The Eggs of Mammals (1936) 2

From Embryology

Chapter II The Origin of the Definitive Ova

The Eggs of Mammals (1936): Introduction | The Origin of the Definitive Ova | The Growth of the Ovum | The Development and Atresia of Full-Grown Ova and the Problem of Ovarian Parthenogenesis | Methods Employed in the Experimental Manipulation of Mammalian Ova | The Tubal History of Unfertilized Eggs | Fertilization and Cleavage | The Activation of Unfertilized Eggs | The Growth and Implantation of the Blastodermic Vesicle | Summary and Recapitulation | Bibliography | Figures | Historic Disclaimer
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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 theoretical 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 phenomenon, it is no longer necessary to postulate the transmission 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 development of the ovary and its germ cells from early embryonic life through sexual maturity. The second is concerned 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 available a fairly detailed description of oogenesis in embryonic life. It is unnecessary here to enter into a detailed description of the embryogeny of the mammalian ovary (see Jenkinson, 1913, de Winiwarter, 1901, de Winiwarter et Sainmont, 1909, Brambell, 1927 and esp. 1930). Our interest lies in the so-called "primordial" germ cells of the embryo, 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 epithelium and the primordial germ cells complete their migration when they become arranged beneath this epithelium which then proliferates medullary tissue into the germ cells. The underlying mesenchyme forms connective tissue trabeculae 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 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) indicating 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 assumption 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 epithelium 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 proliferations during embryonic or prepubertal life. Thus Rubaschkin (1908, 1910, 1912) decided that the large differentially staining primordial germ cells with their prominent attraction spheres degenerate in the early guinea pig embryo and are replaced by two successive proliferations from the germinal epithelium. De Winiwarter and Sainmont (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 proliferation begun at three to four days before birth and lasting 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 human 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 germinal epithelium throughout life. The chief modern protagonists 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; Schron, 1863 — cat and rabbit; Koster, 1868 — man; Slawinsky, 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 definite 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 evidences 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 unIn some cases these signs of operated controls. Abscissae are activity were associated with stages of oestrus cycle; l, early prooestrus; 2, late prooestrus; 3, prooestrus to oestrus; 4, early oestrus; 5, oestrus; 6, early metoestrus; 7, metoestrus; 8, dioestrus. Ordinates are average number of 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).

the period of heat.

Allen (1923) whose investigations are perhaps pioneer to

the most recent developments mitoses per mouse. (From the distinguished four stages in the American Journal of Anatomy.)

behavior of the germinal epithelium of the adult mouse during the oestrus cycle. The first, characterized by extensive 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 epithelium

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 connective 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 observed. 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 (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. 1416) synizesis stages are rare or missing, the pachytene modified 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 modifications, and in the adult the new ova derived from the germinal epithelium contain mature nuclei (Plate II, Figs. 2324) 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 disappearance 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.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

Fig. 10

Fig. 11

Fig. 12

Fig. 13

Fig. 14

Fig. 15

Fig. 16

Plate I. (From the Journal of Morphology)

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, Diplonema.

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.

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, Deutobroch nucleus. 18, Beginning of the formation of clumps shown in next figure. 19, Modified pachynema. 20, Later stage showing characters of diplonema. 21, Nucleus 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.

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 production 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 throughout 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 mammalian 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) bilateral ovariectomy, (2) partial ovariectomy, (3) ovarian 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 ovariectomized mice with apparently functional ovarian tissue appearing 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 fragments remaining hypertrophy and attain dimensions sufficient 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 germplasm continuity, it does not necessarily inform us about the normal process of egg production.

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 Review of Biology.)

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 encapsulated rat and mouse ovaries, or even after careful excision to drop very small crushed fragments. A number of investigators 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 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 onehalf 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 tissue in eight of these eleven. Haterius (1928) also found apparent regeneration in 10 per cent of the mice he ovariectomized, and attributed the regeneration to incomplete extirpation. Pencharz (1929) reported return of oestrus in only three of 118 ovariectomized rats and mice, and demonstrated by serial sections of the ovarian region that incomplete removal had been made in the case of these three. Heys (1929 and 1931), in an extremely careful analysis of 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 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 embedded 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 consisting of eighty-five females under forty days of age and twenty-three older females. Three of the older animals regenerated 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 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 regeneration 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.

Fig. 5. Section through the ovary of mature rat showing the iobed condition. YF, young follicle; F, follicle; FA, fatty tissue. (From the Quarterly Review of Biology.)

It is clear, therefore, that regeneration of ovogenetic tissue from somatic tissue is improbable in mammals. And certainly the definitive ova are normally not recruited from somatic cells. We must turn to other experimental procedures 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 hypertrophy 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 established both clinically (Robertson, 1890; Gordon, 1896; Sutton, 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; Lipschiitz, 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 unilaterally ovariectomized rats found not a doubling in weight, 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 incomplete ovariectomy.

Arai (19205) found definitely that the compensatory hypertrophy 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 increase after semi-spaying. In this Arai was confirmed by Alien (1923) who found that in semi-spayed mice the number 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 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 retention 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 protocols describe a partially preserved or "flattened" (degenerating?) germinal epithelium in the experimental group whereas the germinal epithelium in the control fragments is apparently 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 activity 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 ovogenesis 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 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 transplanted 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 follicular tissue by cellular proliferations from the germinal epithelium in the irradiated ovaries of mice. These proliferations never give rise to ova, however, though the ovaries seem to retain their hormone-producing capacities as evidenced 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 capacities of ovarian tissue. It is notable therefore that the proliferation 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 injections 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 abundantly 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 hypophysectomy. Swezy (19336) has presented quantitative measures 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 summary 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 Pseudopregnancy, and after Hypophysectomy and Thyroidectomy. (From Swezy, 1933b)

Day of






Cycle (or Days



Age, Days

Number of Ova


Primary Follicles

Average Number




Average Number


Corpora Lutea


Oestrus cycle


2nd(l), 4th (4)






Pregnant and pseudopregnant


5 to 22








12 to 90







36 to 42






  • 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 hypophysectomized 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 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 pseudopregnancy maturity hormone is secreted only in subthreshold amount, as evidenced by cyclic ovarian changes 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 eldest of the lot and Arai (1920a) has demonstrated a small decline of ovogenesis with age in adult females (see Figure 6).



ER 60 "



— X

— xB


.....X.— J

  • ^








900 1.000

400 500 600


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.)

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 hypertrophy 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 pituitary 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 between 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 Single Ovaries of Rats Subjected to Various Hormone Treatments. (From Swezy, 19336)


(1) Rat hypophysis

(2) Beef Vsc.c. hypophysis

(3) Beef Vs c.c. hypophysis

plus rat hypophysis

(4) Pregnancy 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 hypophysis











Primordial Follicles

Large Follicles





























































60 and 394





2 1






1952 7225






24- 26

2- 8


present in some









subnormal (3) and hypophysectomized types

76 maturity type


(mixed type) 9.5


  • Varied with amount of hypophysis.

t Average of three.

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 observed (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 contained 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 materials. 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 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, although 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 succeeding 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 separation 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 luteinizing 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 stimulating 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 addition to the ovogenetic activity which is independent of the hypophysis {e.g.^ the ovogenesis seen in hypophysectomized animals) a stimulation to ovogenesis may be engendered by the follicle stimulating hormone. Hisaw and his collaborators find that corporin (the hormone of the corpus luteum) exerts effects on the ovary like those of the follicle stimulating hormone while oestrin decreases the secretion of follicle stimulating hormone and stimulates luteinizing hormone production from the hypophysis. Pregnant and pseudopregnant animals may therefore exhibit an increase in ovogenesis 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 estimates of ovogenesis in its relation to atresia, and particularly an inquiry into the nature of the atresia of young ova and follicles. We are completely unaware of the intimate nature of the intrinsic proliferative capacity of the germinal epithelium. 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 ovogenetic processes in prepubertal and post-pubertal animals little doubt remains that the definitive ova are proliferated 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 embryonic gonads. The experimental manipulation of mammalian 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 (Reagan, 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 Amblystoma 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 epithelium. '

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 disappear by the third week after hatching) a sterile testis develops.

Willier (1933a and h) has demonstrated by means of 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 developing 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 fertilization) 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 germinal 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 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 morphologically indifferent. Morphological differentiation may be considered as initiated when these primary cords become isolated 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 morphologically 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.

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The Eggs of Mammals (1936): Introduction | The Origin of the Definitive Ova | The Growth of the Ovum | The Development and Atresia of Full-Grown Ova and the Problem of Ovarian Parthenogenesis | Methods Employed in the Experimental Manipulation of Mammalian Ova | The Tubal History of Unfertilized Eggs | Fertilization and Cleavage | The Activation of Unfertilized Eggs | The Growth and Implantation of the Blastodermic Vesicle | Summary and Recapitulation | Bibliography | Figures | Historic Disclaimer

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