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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 












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119 



TABLE XXIV 
The Effect of Various Types of Oestrone Injections during the 
Preimplantation Period upon the Implantation Ratio. (From 
Pincus and Kirsch, 1936) 





Days 


Rat 


Total 


Number 


Number 




Animal 


AFTER 


Units 


Number 


OF 


OF Im- 




Number 


Mating 


Injected 


OF Rat 


Corpora 


planta- 


Remarks 




Injected 


Daily 


Units 


Lutea 


tions 




16 


1 


200* 


200 


9 


9 


Implantations normal 


17 


1-2 


200* 


400 


9 


2 


" " 


18 


1-3 


200* 


600 


7 


1 


y) >> 


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