lewis, 1931). The limitations of the ordinary methods of tissue culture are discussed further in Chapter IX in connection with the investigation of the normal physiological environment of developing ova. Nicholas and Rudnick (1933) have cultivated rat embryos upon the chorioallantois of the chick, but ovum development has not been studied. The embryos survive and differenti- ate over a considerable period of time in the foreign environ- ment. Cinematography of developing ova has been undertaken in a number of recent investigations. Standard motion picture cameras adapted for microphotography are employed. For a study of the comparative behavior of ova in vivo and in vitro the writer has transplanted cultured ova into the fallopian tubes of rabbit does (see Pincus and Enzmann, 1934). The operative technique requires the use of a light anaesthesia, e.g., either ether preceded by atropine sulphate injection to inhibit excessive mucous secretion or simple urethane anaesthesia. The exposure of both tubes and ovaries is had by a simple laparotomy. The ova are held in a special pipette with an opening in the tube above the capillary. This type of pipette permits one to take up a minimum amount of fluid with the eggs, and also prevents the ova from being drawn into the wide-bored portion of the pipette. The capillary portion is inserted into the upper 3^ of the tubes and the ova expelled by gentle pressure METHODS FOR THE MANIPULATION OF OVA 67 on the bulb when the opening in the tube is closed over. No amount of pressure on the bulb will expel the ova if the opening is not closed. Extreme care should be taken to expel only the ova and the fluid containing them. If air is also pumped into the tubes it often blows the eggs down too far into the tubes or even into the uterus. Excessive fluid acts in the same way. The writer (in collaboration with Dr. E. V. Enzmann) has also transplanted mouse ova into the fallopian tubes. Here it is necessary to slit the capsule and expose the tubal opening, which is slightly wider than at the ampulla, but not as wide as the rabbit's fimbriated opening. The tubes are observed under a dissecting microscope and the opening exposed by manipulation with iris or watchmaker's forceps. The delicate mouse ova are best handled in warm Ringer- Locke solution plus serum. Nicholas (1933a) has transplanted rat ova from the fal- lopian tubes into the uterus. In this case the tubes are excised at the isthmus and the ova expelled from a capillary pipette into the uterine lumen. CHAPTER VI THE TUBAL HISTORY OF UNFERTILIZED EGGS When ovulation occurs without fertilization the liberated ova enter the Fallopian tubes and eventually degenerate. In most polyovular mammals the ova are shed surrounded by an apparently sticky cumulus ovigerus so that a sort of plug is formed due to the adhesion of the various separate cumuli (see Plate VI, Figure 1). This cumulus mass remains more or less intact for some time and then the cumulus cells gradually become detached so that the ova finally float free. The opossum (Hartman, 1925) and sheep (Clark, 1934) appear to be exceptions since very few follicle cells surround the newly shed ova. The chronology of egg passage in the tubes is best had in the rabbit where ovulation occurs at 9 J^ to 10}^ hours after copulation. The freshly ovulated ova enter the tubes and become massed together, due to the adherence of the sticky masses of cumulus cells. By 11 hours after copulation (about 1 hour after ovulation) this mass of cumulus cells containing the ova becomes securely lodged in the narrower portion of the tubes just below the broad, fimbriated end. On washing from the uterine end of the tubes this mass (see Figure 1, Plate VI) is first ejected, then the washing fluid. The ova remain thus massed together until about 17 hours after copulation, an occasional ovum separating out of the mass as early as 16 hours after copulation. Figure 2, Plate VI, is the photograph of an ovum still embedded in the mass of follicle cells at 16 hours after copulation. Figure 3 is the photograph of the single one of the 10 ova removed at the same time as that of Figure 2 that had separated out of the mass. Note a number of follicle cells still clinging to the egg. 68 Fig. 1 m^' : m Fig. 2 Fig. F" # Fig. -4 Fig. 5 Fig. 6 ^^r??^S^-' >r: -'•:,' ,* Fig. 7 Fig. 8 Plate VI. (From the Proceedings of the Royal Society.) Fig. 1, Three ova in the cumulus mass recovered from the fallopian tubes of rabbit doe 123^ hours after a sterile mating. Fig. 2, An unfertilized ovum still in the cumulus mass 16 hours after a sterile mating. Fig. 3, Another 16-hour ovum free of the cumulus mass. Fig. 4, An ovum recovered 19 hours and 5 minutes after a sterile mating with no adherent follicle cells. Fig. 5, An ovum recovered 243^ hours after a sterile mating showing a definite albumin coating. Figs. 6-9, All from sterile matings at the following intervals after sterile copulation: 6. 43 hours, 30 minutes, 7. 73 hours, 40 minutes, 8 and 9. 96 hours, 45 minutes. 69 70 THE EGGS OF MAMMALS As they separate out of the cuinuhis mass the egjj;s emerge surrounded more or less by a few adherent folUele cells, and proceed down the tubes where these few adherent cells are lost. At 20 hours after copulation all the adherent cells are gone and a thin layer of albumen is laid down about the zona pellucida. Eggs washed out at this time show very clearly the transparent, shining zona pellucida about the yolky, granular egg cytoplasm, with an extremely thin al- bumen layer surrounding the zona (see Figure 4). The process in\'oh'ing the separation of the eggs out of the cumulus mass and the clearing off of adherent cells thus involves a period of about 3 hours. When eggs are washed out during this period one observes in a single washing all the stages described, eggs completely clear of adherent cells being preponderant toward the end of the period. One may even find an occasional egg still surrounded by adherent cells as late as 20 hours after cop- ulation. It is important for reasons that will be obvious later, to note that by 20 hours after copulation all rabbit ova are free of follicle cells and have begun to accumulate a layer of albumen. By 24 hours after copulation this albumen lajTr is quite appreciable (see Figure 5). Subsequently the ova descend to the uterine end of the tubes acquiring in their passage successive layers of albumen so that the albumen layer may eventually become several times the thickness of the egg itself (see Figures 6 to 9). The zona pellucida no longer presents the clear, shining appearance observed before the deposition of albumen. Most of the ova recovered from the tubes contain at least one polar body, occasionally two or even three. In some cases none ha\'e been observed but this may be ascribed to faulty observation as the eggs often come to rest with the polar body hidden. The eggs enter the uterus between 72 and 96 hours after copulation. No more albumen is added and the eggs undergo rapid disintegration. It is, in fact, very difficult to recover unfertilized ova from the uterus. Pincus (1930) was unable TUBAL HISTORY OF UXFKRTILIZl^D EGGS 71 to obtain the full complement as indicated by the corpora lutea count. They are either rapidly resorbed or washed out into the vagina. The cytoplasm of eggs recovered from the uterus shows distinct evidences of degeneration (Fig- ures 8 and 9). The persistence of the corona radiata for some time after ovulation occurs regularly not only in the rabbit (cj. Yamane, 1930, 1935) but also in the mouse CLong, 1912), the rat (Gilchrist and Pincus, 1932), the dog fEvans and Cole, 1931), and man (Allen, Pratt, Newell and Bland, 1930a). It is notable that opossum ova with no surrounding cumulus mass enter the uterine portion of the oviduct in approx- imately twenty-four hours, whereas all available information indicates that in the higher mammals unfertilized ova enter- ing the uterus do so at approximately 3 H days after ovula- tion. In the rat (and probably also the mouse) unfertilized ova apparently degenerate in the uterine portion of the tubes (Long and Evans, 1922; Mann, 1924). Albumen deposition about tubal ova occurs in the rabbit and opos- sum; in most other mammals the ovum traverses the tube surrounded only by the zona pellucida. The dissolution of the cumulus mass surrounding newly liberated ova seems to involve a definite process in the tubes and is in all probabiUty not due to an autogenous change in the cumulus cells themselves. In guinea pigs the fresh cumulus mass is so tenaciously adherent that it cannot be completely removed by dissection (Squier, 1932). Gil- christ and Pincus (1932) found that rat ova incubated in Ringer's solution did not become free of adherent cells even after many hours. In rabbit ova grown in blood plasma a fibroblast-like outgrowth of the cumulus cells occurs but nonetheless the radial connections to the zona pellucida are not lost (Pincus, 1930). The writer has also observed a similar outgrowth from the cells surrounding cultured hu- man ova, but the extremely tenacious covering of follicle cells is not lost. The likelihood that a slow enzymatic process is involved in the freeing of the adherent cells is substanti- 72 THE EGGS OF MAMMALS ated by the great acceleration of this dehiscence in the presence of sperm (see Chapter VII). The unfertihzed ova of most manamals begin to show- signs of degeneration when they reach the distal portion of the tubes. In the opossum clear evidences of degeneration are observed by twenty-four hours after ovulation when the Fig. 20. Fragmenting opossum egg seven days after arriving in the uterus. Section of one of the eggs shown at A, containing three large chromatin masses almost free of cyto- plasm. (From the American Journal of Anat- omy.) ova enter the uterus (Hartman, 1924). The degenerative changes have been described in detail by Smith (1925). The ovum may remain intact but develop a well vacuolized cytoplasm with clumped or fragmented chromatin. Ordi- narily, a definite fragmentation of the whole ovum occurs (Figure 20), and the irregular blastomere-like formations may contain bits of fragmented chromatin or lack chromatin entirely. In some 300 opossum ovum sectioned and ex- amined Smith never observed a true cleavage spindle, and TUBAL HISTORY OF UNFERTILIZED EGGS 73 appropriately concludes that parthenogenetic development never occurs. Her statement that pregnant (or psuedo- pregnant) condition of the animals should favor par- thenogenesis is not neces- sarily correct since activa- tion may require special physiological conditions. In the unmated mouse, how- ever, Charlton (1917) has described identical modes of degeneration in tubal ova with scarcely an ap- proach to normal cleavage, and out of 152 tubal ova in the unmated rat Mann (1924) found only three which appeared to have undergone a belated parthenogenetic development (see Table VIII). In the rabbit (Pincus, 1930) fragmentation occurs rarely; an o\aim of the type shown in Figure 21 is occasionally encountered. The fragmenting ova found in the tubes of rats and mice Fig. 21. Rabbit ovum recovered from the tubes 411^ hours after ster- ile copulation showing polar fragmen- tation. (From the Proceedings of the Royal Society.) TABLE VIII The Conditions of Tubal Ova in Various Portions of the Oviduct IN THE Rat. (From Mann, 1924) z z z , o , o O U ?^ h>- hS < as J ^r, n ^H g > ^ P < ^ ss p £ a 03 s < o < ^ S! TD H a h O h OS o ■t a z a a ^ 5 a ^ z < < < J < H < s „ ^ 3 S 3 a ?^ ^ a Q c^ IrH r\ H O y^ ^ ra '^^ O ■^■^ K «S s u Z P ^ So J ° « >J o > m a E 9 lis ii o z m a z ■< ti ij a Oh < o z a a a H O Q Z OS 2 a a o PhO aixas xnm'A Ui'J-jU Um P^O c^So fe hS QUO ^ 1-3.5 34 4 2 15 2 4-5 6 16 2 1 1 5-8.5 1 7 14 13 1 1 8.5-9 2 6 22 2 Totals 41 4 2 40 16 20 22 2 2 3 74 THE EGGS OF MAMMALS finally disappear either through complete disintegration or, what is more hkely, by phagocytosis (Figure 22). They usually disappear before the succeeding ovulation, although Hensen (1869) has described the retention in a blocked tube of about 100 rabbit eggs apparently from several ovulations. Fig. 22. Section through a fragmented mouse ovum recovered 81 hours after a sterile mating. Phagocytes (C) absorb the degenerated cytoplas- mic particles (E). (From the Biological Bulletin.) The rate of passage of ova in the tubes and the method of transport have been the subject of considerable contro- versy and discussion (see Parker, 1931 and Hartman, 1932&). It is generally acknowledged that the passage through the upper portions of the tubes is relatively rapid (Anderson, 1927; Lewis and Wright, 1935) since except shortly after ovulation both unfertilized and fertilized ova are found for the most part in the lower two-thirds of the tube. The method of propulsion of the ova by ciliary and other tubal movements is adequately discussed by both Parker and Hartman and will not be entered into here. CHAPTER VII FERTILIZATION AND CLEAVAGE The events occurring at fertilization in the fallopian tubes have been subject to detailed examination chiefly in poly- ovular mammals, e.g., the rabbit, rat, mouse, ferret, etc. In all cases the sperm surround the ova embedded in the mass of follicle cells, and penetrate to the ova causing the follicle cells to fall away at the same time. That the sperm swarm present in the tubes is actively responsible for the fall- ing away of the follicle cell mass is abundantly evident from numerous recent observations of fertilization in the rabbit (Pincus, 1930; Yamane, 1930, 1935; Pincus and Enzmann, 1932, 1935). As described previously rabbit ova in does mated to sterile bucks begin to separate out of the follicle cell mass by 16 hours after copulation at the earliest, and the process is normally completed between the 17th and 19th hours. In fertile matings free ova have been observed as early as 113/^ hours after coitus, and all ova are invariably free by the 14th hour. Furthermore, when freshly o\ailated ova from sterile matings are placed in vitro with sperm suspensions there is a rapid dispersion of the surrounding follicle cells which does not occur in control cultures of ova in sperm-free media. Similar phenomena have been observed by Gilchrist and Pincus (1932) in the rat (Figures 23 to 25) and by Pincus (unpublished observations) in the mouse. 75 Fig. 23, Rat ovum recov- ered from the tubes at 16 hours after a sterile mating. Note surrounding folUcle cells. (From the Anatomical Record.) 76 THE EGGS OF MAMMALS Yamane (1930) has ascribed the phenomenon of folHcle cell dispersion to the presence of a proteolytic enzyme in the spermatozoa. He was able to secure a similar dispersal of follicle cells from sperm sus- pensions heated to 60° C. and from preparations of pancrea- tin containing trypsin. Yam- ane (1930) believes that this J "^ '^^^^P^^^' J^^H proteolytic enzyme is also re- f f^&^S^X€jli^S sponsible for the activation of the egg since he observed ''polar" bodies formed in rabbit ova exposed to the suspensions of dead sperm and to the enzyme prepara- tions. Pincus and Enzmann (1935) have examined this situation in some detail. Sperm suspensions free of seminal fluid were obtained from the vas deferens of adult rabbit males. Dilutions were made with a buffered Ringer- Locke solution at pH 7.3 — 7.5. The ova were taken at 12 H to 153^ hours after copulation from rabbit does mated to sterile (vasecto- mized) males ; these ova were invariably well embedded in the massed follicle cells. The procedure followed was to place the massed ova in the sperm suspension and incubate for at least two hours. All ova were examined at two hours after semination and in some instances where no obvious signs of fertilization were observed incubated for 12 hours. Fig. 24. Rat ovum of Fig. 23 after 2 hours with Hving sperm. Note absence of folhcle cells and protrusion resembling a polar body. (From the Aiia- tomical Record.) Fig. 25. R.it ovum recovered 11 hours after sterile mating and in- cubated with living sperm for 2 hours. Note shrunken vitellus and two polar bodies. (From the Ana- tomical Record.) FERTILIZATION AND CLEAVAGE 77 In most instances the ova were fixed in Bouin's solution and sectioned in order to determine the nuclear condition. The TABLE IX The Effect of Various Concentrations of Live Sperm upon Freshly Ovulated Rabbit Ova. (From the Journal of Experimental Zoology) Concentra- tion OF Effect on Cumulus Date Sperm per MM. 3 Cell Mass Effect on Eggs 6/II/34 (undiluted) Destroyed in 2 to 2 polar bodies; ova completely 185,000 3 minutes dissolved after 24 hours 6/II/34 92,500 Destroyed in sev- eral minutes 2 polar bodies 20/1/34 (undiluted) Destroyed very 1 polar body after two hours; 90,000 rapidly polyspermy probable because of very active sperm suspen- sion 10/III/34 80,000 Destroyed 2 polyspermic ova, one polar body; one monospermic with 2 polar bodies 16/1/34 (undiluted) 62,500 2 polar bodies; fertilized 20/1/34 55,000 }f 1 polar body 20/1/34 40,000 " 1 polar body 26/1/34 38,400 }j 2 polar bodies; fertihzed 10/III/34 30,000 Destroyed in 20 1 egg with 3 sperm attached and minutes 2 polar bodies; 2 eggs with single sperm attached and 2 polar bodies; not incubated 6/II/34 32,200 Destroyed 2 polar bodies; fertilized 6/II/34 30,000 >) 1 polar body; no sperm entry 26/T/34 25,000 }f 2 polar bodies; fertihzed 26/1/34 14,300 Partly destroyed 1 polar body; not fertilized 26/1/34 10,700 " " 6/II/34 10,100 " " 6/II/34 8,000 n ;> 26/1/34 7,200 )} ft 6/II/34 4,000 " " 26/1/34 3,600 >> }f 2/II/34 6,000 Destroyed almost 1 polar body; \ at once no fertilization) 2/II/34 3,000 Destroyed in 1 polar body; ( rat 2 minutes no fertilization/ sperm 2/II/34 1,000 Destroyed in 1 polar body; \ 33^ minutes no fertilization/ sectioned ova of the experiments listed in Table IX invari- ably showed true polar bodies; no achromatic extrusions were observed. Furthermore, all ova with two polar bodies 78 THE EGGS OF MAMMALS contained either attached sperm or male pronuclei, whereas all ova with single polar bodies showed no signs of sperm entry with the exception of two heavily polyspermic ova. Polyspermy may prevent the second polar division, but probably only when extremely active and dense sperm sus- pensions are used. The presence of two polar bodies may therefore ordinarily be taken as a sign of activation. It is evident from the data of Table IX that both the degree and speed of dispersion of the follicle cell mass is roughly proportional to the concentration of the sperm sus- pensions used and that those sperm concentrations which fail to effect a complete dispersion of the follicle cell mass also fail to cause second polar body formation. But rat sperm as well as rabbit sperm can effect complete dispersal of the folhcle cells about rabbit ova and yet no polar body forma- tion occurs. This seems to indicate that the activation of the o\aim and follicle cell dispersion involve distinct and separate reactions. The data of Table X substantiate this conclusion for they show that sperm-free fluid from the vas deferens and sperm suspensions heated to 60° C. for a few minutes cause typical follicle cell dispersion but no polar body formation. That a heat-labile substance is involved in the follicle cell disper- sion is evidenced by the data on ova exposed to boiled sperm suspensions. This substance is probably carried by the sperm since similar follicle cell dispersion in vivo is brought about by sperm that have travelled the length of the oviducts. Yamane (1930) found that both rat and horse spermatozoa caused second polar body formation in rabbit ova, and since his pancreatin solutions also caused the same result he con- cluded that a non-species-specific sperm-borne tryptase was involved. As shown in Table IX above rat sperm were ineffective in causing second polar body formation, but they were more potent than rabbit sperm suspensions in causing follicle cell dispersion. Accordingly Pincus and Enzmann (1936a) undertook the experiments with trypsin preparations presented in Table XL TABLE X The Effect of Dead Sperm Preparations and Sperm-Free Seminal Fluid upon Freshly Ovulated Rabbit Ova. Experimental Zoology) (From the Journal of Treatment of Sperm Effect on TTimrr^'T' m>j T^^nnci Dilution of Date Suspensions Cumulus Mass Hjr r liiK^x KJj^t xjouo Preparation 10/1/34 Heated to 60° C. Destroyed in 1 polar body; Undiluted all sperm dead 10 minutes no fertiliza- tion 30/III/34 Completely dessi- Destroyed in 1 polar body Made up to cated at room 2 minutes original vol- temperature; all ume sperm dead 30/III/34 t) Destroyed in 5 minutes )) Made up to original vol- ume and di- luted 3^ 30/III/34 )) Destroyed in 11 minutes >f Made up to original vol- ume and di- luted 34 30/III/34 >» Destroyed in 21 minutes >> Made up to original vol- ume and di- luted Vs 12/1/34 Centrifuged at 3000 R.P.M. for 5 min- utes; heated to 60° C; superna- tant fluid used Destroyed >j Undiluted 17/111/34 Centrifuged at 3000 R.P.M. for 40 minutes; heated to 60° C; super- natant fluid used Destroyed in 3 minutes )) Diluted 1/40 17/III/34 }} Destroyed in 43/2 minutes }f Diluted 1/80 17/111/34 n Destroyed in 73^2 minutes Diluted 1/120 17/III/34 j> Destroyed in 8 minutes )) Diluted 1/160 12/1/34 Centrifuged at 3000 R.P.M. for 5 min- Destroyed 3 eggs out of 9 with sec- Undiluted utes; not heated; - ond polar supernatant fluid body and used; a few sperm sperm present 17/III/34 Centrifuged at 3000 R.P.M. for50min- utes; not heated; no sperm present Destroyed in 13^ minutes 1 polar body Diluted 1/20 20/IX/35 Boiled for 12 min- utes; all sperm dead Left intact af- ter 1 hour )> Diluted H 79 80 THE EGGS OF MAMMALS TABLE XI The Effects of Exposing Freshly Ovulated Rabbit Ova to Various Solutions of Trypsin. (From the Journal of Experimental Zoology) Trypsin Con- Date centration (Dry Trypsin PER 100 c.c. Effect on Cumulus Cell .Mass Effect on Eggs Ringer-Locke Solution) 10/11/34 0.50 Destroyed 3 "polar" bodies in 10 minutes 10/11/34 0.25 M Egg shrunken 10/11/34 0.125 " >) 10/11/34 0.062 Partly destroyed M 10/11/34 0.032 »» " 6/II/34 25.00 Destroyed almost 6 to 10 "polar" bodies followed immediately by partial digestion of ova 6/II/34 21.00 " " 17/11/34 1.00 Destroyed in 1 minute Egg partly digested 17/11/34 0.50 Destro3^ed in 13^ minutes 1st polar body digested 17/11/34 0.25 Destroyed in 3 minutes 1 polar body, egg shrunken * 17/11/34 0.125 Destroyed in 6 minutes >) M :tc 17/11/34 0.062 Destroyed in 14 minutes )) i1 * 17/11/34 0.032 Destroyed in 31 minutes >> n * * All these ova showed irregular masses of webbed tissue in the perivitelline space. The data of these experiments show typical folUcle cell dispersion and also ^^ polar body" formation (Figure 26). These are, however, not true polar bodies but rounded cyto- plasmic masses caused by the action of the enzyme prepara- tion upon the egg surface. Sections of the ova of these experiments showed the ''polar bodies" to be chromatin free. The polar bodies observed by Yamane in his pan- creatin experiments were probably of this nature. The polar bodies formed in his experiments with rat and horse sperm may have also have been false polar bodies due to the strongly digestive action of the heterologous sperm sus- pensions, for, as we have seen, rat sperm suspensions are extremely effective as follicle cell dispersing agents even in very low concentrations. Krasovskaja (19356) believes that FERTILIZATION AND CLEAVAGE 81 actual penetration and pronucleus formation occurred in his attempts to fertilize rabbit eggs with rat sperm. No figures showing actual sperm penetration are given in this paper. The nuclear configurations shown may, in fact, occur in Fig. 26. Rabbit ovum from sterile mat- ing treated with trypsin solution. Note many "polar" bodies. See text. ova cultured in vitro with no sperm added (see Chapter VIII). The inamediate effect of semination (and fertilization) upon mammalian ova is a definite shrinkage of the vitellus (Pincus and Enzmann, 1932). Quantitative estimates of this shrinkage in rat eggs have been made by Gilchrist and Pincus (1932). In Table XII are presented their data on folhcular and tubal ova. They show that a 14 per cent reduc- tion in volume occurs in fertilized tubal ova. Furthermore, when unfertilized ova are exposed to sperm suspensions a similar shrinkage occurs (Table XIII). This shrink- age is not due to polar body extrusion since it occurs in vitro within 5 to 10 minutes, and polar bodies are normally ex- truded at 45 minutes to 1 hour after semination in vitro (Long, 1912). The ova apparently increase somewhat in volume after this initial shrinkage. Krasovskaja (1935a) 82 THE EGGS OF MAMMALS has observed an exactly similar initial shrinkage followed by a return to normal in rabbit ova seminated in vitro. TABLE XII The Volume of Rat Eggs in Three Stages of Development. Gilchrist and Pincus, 1932) (From Stage Follicular Tubal, un- fertilized 1-cell Average Volume of Round Eggs, cu. mm. 0.000333 (1) 0.000251 0.000202 0.000023 0.000009 Average Volume of Elongated Eggs, 0.000339 ± 0.000017 0.000226 0.000200 0.000013 0.000010 Average Volume of All Eggs, cu. mm. 0.000337 ^ 0.000010 0.000234 0.000201 0.000018 0.000010 TABLE XIII The Size of Rat Eggs under Various Conditions of Culture. (From Gilchrist and Pincus, 1932) Treatment Incubated in Ring- er's solution alone Incubated with live sperm Incubated with dead sperm Num- ber OF Eggs Average Diameter Immediately AFTER Putting Eggs on Slide, Microns 74.4 ± 1.4 77.9 ± 1.4 72.8 ± 1.1 Average Volume Calcu- lated, cu. mm. 0.0002 IG 0.000248 0.000204 Average Diameter Some Time AFTER Incubation, Microns 76.3 ± 0.4 72.7 ± 1.4 69.7 ± 1.3 Average Volume, Calcu- lated cu. mm. 0.000232 0.000205 0.000179 Shrink age. Per Cent 17 12 Sperm penetration into living ova has been observed only once (Pincus, 1930); a modified fertilization cone ap- pears to form at the point of contact. This cone very quickly subsides as is apparent also from fixed preparations of mammalian ova in the tubes {e.g., Lams and Doorme, 1908; Sobotta and Burkhard, 1911 ; Lams, 1913; and others). The length of time that the mammalian ovum remains capable of fertilization has been largely a matter of specu- lation. Exact experimental inquiry has, however, been undertaken in the rabbit (Hammond and Marshall, 1925; Hammond, 1928 and 1934) and in the ferret (Hammond and Walton, 1934). Taking advantage of the fact that the FERTILIZATION AND CLEAVAGE 83 Litter Size and Fertility TABLE XIV IN Timed Matings of Rabbit Does. (From Hammond, 1934) No. OF Matings Matings at Hours after Sterile Coitus Hours before ( + ) or after (— ) Ovulation Average Litter Size Matings Fertile, Per Cent No. OF YOUNQ PER Mating Made (a) All strains together (52 different does used) 323 Normal + 10 6.4 79.6 5.3 6 5 + 5 6.4 82.3 5.3 65 6 + 4 4.7 64.6 3.0 55 7 + 3 4.4 58.2 2.5 81 8 + 2 4.2 42.0 1.8 85 9 + 1 3.6 37.6 1.4 68 10 4.5 22.1 1.0 57 11 - 1 3.4 12.3 0.4 63 12 - 2 3.2 6.3 0.2 (b) C strain (17 different does used) 131 Normal + 10 7.4 75.0 5.0 25 6 + 4 5.4 52.0 2.8 18 7 + 3 3.7 55.6 2.1 22 8 + 2 2.8 27.3 0.8 21 9 + 1 4.3 28.6 1.2 20 10 4.5 10.0 0.4 18 11 - 1 19 12 _ 2 (c) E strain (21 different does used) 90 Normal + 10 8.1 80.0 6.5 3 5 + 5 7.0 100.0 7.0 19 6 + 4 5.8 63.2 3.7 23 7 + 3 5.6 65.2 3.8 37 8 + 2 4.9 48.6 2.4 48 9 + 1 3.6 41.7 1.5 25 10 5.4 36.0 2.0 21 11 - 1 4.2 23.8 1.0 21 12 - 2 4.0 9.5 0.4 (d) F strain (14 different does used) 102 Normal + 10 4.0 84.3 3.4 3 o + 5 5.5 66.6 3.7 21 6 + 4 3.4 81.0 2.7 14 7 + 3 2.7 50.0 1.4 22 8 + 2 3.8 5.5 1.7 16 9 + 1 2.7 47.5 1.0 23 10 2.2 37.4 0.4 18 11 - 1 1.5 11.1 0.2 23 12 - 2 2.5 18.7 0.2 84 THE EGGS OF MAMMALS rabbit OMilates at 10 hours after copulation and the ferret at about 30 hours, Hammond and his coworkers undertook a series of matings using an initial sterile mating to initiate the o\ailation stimulus and then fertile mating to permit sperm access to ova at successively later intervals. In the 80 - 8 - , — y 70 - 7 _ \ W \ i-J .......... ......—.-•••, y 1-1 geo -|6 - \ \ u K '. \ u. 2 ___. - — — > \ \ ^50 -h5 _ \ \ \ a Ed \ '--A A H 9 \ "V / \ <40 -^4 — \ X/ \ S > \ X V Ui < O30 - 3 — \ ^ \ \ 20 - 2 ~ \\ 10 - 1 ' s \ \ \ fi It I 1 1 1 1 1 1 1 1 +10 + 5 +4 +3 +2 +1 -1 -2 OVULATION AVERAGE UTTER SIZE ^c OF MATINGS WHICH WERE FERTILE NUMBER OF YOUNG PER MATING HOURS INTERVAL BEFORE (I-) OR AFTER (-) OVULATION Fig. 27. Fertility of matings made at different intervals of time before or after mating (all strains). (From the Journal of Experimental Biology.) most extensive series of rabbit matings (Hammond, 1934) employed three inbred strains of rabbits in order that homo- geneous conditions of fertility might exist in his experi- ments. The data of his experiments are given in Table XIV, and a graphical representation in Figure 27. It is at once obvious from these data that matings to fertile bucks made after the 5th hour following a sterile mating show a decline both in absolute (per cent of fertile matings) and relative fertihty (number of young produced). WTien matings are made to fertile bucks at twelve hours after the sterile copulation, i.e., at two hours after o\ailation minimum fertility is attained. FERTILIZATION AND CLEAVAGE 85 In order to make quite certain that the cause of the smaller litters produced after the experimental matings made late in relation to ovulation was due to the ova not being fertilized and not to any interference with the process of ovulation or other causes, a few does so mated were killed during pregnancy and the number of corpora lutea {i.e., ova shed) compared with the number of foetuses present. The results are given in Table XV, and demonstrate that there is a decrease in the number of ova fertilized in the later matings. This implies that the sperm reach the portion of the tubes containing the ova at a time when these ova are for some reason no longer fertilizable. TABLE XV The Percentages of Rabbit Ova Fertilized in Matings Made at Vari- ous Times before and after Ovulation. (From Hammond, 1934) Matings at Does Number of Ova Not Hours before ( + ) or after (-) Ovulation Hours after Sterile Coitas Number Strains Ova Shed Normal Foetuses Atrophic Foetuses Ova Not Ferti- lized LIZED, Per Cent 6 7 8 9 11 + 4 + 3 + 2 + 1 - 1 2 2 3 2 2 E E,F E E E, F 25 23 41 25 19 15 10 18 6 4 2 3 5 8 7 18 19 15 32 35 44 76 79 On the basis of Heape's (1905) observations that rabbit sperm reach the tops of the tubes in about 4 hours after coitus, Hammond concludes that rabbit ova can remain fertilizable for at most 6 hours after o^Tllation, by allowing a 2-hour postovulatory interval in the matings made at 12 hours after the ovulation-inducing mating. This period coincides approximately with the time {i.e., 7 hours) that it takes for the ova of sterile matings to begin to separate from the follicle cell mass and start their free travel down the tubes. Hammond concludes therefore that the presence of the plug of massed ova is necessary for fertiUzation. He reasons as follows: '^The plug, of liquor folliculi and detritus, containing the ova dams up the top of the Fallopian tube and remains there 86 THE EGGS OF MAMMALS for some 4 (in fertile matings) to 7 (in infertile matings) hours, during which time the ascending sperms are collect- ing in its lower layers (see Figure 28). The accumulation of sperms so effected ensures that sufficient shall be available to fertilise the ova as they emerge from the plug. As the sperms are put in progressively later than normal in relation to the time of ovulation, the accumulation of sperms be- comes progressively less and the chances of all the ova FALLOPIAN TUBE PLUG CONTAINING OVA Fig. 28. Diagram illustrating how the chances of the ova becoming fertilized are reduced as the interval between mating and ovulation is reduced, a = amount of sperm swarm which would accumulate if mating were made at the ordinary time — 10 hours before ovulation, b = amount of sperm swarm which would accumulate if mating were made 4 hours before ovulation. (From the Journal of Ex-perimental Biology.) becoming fertilised are reduced in proportion to the time the fertile mating is delayed with reference to the time of o\ailation. ^^The ascent of the sperms can be represented as a curve (see Figure 28 and Hammond and Asdell, 1926) or as a swarm (in the statistical sense). The apex of the sperm swarm (shown, in order to assist visualisation of the prob- lem, very diagrammatically in Figure 28) reaches the top of the tube just at the time the plug is formed, i.e., at ovu- lation, and so during the time that the plug exists (about 4 hours) it dams up but few sperms as compared with a normal mating made 10 hours before ovulation when the sperm swarm has ascended further (to the point a in Fig- ure 28).'^ FERTILIZATION AND CLEAVAGE 87 While Hammond's deductions are entirely reasonable, it is possible that the 6 hours of fertilizable life allotted to rabbit ova is possibly too short since in normal matings 13^ to 3 hours are required by the sperm to reach the ova. This would make the critical period some 73^ to 9 hours long. Furthermore it is not the arrival of the first sperm that is effective, since as we have previously seen (pages 77 to 78) a definite minimal sperm concentration is necessary for both folUcle cell dispersion and fertilization. If the critical period were thereby further lengthened by 1 to 2 hours it would coincide almost exactly with the time when the ova separating out of the tubal plug begin to ac- quire a coating of albumen. This coating is impervious to sperm (Pincus, 1930). Similar experiments of Hammond and Walton (1934) with the ferret show that fertile matings made as late as 30 hours after ovulation result in the production of young. The rea- sons for the maintenance of the fertilizing capacity of ferret ova for as long as 30 hours are not deducible in detail since the exact tubal history of ferret ova is not known. Hammond and Walton attribute the greater length of fertilizable life in this case to the longer time it takes for the ova to trav- erse the oviduct, e.g., 5 to 6 days in. the ferret compared with 3H days in the rabbit and the presumably correlated slower dissolution of the plug of massed ova. In the spontaneously o\ailating mammals the fertilizable life of the ova is also of short duration, but exact data are not available since it is ordinarily difficult to ascertain the specific time of ovulation. Hartman (1924) has shown that opossum ova traverse the tubal portion of the oviduct in 24 hours and that upon entry into the uterus unfertilized ova are definitely degenerated. Charlton (1917) found clear signs of degeneration in unfertilized tubal mouse ova by two days after parturition. Since post-partum ovulation occurs in the mouse at about 14 hours after parturition (Long and Mark, 1911) mouse ova may be said to retain cyto- logical normality for about 35 hours. In the rat ova present 88 THE EGGS OF MAMMALS in the first third of the oviduct appear cytologically normal (Mann, 1924). According to the data of Long and Evans (1922) the ova remain in this portion of the oviduct for about 33 hours. Hartman's (1932a) data on timed matings in Macacus show that fertile matings occur only between the 9th and 18th days of the menstrual cycle with maximum between days 11 and 16. This, of course, does not imply that the ova are fertilizable for several days, but presumably that ovulation may occur at any time during the critical 9 day period. Matings time in relation to the onset of oestrus in the sheep (Quinlan, Mare and Roux, 1932) and the pig (Lewis, 1911) indicate a maximum period of fertility of 48 hours. It is unnecessary in this monograph to discuss the cyto- logical details of fertilization and cleavage in mammalian ova, since these are now textbook commonplaces. Our inter- est is primarily in the physiological mechanisms underlying these events and their relation to the dynamics of growth and development. We shall again discuss certain aspects of the fertilization process in the chapter dealing with the activation of unfertilized eggs. Now we shall turn our attention to the relatively scant data that deal with the mechanism of cleavage in tubal ova. Until fairly recently no very accurate data on the rate of cleavage in tubal ova have been available. This has been due in part to the difficulty of timing ovulation. Even now it is possible to construct only approximate growth curves for a limited number of species. These curves are presented in Figure 29. It will be noted that rabbit ova cleave much more rapidly than those of the other species (see Plate VII). It is a matter of some interest to ascertain whether this difference in the cleavage rate is the result of an especially stimulating tubal environment in rabbits, or whether the cleavage rate is an inherent property of the ova. The data on the monkey were, in fact, deduced from Lewis and Hartman's (1933) observations of cleavage in vitro, and may be taken to indicate that segregation from the tubes FERTILIZATION AND CLEAVAGE 89 results in no great acceleration of cleavage since the growth rate remains at about the level of the other slow-cleaving species. The writer has transplanted mouse ova into the 28 20 1 1 1 RABBIT / / ++++-t- MONKEY GUINEA PIG / / MOUSE ■DAT' f / f / f / / 1 KAl PIG I / / f 1 •i 1 — / 1 / / / /A — / J:£:^ J< ^^ 'i^^-^^-^'^'^^^^y^ ^ ^^5-i 'J^, ..^^ ^^^^^S^r *---- „--' 20 40 60 80 Fig. 29. Showing the cleavage rates of tubal ova in various species of mam- mals. Abscissa: time in hours after copulation. Ordinate: number of cells. The rabbit = data of Gregory, 1930, and Pincus, 1930. The monkey = data of Lewis and Hartman, 1933. The guinea pig = data of Squier, 1932. The mouse = data of Lewis and Wright, 1935. The rat = data of Gilchrist and Pincus, 1932. The pig = data of Heuser and Streeter, 1929. fallopian tubes of the rabbit and has noted no increase in the cleavage rate over a period of 72 hours. Castle and Gregory (1929; also Gregory and Castle, 1931) have, in fact, found certain definite congenital differences in cleavage rate between different races of rabbits. A resume of their data is given in Table XVI. The animals of their large race (A) attain an average adult weight of about 5500 grams in females and 5400 grams in males. The cor- ^ r^- Mm Fig. 4 «^"^ ti Fig. 5 1m y' « ^^ K^ Fig. 6 !^ Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 :^ vf f':- ^'^ Fig. 14 Fig. 15 )i .^. # Fig. 16 Fig. 1- Fig. 18 Fig. 19 m ♦ ^.::-i?^|^ '.T-^^ ^#** ♦ 4 Fig. 20 Fig. 21 Plate VII (Caption on facing page.) 90 Fig. 22 FERTILIZATION AND CLEAVAGE 91 responding adult weights in the small race (B) are 1500 grams for females and 1400 grams for males. The various hybrid combinations show roughly intermediate adult weights. Their data show clearly that certainly beyond the 32nd hour after copulation the cleavage rate is fastest in the large race animals and the expected sort of intermediate rates occurs in the various hybrid combinations. It is entirely possible that even the earliest cleavages do actually occur sooner in large race animals since large does ovulate later than small does and therefore their ova should be fertilized later. The number of mitoses in cleaving eggs of the large races also exceeds those in the small race, as the data in the columns labelled ''prospective" indicate. Since this differ- ence is consistently present in reciprocal hybrids between the races the implication is that the sperm nuclei also participate in the control of the cleavage rate. In spite of the inherent differences in the speed of segmen- tation the processes of differentiation occur at the same time in the large and small size rabbits. Thus the blast o- Plate VII. All photographs on this plate were made from the living rabbit eggs in Locke's solution, as soon as possible after removal from oviduct or uterus at an enlargement of 180 diameters (apochromatic objective 16 mm., compensating ocular 8). They are arranged in order of development and show the principal features of cleavage and formation of segmentation cavity. It will be noted that the trophoblast is precocious in its differentiation as com- pared with the remainder of the egg, and as soon as the trophoblast becomes histologically different one sees fluid begin to accumulate within the egg, thereby forming the segmentation cavity. Figs. 4 to 9, Litter C 43, 25 hours after coitus. Fig. 4, one-cell stage with two polar bodies; Fig. 5, one cell, with coarse granules, perhaps abnormal; Figs. G to 9, showing two primary blastomeres, one tending to be larger than other. Figs. 10 and 11, Litter C 36, 28^:^ hours after coitus. Four-cell stage with crossed arrange- ment of blastomeres. Figs. 12 to 14, Litter C 45, 32 hours after coitus. 5, 6 and 8-cell stages. In Fig. 13 the cell at top is just dividing. Fig. 15, Litter C 35. 16-cell stage. Fig. 16, Litter C 41, 55 hours. Morula of about 32 cells. Fig. 17, Litter C 32, Q9% hours. Smooth surfaced morula. Fig. 18, Litter C 38, 71^ hours. Differen- tiated trophoblast cells on surface. Fig. 19, Litter C 33, 76^ hours. Fluid beginning to collect in cleft between trophoblast and inner-cell mass. At this time the albumen coat is at its maximum. Fig. 20, Litter C 33, 76^ hours. Subtrophoblastic lakelets of fluid determining early appearance of segmentation cavity. Fig. 21, Litter C 34, 90 hours. Definite segmentation cavity. Note demarcation between trophoblast and inner-cell mass. Fig. 22, Litter C 42, 92 hours. Zona much stretched and layer of albumen much thinned out. Inner-cell mass flattening into typical germ-disc. From Gregory, 1930. 92 THE EGGS OF MAMMALS TABLE XVI The Mean Number of Blastomeres per Ovum at Various Times after Copulation in Large and Small Rabbits and in Certain Hybrids BETWEEN Them. (From Castle and Gregory, 1929, and Gregory and Castle, 1931) Hours AITEK Number Number Mean Num- ber OF Probable Copula- Race OF Does OF Eggs Blasto- Error tion meres 32M A (actual) 3 31 4.06 — >> A (prospective) 3 31 4.29 — tf B (actual and prospective) 3 12 4.41 — 40 A (actual) 4 45 9.94 ±0.24 A (prospective) 4 45 10.82 — B (actual) 8 27 8.29 ±0.19 B (prospective) 8 27 8.37 — AB (actual) 1 9 8.44 — AB (prospective) 1 9 8.60 — 41 A (actual) 3 22 11.64 ±0.44 A (prospective) 3 22 12.68 — B (actual) () 21 8.62 ±0.47 B (prospective) G 21 9.09 — BD (actual) 2 11 8.63 — BD (prospective) B and BD combined 2 11 9.18 — (actual) 8 32 8.62 — AD (actual) 3 20 9.25 — AD (prospective) 3 20 9.55 ±0.36 48 F (actual) 4 28 21.75 — F (prospective) 4 28 22.80 — B (actual) 4 15 14.00 — B (prospective) 4 15 14.50 — A = large race. B = small race. AB = Fi hybrid. BD = seven-eights small (D = AB XB). F = three-quarters large (AB X A). actual = number of blastomeres observed. prospective = number of blastomeres observed plus the number of mitoses. dermic vesicle forms at the end of the 3d day (Plate VII, Figs. 18-20), and the embryonic disc by the 168th hour after coitus. Castle and Gregory therefore attribute large size to an inherent mitotic intensity independent of dif- ferentiation potentials. The ova of the rabbit begin their differentiation early in comparison with the eggs of other species. Thus Gregory (1930) detected the beginning of the formation of the inner cell mass just after the 16-cell stage at about 47 hours after FERTILIZATION AND CLEAVAGE 93 coitus (37 hours after ovulation) and the cavity of the blastodermic vesicle may begin to form while the ova are still in the tubes. Guinea pig (Squier, 1932) ova enter the uterus in the 8-cell stage at the end of the 3d day after copulation and the blastodermic vesicles form only in the uterus at about 43^2 days after coitus. In the rat (Huber, 1915) the ova enter the uterus during the 4th day after coitus in about 12 cells and start to form the blastodermic vesicle during the 4th to 5th days post coitum, and in the mouse (Enzmann, Saphir and Pincus, 1932; Lewis and Wright, 1935) blastocyst formation occurs in the uterus during the 4th day after copulation. The physiological factors governing the cleavage of mam- malian ova have been scarcely examined. It has already been stated that the whole course of cleavage of rabbit eggs may proceed normally in vitro and in heterologous as well as homologous blood plasma (Pincus, 1930). This would seem to imply that no special environmental factors supervene in the tubes. On the other hand the ova of mice, rats and guinea pigs do not cleave under the ordinary (or a variety of) tissue culture conditions. The reasons for this species difference are not known though the superior vitality of rabbit ova has been attributed to their unique albumen coating; but Lewis and Hartman (1933) have over a period of approximately 24 hours, observed the regular cleavage in vitro of a monkey o\aim which lacks an albumen coating. In the case of those ova which have not undergone cleav- age in vitro one can only deduce that some limiting factor obtaining in vivo has not been duplicated. Since it is known that the secretory activity of the tubal epithelium is under hormonal control of the ovary (c/. Snyder, 1923) it is pos- sible that a special contribution to the economy of cleaving ova is made by a hormonally induced secretion. The cleaving ova of all mammals journey through the tubes during the early life of the corpus luteum. The secretory activity of the tubal epithelium changes markedly during the transi- 94 THE EGGS OF MAMMALS tion from the oestral to the luteal phase. Furthermore, it is possible that the ovarian hormones themselves may di- rectly affect the cleavage process. Oestrin, for example, definitely stimulates the mitotic activity of the vaginal epithelium, progestin inhibits uterine mitoses, etc. Accordingly Burdick and Pincus (1935; also Pincus and Kirsch, 1936) have investigated the effect of ovarian hor- mones upon the development of rabbit and mouse ova. They found that the injection of large amounts of oestrin in no way affected the cleavage process although ova in the early uterine stages degenerate and die when only moderate amounts of this hormone are injected (see Tables XXIII to XXV, pages 118-120, 122). That the hormone injected definitely affected the tubal tissue was evidenced by the fact that in both mice and rabbits an effective closure of the tubo-uterine junction was attained, and in rabbits both the contractile activity and the histological appearance of the tubal tissue were definitely altered to the oestrus type. In addition (Pincus and Kirsch, 1936) it was found that rabbit ova grow^n in cultures containing appreciable amounts of oestrin continued to cleave at the normal rate. Finally fertilized rabbit ova in 1- and 2-cell stages were injected into the fallopian tubes of does on heat (and therefore lacking corpora lutea), and these were found to develop normally up to the early blastocyst stage. Corner (1928) had already shown that in bilaterally ovariectomized rabbit does egg development stops at the early blastocyst stage. The segmentation processes appear, therefore, to be inde- pendent of the secretory activity of the ovaries, and of any effect that the ovarian condition may have upon tubal secretion. Rabbit ova will, indeed, go through the morula stage in a carefully balanced buffered Ringer-Locke solu- tion, indicating a fairly complete lack of dependence upon any special organic nutrition. It has, of course, been re- peatedly noted by observers of living material {e.g., van Ben- eden, 1875; Gregory, 1930; Gilchrist and Pincus, 1932; Squier, 1932) and by those who have examined fixed speci- FERTILIZATION AND CLEAVAGE 95 mens (Sobotta, 1895; Huber, 1915; and others) that mam- malian ova show no appreciable increase in size until the blastocyst stage. The most convenient approach to the study of the physio- logical processes underlying segmentation has involved the study of the respiratory processes (Warburg, 1908-14 ; J. Loeb and Wasteneys, 1912-15; J. Loeb, 1913; Runnstrom, 1930; Whitaker, 1933; and others). Mammalian ova are available in such small numbers that exact quantitative measurements of respiratory activity are difficult to make and have not been made. Nonetheless some indication of the nature of the underlying processes may be had by the use of specific poisons known to combine with and inhibit the reactions of definite components of the chain of reactions involved in respiration. Thus HCN is known to combine with iron- containing enzyme phaeohemin which is the initial activator in the aerobic phaeohemin-cytochrome chain (Warburg, 1932) and so to inhibit the respiration involving phaeo- hemin activity. Cyanide also inhibits the cleavage of ova of non-mammalian forms (Lyon, 1902; J. Loeb, 1906; see Needham, 1932), as does an oxygen-free medium (J. Loeb, 1895). Runnstrom (1935) has demonstrated that the mitotic process at segmentation in sea-urchin eggs is not dependent upon the level of respiration since the addition of pyocyanine to cyanide-inhibited egg suspensions restored oxygen con- sumption to normal levels but no division ensued. Rabbit ova presumably develop in a medium relatively low in oxygen, since the oxygen tension of the abdominal cavity, and by inference that of the tubes (which have free access to abdominal fluids), is 40 mni. Hg (Campbell, 1924) as compared with 150 mm. Hg, the oxygen tension of the air. It is of interest to inquire whether the segmentation of rabbit ova is Hnked with the aerobic phaeohemin system. Pincus and Enzmann (19366) have added KCN in appropri- ate concentration to cultures of cleaving rabbit eggs and the segmentation has ceased. Cinematographs of these ova indicated that the eggs were not ''killed" by the poison 96 THE EGGS OF MAMMALS since they exhibited the cyclosis (cytoplasmic movements) typical of living ova. Similar experiments with iodoacet- amide added to the cultures showed normal cytoplasmic activity of the ova but a limited amount of cleavage. lodo- acetamide presumably combines with the coenzyme con- cerned in the reduction of pyruvic to lactic acid (Meyerhof and Kiesling, 1933) so that the inhibition of both the oxygen- activating system and its presumable substrate system re- sults in the arrest of cleavage. While the exact coupling of the respiratory system with the mitotic mechanism has yet to be delineated these data do demonstrate that the fundamental processes are aUke in manomalian and non- mammahan ova. We have seen that rabbit ova may be fertilized and cul- tured in vitro. It is a matter of some importance' to deter- mine whether such ova may give rise to normal rabbits. Accordingly the writer (see Pincus and Enzmann, 1934) undertook the transplantation of such ova into the oviducts of pseudopregnant rabbit does and found that ova fertilized in vitro and also normally fertilized ova kept in culture during the cleavage period apparently resumed normal development after transplantation as evidenced by the production of normal young at term. It is a matter of some interest to note that one set of ova had failed to cleave during 20 hours in culture but nonetheless young were obtained. The development of a technique for the transplantation of mammalian ova into the oviducts makes possible the testing of a number of problems of development hitherto inaccessible. As we shall see later (Chapter IX) it is neces- sary that a progestational uterus be available for ensuring differentiation of uterine stages. Thus Biedl, Peters and Hof stater (1922) transplanted rabbit ova into non-pregnant uteri in some 70 experiments and in only one doubtful case were young recovered. Nicholas (19336) transplanted the isolated blastomeres of the 2-cell stage in the rat under the kidney capsule and observed varying degrees of development of the three germ layers and their various derivatives. The FERTILIZATION AND CLEAVAGE 97 writer has transplanted single blastomeres of 2-cell rabbit embryos into the tubes and obtained normally differentiat- ing, but small sized blastodermic vesicles from the pseudo- pregnant uteri of the recipient does. The physiological processes occurring in such embryos are of extraordinary interest and certainly deserve further investigation. CHAPTER VIII THE ACTIVATION OF UNFERTILIZED EGGS We have seen that the fundamental control of the cleavage mitoses is alike in rabbit and sea-urchin ova. We shall now inquire whether the activation of mammalian eggs is also similar to that of other forms. With the exception of the three 2-cell rat eggs described by Mann (1924) there are no observations of a possible normal parthenogenetic development of unfertilized tubal eggs in vivo. With the exception of a single observation by Champy (1927), the first investigation of the behavior of unfertilized tubal ova placed in tissue culture is that of Pincus (1930). His data are presented in Table XVII. TABLE XVII The Development OF Unfertilized Rabbit Ova in Culture. (From Pincus , 1930) Age of Ova (Hours AFTER Cop- Num- ber Medium Exam- ined (Hours in Cul- Description Num- ber Di- vided Num- ber Undi- ulation) ture) (1) 1 RPRE 44 1 — unsegmented 1 (2) 4 RPCE 48 3 — unsegmented 1 — in 3 regular cells 1 3 (3) 4 RPCE 48 4— unsegmented 4 (4) 11 9 1 CPCE 48 1 — several polar bodies (?) 1 CO (?) (5) 11 25 5 RPRE 24 3 — unsegmented 1 — 8 regular cells 1 — 4 regular cells 2 3 (6) 11 40 2 RPRE 48 2 — unsegmented 2 (7) 12 5 5 RPRE 47 3 — unsegmented 2— in 12 to 16 regular cells 2 3 (8) 12 30 6 RCPCE 27 1 — in 2 regular cells 1 — in 3 regular cells 1 — in 4 regular cells 3 — in 5 to 6 regular cells 6 (C = Chicken. R = Rabbit. P = Plasma. E = Embryo Extract.) THE ACTIVATION OF UNFERTILIZED EGGS 99 TABLE XVII (Continued) The Development of Unfertilized Rabbit Ova in Culture. (From Pincus, 1930) Age of Ova (Hours AFTER Cop- ulation) 13 15 13 35 (11) (12) 13 14 50 (13) 14 35 (14) 15 (15) 15 15 (16) 16 (17) 17 10 (18) 17 33 (19) 17 45 Num- ber Medium RPCE RPRE RPRE RPRE RPCE CPCE RPRE RPCE RPCE CPCE RPCE Exam- ined (Hours IN Cul- ture) 47 43 25 27 30 27 22 23 48 26 Description 2 — unsegmented 1—16 to 20 cells 1 — in 2 cells and 2 to 5 polar bodies 3 — with about 5 polar bodies 7 — unsegmented 2 — unsegmented 1 — in 4 regular cells and 2 polar bodies 1 — in morula 1 — unsegmented 2 — in 3 regular cells 2 — in 4 regular cells 2— in 36 to 40 regular cells 2 — in about 4 cells regular 1 — with multiple polar bodies 1 — unsegmented 1 — in 4 regular cells 1 — in about 16 cells 2 — in 1 very large cell and 10 to 12 small ones 2 — in about 16 cells 1— in 32 to 48 cells 1 — in 2 regular cells and 2 polar bodies 1 — in 3 to 4 large cells and 10 small cells 1 — in 1 large cell and 16 small cells 2 — no segmentation 1 — 2 large, 2 small cells and several polar bodies 1 — in 16 very regular cells 2— in 20 to 32 cells Num- ber Di- vided Num- ber Undi- vided (C = Chicken. R = Rabbit. P = Plasma. E = Embryo Extract.) 100 THE EGGS OF MAMMALS TABLE XVII (Continued) The Development of Unfertilized Rabbit Ova in Culture. Pincus, 1930) (From (20) (21) (22) (23) (24) (25) (26) (27) (28) Age of Ova (Hours AFTER Cop- ulation 18 10 18 li 18 20 18 25 18 30 18 50 19 5 19 30 19 45 Num- ber Medium RPCE RPCE CPCE RPCE RPRE RPRE RPCE RPCE RPCE Exam- ined (Hours IN Cul- ture) 48 29 47 24 48 47 22 27 22 Description 3 — unsegmented 1 — 2 unequal cells and 7 to 8 polar bodies 1 — 3 cells and several polar bodies 1 — 4 regular cells 1 — 10 small cells and 1 large cell 4 — unsegmented 1 — 3 cells regular 2 — about 4 regular cells, but shrunken 1 — in 12 regular cells 1 — in about 8 cells, but shrunken 1 — unsegmented 1 — 3 polar bodies 2 — unsegmented 1 — 1 large cell and 2 to 3 small cells 1 — 2 regular cells and 2 polar bodies 1 — 4 regular cells 1 — 7 regular cells 4 — about 8 cells 2 — unsegmented 1 — in 3 cells 2 — in 8 regular cells 1 — in 10 regular cells 2 — unsegmented 2 — in 2 regular cells 4 — in 4 regular cells 1 — in 7 cells 1 — 2 unequal cells and 3 polar bodies 1 — unsegmented 1 — 2 regular cells 2 — 4 regular cells 1 — unsegmented 3— in 2 cells 2 — in 4 cells 1 — in 6 cells 1 — in 8 cells Num- ber Di VIDEO 1(?) 8 Num- ber Undi- vided (C = Chicken R = Rabbit. P = Plasma. E = Embryo Extract.) THE ACTIVATION OF UNFERTILIZED EGGS 101 The Development of TABLE XVII (Continued) Unfertilized Rabbit Ova Pincus, 1930) IN Culture. (From Age of Ova Exam- Num- (Hours after Cop- Num- ber Medium ined (Hours IN Cul- Description Num- ber Di- vided ber Undi- ulation ) ture) vided (29) 20 2 CPCE 22 2 — many polar bodies 2 (30) 20 10 3 RPRE 49 3 — in many cells 3 (31) 20 20 4 RPCE 47 4 — unsegmented 4 (32) 20 20 2 CPCE 48 1 — unsegmented 1—1 large and 2 to 3 small cells 1 1 (33) 24 25 5 RPCE 27 1 — 2 unequal cells 3—2 regular cells 1 — 3 regular cells 5 (34) 24 45 2 RPRE 44 1 — 1 large cell and sev- eral polar bodies 1—16 to 20 regular cells and a few po- lar bodies 2 (35) 27 35 5 CPCE 46 1 — unsegmented 2 — about 8 cells and many polar bod- ies 2 — one large cell and many polar bodies 4 1 (36) 28 35 9 RPCE 47 4 — unsegment ed 1 — 4 regular cells 1 — 6 unequal cells 3— about 8 cells 5 4 (37) 37 20 2 RCPCE 27 2 — in many cells and degenerate 2 (38) 40 40 3 CPCE 45 3 — in many small cells 3 (39) 43 10 6 CPCE 46 1— in 2 cells 1 — in 4 cells and many polar bodies 2 — in 1 cell and many polar bodies 2 — in many small cells 6 (40) 47 30 6 RPCE 22 3 — unsegmented 1 — in 2 unequal cells 2 — with many polar bodies 3 3 (41) 48 30 8 RPCE 48 5 — unsegmented 3 — with many polar bodies 3 5 (42) 48 47 6 RPRE 52 1 — about 8 large cells 1 — 3 unequal cells and many polar bodies 6 (C = Chicken. R = Rabbit. P = Plasma. E = Embryo Extract.) 102 THE EGGS OF MAMMALS TABLE XVII (Continued) The Development of Unfertilized Rabbit Ova in Culture. (From Pincus, 1930) Age of Ova (Hours AFTER Cop- ulation) Num- ber Medium Exam- ined (Hours in Cul- ture) Description Num- ber Di- vided Num- ber Undi- vided 4— with many polar bodies (43) (44) (45) 50 30 68 33 72 5 3 4 RPRE RPRE RPCE 45 45 22 1 — unsegmented 4 — in many cells 3— unsegmented and degenerate 2 — unsegmented and shrunken 2 — about 10 polar bod- ies and shrunken 4 2 1 3 2 (46) 73 40 7 RPCE 45 4 — unsegmented 2—16 regular (?) cells 1 — 5 cells and 3 polar bodies 3 4 (47) 96 45 2 RPCE 46 2— unsegmented 2 (C = Chicken. R = Rabbit. Plasma. E = Embryo Extract.) The primary and surprising fact evident from the data is that a majority of the ova placed in culture underwent a certain degree of development, so that out of 213 eggs cul- tured, 136 or 63.8 per cent are classified as having ^^ divided," the term ^'divided" including any degree of observable development beyond the 1 -celled state of the ova as re- covered from the animals. It was the primary objective of these investigations to ascertain the nature of the various degrees of development undergone in vitro and to establish any relationship that might exist between the age of the ova and the nature of the development. Before undertaking any detailed analysis of the data it is deemed advisable to describe the various types of development observed. The ova observed in the 2-cell stage varied in appear- ance as shown in Plate VIII, Figs. 1-3. The great major- ity of them resembled that of Figure 1, and showed usually one, sometimes two or three, polar bodies. The ovum of Figure 3 was photographed after the egg had been in cul- f Fig. 1 Fig. 2 Fig. 3 Fig. 4 » Fig. 5 Fig. 6 Fig. 7 Fk.. S Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 Fig. 14 L«, A Fig. 15 Plate VIII. Ova from sterile matings as they appeared after being cul- tured in vitro. (From the Proceedings of the Royal Society.) Recovered at 18 hrs. Recovered at 27 hrs. Recovered at 19 hrs. Recovered at 18 hrs. Recovered at 18 hrs. Recovered at 19 hrs. Fig. 7, Recovered at 19 hrs. Fig. 8, Recovered at 28 hrs. Fig. 9, Recovered at 17 hrs. Fig. 10, Recovered at 24 hrs. Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, 30 mins. 30 mins. 5 mins. 10 mins, 15 mins 5 mins. 5 mins. 35 mins. 10 mins, 45 mins. after sterile copulation cultured for Fig. 11, Recovered at 37 hrs. after sterile copulation cultured for 6 hrs. Fig. covered at 73 hrs. 40 mins. after sterile copulation cultured for 45 hrs. Fig. covered at 73 hrs. 40 mins. after sterile copulation cultured for 45 hrs. Fig. covered from the ovary, and cultured for 28 hrs. Fig. 15, Recovered at 30 mins. after sterile copulation cultured for 24 hrs. after sterile copulation cultured for after sterile copulation cultured for after sterile copulation cultured for after sterile copulation cultured for after sterile copulation cultured for after sterile copulation cultured for ^fter sterile copulation cultured for after sterile copulation cultured for after sterile copulation cultured for 44 hrs. 25 hrs. 22 hrs. 17 hrs. 28 hrs. 22 hrs. 22 hrs. 23 hrs. 23 hrs. 24 hrs. 12, Re- 13, Re- 14, Re- 48 hrs. 103 104 THE EGGS OF MAMMALS ture 22 hours. It was subsequently replaced, and when examined 24 hours later had formed eight cells quite regular in appearance. Note is made of this fact because it indicates that ova segmenting irregularly at the first division may eventually assume an appearance characteristic of ova under- going quite regular division. The ovum of Figure 4 was photographed just as segmentation from two to three cells was being completed. One of the two blastomeres had not quite rounded out at the time of photographing. The segmented ovum of Figure 5 is also in three cells. When first examined after 23 hours of culturing no segmentation had occurred; 5 hours later the ovum had divided as photo- graphed. The ova of Figure 5 were recovered at 18 hours and 15 minutes after copulation and were still surrounded by a number of follicle cells. They were placed vis-a-vis in culture and the out-growing follicle cells of each ovum became intermingled and caused the compression of the ova seen in the photograph. Figure 6 is a photograph of a typical 4-celled stage, exactly comparable to the 4-celled stage of fertilized ova (see Plate VII, Figs. 10 and 11). The num- ber of polar bodies in such ova vary from one to three. Again, the great majority of ova observed in four cells pre- sented the regular appearance of the ovum of Figure 6. Figure 7 represents an ovum containing seven cells in which one of the four blastomeres of the 4-celled stage divided twice while the others remained quiescent. Such differential division may begin after the 2-celled stage as illustrated by Figure 8, in which one of the original two cells has re- mained quiescent while the other divided in two, and one of the two cells formed divided twice to form four small cells. There is also photographed the single polar body of this ovum. Figure 9 represents another case in which one of the early blastomeres has remained quiescent while the others have gone on dividing at a rapid rate. Some such process is responsible for most of the irregular segmentations observed. At the same time segmentation may proceed in a manner comparable to that of normal fertilized ova in vivo, THE ACTIVATION OF UNFERTILIZED EGGS 105 so that one may observe in the same culture the different types described. Figure 10 is a photograph of an ovum segmented to about 20 cells and apparently with a marked degree of regularity. When we come to consider ova seg- mented into 20 and more cells the interpretation of the course of their development becomes difficult because of a peculiar complication. The o\aim of Figure 11 offers a per- tinent illustration. It w^as recovered from the tubes at 37 hours after copulation and was in the 1-cell stage. Six hours later it presented the appearance shown in the photograph. It has apparently segmented into about 36 cells in the course of 6 hours. This means astonishingly rapid segmentation. As a matter of fact what probably occurred was a complex fragmentation of the entire ovum. In the course of filming an ovum recovered at 29 hours and 20 min- utes after copulation the course of such fragmentation was observed. After an initial period of quiescence the ovum underwent a period of activity which resulted in the sudden appearance of many small ^^ blast omeres." This was fol- lowed by a complete quiescence with the cessation of all cytoplasmic movements. The ''cells" of this fragmented ovum, however, were not at all distinct in form or outline. One may observe ''many-celled" ova. in culture that pre- sented this vagueness of cell outline, but we have also seen well advanced ova in which the component blastomeres were as distinct and clear as in the normal fertilized ovum. Inter- pretation must, therefore, proceed slowly until the exact mechanics of division in vitro is thoroughly investigated. A certain amount of light, however, is shed on the problem by the consideration given below to the relation between the age of the ova and the nature of the development observed. Figures 12 and 13 are photographs of two ova recovered at 73 hours and 40 minutes after copulation. They were photographed after having been 45 hours in the same cul- ture. Note the remarkable regularity of the cells of the ovum of Figure 13. The ova of Figures 14 and 15 represent types ordinarily described as "with many polar bodies." Both 106 THE EGGS OF MAMMALS have a very large single cell, beside which lie a number of very small ''cells" comparable in appearance to polar bodies. Very often this group of ''polar bodies" resembles an ir- regular indented cytoplasmic mass, and I have actually seen it formed as such a mass budded or divided off from the main body of the cell. This represents the extreme of irregularity observed. The foregoing account has been given irrespective of the age of the ova figured. It remains for us to ascertain if any relation does exist between the age of the ova cultured and the nature of their development. Before proceeding to a detailed inquiry, however, it must be pointed out that the various types of ova described and figured in the photo- graphs have been observed in ova of all ages so that no absolute correlation exists. Ova have been considered as segmenting regularly only when the cells of the two, four, eight and sixteen cell stages have been of equal size, or when one could obviously trace the regular descent of the cells in ova exhibiting intermediate stages. In the cases of ova exhibiting many cells only those showing clear cell outlines and cells of equal size have been classified as "regular." TABLE XVIII Effect of Age of Ova when Removed from Doe on Subsequent Regularity of Division in Vitro. (From Pincus, 1930) Group Number Age of Ova (Hours after Copulation) Regular Irregular Percentage Regular (1) (2) (3) 11 to 17 17 to 21 24 to 96 All ova 26 37 10(?) 73 8 16 26 50 76.4 69.8 27.7 59.3 In Table XVIII the data are collected into three groups as follows: (1) Ova recovered when practically all were in the cumulus mass; (2) ova separating out of cumulus mass and not yet covered with albumen ; (3) ova covered with the albumen deposit. It is obvious from these data that the THE ACTIVATION OF UNFERTILIZED EGGS 107 percentage of ova segmenting with any semblance of reg- ularity decreased perceptibly with the age of the ova. In the group of ova recovered at 24 to 96 hours after copulation 16 of the ova classified as irregular exhibited one large cell and ''many polar bodies." In fact, 23 or about half of all the ova called ''irregular" are of this type. A number of ova, particularly in the 24 to 96 hour group exhibited "many polar bodies" and a varying number of larger cells. The rest of the ova classified as irregular were either "many- celled" with indistinct cell outlines, or contained cells of unequal size traceable, probably, to the differential division of early blastomeres. Now this fact that the younger ova tend to segment regularly is presumably related to the state of the egg cyto- plasm. The older ova undoubtedly undergo a certain degree of degeneration as they progress down the tubes, and the degree of cytoplasmic degeneration is probably related to the regularity of the subsequent development in culture. The problem is unfortunately complicated by the fact that all ova in culture stop segmenting and degenerate after some time. In these experiments it is probable that prac- tically no development occurs after the ova have been in culture for 36 hours. The time in which the ova may exhibit their potentialities for parthenogenetic development is, un- der the conditions of these experiments, therefore extremely limited. The surprising fact is that such a large proportion of the ova do exhibit a degree of development that must be classified as parthenogenetic. The morphology and cytology of parthenogenetic ova have been studied in a number of invertebrate forms where parthenogenetic development has been induced by various methods of treatment. In almost all cases a very large proportion of the parthenogenetic ova exhibit marked ir- regularities in development {e.g., Wilson, 1901; Scott, 1906; Morris, 1917). In fact all the irregular types described here have been observed in artificially parthenogenetic inverte- brate ova. The proportion of regular divisions observed 108 THE EGGS OF MAMMALS in these ova compares favorably with those observed in invertebrate ova, with the possible exception of the sea- urchin eggs, a very large proportion of which (as much as 100 per cent) may develop regularly into swinaming larvae (Hindle, 1910; Loeb, 1913). It was not possible to make any extensive cytological study of the ova described. The few sectioned and stained eggs obtained, indicate that in ova segmenting regularly the nuclei and cytoplasm are normal in appearance. In ova segmenting irregularly the situation is apparently rather comphcated. There are obvious evidences of degeneration. Some cells contain nuclei, others do not, and the cytoplasm is often quite degenerate. One observes ova with several nuclei and no distinct cell divisions. In the case of one fairly regular ovum there were at least 37 chromosomes in an incomplete metaphase plate. Upon consideration of the various factors involved in the technique of explanting the ova it seemed most likely that those young ova which underwent a normal partheno- genetic cleavage were stimulated by a gradually developed hypertonicity of the culture medium. For in these experi- ments the ova were cultured in watch glasses in a moist chamber, where the evaporation of a small amount of water from the plasma culture was possible. If this conclusion is true then at least one of the many types of parthenogenetic stimuli known to be effective with non-mammalian ova is similarly stimulating to mammalian eggs. In order to examine this question further the writer and Dr. E. V. Enzmann (Pincus and Enzmann, 1936a) have studied the effect of known methods of parthenogenetic stimulation upon rabbit ova. We took as our criterion of activation the production of the second polar body, which, as we have seen in the experiments with semination in vitro, is entirely adequate. The data of these experiments are given in Table XIX. They demonstrate that short treatment with solutions of relatively low hypertonicity are certainly effective in in- THE ACTIVATION OF UNFERTILIZED EGGS 109 TABLE XIX The Effect of Various Treatments upon the Activation of Rabbit Ova IX Vitro. (From the Journal of Experimental Zoology) Date 18/1/34 24/1/34 24/1/34 24/1/34 20/IX/35 20/IX/35 21/IX/35 21/IX/35 21/IX/35 20/IX/35 20/IX/35 21/IX/35 21/IX/35 18/IX/35 Treatment 3 minutes in 2.8 c.c. H/10 butyric acid + 50 c.c. Ringer-Locke fol- lowed by 3 mins. in 8 c.c. 2.5% NaCl -f 50 c.c. Ringer-Locke followed by plasma culture 3 minutes in 5 c.c. N/10 butyric acid -f 100 c.c. Ringer-Locke followed by hypertonic solution as above 3 minutes in 7.5 c.c. N/10 butyric acid -|- 100 c.c. Ringer-Locke followed by hypertonic solution as above 3 minutes in 10 c.c. N/10 butyric acid -]- 100 c.c. Ringer-Locke followed by hypertonic solution as above 10 minutes in 1.8% Ringer-Locke 5 minutes in 1.8% Ringer-Locke 8 minutes in 1.8% Ringer-Locke 8 minutes in 1.6% Ringer-Locke 8 minutes in 2.0% Ringer-Locke 2 minutes exposure to 45.5° C. 3 minutes exposure to 45.5° C. 2^ minutes exposure to 45.5° C. 3 minutes exposure to 45.5° C. 2 minutes exposure to 60° C. Result Cumulus partly dispersed ; one ovum with 2 polar bodies; 7 with 1 polar body; much shrinkage Cumulus partly dispersed; 2 ova with 2 polar bodies; with 1 polar body; much shrinkage Plasmolysis of ova Plasmolysis of ova Cumulus intact; only 1st polar body Cumulus intact; only 1st polar body 3 polar bodies in 5 hours 1 egg with 2 polar bodies; 1 egg with 3 polar bodies 2 polar bodies in 3 hours 3^ with 2 polar bodies 2 or 3 polar bodies per egg 2 polar bodies formed 2 or 3 polar bodies per egg No polar body formation ducing activation, and that more drastic treatment (e.g., longer treatment, or Loeb's treatment) is only occasionally effective. This indicates that the optimum conditions for the activation of rabbit ova are different from those em- ployed with sea-urchin eggs. - The data on the experiments with ova heated to 45° to 47° show that this heat treatment is most effectively activating. We may conclude therefore that certain of the methods ordinarily employed in the artificial activation of non- mammalian ova are also effective in activating mammalian eggs. In a preliminary group of experiments (unpublished 110 THE EGGS OF MAMMALS data) the writer has transplanted ova so activated into the fallopian tubes of pseudopregnant rabbit does and has later recovered the transplanted ova. A number had undergone normal but obviously belated cleavage. A few cleaved at the normal rate and about 10% of the total attained the blastula stage. In order to obviate any undetected effects of the manipula- tion of ova in vitro Pincus and Enzmann (1936a) undertook the activation of ova in vivo by injecting into the tops of rabbit fallopian tubes sperm suspensions previously irradi- ated with ultraviolet light of 2357 A° wavelength. The does used in these experiments had been mated to sterile bucks 12 to 13 hours previously so that their ovulated ova were embedded in the follicle cell plug. Into one oviduct the rayed sperm were injected, into the other an identical sample of unrayed sperm. It was found that ova from the tubes receiving unrayed sperm suspensions were for the most part normally fertilized and cleaved at the normal rate. Ova seminated with rayed sperm showed varying proportions of normally cleavage stages depending upon the time of exposure of the sperm to the ultraviolet light. Long exposures resulted in a preponderance of irregularly cleaved ova. But even the regularly cleaved ova resulting from seminations of sperm given short exposures were markedly re- tarded when compared with the control ova in the other tube. The ultraviolet treatment with the particular wavelength used results presumably in the inactivation of the sperm chromatin (see Swann and del Rosario, 1932), and depend- ing on the time of exposure (e.g., intensity of radiation) leaves the non-chromatic portions of the sperm relatively unaffected. Dalq and Simon (1931) have shown that sperm treated with ultraviolet light penetrate into the egg cyto- plasm but pronucleus formation does not occur and the chromatin disintegrates. If the sperm centrosome apparatus is not inactivated normal cleavage occurs, otherwise irregular development ensues. The data of Pincus (1930) indicate that parthenogenetic THE ACTIVATION OF UNFERTILIZED EGGS 111 cleavages occur later than normal cleavages (although the time taken for the segmentation process itself is the same in fertilized and unfertilized eggs) . It thus appears that the re- tarded cleavages observed in vivo as the result of semination with irradiated sperm are parthenogenetic in the sense that the sperm chromatin did not participate in the mitoses. Novak and Eisinger (1923) attempted to activate rabbit eggs by tying off the tubes at the isthmus to prevent entry of the ova into the uterus. The ova that they recovered were either irregularly cleaved or fragmented with perhaps one or two normal cleavages. Their data thus resemble those of Mann (1924) on rat ova (see Table VIII) which do not descend into the uterus in unmated animals. Grusdew (1896) who injected sperm into the tops of rabbit tubes together with ova from punctured follicles also tied the tubes off at the isthmus and in a number of ova which gave no evidence of sperm penetration he observed ordinarily ir- regular but occasionally regular development. It would seem then that parthenogenetic development may be in- duced in vivo but that extensive embryonic differentiation has not been demonstrated. It is obvious, of course, that a mere beginning has been made in the investigation of the parthenogenetic potencies of tubal ova. Presumably normal embryos might develop if a diploid cleavage nucleus could be induced to form. Pincus and Enzmann (1935) have, in fact, found indications that such a process may occur in activated rabbit eggs noting, again, after a rather long latent period, two fusion nuclei in unfertilized ova. The writer has observed an initial nuclear division without cytoplasmic cleavage in a primate ovarian o\nim cultured t/i vitro. For full development in vivo it seems necessary that parthenogenetic ova should duplicate with some exactitude not only the normal morphological changes but also the rate of these processes. For the dif- ferentiating embryo is dependent upon an uterine environ- ment the optimum development of which involves a fairly definite time schedule. CHAPTER IX THE GROWTH AND IMPLANTATION OF THE BLASTODERMIC VESICLE In the cinematographs of Lewis and Gregory (1929) the regular cleavage of rabbit ova in vitro is shown to occur at approximately the same rate as in vivo and the formation of the blastocyst is initiated. The rapid expansion of the blastocyst into the typical large blastodermic vesicle (see Plate VII, Fig. 21) does not, however, occur. The attempted expansion is apparently barred by the presence of the rela- tively rigid zona pellucida and albumen coating so that the blastocyst alternately expands and collapses over a period of many hours until degeneration finally ensues. Br ache t (1912, 1913) had previously shown that ova recovered from the uterus of the rabbit at 5 to 6 days after coitus will develop normally for 24 hours to 48 hours, passing from the tridermic stage to the stage of the primitive streak, with normal development of the ectoplacenta. Rabbit ova enter the uterus between 72 and 75 hours after copulation (Cruikshank, 1797; Assheton, 1894; Gregory, 1930) in the early blastocyst stage and still surrounded by the zona pellucida and the albumen coat. There is a rapid expansion of the ovum at this time due to the infiltration of fluid into the vesicle cavity so that by 96 hours after copulation the blastocyst is easily three times the diameter of the tubal egg. Very soon after the entry of the ovum into the uterus the viscosity of the stretched albumen layer appears to decrease so that its persistence about the large pre-primitive streak vesicle of the 6th day must be due to a marked soften- ing. By the end of the 6th day to the 7th day it disappears completely due probably to its digestion by uterine fluids since it does not disappear in culture-grown ova. The growth 112 VESICLE GROWTH AND IMPLANTATION 113 in culture of whole vesicles during the period when the albumen and zona coverings still remain is extremely diffi- cult for the ova soon degenerate and often collapse (Water- man, 1932, 1934). As soon as the early primitive streak stage is reached, explantation results in a moderate degree of de- velopment. Waddington and Waterman (1933) explanted the \ ,!^ ' / ■^-. Fig. 30. Camera lucida drawings of embryonic areas of the rabbit at the stages of explantation. XG, late pre-primitive streak. XE, stage of pos- terior thickening. XL, medium primitive streak. XK, pre-somite. XB, three somite, p.st., prim- itive streak; c.pl, chorda plate; p.kt., primitive knot; p. pi., prochordal plate; p.m.s., pre-meso- dermal somite; s., somite. (From the Journal of Experimental Biology.) embryonic portion of the blastodermic vesicles upon a me- dium of chicken plasma plus chicken embryo extract and found that the older and more differentiated the embryo at the time of explantation the greater the degree of differenti- ation in culture. Using the five stages illustrated in Figure 30, the development observed was as follows : (a) The stage of late pre-primitive streak gives no appar- 114 THE EGGS OF MAMMALS ent differentiation as seen in whole mount preparations. Localized thickenings only occur. (b) The stage of posterior thickening and initial elongation of the embryonic disc develops one or two beating hearts, and localized thickenings after 4-5 days' growth in vitro. (c) The stage of short primitive streak undergoes marked elongation of the primitive streak and embryonic disc on the 2nd day; two, and in one case three, beating hearts appeared after 2-3 days of culture. (d) The stage of medium primitive streak gives results comparable to (c) . In several instances brain, hearts, neural tube and somites appear. (e) The stage of long primitive streak gave rise to em- bryos with as many as six pairs of somites after 1 day of culture, and the pre-somite and two-somite stages give only slightly, if at all, better development. Nicholas and Rudnick (1934) similarly were unable to obtain any adequate development of rat blastocysts in stages earlier than the pre-somite or 5-7 somite. But vesicles in the latter stages developed markedly in a medium consisting of equal parts of rat plasma and 14-15 day rat embryo extract. They report that growth occurs during the first twenty-four hours in vitro gradually slowing and ceasing by the 36th hour. ''At 48 hours or earlier, differentiation in the embryo has reached a maximum, at which it may be maintained for another 24 hours. ''During this period the embryos in the best cases have differentiated from 2 to 16 somites. The allantoic bud has grown from a small lump of tissue at the angle between the amnion and the posterior part of the embryo to join with the superior surface of the ectoplacental cone. The heart, un- formed at the time of implantation, has differentiated a two chambered structure and has initiated its beat, the blood islands have developed in the yolk sac epithelium, and cir- culation has commenced, both in the yolk sac and in the embryo. The nervous system has differentiated consider- ably; eyes and ears have differentiated and the embryo as VESICLE GROWTH AND IMPLANTATION 115 a whole has gone through a primary torsion, separating it from the embryonic membranes in the region of the intestinal portal and contributing to its apparent reversal of posture. "The total growth attained in the 48 hour period is less than half that attained by the normal embryo during the same period. The maximum differentiation is nearly three- quarters of that undergone by the normal. The factors limiting growth are affected earlier than those limiting differentiation. '^ Apparently respiratory interchange is the most important functional necessity at this stage. The efficiency of this mechanism is not only lowered by the total absence of maternal circulation but even further prevented by the growth of a new enveloping membrane in the nature of a decidua from the marginal cells of the ectoplacental cone. The accumulation of break-down products due to metabolic activity is another checking factor. A few preliminary experiments have shown that these can be removed by washing the entire culture in sterile Ringer's solution and adding fresh embryonic extract. By using this method embryos have been kept alive for 96 hours although growth and differentiation occur only at a low rate during the last 24 hours." Nicholas (1934) has also observed a few cases of the development of rat embryos from ova dropped into the uterine cavity, and extra-uterine pregnancies in man are of course well known. In the rat the removal of the entire gestation sac from the uterus into the peritoneal cavity may be performed without hindering fairly advanced em- bryo development in the extra-uterine environment (Selye, Collip and Thomson, 19356).^ It therefore appears that some somatic influence carries the ova through the critical early blastocyst stages and that this influence does not operate in the ordinary tissue culture media. It will be recalled that this critical stage occurs at the time of the disappearance of the egg envelopes and Hall (1935) has recently presented data offering a possible clue 116 THE EGGS OF MAMMALS to the critical events. He found that the zona pellucida of rat and mouse ova placed in fluids of low acidity quickly disappeared (at pH 3.7 or below). In a few cases the zona pellucida was dissolved in Ringer's solution with a pH as high as 5.4. Deciduomata of the rat have shown pH values as low as 5.7, which are, however, not below the critical levels of the in vitro experiments. Pincus and Enzmann (unpublished data) have taken a number of measurements of the pH of pseudopregnant and pregnant endometria and Fig. 31. Left, normal rabbit blastocysts of the 5th day of pregnancy. Right, blastocysts of the 5th day of pregnancy from rabbit doe ovariectomized 18 hours after mating. (From the American Journal of Physiology.) have never observed pH values below 6.5. Nonetheless it is possible that in the small decidual crypts into which the ova fall the critical acidity may be attained. Burdick and Pincus (1935) and Pincus and Kirsch (1936) have examined this critical stage of development from a somewhat different angle. Corner (1928) had noted that in rabbit does in which both ovaries or all the corpora lutea were removed shortly after fertilization the uterine ova remained in the early blastocyst stage (see Figure 31 and Tables XX to XXII), whereas in control rabbits with corpora lutea normal development occurred. The degenerating blastocysts were associated with an oestrus type of endo- metrium, and normal growth of a progestational endome- trium with implantation of embryos occurred when corpus VESICLE GROWTH AND IMPLANTATION 117 luteum extracts were injected daily after ovariectomy (Allen and Corner, 1929). Burdick and Pincus (1935) observed that the daily in- jection of oestrone begun one or two days after copu- lation in unoperated rabbits (100-150 rat units per day) and mice (5 rat units per day) resulted in the degen- eration of rabbit ova in the early blastocyst stages and of mouse ova in late morula stages, i.e., at the stages during which uterine entry occurs. Pincus and Kirsch (1936) extended these ob- servations in order to fix the critical time of action of the hormone. Injections of oes- trone were made at various periods both before and after ovulation, and in the case of the post-0 vulatory injec- tions the uteri were exam- ined at the 10th to 12th days to determine the extent of implantation. Their data presented in Tables XXIII and XXIV indicate clearly that the minimum sterilizing dosage can be given on days 3 to 4 post coitum. These days cover the period of early blastocyst development. The TABLE XX GROUP I BOTH OVARIES REMOVED AT 14-lH HR8. | NO. AUTOPSIED STATE OF EMBRYOS PROLIF. 1 4Hd DEGENERATED 0.2 MM. DIAM. 18 4Kd 0.2 '< 34 4^d 0.15-0.2" 3 5Hd 0.2 " 2 7Hd 0.4 «< 4 7%d 0.3 .. 38 5%d 0,45 " TABLE XXI UKoUf U CONTROL OPERATIONS AT 1 j-18 V- HKS. NO. OP. AUTOPSIED STATE OF EMBRYOS PROLIF. 33 oj- ei^ 7 NORMAL 0.5 MM. + 24 01 eid 7 NORMAL O.C MM. 4 DEQEN. ■¥ 27 m 5 rid 1 ABNORMAL 1 MM. + 37 00 5Kd 7 NORMAL 2 MM. + 23 08 G%d 3 NORMAL, SHIELD STA3E + 5 so l%d 5 NORMAL, iX SOMITES + 21 SQ md 1 NORMAL, SOMITE 8TA0E + TABLE XXII GROUP lU ALL CORPORA LUTEA EXCISED AT 15-20 HR3. | NO. OP. AUTOPStED STATE OF EMBRYOS PROUF. 16 R. L. m 4Hd 4 EARLY DEGEN. 0.4 MM. \9 Qe A%d 4UN8EQ.0VA IN TUBE 30 fi§ h%d NO EMB. (OVULATION -(-) '31 se 5Kd .. 32 so h%d .. 10 08 V/2d 4 DEG. BLASTOCYSTS 0.2 MM. IN TUBE -u r- D •- a o s .S M ^ o3 c3 O 03 i^§i HH H HHHH b^^^ S o Q C K 32 S " S a fil CQ c w P i- o ?^ o w ^ 2 o to t^ o o O O CO a o *; Ch fe « < . <5 ruMBE: OF ORPOR LuTEA > 00 o o O 00 lO o O r-H GO O t« /^ o M X! W W ^ :2; o Hours AFTER Coitus Killed \^ :^ PQ cu Tf O 't' 'f t^ lO LO »0 CD O < t:) O CO (M J (M CV> 00 t^ H o E-i O Pi o o Q o o o o o o o oo H Q 1-5 ^ o l> t>. O i-O o CS (M -^ CO o o o O LO o o o o o o o 1-H (M Ci r-H (M CS EC a< o o bC H G W) b Q ^ - ^ .£ b u S o - i ^ S 3 1 ■< Q 2^ ^ ^ ^ ^ 03 3 8 3 CO CO CO CO "^ Lo o >0 O c ^^ •t; +j 03 03 o 2 s 1 i2 03 a oe c o O r O C^ ^ X 2 -2 c li C 5 c ■^ < o 3 w ^ SP 5 OUND DBES OR ERUS HH H u H ^ h;::^ ^ feh 5 Q a (kS w c « K C « o 00 -* '"' .—1 CO r^ t^ 05 ^ o a '"' « < w « < Numb OF CoRPO Lute O GO o o: T-( t^. Oi 1—1 1— t 1-H un \?^ m^2 00 c^ o O O CC CO CO o GO 00 00 00 00 i^i m ci ci m 11 § o o "^ d ■^ rt^ CC 05 05 §!« * * * § * O Lt o '^ o A, (N CO 8 c3 coSi bC Q a '-3 :; ^ ^ ^ ^ ^ u 03 a "J s t^ ?= Bz «-<-i ^ ^ ~ ^ ^ ^ •^ - •^ " " /-« r^ —1 c^ CO CO CO CO CO n n T-l C^ CO -^ > 20 1-3 200* 600 5 2 j> >> 19 1-4 200* 800 7 1 M >> 25 1-5 200 * 1000 10 24 4 200* 200 10 8 Implantations normal 38 4 400 400 7 1 )y >> 21 4-5 200 400 7 41 4-5 100 200 9 8 3 dying; 5 normal 26 4-6 200 600 8 44 5-6 200 400 12 7 Implantations subnormal in size 48 5-6 200 400 To term No litter 47 3-4 100 200 " " Litter of four 37 3-4 200 400 12 45 3-4 150 300 10 1 100% 71 3-4 150 t 300 11 dead 69 3-4 150 § 300 8 6 Implantations normal 40 3-4 100 200 14 5 Implantations normal 60.3% dead 52 3-4 100 200 13 6 2 embryos subnormal 60 3-4 75 150 8 1 Implantations normal 87.5% dead 56 3-4 373^ 75 11 1 Implantations normal 61 66 3-4 3-4 37H 371^ 75 75 6 5 5 Implantations normal 72.7% dead 58 3-4 30 60 10 3 Implantations normal 33.3% 65 3-4 30 60 11 11 Average diameter of egg chambers 1.43 dead 59 3-4 25 50 9 7 X 1.07 Implantations normal 62 3-4 25 50 10 10 Implantations normal 11.1% dead 64 3-4 25 50 8 7 Implantations normal 13 1-5 3c.c. .005% 15C.C. 6 5 Implantations normal 54 No inj NaOH ections 10 10 1 subnormal in size 9.8% dead 55a " " 5 5 Implantations normal 55b n n 11 8 ,. 63 }f >> 9 9 " * Oestrone in aqueous solution (Parke-Davis Theelin). t Crystalline oestrone in oily solution. § Crystalline oestrone in aqueous solution. 120 VESICLE GROWTH AND IMPLANTATION 121 minimum daily sterilizing dosage for days 3 and 4 is 150 rat units of oestrone-in-oil. When lesser dosages are injected a partially sterilizing effect is observed. This partially steriUz- ing effect is measured by observing the ratio between the num- ber of corpora lutea and the number of implantations. The re- lation of the implantation ratio to the hormone dosage is given in Figure 32. It will be seen that even relatively low hormone dosages have a lethal effect upon a number of the embryos. This effect may be due either to prevention of implantation of vesicles developing normally till implantation time or to a 120 150 Fig. 32. Abscissa: oestrone dosage in R.U. per day. Ordinates: A, per cent of embryos unimplanted; B, number of unimplanted embryos per female. (From the American Journal of Physiology.) degeneration before implantation. The latter alternative seems most likely when one observes the degenerated condi- tion of the preimplantation blastocysts. In addition prac- tically all the embryos that do become implanted are normal in appearance, and, in fact, give rise to normal young at term (rabbit no. 47). W^en eggs in the blastocyst stage are placed in culture they will develop normally for 24 to 36 hours (Brachet, 1913; Pincus, 1930). Cleaving ova will, as we have seen, develop for several days and collapse when the blastocyst stage is reached and presomite stages continue development for 3 to 9 days. This implies that the explanted blastocyst either carries with it from the uterine environment a limited supply of necessary nutrition or that it rapidly exhausts the necessary materials from the ordinary culture medium. 122 THE EGGS OF MAMMALS If oestrone in some way directly interferes with the assimila- tion or metabolism of this critical nutrition then blastocysts cultured with this hormone should show inhibited develop- ment compared to that of controls in a normal medium. Pincus and Kirsch (1936) cultured early blastocysts taken from the uterus of rabbit does with varying amounts of oestriol (12.5 to 25.2 y per culture) and found that control blastocysts developed at the same time as those in the oestriol-containing media. Oestriol was used instead of oestrone because the former is much more soluble in aqueous media and it also has a lethal effect upon developing blasto- cysts when injected in vivo (see Table XXV). These experi- ments show that the lethal effect of the hormone is not due to the direct action of the hormone upon the developing blastocyst. The sterilizing effects of oestriol and dihydro- oestrone (Table XXV) indicate that the lethal effect is not oestrone specific, and point again to the disturbance of a needed nutritive condition. TABLE XXV The Effect of Various Injections of Oestriol and Dihydrooestrone UPON the Implantation Ratio. (From Pincus and Kirsch, 1936) Animal Number Days AFTER Mating Injected Amount Injected Daily (IN Gamma) Total Amount (in Gamma) Number OF Corpora Lutea Number of Implan- tations Remarks 46 70 78 3-4 3-4 3-4 3-4 3-4 4-5 4-5 4-5 3-4 3-4 3-4 3-4 16.7* 16.7t 18.0t 22.2* 22.2t 11.1* 5.5* 11.1* 66.0§ 100.0§ 150.0§ 225.0§ 33.3 33.3 36.0 44.4 44.4 22.2 11.0 22.2 132.0 200.0 300.0 450.0 9 9 8 12 10 16 10 Tot 6 7 9 8 8 6 16 10 erm 6 3 2 Implantations normal 51 7."^ 42 43 49 68 74 76 77 Implantations subnormal in size Implantations normal No litter Implantations normal Average diameters of egg chambers 1.90 X 1.43 cm. Egg chambers = .8 X 1.0 and .9 X 1.1 * Dihydrooestrone in aqueous solution, t Dihydrooestrone in oily solution. § Oestriol in oily solution. VESICLE GROWTH AND IMPLANTATION 123 Just what special conditions are needed for carrying the blastodermic vesicle over this critical stage cannot be ex- plicitly stated. It is obvious that corpus luteum activity is necessary for the establishment of these conditions, and the oestrone effect is due to an inhibition of this activity. Thus it is possible to overcome the partially sterilizing ef- fect of low oestrone dosages by the simultaneous injection of a corpus luteum hormone preparation (Pincus and Kirsch, unpublished data). Other substances {e.g., vitamins A and C) are ineffective as inhibitors of complete sterilization. There seem to be tw^o alternatives: either (1) progesterone or some corpus luteum product act directly upon the blasto- cysts or (2) corpus luteum secretions induce a special uterine environment through their action upon the endometrium. Pincus and Enzmann (unpubhshed data) have made crude extracts of the endometrium of pseudopregnant rabbit does, and have cultured blastocysts in media containing these extracts. No marked effect was obtained with the particular preparations employed, but further investigation may dis- close the presence of an active substance. It is certain that blastocyst death due to oestrone action occurs in a uterus the endometrium of which still shows at least partial pseudo^ pregnant proliferation. The minimum sterilizing dosage employed by Pincus and Kirsch is insufficient to abolish pseudopregnant growth completely (Leonard, Hisaw and Fevold, 1931; Courrier and Raynaud, 1933). Courrier and Raynaud (1934) have also found that dosages sufficient to prevent implantation are below the level necessary for the abolition of pseudopregnant growth. The data presented here on sub-sterilizing dosages demonstrate explicitly that a certain number of vesicles fail to develop in a uterus in which others proceed normally. We may consider therefore that there is necessary at least a threshold amount of a necessary active substance, or an optimum-hydrogen ion concentration alterations of which differentially affect the various blastocysts, or a rate of uterine contraction which causes the proper lodging of the blastocysts in the endo- 124 THE EGGS OF MAMMALS metrium thus preventing their injury. The fact that blasto- cysts in culture also show unusual sensitivity leaves the first two of these alternatives. The behavior and differentiation of the blastodermic ves- icle at the time of implantation have been the object of extensive investigation by mammalian embryologists since the publication of Bischoff's (1852) classical memoir on the subject. These investigations have been concerned chiefly with presenting exact descriptions of the mode of implanta- tion in various classes of mammals (see Robinson, 1904; Grosser, 1909; Bonnet, 1903; Spee, 1915; Wilson, 1928; Sansom and Hill, 1930) and the accompanying differentia- tion of the vesicle. The physiological processes underlying these phenomena have been scarcely investigated. The writer has been interested in the phenomenon of the delayed pregnancy which seems to offer an opportunity to exploit the processes occurring at implantation. Delayed pregnancy, or late parturition, occurs notably in the lactating mouse or rat which is carrying a set of fertilized eggs during lactation. This is a result of the fact that mice and rats have an oestrus period within 48 hours of parturition in which normal mating and fertilization take place. Enzmann, Saphir and Pincus (1932) have analyzed all the available data in the literature and found that in mice and rats each suckling young on the average prolonged pregnancy by about 21 hours (see Figure 33), though this time of prolonga- tion seemed to vary somewhat from strain to strain. An examination of mated mice in a series of timed matings disclosed the fact that the preimplantation vesicle in suckling females failed to implant at the normal time but some time later depending upon the number of young suckling (see Kirkham, 1916, 1918). Once implantation occurs the growth of the embryo proceeds at the rate characteristic of normal embryos (Enzmann, 1935). Obviously the lactation process results in the establishment of conditions in uUro which inhibit implantation, and the rather exact relationship be- tween the degree of delay of pregnancy and the number of VESICLE GROWTH AND IMPLANTATION 125 young suckling suggests that definite quantities of necessary substances are withdrawn from the uterus as the result of mammary gland activity. Teel (1926) found that the daily injection of a NaOH extract of the anterior hypophysis delayed implantation in rats when injections were begun on the day of mating. Injections on days 1 to 6 caused delayed implantation with parturition occurring in normal fashion but several days 10 — •4 9 — ^^ 8 — ■ • • 5' — ■ • ! t W 4 — 14* ▲ SYMBOLS 3 •■ •■ 1 AVERAGES OF ALL DATA_ # | A GENE TIMEI RAL STOCK ▲ 2 ■> MATINGS ■ 1 n 1 f ( 1 1 1 1 I 1 1 1 I 4 5 6 7 8 9 10 11 NUMBER OF YOUNG SUCKLED 12 13 14 Fig. 33. Showing the relationship between the degree of delay of pregnancy and the number of suckling young. (From the Anatomical Record.) after term; injections on days 1 to 12 also caused delayed implantation but a definite interference in the birth mecha- nism so that only one of a series of females produced normal hving young in a late parturition; injections over a longer period resulted not only in delayed implantation but also in stillbirths 5 to 7 days after normal term. The impairment of the birth mechanism can therefore be avoided by early injection and is presumably a phenomenon distinct from that of delayed implantation. The inhibition of parturition can be caused not only by alkaline pituitary extracts (Evans and Simpson, 19296; Snyder, 1934) but also by corpus 126 THE EGGS OF MAMMALS luteum extracts (Nelson, PMner and Haterius, 1930). Since the pituitary extracts employed by Teel caused marked luteinization of the ovaries of injected animals it is possible that the delay in implantation may be due to excessive corpus luteum secretion. Selye, CoUip and Thomson (1935??) have ingeniously demonstrated that the rat ovary during lactation presumably produces little or no oestrin, so that the hormone-producing tissue of the ovary during lactation is predominantly the luteal tissue. One need not postulate hypersecretion by the corpus luteum during lactation but merely an unbalance in which corpus luteum hormone pre- dominates (thus Selye, Collip and Thomson actually obtain larger corpora lutea in lactating mice when oestrin is injected). Wislocki and Goodman (1934) injected a preparation of progestin (after Allen, 1930), for 8 days after mating into two rabbits but no delay of pregnancy ensued. Antuitrin-S and antuitrin-G injected in fairly large amounts during early pregnancy were also ineffective although these prepara- tions induced a fresh ovulation and new corpus luteum formation. The ineffectiveness of progestin in the two experi- ments of Wislocki and Goodman may have been due to an insufficient dosage. On the other hand it is possible that delayed pregnancy is due to an insufficiency of corpus luteum secretion, so that the immediate effect of Teel's extract may be considered the stimulation of oestrin production with inhibition of luteal secretion followed by corpus luteum activity which induced or completed the implantation proc- ess. Hamlett (1935) is in fact of the opinion that delayed implantation is due to hyposecretion of the corpus luteum. He has found (1932) that copulation and cleavage occur in the nine-banded armadillo of Texas during July, and the unimplanted vesicle lies free in the uterine lumen until early November when implantation takes place. Correlated with the quiescent period is a large corpus luteum the cells of which contain few or no secretory droplets or granules. Shortly before implantation vacuolization and lipoidal secre- VESICLE GROWTH AND IMPLANTATION 127 tion occurs in the luteal cell cytoplasm, and the removal of such corpora lutea leads to abortion whereas removal during the free vesicle period has no discernible effect upon the uterus or ovum. Hamlett (1935) quotes a number of instances of naturally-occurring delayed implantation of a presumably similar nature. This possibility has been tested by injecting oest rone-free corpus luteum extracts into lactating pregnant mice during the early part of pregnancy (unpublished data). Injections of approximately l/20th of a Corner- Allen rabbit unit were made over a 5 to 8 day period. A number of the mice failed to produce any young but seven females gave birth to normal litters. These were born not at term but much later; in fact, the average date of birth was 4 days later than would occur in delayed pregnancy if the expected delay is calculated on the basis of 21 hours per suckling young. The implication is clear that excessive corpus luteum secretion caused a delay of pregnancy in mice. Since Teel (1926) found that deciduomata formation could be readily induced in the uteri of unmated females treated with his extracts corpus luteum activity undoubtedly occurred as a result of luteinizing hormone injection. The act of suckling then, by prolonging corpus luteum activity (which it does — see Parkes, 1929; Turner, 1932), results in a delay of im- plantation. Selye and McKeown (1934a) have in fact shown that suckling in rats prolongs pseudopregnancy and that the effects of suckling do not occur in the absence of the ovary (Selye and McKeown, 19346). The fact that Teel obtained definite deciduomata in ani- mals subjected to a treatment that produces delayed preg- nancy indicates either: (1) that mechanical irritation is more effective than ovum contact and that therefore the corpus luteum effect is really subnormal or (2) that excessive corpus luteum activity in some way inhibits the actual process of implantation of the blastocysts. The problem is an interesting one and is receiving further investigation. CHAPTER X SUMMARY AND RECAPITULATION For the purposes of this monograph an ovum is considered as such from the moment of its functional differentiation in the ovary until its implantation in the uterine endometrium. An examination has been made of the experimental investi- gations of the growth and development of the mammahan ovum during the various stages of its life history in the ovary and oviducts. The problem of the origin of the definitive ova has re- ceived much attention, but it cannot be said to have been completely resolved. If we are to judge by evidence from non-mammalian forms the large amoeboid primordial germ cells must enter the embryonic gonad if it is to differentiate as a functional organ. A functional ovary develops only from embryonic gonads in which the secondary sex cords proliferate to form a true ovarian cortex associated with the germinal epithelium. The ovaries of young mammals contain large numbers of primitive oocytes. The conception that these oocytes are the only precursors of the definitive ova is controverted by a large body of recent evidence which indicates that new ova are .proliferated from the germinal epithelium and that the rate of this proliferation varies with the various stages of the oestrus and pregnancy cycles. Ovogenesis in the adult seems to be partially inhibited by certain secretions of the anterior pituitary, the gonad-stimulating hormones affect- ing follicle growth primarily. The exact relation of the gonad-stimulating hormones to the ovogenetic processes is not at all obvious. It seems certain that the prophase stages of the oocyte nuclei occur independent of pituitary hormone activity. 128 SUMMARY AND RECAPITULATION 129 Pituitary hormones are definitely concerned in the final stage of ovum maturation, the first polar division which normally occurs in the ovary of most mammals. The pi- tuitary secretions do not affect the eggs directly but initiate changes in the follicles which make for maturation in the ova. Similar changes occur in atretic follicles with a resulting "pseudomaturation" in the ova of such follicles. The initia- tion of ovum activation represented by the first maturation division occurs in vitro simply upon the explantation of ovarian eggs. Maturation in vivo and in vitro can be ex- plained as the result of a functional isolation of the ovum from the follicular epithelium. It is held probable therefore that the parthenogenetic development of ova observed in mammalian ovaries occurs as the result of the establishment in the follicle of special activating conditions. Parthenogenetic development of unfertilized tubal ova rarely if ever occurs in vivo. In most eutherian mammals the eggs are shed surrounded by follicle cells. If sperm are not present the surrounding cells slowly fall away, and the naked ova descend into the lower portion of the tubes where they degenerate and are eventually either resorbed or washed out into the uterus. When sperm are present there is a rapid dissolution of the surrounding follicle cells due to the action of a heat labile substance carried by the sperm. It has been claimed that this same substance acti- vates the ova into forming the second polar body, but the available evidence is contradictory. Tubal eggs remain fertilizable for a few hours in the rabbit, and for thirty hours in the ferret. Manamalian ova may be fertilized in vitro and normal cleavage ensues. This is most readily demonstrated with rabbit ova, for the ova of most of the other forms examined do not cleave or develop appreciably under the ordinary conditions of tissue culture. Segmentation in vivo occurs at fairly characteristic rates in the various species of mammals. The cleavage rate in rabbits is definitely correlated with the adult size of the strain employed. The cleavage process 130 THE EGGS OF MAMMALS itself is under the control of a cyanide-labile system. The process of cleavage is apparently independent of the activity of the primary sex hormones, oestrin and progestin. Tubal rabbit ova readily exhibit parthenogenetic cleav- ages under certain conditions of explantation in vitro. Parthe- nogenetic activation can be initiated experimentally by treat- ment with cytolytic agents, by exposure to hypertonic solutions, and by heat treatment. The development of the blastodermic vesicle in vivo is conditioned by the activity of corpus luteum secretions. In the absence of the corpus luteum development does not occur beyond that stage in which ova just entering the uterus are found. The evidence indicates that the corpus luteum secretions either stimulate the eggs directly or provide through stimulation of the uterine endometrium a suitable environment for the developing blastocysts. Oestrin and allied compounds prevent blastocyst growth by inhibiting the corpus luteum effect, the ova being most sensitive to this inhibition during the early blastocyst stages. The implantation process itself is also under hormonal control. In the rat and mouse ovum implantation is delayed during lactation. This delay appears to be due to excessive corpus luteum secretion. The development of various techniques for the explanta- tion of ova both in vivo and in vitro makes available a variety of experimental investigations of the manmaalian ovum. The ova of certain forms are particularly adapted to experi- mental manipulation. Mammalian ova normally develop in a homeostatic environment. Certain components of this homeostasis sharply limit the extent and nature of ovum development at certain stages. During other phases of its growth the ovum appears to be a relatively independent organism. Careful investigation of the physiological proc- esses occurring in the ovum itself and in its homeostatic environment is made possible by the various explantation and transplantation techniques. BIBLIOGRAPHY Addison, W. H. F. 1917. Fragmentation of the ovum within the Graafian follicle. Proc. Path. Soc. Phila. 37, 1. Allen, B. M. 1904. Embryonic development of the ovary and testis of the mammals. Am. J. Anat. 3, 89. Allen, E. 1923. Ovogenesis during sexual maturity. Am. J. Anat. 31,4:39. . 1932. The ovarian follicular hormone, theelin; animal reactions. In: Sex and internal secretions. Williams and Wilkins. Baltimore. Allen, E., Francis, B. F., Robertson, L. L., Colgate, C. E., Johnston, C. G., DoisY, E. A., KouNTz, W. B. and Gibson, H. V. 1924. The hormone of the ovarian follicle; its localization and action in test animals and additional points bearing upon the internal secretion of the ovary. Am. J. Anat. 34, 133. Allen, E., Kountz, W. B. and Francis, B. F. 1925. Selective elimination of ova in the adult ovary. Am. J. Anat. 34, 445. Allen, E., Pratt, J. P., Newell, Q. U. and Bland, L. J. 1930a. Human tubal ova; related early corpora lutea and uterine tubes. Carnegie Inst. Wash. Contrib. Emhryol. 22, 45. . 19306. Human ova from large follicles; including a search for mat-. uration divisions and observations on atresia. Am. J. Anat. 46, 1. Allen, W. M. 1930. Physiology of the corpus luteum; preparation and some chemical properties of progestin, hormone of corpus luteum which produces progestational proliferation. Am. J. Physiol. 92, 174. Allen, W. M. and Corner, G. W. 1929. Physiology of the corpus luteum III. Normal growth and implantation of embryos after very early ablation of the ovaries, under the influence of extracts of the corpus luteum. Am. J. Physiol. 88, 340. Amann, J. A. 1899. Ueber Bildung von Ureiern und primarfollikelahn- lichen Gebilden in senilen Ovarium. Festschr. zum siebzigsten Ge- burtstag C. Kupfer. Anderson, D. H. 1927. The rate of passage of the mammalian ovum through various portions of the Fallopian tube. Am. J. Physiol. 82, 557. Arai, H. 1920a. Post natal development of the ovary in the white rat. Am. J. Anat. 27, 405. . 19206. On the cause of the hypertrophy of the surviving ovary 131 132 THE EGGS OF MAMMALS after semi-spaying and the number of ova in it. Am. J. Anat. 28, 59. AsAMi, G. 1920. Observations on follicular atresia in the rabbit ovary. Anat. Rec. 18, 323. Aschner, B. 1914. Ueber den Kampf der Teile im Ovarium. Arch. Ent. Mech. 40, 565. AsDELL, S. A. 1924. Some effects of unilateral ovariectomy in rabbits. Brit. J. Exp. Biol. 1, 473. AssHETON, R. 1894. A re-investigation into the early stages of the de- velopment of the rabbit. Quar. J. Micr. Sc. 37, 113. Athias, M. 1909. Les phenomenes de division de I'ovule dans les follicules de De Graaf en voie d'atresie chez le Lerot. Anat. Anz. 34, 1. . 1920. Invagination de I'epithelium superficiel et neoformation ovulaire dans I'ovaire transplante chez le cobaye. Compt. rend. Soc. biol. 83, 1647. VON Baer, K. E. 1827. De ovi mammalium et hominis genesi. Epistola. Leopoldi Vossi. Leipzig. Balfour, F. M. 1878. On the structure and development of the verte- brate ovary. Quar. J. Micr. Sc. 18, 383. . 1882. Handbuch der vergleichenden Embryologie. Trans, of Vetter. Jena. Barry, M. 1838. Researches in embryology, first series. Phil. Trans. Roy. Soc. London, p. 301. . 1839. Researches in embryology, second series. Phil. Trans. Roy. Soc. London, p. 307. Bellerby, C. W. 1929. The relation of the anterior lobe of the pituitary to ovulation. /. Physiol. 67, xxxiii. Benoit, J. 1930. Contribution a I'etude de la lignee germinale chez le poulet. Destruction precoce des gonocytes primaires par les rayons ultraviolets. Compt. rend. Soc. biol. 104, 1329. BiEDL, A., Peters, H. and Hofstatler, R. 1922. Transplantation befruchteter Eier bei Kaninchen. Z. Geburtsh. 84, 60. BiscHOFF, T. L. W. 1842. Entwicklungsgeschichte der Saugethiere und des Menschen. Voss. Leipzig. . 1845. Entwicklungsgeschichte des Hundeeies. Freidrich Vie wag und Sohn. Braunschweig. . 1852. Entwicklungsgeschichte des Meerschweinchens. J. Rickersche Buchhandlung. Giessen. . 1854. Entwicklungsgeschichte des Rehes. J. Rickersche Buch- handlung. Giessen. BIBLIOGRAPHY 133 Bond, C. S. 1906. Some points in uterine and ovarian physiology and pathology in rabbits. Brit. Med. J. 2, 121. Bonnet, R. 1884. Beitrage zur Embryologie der Wiederkauer, gewonnen am Schafei. Arch. Anal. u. Physiol. 170. . 1891. Grundriss der Entwickelungsgeschichte der Haussaugethiere. Verlag von Paul Parey. Berlin. . 1899. Gibt es bei Wirbeltieren Parthenogenesis? Ergeb. Anat. Entwcklngsgesch. 9, 820. . 1903. Uber Syncytien, Plasmodien und Symplasma in der Placenta der Saugetiere und des Menschen. Monats. Geburtsh. u. Gynak. 18, I. BosAEUs, W. 1926. Beitrage zur Kenntnis der Genese der Ovarialem- bryoma. Almquist und Wiksells. Upsala. Bracket, A. 1912. Developpement in vitro de blastodermes et de jeunes embryons de mammiferes. Compt. rend. Soc. biol. 155, 1191. . 1913. Recherches sur le determinisme hereditaire de Toeuf des Mammiferes. Developpement "in vitro" de jeunes vesicules blasto- dermiques du lapin. Arch. Biol. 28, 447. Brambell, F. W. R. 1927. The development and morphology of the gonads of the mouse. Part I. The morphogenesis of the indifferent gonad and of the ovary. Proc. Roy. Soc. B 101, 391. . 1927. The development and morphology of the gonads of the mouse. Part II. The development of the Wolffian body and ducts. Proc. Roy. Soc. B 102, 206. . 1928. The development and morphology of the gonads of the mouse. Part III. The growth of the follicles. Proc. Roy. Soc. B 103, 258. . 1930. The development of sex in vertebrates. Macmillan Co. New York. Brambell, F. W. R., Parkes, A. S. and Fielding, V. 1927a. Changes in the ovary of the mouse following exposures to x-rays. I. Irradiation at three weeks old. Proc. Roy. Soc. B 101, 29. . 1927b. Changes in the ovary of the mouse following exposures to x-rays. II. Irradiation at or before birth. Proc. Roy. Soc. B 101, 95. Branca, A. 1925. L'ovocyte atresique et son involution. Arch. Biol. 35, 325. Buhler, a. 1894. Beitrage zur Kentniss der Eibildung beim Kaninchen und der Markstrange des Eierstockes beim Fuchs und Menschen. Z. wissensch. Zool. 58, 314. BuRDicK, H. 0. and Pincus, G. 1935. The effect of oestrin injections upon the developing ova of mice and rabbits. Am. J. Physiol. 111,201. 134 THE EGGS OF MAMMALS Butcher, Earl 0. 1927. The origin of the definitive ova in the white rat (Mus norvegicus albinus). Anat. Rec. 37, 13. . 1932. Regeneration in hgated ovaries and transpla^.ted ovarian fragments of the white rat (Mus norvegicus albinus). Anat. Rec. 54, 87. BuYSE, Adrian. 1935. The differentiation of transplanted mammalian gonad primordia. J. Exp. Zool. 70, 1. Caldwell, W. H. 1887. The embryology of the Monotremata and Mar- supalia. Part I. Phil. Trans. Roy. Soc. B 178, 463. Campbell, J. A. 1924. Changes in the tensions of CO2 and O2 in gases injected under the skin and into the abdominal cavity. J. Physiol. 59, 1. Carmichael, E. S. and Marshall, F. H. A. 1908. On the occurrence of compensatory hypertrophy in the ovary. J. Physiol. 36, 431. Carrel, A. 1924. Leucocytic trephones. /. Am. Med. Assoc. 82, 255. Casida, L. E. 1935. Prepuberal development of the pig ovary and its re- lation to stimulation with gonadotropic hormones. Anat. Rec. 61, 389. Castle, W. E. and Gregory, P. W. 1929. The embryological basis of size inheritance in the rabbit. J. Morph. and Physiol. 48, 81. Champy, C. 1927. Parthenogenese experimentale chez le Lapin. Compt. rend. Soc. hiol. 96, 1108. Chapin, Catherine L. 1917. A microscopic study of the reproductive system of foetal free-martins. /. Exp. Zool. 23, 453. Charlton, H. H. 1917. The fate of the unfertilized egg in the white mouse. Biol. Bull. 33, 321. Clark, E. B. 1923. Observations on the ova and ovaries of the guinea- pig, Cavia cobaya. Anat. Rec. 25, 313. Clark, R. T. 1934. Studies on the physiology of reproduction in the sheep. II. The cleavage stages of the ovum. Anat, Rec. 60, 135. Coert, H. J. 1898. Over de ontwikeling en den bouw van den geslacht- sklier bijde zoogdiern meer in het bijzonder van den eierstock. Akad. Proefschrift. Leiden. Corey, E. L. 1928. Effect of prenatal and postnatal injections of the pituitary gland in the white rat. Proc. Soc. Exp. Biol. & Med. 25, 498. Corner, G. W. 1928. Physiology of the corpus luteum. I. The effect of very early ablation of the corpus luteum upon embryos and uterus. Am. J. Physiol. 86, 74. Courrier, R. 1923. Vesicule blastodermique parthenogenetique dans un ovaire de cobaye impubere. Arch. Anat., Histol. et Embryol. 2, 455. BIBLIOGRAPHY 135 CouRRiER, R. et Oberling, C. 1923. Parthenogenese spontanee dans I'ovaire du cobaye. Bull. Soc. Anat. Paris 93, 724. CouRRiER, R. et Raynaud, R. 1933. Experiences d'antagonisme hu- moral ovarien realisees avec I'etalon international de folliculine cris- tallisee. Compt. rend. Soc. biol. 115, 299. . 1934. Etude quantitative de Tavortement folliculinique provoque chez la lapine, par Thormone cristallisee. Realisation d'un avortement partiel. Compt. rend. Soc. biol. 116, 1078. CowPERTHWAiTE, Marian M. 1925. Observations on pre- and post- pubertal oogenesis in the white rat (Mus norvegicus albinus). Am. J. Anat. 36, 69. Crew, F. A. E. 1927. On the effects of unilateral ovariotomy and sal- pingectomy in the rat. Biol. Generalis 3, 207. Cruikshank, W. 1797. Experiments in which, on the third day after impregnation the ova of rabbits were found in the fallopian tubes and on the fourth day after impregnation in the uterus itself; with the first appearance of the foetus. Phil. Trans. Roy. Soc. London 18, 197. Dalq, a. et Simon, S. 1931. Contribution a I'analyse des fonctions nucleaires dans I'ontogenese de la Grenouille. III. Etude statistique et cytologique des effets de I'irradiation d'un des gametes sur la gastrulation chez Rana Fusca. Arch. Biol. 1^2, 107. Davenport, C. B. 1925. Regeneration of ovaries in mice. J . Exp. Zool. Deansley, R., Fee, A. R. and Parkes, A. S. 1930. Studies on ovulation. II. The effect of hypophysectomy on the formation of the corpus luteum. /. Physiol. 70, 38. Defrise, a. 1933. Some observations on living eggs and blastulae of the albino rat. Anat. Rec. 57, 239. DoiSY, E. A., Curtis, J. and Collier, W. D. 1931. Effect of theelin upon the developing ovary of the rat. Proc. Soc. Exp. Biol. & Med. 28, 885. DoMM, L. V. 1929. Spermatogenesis' following early ovariotomy in the brown leghorn fowl. Arch. Eritwcklngsmech. Org. 119, 171. DoRAN, M. A. 1902. Pregnancy after removal of both ovaries for cystic tumor. /. Ohst. a7id Gynec. Brit. Emp. 2, 1. Emery, F. E. 1931. Changes in the ovary and oestrus cycle following the removal of one ovary in albino rats. Physiol. Zool. 4, 101- Engle, E. T. 1927a. Polyovular follicles and polynuclear ova in the mouse. Anat. Rec. 35, 341. 136 THE EGGS OF MAMMALS Engle, E. T. 19276. A quantitative study of follicular atresia in the mouse. Am. J. Anat. 39, 187. . 1928. The role of the anterior pituitary in compensatory ovarian hypertrophy. Anat. Rec. 37, 275. . 1929. Ovarian responses: differences elicited by treatment with urine from pregnant women and by freshly implanted anterior lobe. J. Am. Med. Assoc. 93, 276. . 1931a. Prepubertal growth of the ovarian follicle in the albino mouse. Anat. Rec. 4-8, 341. . 19316. The pituitary-gonadal relationship and the problem of pre- cocious sexual maturity. Endocrinology 15, 405. Enzmann, E. V. 1935. Intrauterine growth of albino mice in normal and in delayed pregnancy. Anat. Rec. 62, 31. Enzmann, E. V., Saphir, N. R. and Pincus, G. 1932. Delayed preg- nancy in mice. Anat. Rec. 5^, 325. Evans, H. M. and Cole, H. H. 1931. An introduction to the study of the oestrus cycle in the dog. Mem. Univ. Calif. 9, 65. Evans, H. M., Meyer, K. and Simpson, M. E. 1932. Relation of prolan to the anterior hypophyseal hormones. Am. J. Physiol. 100, 141. Evans, H. M., Meyer, K., Simpson, M. E., Szarka, A., Pencharz, R. I., Cornish, R. W. and Reichert, F. L. 1933. The growth and gonad- stimulating hormones of the anterior hypophysis. Mem. Univ. Calif. 11,1. Evans, H. M. and Simpson, M. E. 1928. Antagonism of growth and sex hormones of the anterior hypophysis. /. Am. Med. Assoc. 91, 1337. . 1929a. A comparison of the ovarian changes produced in immature animals by implants of hypophyseal tissue and hormone from the urine of pregnant women. Am. J. Physiol. 89, 381. ' . 19296. Impairment of the birth mechanism due to hormones from the anterior hypophysis. Proc. Soc. Exp. Biol. & Med. 26, 595. Evans, H. M. and Swezy, 0. 1931. Ovogenesis and the normal follicular cycle in adult mammalia. Mem. Univ. Calif. 9, 119. Feltx, W. 1912. Development of the urogenital organs, in: Manual of human embryology, 2, 752. Keibel and Mall. Philadelphia. Fellner, 0. 0. 1909. Histologie des Ovariums in der Schwangerschaft. Arch. mikr. Anat. 73, 288. Fevold, H. L. and Hisaw, F. L. 1934. Interactions of gonad stimulating hormones in ovarian development. Am. J. Physiol. 109, 655. Fevold, H. L., Hisaw, F. L. and Greep, O. 1934. Factors which govern ovarian development. Anat. Rec. 60 Supp., 51. BIBLIOGRAPHY 137 Fevold, H. L., Hisaw, F. L. and Leonard, S. L. 193 L The gonad- stimulating and the luteinizing hormones of the anterior lobe of the hypophysis. A??i. J. Physiol 97, 291. FiRKET, J. 1920. On the origin of germ cells in higher vertebrates. Anat. Rec. IS, 309. Fischer, Albert. 1925. Tissue culture. Levin and Munksgaard. Copen- hagen. Flemming, W. 1885. Tiber die Bildung von Richtungsfiguren in Sauge- tiereiern beim Untergang Graafscher Follikel. Arch. Anat. u. Eni- wickl. 221. FouLis, J. 1876. The ova and ovary in man and other mammalia. Quar. J. Micr. Sc. 16, 190. Friedgood, H. and Pincus, G. 1935. Studies on the conditions of activ- ity in endocrine organs. XXX. The nervous control of the anterior pituitary as indicated by maturation of ova and ovulation after stimulation of cervical sympathetics. Endocrinology 19, 710. Friedman, M. H. 1929. Mechanism of ovulation in the rabbit. II. Ovu- lation produced by the injection of urine from pregnant women. Am. J. Phijsiol. 90, 617. Fuss, A. 1911. Uber extraregioniaire Geschlechtzellen bei einem Men- schhchen Embryo von vier wochen. Anat. Am. 39, 407. . 1913. Uber die Geschlechtzellen des Menschen und der Saugetiere. Arch. mikr. Anat. 81, 1. Genther, I. 1931. Irradiation of the ovaries of guinea pigs and its effect on the oestrus cycle. Am. J. Anat. 48, 99. . 1934. X-irradiation of the ovaries of guinea pigs and its effect on subsequent pregnancies. Am. J. Anat. 55, 1. Gerard, P. 1920. Contribution a I'etude de Fovaire des mammiferes. L'ovaire de Galago mossambicus (Young). Arch. Biol. 30, 357. Gilchrist, F. and Pincus, G. 1932. Living rat eggs. Anat. Rec. 54, 275. Gordon, C. S. 1896. Two pregnancies following removal of both ovaries and ligation of tubes. Trans. Am. Gynec. Soc. 21, 104. Gregory, P. W. 1930. The early embryology of the rabbit. Carnegie Inst. Wash. Contrib. to Embryol. 21, 141. Gregory, P. W. and Castle, W. E. 1931. Further studies on the em- bryological basis of size inheritance in the rabbit. /. Exp. Zool. 59, 199. Grosser, 0. 1909. Vergl. Anat. u. Entwicklung der Eihaute und der Placenta. Wilh. Braumiiller. Wien u. Leipzig. 138 THE EGGS OF MAMMALS Grusdew, W. S. 1896. Versuche liber die kiinstliche Befruchtung von Kanincheneiern. Arch. Anat. u. Physiol., Anat. Abt. 269. GuRWiTSCH, A. 1900. Idiozom und Centralkorper im Ovarialei der Saugetiere. Arch. mikr. Anat. 66, 377. GuTHERZ, S. 1925". Tiber vorzeitige Chromatinreifung an physiologisch degenerierenden Saugeoozyten des friihen Wachstumsperiode. Z. mikr. Anat. Forsch. 2, 1. Haberlandt, G. 1922. Uber Zellteilungshormone und ihre Beziehungen zur Wimdheilung, Befruchtung, Parthenogenesis und Adventivem- bryone. Biol. Zentr. Jf2, 145. Haggstrom, p. 1922. Uber degenerative parthenogenetische Teilungen von Eizellen in normalen Ovarien des Menschen. Acta Gynecol. Scandinav. 1, 137. Hall, B. V. 1935. The reactions of rat and mouse eggs to hydrogen ions. Proc. Soc. Exp. Biol. & Med. 32, 747. Hamlett, G. W. D. 1932. The reproductive cycle in the armadillo. Z. wissensch. Zool. 1^1, 143. . 1935. Delayed implantation and discontinuous development in the mammals. Quar. Rev. Biol. 10, 432. Hammond, J. 1928. Die Kontrolle der Fruchtbarkeit bei Tieren. Zilch- tungskunde 3, 523. . 1934. The fertilization of rabbit ova in relation to time. A method of controlling the litter size, the duration of pregnancy, and the weight of the young at birth. J. Exp. Biol. 11, 140. Hammond, J. and Asdell, S. A. 1926. The vitahty of spermatozoa in the male and female reproductive tract. Brit. J. Exp. Biol. 4, 155. Hammond, J. and Marshall, F. H. A. 1925. Reproduction in the rabbit. Oliver and Boyd. London. Hammond, J. and Walton, A. 1934. Notes on ovulation and fertilization in the ferret. /. Exp. Biol. 11, 307. Hanson, F. B. and Boone, C. 1926. On the migration of ova from one uterine horn to the other in the albino rat. Am. Nat. 60, 257. Hargitt, G. T. 1925. The formation of the sex glands and germ cells of mammals. 1. The origin of the germ cells in the albino rat. /. Morphol. and Physiol. Jfi, 517. . 1930. The formation of the sex glands and germ cells of mammals. V. Germ cells in the ovaries of adult pregnant and senile albino rats. /. Morphol. and Physiol. 50, 453. Harms, J. W. 1926. Korper und Keimzellen. J. Springer. Berlin. Hartman, C. G. 1916. Studies in the development of the opossum, BIBLIOGRAPHY 139 Didelphys virginiana L. I. History of early cleavage. 11. Formation of the blastocyst. /. Morphol. 27, 1. Hartman, C. G. 1919. Studies in the development of the opossum, Didelphys virginiana L. HI. Description of new material on matura- tion, cleavage, and endoderm formation. IV. The bilaminar blas- tocyst. /. Morphol. 32, 1. . 1924. Observations on the viability of the mammalian ovum. A7n. J. Obst. and Gij.iec. 7, 1. . 1925. Observations on the functional compensatory hypertrophy of the opossum ovary. Am. J. Anat. 35, 1. . 1929. How large is the mammalian egg? A review. Quar. Rev. Biol. 4, 373. . 1932a. Studies in the reproduction of the monkey, Macacus (Pithe- cus) rhesus, with special reference to menstruation and pregnancy. Carnegie Inst. Wash. Contrib. Embryol. 23, 1. . 19326. Ovulation and the transport and viabihty of ova and sperm in the female genital tract. In Allen: Sex and internal secretions. Williams and Wilkins. Baltimore. Harz, W. 1883. Beitrage zur Histologie des Ovariums der Siiugetiere. Arch. mikr. Anat. 22, 374. Hatai, S. 1913. The effect of castration, spaying, or semi-spaying on the weight of the central nervous system and of the hypophysis of the albino rat; also the effect of semi-spaying on the remaining ovary. /. Exp. Zool. 15, 297. . 1915. The growth of organs in the albino rat as affected by gonad- ectomy. /. Exp. Zool. 18,1. Haterius, H. 0. 1928. An experimental study of ovarian regeneration in mice. Physiol. Zool. 1, 45. Heape, W. 1883. The development of the mole, Talpa Europea. The formation of the germ layers and early development of the medullary groove and notochord. Qttar. J. Micr. Sc. 23, 412. . 1886. The development of the mole (Talpa Europea) ; the ovarian ovum and segmentation of the ovum. Quar. J. Micr. Sc. 26, 157. . 1905. Ovulation and degeneration of ova in the rabbit. Proc. Roy. Soc. B 76, 260. Hegner, R. W. 1914. The germ-cell cycle in animals. Macmillan Co. New York. Henneguy, F. 1893. Sur la fragmentation parthenogenesique des ovules des vertebres pendant I'atresie des follicules de Graaf. Compt. rend. Soc. biol. 45, 500. 140 THK rXlGS OF MAMMALS Hensen, V. 1869. Uber die Ziiclitunfi; unhernichteter Eier. Centr. med. Wissemch. 7, 403. . 1870. Beobachtung fiber die Befruchtung iind Entvvicklung des Kaninchens und Meerschweinchens. Z. Anal. Eniwckimjs. 1, 213; 851. Hertz, R. and IIisaw, F. L. 1934. l*]ffects of follicle-stinuilating and luteinizing pituitary extracts on the ovaries of the infantile and juvenile rabbit, jlm. J. Physiol. lOS, 1. Heuser, C. H. and Streeter, G. L. 1929. Early stages in the develop- ment of pig embryos. Carnegie Inst. Wash. Conirih. Embryol. 20, 1. IIeys, Florence. 1929. Does regeneration follow complete ovariotomy in the albino rat? Science 70, 289. . 1931. The problem of the origin of the germ cells. Quar. Rev. Biol. 6, 1. Hill, J. P. 1910. The early development of the Marsupalia with especial reference to the native cat (Dasyurus viverrinus). Quar. J. Micr. Sc. 56, 1. . 1918. Some observations on the early development of Didelphys aurita. Quar. J. Micr. Sc. OS, 91. Hill, J. P. and Tuihe, M. 1924. The early development of the cat (Felis domestica). Quar. J. Micr. Sc. 6S, 513. Hill, M. and Parkes, A. S. 1931. Attempts to promote the reformation of germ cells. /. Anat. 65, 212. HiNDLE, E. 1910. A cytological study of artificial parthenogenesis in Strongylocentrotus purpuratus. Arch. Entwcklngsmechn, 31, 145. HiNSEY, J. C. and Markee, J. Vj. 1933. Studios on prolan-induced ovula- tion in midbrain and midbrain-hypophysectomized rabbits. Am. J. Physiol. 106, 48. HiSAW, F. L., Fevold, H. L., Foster, M. A. and Hellbaum, A. A. 1934. A physiological explanation of the oestrus cycle of the rat. Anat, Rec. 60 Sup p., 52. HuBER, G. C. 1915. The development of the albino rat, Mus norvegicus albinus. I. From the pronuclear stage to the stage of the mesoderm anlage; end of the first to end of the ninth day. /. Morphol. 26, 247. HuBRECHT, A. A. W. 1912. Friihe Entwickhmgsstadien des Igels und ihre Bedeutung fiir die Vorgeschichte (Phylogenese) des Amnions. Zool. Jahrb. Supp. XV, 2, 739. Humphrey, R. R. 1928. The developmental potencies of the intermediate mesoderm of amblystoma when transplanted into ventro-lateral sites in other embryos: the primordial germ cells of such grafts and their role in the development of a gonad. Anat. Rec. Jfi, 67. BIBLIOGRAPHY 141 Janosik, J. 1897. Die Atrophic der FoIIikel und ein seltsames Verhalten der Eizelle. Arch. mikr. Anal. 48, 169. Jenkinson, J. W. 1900. A reinvestigation of the early stages of the de- velopment of the mouse. Quar. J. Micr. Sc. 43, Gl. . 1913. Vertebrate embryology. Oxford. Jones, T. W. 1837. On the first changes in the ova of Mammifera in consequence of impregnation, and the mode of origin of the chorion. Phil. Trans. Roy. Sac. 2, 339. . 1838. On the ova of man and mammiferous brutes as they exist in the ovaries l^efore impregnation and on the discovery in them of a vesicle. London Med. Gaz., 680. . 1885. On the ova of man and mammals before and after fecunda- tion. Lancet 2, 283. Just, E. E. 1928. Cortical reactions and attendant physico-chemical changes in ova following fertilization. In Alexander: Colloid Chem- istry. The Chemical Catalog Co. Inc. New York. Kampmeier, O. F. 1929. On the problem of "parthenogenesis" in the mammalian ovary. Am. J. Anal. 43, 45. Kanel, V. Y. 1901. Regeneration processes in the ovaries of rabbits. Mattissen. Kieff. Keibel, F. 1888. Zur Entwicklungsgeschichte des Igels. Anat. Am. 3, 632. . 1894. Studien zur Entwicklungsgeschichte des Schweines (Sus scrofa dom.). Schwalbe's Morphol. Arb. 3, 1. . 1899. Zur Entwicklungsgeschichte des Rehes. Verh. Anat. Ges., Anat. Anz. Ergdnz. 16, 64. . 1901. Frlihe Entwicklungsstudien des Rehes und die Gastrulation der Siluger. Verh. Anat. Ges. Bonn, Anat. Anz. Ergdnz. 19, 184. . 1902. Die Entwicklung des Rehes bis zur Anlage des Mesoblast. Arch. Anat. u. Physiol,, Anat. Abt., 292. Kingery, H. M. 1914. So-called parthenogenesis in the white mouse. Biol. Bull. 27, 240. , 1917. Oogenesis in the white mouse. /. Morphol. SO, 261. Kingsbury, B. F. 1913. The morphogenesis of the mammalian ovary: Felis domestica. Am. J. Anat. 15, 345. . 1914. Interstitial cells of the mammalian ovary. Am. J. Anat. 16, 59. KiRKHAM, W. B. 1915. The germ cell cycle in the mouse. Proc. Am. Assoc. Anat., Anat. Rec. 10, 217. 142 THE EGGS OF MAMMALS KiRKHAM, W. B. 1916. The prolonged gestation period in suckling mice. Anat. Rec. 11, 31. . 1918. Observations on the relation between suckling ai. le rate of embryonic development in mice. J. Exp. Zool. 27, 49. KoHNO, S. 1925. Zur Kenntnis der Keimbahn des Menschen. Arch. Gyjiak. 126, 310. KuscHAKEWiTscH, S. 1910. Die Entwicklungsgeschichte der Keim- drusen von Rana esculenta. Festschr. f. R. Hertwig 2, 61. Krasovskaja, 0. V. 1935a. Fertilization of the rabbit egg outside the organism. Contribution Z'^. Variations of size of the rabbit eggs before and after fertilization (in Russian). Biol. Jour. 4, 262. . 19356. Cytological study of the heterogeneous fertilization of the egg of the rabbit outside the organism. Acta Zoologica 16, 449. Kynoch, J. A. C. 1902. Repeated ovariotomy. /. Obst. and Gynec. Brit. Emp. 2, 366. Lams, H. 1910. Recherches sur I'oeuf de cobaye (Cavia cobaya), matura- tion, fecondation, segmentation. Compt. rend. Assoc. Anat. Bruxelles 12, 119. . 1913. fitude de I'oeuf de cobaye aux premiers stades de Fembryo- genese. Arch. Biol. 28, 229. . 1924. L'oeuf de la rate pendant les premieres phases de son de- veloppement avant son arrivee dans Futerus. Compt. rend. Assoc. Anat. Bruxelles, 195. Lams, H. and Doorme, J. 1908. Nouvelles recherches sur la maturation et la fecondation de Foeuf des mammiferes. Arch. Biol. 23, 259. Lane-Claypon, J. E. 1905. On the origin and life history of the inter- stitial cells of the ovary of the rabbit. Proc. Roy. Soc. B 77 ^ 32. . 1907. On ovogenesis and the formation of the interstitial cells of the ovary. /. Ohst. and Gynec. 11, 205. Lane, C. E. 1935. Some influences of oestrin on the hypophyseal-gonad complex of the immature female rat. Am. J. Physiol 110, 681. Lane, C. E. and Hisaw, F. L. 1934. The follicular apparatus of the ovary of the immature rat and some factors which influence it. Anat. Rec. 60 Supp. 52. Lange, J. 1896. Die Bildung der Eier und der Graafschen Follikel bei der Maus. Verb. phys. med. Gesell. Wurzburg N. F. 30, 57. League, B. and Hartman, C. G. 1925. Anovular Graafian follicles in mammalian ovaries. Anat. Rec. 30, 1. Lee, F. C. 1928. The tubo-uterine junction in various mammals. Bull. Johns Hopkins Hosp. 42, 335. BIBLIOGRAPHY 143 Lelievre, Peyron et Corsy. 1927. La parthenogenese dans I'ovaire des mammiferes et le probleme de I'origine des embryonies. Bull. pf etude de Cancer 16, 71L Leonard, S. L., Hisaw, F. L. and Fevold, H. L. 193L Further studies of the folHcular-corpus luteum hormone relationship in the rabbit. Am. J. Physiol. 100, UL Leonard, S. L., Meyer, R. K. and Hisaw, F. L. 193 L The effects of oestrin on the development of the ovary in immature female rats. Endocrinology IS, 17. Lewis, L. L. 1911. The vitality of reproductive cells. Okla. Agr. Exp. Sta, Bull. 96. Lewis, W. H. 1931. Living mouse eggs. Anat. Rec. 48, 52. Lewis, W. H. and Gregory, P. W. 1929. Cinematographs of living developing rabbit eggs. Science 69, 226. Lewis, W. H. and Hartman, C. G. 1933. Early cleavage stages of the egg of the monkey. Carnegie Inst. Wash. Contrib. Embryol. 24, 187. Lewis, W. H. and Wright, E. S. 1935. On the early development of the mouse egg. Carnegie Inst. Wash. Contrib. Embryol. 25, 113. LiLLiE, F. 1919. Problems of fertilization. University of Chicago Press. Chicago. LiLLiE, R. S. 1934. The influence of hypertonic and hypotonic sea water on artificial activation of starfish eggs. Biol. Bull. 66, 361. LiPSCHtJTZ, A. 1924. The internal secretions of the sex glands. Williams and Wilkins. Baltimore. . 1928. New developments in ovarian dynamics and the law of follicular constancy. Brit. J. Exp. Biol. 5, 283. LiPSCHUTz, A., Kallas, H. and Pabz, R. 1929. Hypophyse und Gesetz der Pubertat. Arch. ges. Physiol. 221, 695. LiPSCHUTz, A. and Voss, H. E. 1925. Further developments on the dy- namics of ovarian hypertrophy. Brit. J. Exp. Biol. 3, 35. LoEB, J. 1895. Untersuchungen iiber die physiologischen Wirkungen des Sauerstoffmangels. Arch. ges. Physiol. 62, 249. . 1906. Versuchen iiber den Qhemischen Charakter des Befruch- tungsvorgangs. Biochem. Z. 1, 183. . 1913. Artificial parthenogenesis and fertilization. University of Chicago Press. Chicago. LoEB, J. and Wastenys, H. 1912. Die Oxydationsvorgange im befruch- teten und unbefruchteten Seesternei. Arch. Entwcklngsmech. 35, 555. LoEB, L. 1901. On progressive changes in the ova in mammalian ovaries. J. Med. Res. 6, 39. 144 THE EGGS OF MAMMALS LoEB, L. 1905. Uber hypertropische Vorgange bei den Follikelatresie nebst Bemerkungen liber die oocyten in den Markstrangen und liber Teilungserscheinungen am Ei im ovarium des Meerschweinchen. Arch. mikr. Anat. 65. 728. . 1911a. Beitrage zur Analyse des Gewebewachstums. VII. Uber einige Bedingungen des Wachstums der embryonalen Placenta. Arch. Entwcklngsmech. 32, 662. . 19116. The parthenogenetic development of ova in the mammalian ovary and the origin of ovarian teratomata and chorioepitheliomata. /. Am. Med. Assoc. 56, 1327. . 1912. Uber chorionepitheliomartige Gebilde im Ovariums des Meerschweinchens und liber ihre warseheinliche Entstehung aus parthenogenetisch sich entwickelnden Eiern. Z. Krebsforsch. 11,1. . 1915. An early stage and an experimentally produced extrauterine pregnancy and the spontaneous parthenogenesis of the eggs in the ovary of the guinea pig. Biol. Bull. 28, 59. . 1917. Factors in the growth and sterility of the mammahan ovary. Science JfS, 591. . 1923. The parthenogenetic development of eggs in the ovary of the guinea pig. Science 58, 35. . 1932. The parthenogenetic development of eggs in the ovary of the guinea pig. Anat. Rec. 51, 373. Long, J. A. 1912. Studies on early stages of development in rats and mice. Univ. Calif. Pub. Zool. 6, 105. Long, J. A. and Evans, H. M. 1922. The oestrus cycle in the rat and associated phenomena. Mem. Univ. Calif. 6, 1. Long, J. A. and Mark, E. L. 1911. The maturation of the egg of the mouse. Carnegie Inst. Wash. Publ. 1^2, 1. Lowenthal, N. 1888. Zur Kenntnis des Keimfieckes im Urei einiger Sanger. Anat. Am. 3, 363. Lyon, E. P. 1902. Effects of KCN and of lack of O2 upon the fertilized eggs and the embryos of the sea-urchin. Am. J. Physiol. 7, 56. MacDowell, E. C, Allen, E. and MacDowell, C. G. 1929. The rela- tion of parity, age, and body weight to the number of corpora lutea in mice. Anat. Rec. 4I, 267. MacDowell, E. C. and Lord, E. M. 1925. The number of corpora lutea in successive pregnancies. Anat. Rec. 31, 131. Mann, M. C. 1924. Cytological changes in unfertilized tubal eggs of the rat. Biol. Bull. 46, 316. BIBLIOGRAPHY 145 Marshak, a. 1935. The effect of x-rays on chromosomes in different stages of meiosis. /. Gen. Physiol. 19, 179. Marshall, F. H. A. and Jolly, W. A. 1907. Results of removal and transplantation of ovaries. Trans. Roy. Soc. Edinburgh 4^, 589. . 1908. On the results of heteroplastic transplantation as compared with those produced by transplantation in the same individual. Quar. J. Exp. Physiol. 1, 115. Melissinos, K. 1907. Die Entwicklung des Eies der Mause von den ersten Furchung-Phanomenen bis zur Festsetzung der Allantois an der Etoplacentasplatte. Arch, niikr. Anal. 70, 577. Meredith, W. A. 1904. Pregnancy after removal of both ovaries for dermoid tumor. Brit. Med. J. 1, 1360. Meyer, R. 1913. Ueber die Beziehung der Eizelle und des befruchteter Eies zum FoUikelapparat, sowie des Corpus luteum zur menstruation. Arch. Gyndk. 100, 1. Meyer, R. K., Leonard, S. L., Hisaw, F. L. and Martin, S. J. 1932. The influence of oestrin on the gonad-stimulating complex of the anterior pituitary of castrated male and female rats. Endocrinology 16, 655. Meyerhof, 0. und Kiessling, W. 1933. Uber das Auftreten und den Umsatz der a-glycerinphosphorsaure bei der Enzymatischen Kohlen- hydratspaltung. Biochem. Z. 264, 40. Minot, C. S. 1889. Segmentation of the ovum with special reference to the Manomalia. Am. Nat. 23, 463. Morris, M 1917. A cytological study of artificial parthenogenesis in Cumingia. /. Exp. Zool. 22, 1. Morris, M. M. 1901. Pregnancy following removal of both ovaries and tubes. Boston Med. and Surg. J. 144, 86. McPhail, M. K. 1933. Studies on the hypophysectomized ferret. Proc. Roy. Soc. B 114, 124. Needham, J. 1932. Chemical embryology. Cambridge University Press. Cambridge. Nelson, W. 0., Pfiffner, J. J. and Haterius, H. 0. 1930. The pro- longation of pregnancy by extracts of corpus luteum. Am. J. Physiol. 91, 690. Newman, H. H. 1912. The ovum of the nine-banded armadillo: growth of the ovocytes, maturation, and fertilization. Biol. Bull. 23, 100. . 1913. Parthenogenetic cleavage of the armadillo ovum. Biol. Bull. 25, 54. 146 THE EGGS OF MAMMALS Nicholas, J. S. 1933a. Development of transplanted rat eggs. Proc. Soc. Exp. Biol. & Med. 30,1111. . 19336. The development of rat embryonic tissues after transplanta- tion of the egg to the kidney. Anat. Rec. 55, Supp. 31. . 1934. Experiments on developing rats. I. Limits of foetal regenera- tion; behavior of embryonic material in abnormal environments. Anat. Rec. 58, 387. Nicholas, J. S. and Rudnick, D. 1933. The development of embryonic rat tissues upon the chick chorioallantois. J. Exp. Zool. 66, 193. . 1934. The development of rat embryos in tissue culture. Proc. Nat. Acad. Sc. 20, 656. Novak, J. and Eisinger, K. 1923. Ueber kunstlick bewirkte Teilung des unbefruchteten Saugertiereies. Arch. mikr. Anat. 98, 10. NussBAUM, M. 1880. Zur Differenzierung des Geschlechts im Thierreich. Arch. mikr. Anat. 18, 1. VAN OoRDT, G. J. 1921. Early developmental stages of Manis javanica Desm. Verhandl. kon. Scad, ven Wetensch. Amsterdam, Sec. 2, Part XXI, 1. Palladino, G. 1887. Ulteriori ricerche sulla distruzione e rinovamento continuo del parenchima ovarico dei Mammiferi. Tipi del cav. Antonio Morano. Napoli. . 1894. La destruction et le renouvellement continuel du parenchyme ovarique des mammiferes. Arch. ital. Biol. 21, XV Sec. d'Anat. . 1898. Sur le type du structure de I'ovariere. Arch. ital. Biol. 29, 139. Pallot, G. 1928. A propos de la regeneration ovarienne et des modifica- tions periodiques de I'epithelium vaginal chez le rat blanc. Compt. rend. Soc. biol. 99, 1333. Papanicolou, G. N. 1925. Ovogenesis during sexual maturity as eluci- dated by experimental methods. Proc. Soc. Exp. Biol. & Med. 21, 393. Parker, G. H. 1931. The passage of sperms and eggs through the ovi- ducts in terrestrial vertebrates. Phil. Trans. Roy. Soc. London B 219, 381. Parkes, a. S. 1926. On the occurrence of the oestrus cycle after x-ray sterilization. Part I. Irradiation of mice at three weeks old. Proc. Roy. Soc. B 100, 172. . 1927a. On the occurrence of the oestrus cycle after x-ray steriliza- tion. Part 11. Irradiation at or before birth. Proc. Roy. Soc. B 101, 71. . 19276. On the occurrence of the oestrus cycle after x-ray steriliza- tion. Part III. The periodicity of oestrus after sterilization of the adult. Proc. Roy. Soc. B 101, 421. BIBLIOGRAPHY 147 Parkes, a. S. 1927c. On the occurrence of the oestrus cycle after x-ray sterilization. Part IV. Irradiation of the adult during pregnancy and lactation; and general summary. Proc. Roy. Soc. B 102, 51. . 1929 The internal secretions of the ovary. Longmans, Green and Co. London. . 1931. The reproductive processes of certain mammals. II. The size of the Graafian follicle at ovulation. Proc. Roy. Soc. B 109, 185. Parkes, A. S., Fielding, Una and Brambell, F. W. R. 1927. Ovarian regeneration in the mouse after complete double ovariotomy. Proc. Roy. Soc. B 101, 328. Parkes, A. S., Rowlands, I. W. and Brambell, F. W. R. 1932. Effects of x-ray sterilization on oestrus in the ferret. Proc. Roy. Soc. B 109, 425. Pearl, R. and Schoppe, W. F. 1921. Studies on the physiology of repro- duction in the domestic fowl. /. Exp. Zool. 34, 101. Pencharz, Richard. 1929. Experiments concerning ovarian regeneration in the white rat and white mouse. /. Exp. Zool. 54, 319. PpLtJGER, E. 1863. Die Eierstocke der Saugetiere und der Menschen. Wilhelm Engelmann. Leipzig. PiNCUS, G. 1930. Observations on the living eggs of the rabbit. Proc. Roy. Soc. 107, 132. . 1931. The transplantation of mouse ovaries into the rat. Anat. Rec. 49, 97. PiNCUS, G. and Enzmann, E. V. 1932. Fertilization in the rabbit. /. Exp. Biol 9, 403. . 1934. Can mammalian eggs undergo normal development in vitro? Proc. Nat. Acad. Sc. 20, 121, . 1935. The comparative behavior of mammalian eggs in vivo and in vitro. I. The activation of ovarian eggs. /. Exp. Med. 62, 665. . 1936a. The comparative behavior of mammalian eggs in vivo and in vitro. II. The activation of tubal eggs of the rabbit. J. Exp. Zool. 73, 195. . 19366. The growth, maturation and atresia of the ovarian eggs of the rabbit. (In press.) PiNCUS, G. and Kirsch, R. E. 1936. The sterility in rabbits produced by injections of oestrone and related compounds. Am. J. Physiol. 115, 219. PiNcus, G. and Werthessen, N. 1933. The continued injection of oestrin into young rats. Am. J. Physiol. 103, 631. 148 THE EGGS OF MAMMALS QUINLA.N, J., Mare, G. S. and Roux, L. L. 1932. 18th Rep. Div. Vet. Serv. and Anim. Ind. Union of South Africa, Pt. II, 813. Rabl, H. 1898. Zur Kenntnis der RichtungsspindeLn in degenerierenden Saugetiereiern. Sitzber. Akad. Wiss. Naturiciss. Kl. 106, 95. Reagan, F. P. 1916. Some results and possibilities of early embryonic castration. Anat. Rec. 11, 489. Reichert, K. 1861. Beitrage zur Entwicklungsgeschichte des Meer- schweinchens. Abhandl. K. Akad. Wissensch. 182. Berhn. Rein, G. 1883. Beitrage zur Kentniss der Reifungserscheinungen und Befruchtungsvorgange am Saugethierei. Arch. mikr. Anat. 22, 233. Reiss, M., Selye, H. und Balint, J. 1931a. Uber die Wirkung alkahscher Hypophysenvorderlappenextrakte auf das Genitale der weibhchen Ratte. Endokrinol. 8, 15. . 19316. tjber die Beeinflussung des mannhchen Genitales durch den luteinisierenden Wirkstoff des Hypophysenvorderlappens. Endo- krinol. 5, 81. Robertson, J. A. 1890. Renewal of menstruation and subsequent preg- nancy after removal of both ovaries. Brit. Med. J. 2, 722. Robinson, A. 1892. Observations upon the development of the segmenta- tion cavity, the archenteron, the germinal layers, and the amnion in mammals. Quar. J. Micr. Sc. 33, 369. . 1904. Lectures on the early stages in the development of mamma- lian ova and on the formation of the placenta in different groups of mammals. /. Anat. 38, 186. . 1918. The formation, rupture and closure of ovarian follicles in ferrets and ferret-polecat hybrids, and some associated phenomena. Trans. Roy. Soc. Edinburgh 52, 303. RuBASCHKiN, W. 1906. Uber die Veranderungen der Eier in den zugrunde- gehenden Graafschen Follikeln. Aimt. Heft£ 32, 255. . 1908. Zur frage von der Ent-stehung der Keimzellen bei Sauge- tierembryonen. Anat. Anz. 32, 222. . 1910. Uber das erste Auftreten und Migration der Keimzellen bei Saugetierembryonen. Anat. Hefte J^l, 243. . 1912. Zur lehre von der Keimbahn bei Saugetieren. Uber die Entwicklung der Keimdriisen. Anat. Hefte Jj6, 342. RuNNSTROM, J. 1930. Atmungsmechanismus und entwicklungserregung bei dem Seeigelei. Protopla^ma 10, 106. . 1933. Zur Kenntnis des Stoffwechselvorgange bei der Entwick- lungserregung des Seeigeleies. Biochem. Z. 258, 257. BIBLIOGRAPHY 149 RuNNSTROM, J. 1935. On the influence of pyocyanine on the respiration of the sea urchin egg. Biol. Bull. 68, 327. Sainmont, G. 1905. Recherches relatives a I'organogenese du testicle et de I'ovaire chez le chat. Arch. Biol. 22, 71. Sakurai, T. 1906. Normentafel zur Entwicklungsgeschichte des Rehes (Cervus capreolus). Verlag von Gustav Fischer. Jena. Sansom, G. S. 1920. Parthenogenesis in the water vole. /. Anal. 55, 68. Sansom, G. S. and Hill, J. P. 1930. Observations on the structure and mode of implantation of the blastocyst of Cavia. Tram. Zool. Soc. London 21, 295. Schottlander, G. 1891. Beitrag zur Kenntnis der Follikelatresie nebst einigen Bemerkungen liber die unveriinderten Follikel in den Eier- stocken der Saugetiere. Arch. mikr. Anal. 37, 192. ScHRON, 0. 1863. Beitrag zur Kentniss der Anatomie und Physiologic des Eierstockes der Siiugethiere. Z. ivissensch. Zool. 12, 409. ScHULTZ, W. 1900. Transplantation der Ovarien auf miinnliche Tiere. Zenlr. allg. Path. 2, 200. Scott, J. 1906. Morphology of the parthenogenetic development of Amphitrite. /. Exp. Zool. 3, 49. Selenka, E. 1883. Studien fiber die Entwicklungsgeschichte der Thiere. 1 Heft, Keimbliitter und Primitivorgane der Maus. C. W. Kreidel's Verlag. Wiesbaden. . 1884. Studien fiber die Entwicklungsgeschichte der Thiere. 3 Heft, Die Blatterumkehrung im Ei der Nagethiere. C. W. Kreidel's Verlag. Wiesbaden. . 1887. Studien fiber die Entwicklungsgeschichte der Thiere. 4 Heft. Das Opossum. C. W. Kreidel's Verlag. Wiesbaden. Selye, H. 1933. Effect of hypophysectomy on the ovary of immature rats. Proc. Soc. E.vp. Biol. & Med. 31, 262. Selye, H. and Collip, J. B. 1933. Production of exclusively thecal luteinization and continuous oestrus with anterior-pituitary-liko hor- mone. Proc. Soc. Exp. Biol. & Med. 30, 647. Selye, H., Collip, J. B. and Thomson, D. L. 1935^7. Endocrine inter- relationships during pregnancy. Endocrinology 19, 151. . 1935/;. Effect of oestrin on ovaries and adrenals. Proc. Soc. Exp. Biol. & Med. 32, 1377. Selye, H. and McKeown, T. 1934a. Production of pseudopregnancy by mechanical stimulation of the nipples. Proc. Soc. Exp. Biol. c(' Med. 31, 683. 150 THE EGGS OF MAMMALS Selye, H. and McKeown, T. 19346. Further studies on the influence of suckhng. Anat. Rec. 60, 323. SiMKiNS, C. S. 1923. Origin and migration of the so-called primordial germ cells in the mouse and rat. Acta Zool. 4, 241. . 1928. Origin of sex cells in man. Am. J. Anat. p, 249. Slawinsky, K. 1873. Filaments glandulaires trouves dans Tovaire d'une femme adulte. Bull. Soc. Anat. Paris 4-8, 844. Slonaker, J. R. 1927. Semi-ovariectomy, compensatory hypertrophy of the remaining ovary and migration of the ova in the albino rat. Am. J. Physiol. 81, 620. Smith, P. E. 1930. Hypophysectomy and a replacement therapy in the rat. Am. J. Anat. 1^.5, 205. . 1932. The effect on the reproductive system of ablation and im- plantation of the anterior hypophysis. In Allen: Sex and Internal Secretions, 734. Williams and Wilkins. Baltimore. Smith, P. E. and Engle, E. T. 1927. Experimental evidence regarding the role of the anterior pituitary in the development and regulation of the genital system. Am. J. Anat. Jfi, 159. Smith, P. E. and MacDowell, E. C. 1931. The differential effect of hereditary mouse dwarfism on the anterior-pituitary hormones. Anai. Rec. 50, 85. Smith, P. E. and White, W. E. 1931. The effect of hypophysectomy on ovulation and corpus luteum formation in the rabbit. /. Am. Med. Assoc. 97, 1861. Smith, S. C. 1925. Degenerative changes in the unfertilized uterine eggs of the opossum (Didelphis virginiana), with remarks on the so-called parthenogenesis in mammals. Am. J. Anat. 35, 81. Snyder, F. F. 1923. Changes in the fallopian tube during the ovula- tion cycle and early pregnancy. Bull. Johns Hopkins Hosp. 34-, 121. . 1934. The prolongation of pregnancy and complications of parturi- tion in the rabbit following induction of ovulation near term. Bull. Johns Hopkins Hosp. 54, 1. SoBOTTA, J. 1893. Mitteilungen liber die Vorgange bei der Reifung, Befruchtung und erste Furchung des Eies der Maus. Verhandl. Anat. Ges. Gottingen, 111. . 1895. Die Befruchtung und Furchung des Eies der Maus. Arch. mikr. Anat. 45, 15. . 1899. Uber die Bedeutung der mitotischen Figuren in den Eier- stockseiern der Saugetieren. Wurzburg. BIBLIOGRAPHY 151 SoBOTTA, J. und BuRCKHARD, G. 1911. Rcifung und Befruchtung des Eies der weissen Ratte. Anat. Hefte 42, 433. Spee, F. 1915. Anatomie und Physiologie der Schwangerschaft. 1. Theil in Doderlein: Handbuch der Geburtshilfe 1. Bergmann. Munich. Spencer, J., D'Amour, F. E. and Gustavson, R. G. 1932. Effects of continued oestrin injections on young rats. Am. J. Anat. 50, 129. Spuler, a. 1900. Uber die Teilungserscheinungen der Eizellen in degen- erierenden Follikeln des Saugerovariums. Anat. Hefte 16, 85. Squier, R. R. 1932. The living egg and early stages of its development in the guinea-pig. Carnegie Inst. Wash. Contrih. Emhnjol. 23, 225. Stockard, C. R. and Papanicolou, G. N. 1917. The existence of a typical oestrus cycle in the guinea-pig, with a study of its histological and physiological changes. Am. J. Anat. 22, 225. Stotsenburg, J. M. 1913. The effect of spaying and semi-spaying young albino rats (M. norvegicus albinus) on the growth in body weight and body length. Anat. Rec. 7, 183. Streeter, G. L. 1931. Development of the egg as seen by the embry- ologist. Sc. Month. 32, 495. Sutton, R. S. 1896. Double ovariotomy followed by pregnancy and de- livery at term. Trans. Am. Gynec. Soc. 21, 109. SwANN, W. F. G. and del Rosario, C. 1932. The effect of certain mono- chromatic ultra-violet radiation upon Euglena cells. J. Franklin Inst. 213, 549. SwEZY, 0. 1929. The ovarian chromosome cycle in a mixed rat strain. J. Morphol. & Physiol. 48, 445. . 1933a. The changing concept of ovarian rhythms. Quar. Rev. Biol. 8, 423. . 19336. Ovogenesis and the hypophysis: the effects of pregnancy, hypophysectomy, thyroidectomy and hormone administration on the ovary of the rat. Science Press. Lancaster. SwEZY, 0. and Evans, H. M. 1930. Ovarian changes during pregnancy in the rat. Science 71, 46. Tafani, a. 1889. I primi momenti delb sviluppo dei mammiferi. Rendi- conti R. Accad. Lincei 5, 119. Tamura, Y 1926. The effects of implantation upon ovarian grafts in the male mouse. Proc. Roy. Soc. Edinburgh 47, 148. Teel, H. M. 1926. The effect of injecting anterior hypophysial fluid on the course of gestation in the rat. Am. J. Physiol. 79, 120. Turner, C. W. 1932. The mammary glands. In Allen: Sex and Internal Secretions. Williams and Wilkins. Baltimore. 152 THE EGGS OF MAMMALS Van Beneden, E. 1875. La maturation de I'oeuf, la fecondation et les premieres phases du developpement embryonaire des Mammiferes d'apres les recherches faites sur le Lapin. Bull. Acad. Roy. Sc. Lettres et Beaux Arts Belg. J^O, 686. . 1880. Contribution a la connaissance de I'ovaire des mammiferes. Arch. Biol. 1, 475. . 1899. Recherches sur les premiers stades du developpement du Murin (Vespertilio murinus). Anat. Anz. 16, 307. . 1911. De la segmentation, de la formation de la cavite blasto- dermique et de I'embryon didermique chez le Murin. Arch. Biol. 26, 1. . 1912. Recherches sur Fembryologie des mammiferes. Arch. Biol. 27, 191. Van Beneden, E. et Julin, C. 1880. Observations sur la maturation, la fecondation et la segmentation de I'oeuf chez les Cheiropteres. Arch. Biol. ^ 551. Van der Stricht, 0. 1901. L'atresie ovulaire et I'atresie foUiculaire du follicule de De Graaf dans Fovaire de chauve-souris. Verhandl. anat. Ges. 15, Versamml. Bonn. Vanneman, a. S. 1917. The early history of the germ cells in the armadillo Tatusia novemcineta. Am. J. Anat. 22, 341. Voss, H. E. 1925. Condition de la greffe ovarienne intratesticulaire. Compt. rend. Soc. biol. 93, 1066. Waddington, C. H. and Waterman, A. J. 1933. The development in vitro of young rabbit embryos. /. Anat. 67, 356. Wagener, G. 1879. Bemerkungen iiber den Eierstock und den Gelben Korper. Arch. Anat. u. Physiol. 175. Wagner, R. 1836. Prodromus historiae generationis hominis atque animahum. Leipzig. Waldeyer, W. 1870. Eierstock und Ei. Verlag von Wilhelm Engelmann. Leipzig. . 1906. Die Geschlechtzellen. In: Handbuch der vergleichenden und experimentellen Entwickelunglehre der Wirbeltiere. Oskar Hert- \vig. Jena. Walsh, L. S. M. 1917. The growth of the ovarian follicle in the guinea pig under normal and pathological conditions. J. Exp. Med. 26, 245. Walton, A. and Hammond, J. 1932. Observations on ovulation in the rabbit. Brit. J. Exp. Biol. 6, 190. Wang, G. H. and Guttmacher, A. F. 1927. The effect of ovarian trau- matization on the spontaneous activity and genital tract of the albino BIBLIOGRAPHY 153 rat, correlated with a histological study of the ovaries. Ayn. J. Physiol. 82, 335. Warburg, O. 1908. Beobachtungen fiber die Oxydationsprocesse im Seeigelei. Z. physiol. C/iem. 57, 1. . 1909. Ue})er die Oxydation im Ei: II, Mittheilung. Z. physiol. Chem. 60, 443. . 1910. Ueber die Oxydationen in lebenden Zellen nach Versuchen am Seeigelei. Z. physiol. Chem. 66, 305. . 1914rt. Uel)er die Rolle des Eisens in der Atmung des Seeigeleis nebst Bermerkungen iiber einige durch Eisen beschleunigte Oxyda- tionen. Z. physiol. Chem. 92, 231. . 19146. Zellstruktur und Oxydationsgeschwindigkeit nach Ver- suchen am Seeigelei. Pflug. Arch. ges. Physiol. 158, 189. . 1932. Das sauerstoffiibertragende Ferment der Atmung. Z. angew. Chem. Jf.5, 1. Waterman, A. J. 1932. Culture * in vitro ' and transplantation of young rabbit embryos. Anat. Rec. 54, 72. . 1934. Survival of young rabbit embryos on artificial media. Proc. Nat. Acad. Sc. 20, 145. Weil, C. 1873. Beitrage zur Kentniss der Befruchtung und Entwicklung des Kanincheneies. Strieker's med. Jahrb. Weismann, August. 1883. Enstehung der Sexuallzellen bei den Hydro- medusae. Verlag von Gustav Fischer. Jena. . 1904. Vortrage liber Descendenztheorie. English translation. Edward Arnold. London. Whitaker, D. M. 1933. On the rate of oxygen consumption by fertilized and unfertilized eggs. V. Comparisons and interpretation. /. Gen. Physiol. 16, 497. Williams, W. L. 1909. Veterinary obstetrics including the diseases of the breeding animals and the new-born. Williams. Ithaca. WiLLiER, B. H. 1921. Structures and homologies of free-martin gonads. /. Exp. Zool. 33, 63. . 1932. The embryological foundations of sex in vertebrates. In Allen: Sex and Internal Secretions. Williams and Wilkins. Balti- more. . 1933a. Potencies of the gonad-forming area in the chick as tested in chorio-allantoic grafts. Arch. Entwcklngsmech. Organ. 130, 616. . 19336. On the origin and differentiation of the sexual gland. Am. Nat. 67, 1. Wilson, E. B. 1901. Experimental studies in cytology. I. A cytological 154 THE EGGS OF MAMMALS study of artificial parthenogenesis in sea urchin eggs. Arch. Ent- wcklngsmech. 12, 529. Wilson, J. T. 1928. On the question of the interpretation of the struc- tural features of the early blastocyst of the guinea-pig. /. Anat. 62, 346. Wilson, J. T. and Hill, J. P. 1907. Observations on the development of Ornithorhynchus. Phil. Trans. Roy. Soc. 199 B, 31. DE Winiwarter, H. 1901. Recherches sur Tovogenese et I'organogenese de Fovaire des mammiferes (lapin et homme). Arch. Biol. 17, 33. . 1910. Contributions a I'etude de Fovaire humaine. I. Appareil nerveux et pheochrome. II. Tissue musculaire. III. Cordons medul- laires et corticaux. Arch. Biol. 25, 683. . 1920. Couche corticale definitive au hile de I'ovaire et pseudo- neoformation ovulaire. Compt. rend. Soc. biol. 83, 1406. DE Winiwarter, H. et Sainmont, G. 1909. Nouvelles recherches sur Tovogenese et I'organogenese de Fovaire des mammiferes (chat). Arch. Biol. 24, 1. WiSLOCKi, G. B. and Goodman, L. 1934. The effect of anterior lobe extract or concentrated human urine of pregnancy upon the early part of gestation in the rabbit. Anat. Rec. 69, 375. WiSLOCKi, G. B. and Snyder, F. F. 1931. On the experimental production of superfetation. Bull. Johns Hopkins Hasp. 4-9, 106. WiTscHi, E. 1929. Studies on sex differentiation and sex determination in amphibians. II. Sex reversal in female tadpoles of Rana sylvatica following the application of high temperature. /. Exp. Zool. 52, 267. Yamane, J. 1930. The proteolytic action of mammalian spermatozoa and its bearing upon the second maturation division of ova. Cytologia 1, 394 . 1935. Kausal-analytische Studien iiber die Befruchtung des Kanin- cheneies. I. Die Dispersion der Follikelzellen und die Ablosung der Zellen der Corona radiata des Eies durch Spermatozoen. Cytologia 6, 233. Zondek, B. 1931. Die Hormone des Ovariums und des Hypophysen- vorderlappens. Julius Springer. Berlin. AUTHOR INDEX Addison, W. H. F., 52 Allen, B. M., 30 Allen, E., 8, 9, 10, 14, 19, 42, 44, 45, 57, 63, 71 Allen, W. M., 117 Amann, J. A., 9 Anderson, D. H., 74 Aral, H., 8, 19, 23, 24, 32, 33, 39 Asami, G., 44 Aschner, B., 7 Asdell, S. A., 18, 86 Assheton, R., 112 Athias, M., 21, 52 von Baer, K. E., 1 Balfour, F. M., 8, 52 Balint, J., 26 Barry, M., 1, 2, 62 Bellerby, C. W., 48 Benoit, J., 29 Biedl, A., 96 Bischoff, T. L. W., 2, 62, 124 Bland, L. J., 57, 63, 71 Bond, C. S., 18 Bonnet, R., 2, 52, 124 Boone, C, 18 Bosaeus, W., 53 Brachet, A., 3, 112, 121 Brambell, F. W. R., 6, 7, 11, 16, 21, 30, 32, 34, 36 Branca, A., 53, 54 Buhler, A., 8 Burckhard, G., 82 Burdick, H. O., 94, 116, 117 Butcher, Earl O., 8, 20 Buyse, Adrian, 31 Caldwell, W. H., 2 Campbell, J. A., 95 Carmichael, E. S., 18 Carrel, A., 55 Casida, L. E., 33 Castle, W. E., 89, 92, 93 Champy, C, 98 Chapin, Catherine L., 30 Charlton, H. H., 73 Clark, E. B., 52 Clark, R. T., 68 Coert, H. J., 9 Cole, H. H., 51, 71 Collier, W. D., 26, 27 Collip, J. B., 33, 115, 126 Corey, E. L., 33 Corner, G. W., 94, 116, 117 Corsey, 53 Courrier, R., 53, 61, 123 Cowperthwaite, Marian M., 7, 10 Crew, F. A. E., 18 Cruikshank, W., 62, 112 Curtis, J., 26, 27 Dalq, A., 110 D'Amour, F. E., 26 Davenport, C. B., 15, 16 Deansley, R., 48 Defrise, A., 3, 62, 65 Del Rosario, C, 110 Doisy, E. A., 26, 27 Domm, L. V., 29 Doorme, J., 2, 82 Doran, M. A., 18 Eisinger, K., 2, 111 Emery, F. E., 18, 19 Engle, E. T., 24, 26, 33, 35, 42, 43, 44, 45, 51, 52, 53 Enzmann, E. V., 36, 38, 44, 46, 50, 56, 66, 67, 75, 76, 78, 81, 93, 95, 96, 97, 108, 110, 111, 116, 123, 124 Evans, H. M., 2, 8, 11, 14, 19, 23, 24, 26, 44, 51, 52, 71, 88, 125 Fee, A. R., 48 Fehx, W., 7, 31 Fellner, O. O., 9 Fevold, H. L., 27, 123 Fielding, Una, 16, 21 Firket, J., 8 Fischer, Albert, 55 Flemming, W., 53 Foster, M. A., 27 Foulis, J., 8 155 156 AUTHOR INDEX Francis, B. F., 14, 19, 22 Friedgood, H., 49 Friedman, M. H., 48 Fuss, A., 6 Genther, I., 21 Gerard, P., 10 Gilchrist, F., 62, 71, 75, 81, 82, 89,94 Goodman, L., 126 Gordon, C. S., 18 Greep, O., 27 Gregory, P. W., 3, 65, 89, 91, 92, 93, 94, 112 Grosser, O., 124 Grusdew, W. S., 2, 53, 111 Gm'witsch, A., 53 Gustavson, R. G., 26 Gutherz, S., 55 Guttmacher, A. F., 19 Haberlandt, G., 55 Hiiggstrom, P., 53 Hall, B. v., 115 Hamlett, G. W. D., 126, 127 Hammond, J., 18, 33, 46, 82, 83, 84, 85, 86, 87 Hanson, F. B., 18 Hargitt, G. T., 8, 35 Harms, J. W., 5 Hartman, C. G., 2, 3, 18, 35, 40, 45, 65, 68, 72, 74, 87, 88, 89, 93 Hars^, W., 9 Hatai, S., 18 Haterius, H. O., 15, 16, 126 Heape, W., 2, 46 Hegner, R. W., 7 HcUbaum, A. A., 27 Hcnneguy, F., 53 Hensen, V., 2, 52, 74 Hertz, R., 33 Heuser, C. H, 89 Heys, Florence, 5, 16, 17, 18 Hill, J. P., 2, 11, 124 Hill, M., 2 Hindle, E., 103 Hinsey, J. C., 49 Hisaw, F. L., 26, 27, 28, 33, 54, 123 Hofstatler, R., 96 Huber, G. C., 2, 93, 95 Hubrecht, A. A. W., 2 Humphrey, R. R., 29 Janosik, J., 52 Jenkinson, J. W., 2, 6 Jolly, W. A., 20 Jones, T. W., 1 Julin, C., 2 Just, E. E., 54, 59 Kallas, H., 33 Kampmeier, O. F., 52 Kanel, V. Y., 18 Keibel, F., 2 Kiesling, W., 96 Kingery, H. M., 8, 52 Kingsbury, B. F., 8 Kirkham, W. B., 52, 124 Kirsch, R. E., 94, 95, 116, 117, 118, 119, 120, 122, 123 Kohno, S., 8 Kountz, W. B., 14, 19, 22 Krasovskaja, O. V., 80, 81 Kuschakewitsch, S., 29 Kynoch, J. A. C., 18 Lams, H., 2, 82 Lane, C. E., 27 Lane-Claypon, J. E., 9 Lange, J., 9 League, B., 35 Lee, F. C., 63 Lelievre, 53 Leonard, S. L., 27, 123 Lewis, L. L., 88 Lewis, W. H., 3, 62, 65, 66, 74, 88, 89, 93,94,112 Lillie, F., 54 LiUie, R. S., 54 Lipschutz, A., 18, 20, 33 Loeb, J., 54, 59, 95, 96, 108, 109 Loeb, L., 45, 53, 61 Long,J. A.,2, 52, 71,81,87, 88 Lord, E. M., 44 Lowenthal, N., 53 Lyon, E. P., 95 MacDowell, C. G., 44 MacDowell, E. C., 34, 44 McKeown, T., 127 McPhail, M. K., 48 Mann, M.C., 71,73, 88, 98, 111 Marc, G. S., 88 Mark, E. L., 87 Markee, J. E., 49 Marshak, A., 39 AUTHOR INDEX 157 MarshaU, F. H. A., 18, 20, 33, 82 Martin, S. J., 26 Melissinos, K., 2 Meredith, W. A., 18 Meyer, K., 26 Meyer, R., 45 Mever, R. K., 26 Meyerhof, O., 96 Minot, C. S., 2 Morris, M., 107 Morris, M. M., 18 Needham, J., 54, 95 XeLson, W. O., 126 Newell, Q. U., 57, 63, 71 Xewinan, H. H., 53 Nicholas, J. S., 3, 66, 67, 96, 114, 115 Novak, J., 2, 111 Xussbaum, M., 5, 6 Oberling, C, 53, 61 van Oordt, G. J. 2 Paez, R., 33 Palladino, G., 9, .53 Fallot, G., 16 Fapanicolou, G. X., 2, 8, 52 Farker, G. H., 74 Farkes, A. S., 16, 21, 33, 36, 38, 39, 46, 48, 127 Fearl R., 7 Fencharz, Richard, 16 Feters, H., 96 Fe\Ton, 53 Ff^ner, J. J., 126 Ffluger, E., 8, 53 Fincus, G., 15, 26, 36, 38, 44, 46, 49, 50, 56, 57, 62, 65, 66, 70, 71, 73, 75, 76, 78. 81, 82, 87, 89, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 106, 108, 110, 111, 116, 117, 118, 119, 120, 121, 122, 123, 124 Fratt, J. R, 57, 63, 71 Quinlan, J., 88 Rabl, H., 53 Ra^^laud, R., 123 Reagan, F. F., 29 Reichert, K., 2 Rein, G., 2 Reiss, M., 26 Robertson- J. A., 18 Robinson, A., 2, 8, 47, 124 Roux, L. L., 88 Rowlands, I. W., 21 Rubaschkin, W., 7, 52 Rudnick, D., 3, 66, 114 Runnstrom, J., .54, 59, 95, 96 Sainmont, G., 6, 7, 30 Sakxirai, T., 2 Sansom, G. S., 53, 124 Saphir, X. R., 93, 124 Schoppe, W. F., 7 Schottlander, G., .53 Schron, O., 9 . Schultz, W., 20 Scott, J., 107 Selenka, E., 2 Selye, H., 26, 32, 33, 115, 126, 127 Simkins, C. S., 8 Simon, S., 110 Simpson, M. E., 24, 26, 125 Slawinskv, K., 9 Slonaker, J. R., 18 Smith, F. E., 22, 24, 32, .33. .34, 44, 4.5, 48 Smith, S. C., 72 Snyder, F. F., .50, 93, 125 Sobotta, J., 2, .52, 82, 95 Spee, F., 62, 124 Spencer, J., 26 Spuler, A., .53 Squier, R. R., 3, 62, 66, 71, 89, 93, 94 Stockard, C. R., 2, .52 Stotsenburg, J. M., 18 Streeter, G. L., 41, 89 Subba Ran, A., 11 Sutton, R. S., 18 Swann, W. F. G., 110 Swezv, O., 8, 11, 14, 19, 22, 23, 24, 25, 26,27,32,44 Tafani, A., 2 Tamura, Y., 20, 21 Teel, H. M., 12.5, 126, 127 Thomson, D. L., 11-5, 126 Tribe, M., 2 TmTier, C. W., 127 Van Beneden, E., 2, 9, 94 Van der Stricht, O., .53 Vanneman, A. S., 7 Voss, H. E., 18, 20 Waddington, C. H., 113 Wagener, G., 9 158 AUTHOR INDEX Wagner, R., 1 Waldeyer, W., 7 Walsh, L. S. M., 45 Walton, A., 46, 82, 87 Wang, G. H., 19 Warburg, O., 95, 96 Wastenys, H., 95 Waterman, A. J., 113 Weil, C, 2 Weismann, August, 5 Werthessen, N., 26 Whitaker, D. M., 54, 95 White, W. E., 48 Williams, W. L., 19 Wilher, B. H., 29, 30 Wilson, E. B., 107 Wilson, J. T., 2, 124 de Winiwarter, H., 6, 7, 8, 10 Wislocki, G. B., 50, 126 Witschi, E., 30 Wright, E. S., 3, 74, 89, 93 Yamane, J., 71, 75, 76, 78, 80 Zondek, B., 33 '«? /it SUBJECT INDEX Atresia, in follicles during oestrus cycle and early pregnancy, 42, 43 in hypophysectomized rats, 32 prevention of, 44 Blastocyst, development in vitro, 112 and ff. effect of ovariectomy on, 116 stages in the rabbit, 91 time of formation, 91, 93 Blastomeres, actual and prospe tive in rabbit, 92, 93 transplantation of in rat, 97 Cleavage, and cyanide inhibition, 95, 96 and iodoacetate inhibition, 96 rate in various rabbit strains, 89 and ff. rate of, 88 and ff. relation to tubal and ovarian se- cretions, 94 and ff. stages in the rabbit, 90 Corpus luteum, and delayed im- plantation, 126 and ff. relation to blastocyst growth, 116 and ff. Cortex, gonad, 30, 31 Deciduomata, and delayed preg- nancy, 127 pH of, 116 Embryo, development in vitro, 113 and ff. Endometrium, and ovum growth, 117, 123 Fertilization, effects of sperm dilu- tion, 77, 78 relation to sperm extracts, 79 semination and ac \^a,tion, 75 and ff. with irradiated sperm, 110 Follicle cells, formation, 10 Follicle graafian, antrum develop- ment, 32, 37, 38 earliest response to pituitary hor- mones, 33 growth in relation to ovum growth, 34-39 in dwarf mice, 35 Follicle stimulating hormone and ovogenesis, 26, 27 Follicles, anovular, 35 atresia during oestrus cycle and early pregnancy, 42, 43 types in rabbits, 36, 37 Fragmentation, cinematography of, 105 Germ cells, meiosis, 7 origin, 5 and ff. primordial, 6, 7 theories of origin, 7-9 Germinal epithelium, mitosis fre- quency, 9 origin, 6 Gonadogenesis, 29-31 Gonads, embryogeny, 6 Grafts, gonad, 30, 31 Growth hormone, effects on ovogene- sis, 24, 25 Heat treatment of eggs, 109 Hormones, ovarian, 2, 3 Hypertonicity, and activation, 109 Hypophysectomy, and ovogenesis, 22 Implantation, hormonal control of, 125 and ff. time of, 124 Inner cell mass, 93 Lactation and delayed pregnancy in rats and mice, 124 and ff. Luteinizing hormone and ovogenesis, 26-28 Macacus, fertilizable life of ovum of, 88 159 160 SUBJECT INDEX Oestrin, and sterilization, 120, 121 effect on cleavage and growth stages, 117 and ff. effect on egg cultures, 95, 122 effects on ovaries and ovogenesis, 25, 27 Oestrus cycle, 22 Oocytes, primate, 11 Ova, ovarian morphogenesis, 6 and ff. ovarian, number at various ages, 23 primary, minimum size, 32 Ovariectomy, bilateral, effect on ovaries, 15-20 effect on ovum growth, 116, 117 Ovaries, effects of x-ray on, 21 transplanted, 20, 21 Ovogenesis, effects of pituitary secre- tions, 22 and ff. Ovum, albumin covering in rabbit, 70 atresia and activation, 54, 55 binucleate, 57 condition at ovulation, 68 corona, 71 entry into uterus, 70 and ff. fertilizable life, 82 and ff. fragmentation, 52, 72 and ff. human, recovery of, 63 maturation in rabbit, 46 methods of culture, 64-66 oestrin production, 44 polar body formation, 47-49 rate of passage in tubes, 74 recovery from tubes and uterus, 62-64 sizes in various classes of mam- mals, 40 transplantation into oviduct, 66, 67, 94, 97 tubal, artificial activation of, 190 and ff. tubal, cytology after activation, 107, 108 tubal, effect of age on behavior in vitro, 106 Ovum — Continued tubal, effect of retention in tubes, 111 unfertilized, behavior in vitro, 98 and ff. unfertilized, tubal history of, 68 and ff. vitellus at fertihzation, 80-82 Parthenogenesis, 1 and activation, 53 and ff. ovarian, 57-59 Pituitary hormones, and delayed pregnancy, 125-127 and maturation, 48-50, 56, 57 Pregnancy, and ovogenesis, 22 delayed, 124 and ff. Pronucleus, 56, 110 Pseudomaturation, 33, 42, 43, 51 Pseudoparthenogenesis, 53 Rat, developmental stages, 11-14 Sperm, penetration into ovum, 82 swarm, 86 Suckling, and pseudopregnancy, 127 Superfetation, 50 Superovulation, 44 Tetrad formation in ovarian ova, 47 Tissue culture, 3, 64-66 Thyroidectomy and ovogenesis, 22 Thyroxin and maturation, 50, 51, 56, 57 Trephones, 28, 55 Trypsin, effect on ova, 79-81 Vesicle, germinal, 112 and ff. X-rays, and pachytene stage of meiosis, 39 effects on ovaries, 21 Zona pellucida, formation, 37 loss of, 1 16