the development in vitro of the mammalian gonad. ovary and ovogenesis

The Development in vitro of the Mammalian Gonad. Ovary and OvogenesisAuthor(s): P. N. MartinovitchSource: Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 125, No.839 (Apr. 27, 1938), pp. 232-249Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/82192 .

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232 E. C. Dodds and W. Lawson

Mills, W. H. and Nixon, I. G. 1930 J. Chem. Soc. p. 2520. Norris, J. F., Thomas, R. and Brown, B. M. 1910 Ber. dtsch. chem. Ges. 43, 2958. Robson, J. M. and Schonberg, A. 1937 Nature, Lond., 140, 196. Russanow, A. 1889 Ber. dtsch. chem. Ges. 22, 1944. Schiff, H. 1874 Liebigs Ann. 172, 357. Schlenk, W. 1909 Liebigs Ann. 368, 303. Schmidlin, J. and Garcia-Banus, A. 191a Ber. dtsch. chem. Ges. 45, 3199. Schmidlin, J. and Massini, P. 1909 Ber. dtsch. chem. Ges. 42, 2381. Schneider, F. 1899 Ber. dtsch. chem. Ges. 32, 689. Schopf, M. 1894 Ber. dtsch. chem. Ges. 27, 2324. Serini, A. and Steinruck, K. 1937 Naturwissenschaften, 25, 682. Smith, A. 1893 Ber. dtsch. chem. Ges. 26, 68. Stoermer, R. and Kippe, B. 1903 Ber. dtsch. chem. Ges. 36, 4009. Thiele, J. and Schleussner, K. 1899 Liebigs Ann. 306, 198. Ullmann, F. and Mourawiew-Vinigradoff, A. 1905 Ber. dtsch. chem. Ges. 38, 2218. Ullmann, F. and Stein, A. 1906 Ber. dtsch. chem. Ges. 39, 624. Ullmann, F. and Wurstemberger, R. 1905 Ber. dtsch. chem. Ges. 38, 4105. Werner, G. 1895 Ber. dtsch. chem. Ges. 28, 1999. Wislicenus, W. and Endres, A. 1903 Ber. dtsch. chem. Ges. 36, 1194. Zincke, T. and Munch, S. 1904 Liebigs Ann. 335, 184. Zincke, T. and Muhlhausen, G. 1903 Ber. dtsch. chem. Ges. 36, 131.

611.013.i6:6ii.013.9

The development in vitro of the mammalian gonad.

Ovary and ovogenesis*

By P. N. Martinovitch

Strangeways Research Laboratory, Cambridge

{Communicated by F. H. A. Marshall, F.E.S.?Received 15 December 1937)

[Plates 9-11]

Introduction

Several investigators have tried to cultivate in vitro the mammalian and

avian gonad, usually with the object of obtaining the growth and differenti?

ation of the germ cells under in vitro conditions. The results so far

obtained, however, have been disappointing. Champy (1920) is the only * This work has been made possible by a fellowship granted by the Rockefeller

Foundation.



The development in vitro of the mammalian gonad 233

author, so far as we know, who has definitely claimed to observe some

differentiation of the germ cells in vitro. He states that in testicular ex-

plants from the mature rabbit he observed the transformation of spermato-

gonia into spermatocytes, but the process did not advance beyond the

leptotene phase of meiosis. Champy's findings, however, have not been

confirmed by other investigators, probably owing to the difficulty of

cultivating mature testicular tissue.

In birds, progressive differentiation of the germ cells in vitro has not

been recorded. Cultures of ovarian and testicular tissue of the embryonic fowl have been studied by Fano and Garofolini (1927 and 1928). The

explants formed an epithelial outgrowth which was soon replaced by what

appeared to be fibroblasts. Dantchakoff (1932) cultivated the undif-

ferentiated gonad of 3|-day fowl embryos and described three types of

cells in her cultures: (a) large amoeboid cells?Waldeyer's "Ureier" or "

Urkeimzellen"?which in vitro developed pseudopodia and showed

active amoeboid movement, (b) connective tissue strands, (c) epithelium? like tissue. Wermel (1933) made cultures of the undifferentiated gonad and of embryonic and adult testes and observed growth of both the somatic

and germinal tissue. Muratori (1935 and 1937) cultivated gonad tissue

from embryonic and newly hatched fowls, but although he describes the

nuclear pattern of the germ cells emigrating from the explants, he makes

no mention of any differentiation occurring in the explanted gonocytes. Several workers have grown mammalian gonad tissue in vitro.

Maccabruni (1913) studied cultures of the human foetal ovary and

observed proliferation from the explant of connective tissue only. Wolff

and Zondek (1925) obtained proliferation of both connective tissue and

epithelium from explants of human foetal ovaries and from the ovary of a

45-year old woman. Olivo (1934) obtained successful cultures of the

cumulus oophorus tissue containing the egg from a human embryo, but

failed to cultivate the granulosa tissue without the egg, from the same

ovary. He also noted that the egg enlarged somewhat during cultivation

and two large nuclei appeared in it.

Mjassojedoff (1925) found that the follicular cells of the rabbit ovary when growing in vitro, quickly lose their epithelial character and in

6-8 days become very like fibroblasts. Champy (1920, 1926, 1927) and

Champy and Morita (1928) explanted ovarian and testicular tissue from

mammals (rabbit, rat) and from some of the lower vertebrates, and

observed that the germinal tissue proliferated in vitro in an undifferentiated

form. Champy regarded this observation as evidence in support of his

dedifferentiation theory. As stated above, Champy also described one



234 P. N. Martinovitch

stage of spermatogenesis in vitro which took place in a 9-day culture of

adult rabbit testis after all the spermatocytes present at the time of

explantation had degenerated. Esaki (1928) studied the fate of interstitial

and Sertoli cells in explants of rabbit and guinea-pig testis. He stated that

the interstitial cells are derived from the mesenchyme and that in vitro

they become transformed into spindle-shaped elements indistinguishable from ordinary fibroblasts; the Sertoli cells became rounded and phago-

cytic in culture. Mihailoff (1937) recorded the survival in vitro, for about

70 days, of the seminiferous tubules of the immature rabbit. A few

primitive germ cells (gonocytes) persisted in a more or less normal state

during this period, but failed to differentiate. Both mesenchyme derivative

and Sertoli cells grew and migrated actively. The object of the present investigation was to cultivate the entire ovary

of embryonic and new-born rats and mice and to study its behaviour

in vitro with special reference to the growth and differentiation of the germ cells. It was clear that if the germinal tissue could be made to differentiate

in vitro, it would provide a new method for analysing the complex factors

responsible for the growth and development of the ovary (Martinovitch

1937)- Of recent years something has been learned of the influence of the

gonadotropic hormones on the normal functions of the ovary. Little is

known, however, of the possible influence of the organ as a whole on the

germinal tissue which it contains or of the inherent capacity of the germ cells for independent growth and differentiation. The results of the present

experiments shed some light on both these problems and also on the vexed

question of the length of survival of the mammalian ovum.

Material and technique

Forty ovaries from rats and mice ranging in age from the 16th day of

embryonic life to the 4th day after birth were cultivated in vitro by the

watch-glass method (Fell and Robison 1929). The culture medium was

composed of equal parts of chicken embryo extract prepared in Pannett

and Compton's solution and chicken plasma. In some experiments 1 %

glucose was added to the medium. One ovary from each animal was

explanted in vitro and the corresponding ovary was fixed as a control. In

order to compare the rate of growth of the ovum in vitro and in vivo, the

normal ovaries of 8-, 5- and 2-day-old rats were also fixed and sectioned.

The explants and some of the controls were fixed in Allen's modification

of Bouin's fluid and serially sectioned. The remaining controls were fixed




in unmodified Bouin's fluid. Sections were stained with Meyer's haemat-

oxylin, Heidenhain's haematoxylin or by Feulgen's method counterstained

with light green. Methods of preparation of the ovaries employed in this work were not

of the nature to reveal any of the various cytoplasmic constituents.

Observations

The results showed that the entire process of oogenesis can take place in vitro under appropriate conditions of cultivation. Oogenesis in vitro

does not differ materially from the normal process in vivo, and the series of

development of explanted ova are therefore described in chronological

order, beginning with a stage of development just preceding the appearance of the first meiotic figures. For the sake of convenience, the rat ovaries

only are considered, although the mouse ovaries behave in a similar way.

The development in vitro of ovaries from 15-16-day embryonic rats.

Normal (control) 15-16-day embryonic ovary (fig. 1, Plate 9)

This ovary was taken from one of the same batch of embryos as the

explanted ovaries B XI2 to B XI6 inclusive, and was fixed at the beginning of the culture period as a control.

The dense mass of tissue composing the ovary contains two main types of cell: (a) the oogonia, large cells with big spherical nuclei and (b) the

somatic ovarian cells which are smaller and contain round or oval nuclei.

The oogonia are most abundant towards the centre of the ovary and are

very scarce in the ovarian epithelium. In some regions of the ovary there is no visible sign of a developing

albuginea, the germinal epithelium being continuous with the underlying

tissue, but elsewhere, especially near the site of attachment to the body

wall, some of the mesenchyme cells immediately beneath the epithelium have begun to elongate. Similar elongated mesenchyme cells occur in the

interior of the ovary where they appear in section as single rows indicating the boundaries of the future germinal nests and cords.

The nuclei of the oogonia contain numerous very fine, dust-like particles of chromatin and several larger, usually peripheral, knots of intensely

staining chromatin. Although the particles seem to be connected by very fine protoplasmic bridges, no sign of that formation of chromatin threads

which heralds the onset of meiosis, could be detected. In general appear? ance the oogonia resemble the spermatogonia?"gonies poussiereuses

" of

Regaud?which occur in the testis of a new-born rat, except that the fine

Vol. CXXV. B. 16




particles of chromatin are slightly coarser and the chromatin knots less

distinct than in the spermatogonia. Many germ cells are undergoing mitotic

division, metaphase being much the most numerous stage. The cells of

the mesenchyme and of the germinal epithelium are also dividing actively.

A few germ cells are degenerating. The ovary contains many nucleated red blood cells which in the

explanted organs sometimes persisted for as long as 10 or 12 days.

Explanted ovary after 19 hr. in vitro (explant No. B XIX)

The most conspicuous change in the general structure of the ovary, as

compared with that of the control described above, is the increase in the

number of spindle-shaped cells which now form well-defined parallel single

cell rows running in a dorso-ventral direction with regard to the gonad's

original attachment to the body wall. The future nests and cords are thus

more clearly outlined. The mesenchyme cells beneath the germinal

epithelium, though not yet spindle-shaped, are more numerous, but a

typical stroma has not so far differentiated in this region. The cells of the germinal epithelium are irregular in shape and arrange?

ment, some being only loosely attached to the surface of the explant. The injured surface of the organ is covered by a single, discontinuous

layer of epithelial cells. Many white blood cells adhere to the ovarian wall,

but there is no migration of the ovarian tissue into the medium.

The germ cells, which are very abundant, differ from those of the control.

The powdery particles of chromatin have increased both in number and

staining intensity, and the large chromatin knots have either partially or

completely disappeared. In a few of the germ cells the chromatin granules have a linear arrangement which foreshadows the thread-like formations of

the leptotene phase. The number of degenerate germ cells is about the same as in the control.

No central necrotic area ever forms in cultures of 15-16-day ovaries.

Explanted ovary after 42 hr. in vitro (explant No. B XI2)

The first stages of the meiotic prophase?leptotene and synaptene?-

appear at this period of cultivation. The leptotene phase is represented

by numerous fine threads either scattered evenly through the nucleus

(fig. 2, Plate 9) or slightly polarized (fig. 3, Plate 9). Cells at a stage inter?

mediate between the preceding "dusty" stage and the leptotene are still

fairly common.

Synaptene figures are already numerous. The chromatin threads have

begun to contract towards one end of the nucleus. A double structure was




not observed before the contraction stage, although some of the threads

were distinctly looped forming the familiar bouquet figure. In later stages of contraction, the closely packed threads appear as an almost uniform

mass of chromatin to one side of the otherwise clear nucleus, with merely a few loops protruding at the margin to indicate the filamentous nature of

the mass.

The number of dividing oogonia, mostly in metaphase, is still very great

(fig. 4, Plate 9). The degenerating germ cells appear as an intensely stained, ellipsoid

mass of nuclear material capped with a deeply staining crescent of cyto?

plasm. The connective tissue of the stroma has not differentiated much further,

although in the region of the hilum a narrow band of elongated cells clearly demarcates the ovarian tissue proper from a tiny strip of mesovarian tissue

included in the explant. The germinal epithelium consists of a single layer of flattened cells, separated from the underlying germ cells by a sheet of

rather flattened connective tissue cells which represent the developing

albuginea. Ova do not occur either in the germinal epithelium or in

the albuginea. Cells have now begun to migrate from the surface of

the ovary.


Structures resembling Pfliiger's cords make their first appearance at this

stage of cultivation (fig. 5, Plate 9). The mesenchyme separating groups of germ cells has become compressed and flattened to form well-marked

partitions, thus producing a cord-like structure in the ovarian tissue. The

cords are packed with oocytes and Sertoli cells, the former being the more

numerous. Only a few cells of the oogonial type are present, the proportion of oocytes having greatly increased. Leptotene and synaptene figures are about equally represented. The healthy and normal appearance of the

explanted ovary at this stage is striking. Fewer germ cells are dividing than before, and the mitotic wave has

receded towards the periphery of the organ. It is noteworthy that some

of the metaphase figures are abnormal; the chromosomes are clumped

together in certain germ cells, and in others spherical pycnotic masses of

chromatin of different sizes are scattered throughout the cell body. It is

most unlikely that such cells complete division. On the other hand,

mitotic figures in the somatic cells, both in the germinal epithelium and in

the cords, appear normal (fig. 6, Plate 9) (see Rauh 1926). 16-2




The single-layered germinal epithelium contains an unusually large number of dividing cells. In this explant there is no true albuginea, and the

nests of germ cells lie close to the surface.

A few cells of the germinal epithelium and underlying mesenchyme have emigrated from the organ. In all cultures of 15-16-day embryonic ovaries the zone of outgrowth was found to be small, although its area

could be increased by adding glucose to the medium and was of the

familiar fibroblastic type. In explants from embryos of this age the

emigration of germ cells was never, and the formation of epithelial sheets

only occasionally, observed in the zone of growths, though it should be

emphasized that no special study was made of the nature of the outgrowth in these cultures.


At this stage the vast majority of the oocytes are at the advanced

contraction phase of meiosis (synizesis) (fig. 7, Plate 10). In preparations stained by Feulgen's method, the chromatin appears as a deeply stained, almost impenetrable mass, but in those stained with Meyer's haemat-

oxylin the individual threads are sometimes clearly visible. The leptotene nuclei are distributed along a narrow zone parallel to the surface of the

organ, and intermingled with them are the dividing oogonia, now greatly reduced in number.

The strands and partitions of connective tissue between the nests of

germ cells have increased in both number and size, and the germinal nests

have become correspondingly smaller and more numerous. There is no

appreciable change in the germinal epithelium or underlying stroma.


At this stage many of the oocytes have emerged from the contraction

phase and the chromatin threads have reappeared. The oocytes appear on

the whole less healthy than in the preceding culture, as indicated by the

variations in the appearance, number and distribution of the chromatin

threads and in the size of the nuclei. In some cells the threads are long and

fine and in others short and stubby; sometimes they appear as a continuous,

highly convoluted spireme, filling the whole nucleus, but in others they are

definitely polarized, the spireme-like arrangement occurs in large nuclei

only, but otherwise there seems to be no correlation between the appear? ance of the chromatin threads and the size or shape of the nucleus con?

taining them.




It is often difficult to tell which of the various forms of chromatic thread

represent successive stages in a continuous process, and which are mere

abnormalities. It is also impossible to determine which of the abnormalities

represent an irreparable injury to the cell, and which are merely reactions

to new conditions of life and not necessarily lethal.

Many of the germ cells are obviously necrotic and oocytes are de?

generating at every stage of development; very few are in mitosis and

these appear abnormal. The larger nests of germ cells are breaking up into smaller groups.

The germinal epithelium is no longer a continuous layer, owing to the

continual migration of cells into the surrounding medium and to the failure

of the remaining cells to divide fast enough to fill the gaps thus produced. The underlying mesenchyme cells, many of which are in mitosis, have

become definitely flattened.

A conical mass of connective tissue has begun to differentiate near the

original hilum of the ovary, the base of this cone coincides with the former

area of attachment and the apex is thrust into the centre of the ovary.

Explanted ovary after 6-6*16 days in vitro (explants No. B XI6 and

B XIIM)

Nearly all the germ cells have now evolved from the contraction phase. The chromatin filaments, once more distinct, now form the thick, short,

polarized loops characteristic of the post-contraction "bouquet" phase of

the normal embryonic ovary (fig. 8, Plate 10). The oocytes, though greatly reduced in number, look healthier than in the ovary of the preceding stage. Some diplotene nuclei have already appeared.

The germinal epithelium has almost disappeared and is represented by a

few scattered epithelial cells clinging to the surface of the ovary. After the virtual disappearance of the germinal epithelium, the surface

of the ovary is covered by the albuginea, which is composed of three or

four layers of flattened fibrocytes. There is a striking difference in

appearance between the typical connective tissue cells of the albuginea and the mesenchyme-like cells of the deeper layers of the ovary.

Up to this time the size of the ovary has remained unaltered.

Explanted ovary after 6*21-8 days in vitro (explants Nos. B XII3 4 5 6)

In vitro the pachytene phase of meiosis is brief. As stated above, a few

diplotene nuclei had already appeared at the preceding stage, although most of the oocytes were in the pachytene phase.




Diplotene nuclei become much more numerous by about the 7th day of

incubation (fig. 9, Plate 11). Many nuclei contain discontinuous, double

threads which have been interpreted as representing the homologous chromosomes separating after their intimate contact during the zygotene

phase. Such nuclei were most numerous in a 7-2-day culture, but in sister

cultures obtained from the same litter and fixed a few hours before and

after 7-2 days they were comparatively rare. This indicates that the

separation of the homologous chromosomes occurs fairly quickly. It is

interesting that in many of the oocytes which have passed through the

pachytene phase, the chromatin loops much resemble a fine spireme. These

loops are not so fine as the "smooth" leptotene threads and have a beaded

appearance. In Feulgen preparations the nucleoli of the germ cells are

faintly stained with light green. The explants on the whole seemed quite healthy, although many oocytes

were degenerating. The general structure was the same as in the preceding

stage.

Although no accurate timing of the duration in vitro of synaptene stage has been attempted in these experiments, it has been found that on the

whole it agrees fairly well with that recorded by Pratt and Long (1917) in

normal ovaries.

Later development of the explanted ovary up to the 30th day in vitro

The stages in the development of the explanted ovary subsequent to the

diplotene phase may be described together. In normal development, according to the usual view, the diplotene

phase is followed by a resting stage in which the individuality of the

chromatin threads seems to disappear completely. The nucleus then con?

tains an achromatic network in which are scattered many small chromatin

granules and from one to three larger chromatin nucleoli. Most of the

oocytes remain unchanged in this condition for weeks, months or, according to some authors, even for years, but a few enlarge and eventually become

full-grown ova.

Somewhat similar changes occur in vitro. After the diplotene phase the

nuclear material becomes less chromophil and breaks down into one to

three chromatin nucleoli and a number of smaller particles. Inter?

connecting achromatic strands are often extremely hard to see. The

chromatin granules are coarser and less deeply staining than those of the

normal oocyte. Sometimes the nuclear material consists of a nucleolus and

a loosely connected, amorphous mass of particles with no apparent

organization. The resting ova change little during subsequent cultivation.




As in the normal ovary, some of the resting ova in the explants begin to enlarge. Enlargement of the cytoplasm is accompanied by growth of

the nucleus and increase of the chromatin. In the full-grown ova, the

chromatin particles are large and coarse, and one or two large, spherical

plasmasomes are present. The plasmasomes differ from the normal in

being "less chromophil" and more clearly visible, a difference which is due

to the fact that in the explants the surface of the plasmasomes is often

free from the adherent particles of chromatin by which they are observed

in the normal ovum. In nearly full-grown ova the nucleus assumes an

excentric position. The ova are usually spherical and, unlike the normal

ovum, appear completely devoid of a zona pellucida.

Abnormally shaped ova sometimes occur. Fig. 10 (Plate 11) shows one

that is ring-shaped in section, the interior being occupied by a large, hollow

space. A "peculiarity " of all ova cultivated in vitro is that even in the best

fixed preparations they appear shrunken, so that a clear, ring-like space, sometimes bridged by fine radially distributed strands of protoplasm, intervenes between the ovum and the surrounding tissue. Whether this

appearance is an artifact or whether it also occurs in the living explant has not been ascertained.

Some of the ova degenerate. Large ova do so by a process of karyor- rhexis. The chromatin material breaks down into deeply stained fragments of various sizes, which are at first irregular in outline but gradually become

spherical. No normal maturation division has been observed in the older cultures,

but one abnormal figure was found in a 25-day culture. This dividing ovum

has passed in vitro through all the stages of differentiation and growth from an oogonium to a maturing ovum.

From the foregoing description it is seen that the development of the

explanted ovary during synapsis differs little from that of the normal organ. After this stage, however, it shows a divergence from the normal which may be considered as peculiar to conditions in vitro. Two chief abnormalities

occur in culture: (1) partial or total failure of the Sertoli cells to develop into Graafian follicles; (2) failure of the growing ovum to move towards the

interior of the ovary. As a result of these abnormalities, nearly full-grown

ova, either naked or surrounded by a few follicular cells, are clustered

together near the surface of the ovary in what are really the original

germinal nests and cords (fig. 11, Plate 10). As in vivo only a small number of oocytes at a time begin to grow, the

vast majority remaining unchanged. In vitro, the first batch of growing ova attain their maximum size 18 days after explantation, a period




corresponding to 13 days of extra-uterine life. Measurements show that the

largest size reached in culture is a diameter of 50-55/*. This first batch of

ova soon degenerates and is replaced by others. After 18 days' cultiva?

tion, ova of almost all sizes are to be found in the same ovary. Growth of

the ovum in vitro is somewhat retarded as compared with the normal.

The explanted ovary does not enlarge very much, and after a month's

cultivation is only about double its original volume. It enlarges in three

ways: (1) by the growth of the ova, (2) by the formation of a primary follicle around certain oocytes, (3) by multiplication of the stroma cells.

Many connective tissue cells are lost by migration into the medium and by

degeneration. The ovarian stroma undergoes very slight differentiation in vitro and the

cells retain their primitive, mesenchymal appearance even after several

weeks' cultivation. In the centre of the explant some typical stroma

appears consisting of fairly dense fibrous tissue and a few scattered cells

but elsewhere the intercellular fibres are very scanty. Medullary cords

never develop in the explanted mouse ovary. The germinal epithelium, as stated above, usually disappears after a

week's cultivation, partly owing to migration of cells into the medium

and partly owing to loss through mechanical damage inflicted during sub-

cultivation. Sometimes, however, a few clustered cells of epithelial appear? ance cling to the surface of the explant for several weeks.

As already mentioned, no central area of necrosis appears in explanted ovaries from 16-day embryos, but degeneration changes affecting the entire

organ begin to occur after 26-28 days in vitro. The translucency of the

healthy explant gradually disappears and the organ becomes opaque. After 30 days' cultivation the whole organ is necrotic.

The development in vitro of ovaries from late embryonic and new-born rats

It seemed interesting to compare the development in vitro of the ovaries

from 16-day embryos with that of ovaries from older embryos and newly born rats. Accordingly ovaries from 17-, 18-, 19- and 20-day embryos, new

born and 4-day rats were cultivated in vitro.

It was found that on the whole the more highly developed the ovary at the time of explantation, the further it will differentiate during subsequent cultivation. This is particularly true of the ovarian follicle but only up to a certain stage in its development. Thus an explanted ovary from a 19-20-

day embryonic rat after 3 weeks' cultivation may contain numerous, small Graafian follicles which do not differ essentially from follicles of the same




size in the normal ovary (cf. figs. 12 and 13, Plate 11). Moreover, these

follicles have moved towards the centre of the explant, as in vivo, instead

of retaining their original superficial position as in explants of the 16-day

embryonic ovary (cf. figs. 11 and 12). The amount and arrangement of the

stroma also approaches more closely that of the mature gland than does

that of explants of younger embryonic ovaries.

Another difference between the older and younger ovaries in culture is

the appearance in the former of a central necrotic area. This degenerate

region is seen shortly after explantation but disappears a few days later, the

dead matter being either resorbed or cast out. The formation of the

central necrotic area may be due to poor aeration or to a slow exchange of

metabolites or to both these factors. Its disappearance seems to show

that after an interval the ovary adjusts itself to the conditions in vitro.

A necrotic area may disappear completely, to reappear a second time and

again vanish. Many of the residual ova survive in vitro until the entire

ovary degenerates (fig. 14, Plate 11). In the explanted ovary of a 4-day mouse, a typical maturation-division

figure was observed after 9 days' cultivation (fig. 15, Plate 11). The

appearance of the figure was preceded by an actual growth of the ovum, since measurements showed that in the control ovary the diameter of the

largest ovum was 45/*, while the diameter of the dividing ovum in the

culture was 52/*. The growth and differentiation of the ova is never seriously disturbed

by cutting off the blood supply, and nearly all the young ova, at whatever

stage in the early meiotic prophase they are explanted, continue their

normal development in vitro. There seems to be no phase of meiosis which

is particularly susceptible to injury by the process of explantation; but it is

doubtful whether the different phases are of equal resistance to prolonged cultivation.

Discussion

Phenomena of meiosis and growth of the ovum

The results recorded in this communication show that the primitive

germ cells can undergo in vitro all the normal changes of differentiation and

growth up to the stage of the nearly full-grown ovum. The question arises

as to how far the growth and development of the ovum is controlled by factors intrinsic in the cell and how far it is influenced by external agents.

Two kinds of external agent must be considered: (a) those supplied by the body as a whole, and (b) those supplied by the ovary. The distinction




between the two classes is arbitrary, however, as interaction between all

the factors concerned, both extrinsic and intrinsic, may play an important

part in the development of the germ cells.

Explantation in vitro, while depriving the ovary of its natural environ?

ment, by no means removes it completely from the influence of the body. The two constituents of the culture medium, plasma and embryo extract,

might contain agents produced by the soma, which acted upon the germ cells.

The somatic agents known to influence the differentiation of the gonad are: (1) gonadotropic hormones, and (2) sex hormones.

With regard to the possible influence of gonadotropic hormones in the

medium on the development of the explanted ovary, Pincus (1936) quotes extensive experimental evidence showing fairly conclusively that the

Graafian follicles can only be activated by these hormones after attaining a certain stage of maturity and that growth of the ovum and of the early follicle are independent of the hypophysis. Even if the gonadotropic hormones do stimulate oogenesis under normal conditions, however, it is

unlikely that the plasma and embryo extract contain enough of these

substances to influence the explanted ova (see Robson 1934). On these

grounds, therefore, it seems fairly safe to exclude the gonadotropic hormones as possible somatic agents stimulating oogenesis in the

explants. Whether the sex hormones in the culture medium influence oogenesis

in vitro and if so, in what way, is difficult to determine. The recent

experiments of Dantchakoff (1936) and Willier, Gallagher and Koch (1937) on the embryonic fowl, show that small doses of oestrin cause the histo-

logically undifferentiated gonad of a zygotically determined male embryo to develop into an ovary and the genetically determined male germ cells,

that it contains, to be transformed into female germ cells; whereas the male

sex hormone causes the undifferentiated female gonad to become an ovo-

testis. But these authors make no clear statement whether the sex

hormones may influence the process of gametogenesis under the conditions

of their experiments. It should be emphasized, also, that these results refer

to the avian gonad only, and whether they also apply to mammalian

development is at present unknown.

The part that may, possibly, be played by the ovarian soma in inducing

ovogenesis has received very little attention, and nothing is known of the

possible interrelationship between the germ and other ovarian cells.

That the germinal epithelium is not essential to the growth of the ovum

after synapsis, is proved by the fact that the epithelium usually disappears




after a week's cultivation while the ova may continue to grow for another

20 days. The tissue culture experiments afford no evidence as to whether

the stroma plays any specific part in the development of the ova, as this

tissue persists, though in an undifferentiated form, throughout the culture

period. They also give no information about the epithelial cells of the

cortex in relation to oogenesis.

Very little is known of the ovum-follicle relationship in the early

stages of the normal development of the ovum. In the explants nearly

full-grown ova, 50-55/* in diameter, developed completely devoid of

follicle cells. This showed (a) that the early growth of the ovum is not

dependent on the follicular epithelium, and (b) that in culture, growth of the

follicle is not conditioned by growth of the ovum. Whether the ovum can

complete its growth in the absence of a follicle is doubtful. The only full-

sized ovum found in the explants, a binucleate egg 68/* in diameter, occurred in an ovary from a 19-20 day embryo rat and was covered by a follicular envelope several cell layers thick. Why some growing ova

should develop a follicle and others fail to do so is quite obscure. It was

noted that ovaries explanted at birth formed more and larger follicles

than those removed from embryos, suggesting that the capacity for

follicular development increases with age. With regard to the influence of the soma on oogenesis, the explantation

experiments seem to indicate that in normal development the organism as a whole merely nourishes the germ cells and has no specific effect on

their differentiation. What influence, if any, the ovary itself exerts upon the germ cells remains obscure, because although the explanted ovary

develops a rather simplified structure in vitro, it is still too complex to

allow of any definite conclusions being drawn.

Survival in vitro of the ovum and the problem of neo-formation

of the germ cells

Whether the primitive germ cells persist, stored in the ovary, throughout

post-embryonic life or whether they are continuously (Kingery 1917; Arai 1920; Butcher 1927, 1932; Hargitt 1930 a) or periodically (Allen 1923) renewed, are controversial questions.

The average life of the ovary in vitro is about one month, and in some

explants many, and in others, a few, residual ova persist until the end of

this period. The residual ova succumb only when the entire organ

degenerates. In the explants, new ova are formed only by the mitotic

division of pre-existing oogonia and not by neo-formation from the germinal

epithelium. In the explanted ovaries of late embryos, but never in those




of 15-16 days' gestation, a few ova occur in the germinal epithelium but

they are at the same stage of development as those in the deeper parts of the ovary, and there is no evidence that such cells differentiate from

the germinal epithelium. The germinal epithelium is usually completely lost after a week's cultivation and is thus finally eliminated as a possible source of new germ cells during the last three weeks of the culture period. All the germ cells in older cultures are embryonic in origin, i.e. they are cells which have passed through the early phases of meiosis. But

even though tissue culture experiments show that neo-formation of germ cells from the germinal epithelium does not take place in vitro, they do

not disprove the possibility that such proliferation might occur in normal

post-embryonic life.

With the exception of Firket (1920), who claims that the primary germ cells of the albino rat degenerate 15 days after birth, those who support the

view that the embryonic ova degenerate early agree that they normally survive for a period which exceeds the maximum culture period in the

present experiments. In order to test the storage theory more completely it would be highly desirable, therefore, if a method was found of keeping the resting ova in vitro for longer periods of time than it has been possible heretofore.

On the other hand, these experiments destroy one of the arguments used by

Hargitt and others against the storage theory. Hargitt (1930a) argues that

"if an assumption be made that many latent primary follicles are stored in

the mammalian ovary, a serious difficulty is encountered in explaining the

stimulation of a few to renewed development, while others remain quiescent.

Likely enough, some hormone might stimulate renewed activity, but why of a few not all of the same age?" In the tissue culture experiments the

ovaries were explanted before the ova had begun to enlarge and, from the

evidence given above, the explants throughout the culture period, were

known to contain embryonic ova only. Nevertheless, as in the normal

post-embryonic organ, only a few ova enlarged at any one time and many residual ova persisted unchanged until the end of cultivation.

That only a few out of a large number of apparently similar oocytes should enlarge at one time, is indeed a surprising fact for which no adequate

explanation is at present forthcoming. The factors which cause the growth of any given ovum must be sought in the ovary itself. The position of the

ovum is not concerned, since in the explants nearly full-grown ova often

occur at the periphery of the organ (fig. 11, Plate 10) while on the other

hand resting ova have been observed in the centre of a mouse ovary which

had recovered from permanent ligation (unpublished data). Nor does the




size of the ovary seem to be important in this connexion, since the volume

of the explants is only about one-tenth that of the normal ovary of

corresponding age.

I wish to express my thanks to Dr H. B. Fell, Director of the Strange-

ways Laboratory, for her help and advice throughout the investigation. I am also indebted to Mr V. C. Norfield, chief assistant at the laboratory, who was responsible for the photomicrographs.

Summary of results

1. The ovaries of embryonic and new-born rats and mice when explanted in vitro continue to live and develop for about 4 weeks.

2. Growth and differentiation of the germ cells occur in 100 % of cultures.

3. In explants of 16-day embryonic ovaries the oogonia undergo all but

the final stages of normal differentiation and growth, and form nearly full-sized ova 50-55/* in diameter. The ova in explants obtained from 2-5-

day rats reach full-size and may undergo apparently normal maturation

division.

4. The oocytes in the cultures only enlarge if they pass through the

synaptic phases of meiosis.

5. Growth of the follicle in the explants is partly, and sometimes com?

pletely, suppressed. 6. The egg may enlarge in the absence of a follicular envelope. 7. The formation of a zona pellucida was never observed in ova growing

in vitro.

8. Growth of the ovum is somewhat retarded in vitro.

9. Oocytes in culture multiply by the mitotic division of already differen?

tiated oogonia and not by neo-formation from the germinal epithelium. 10. The germinal epithelium usually persists for about a week and then

disappears owing to the emigration of its component cells into the sur?

rounding culture medium.

11. Emigration of germ cells into the medium was not observed.

12. After 4 weeks' cultivation the embryonic rat ovary has about

doubled its volume.

References

Allen, E. 1923 Amer. J. Anat. 31, 439-70. Arai, H. 1920 Amer. J. Anat. 27, 405-62. Butcher, E. O. 1927 Anat. Bee. 37, 13-25.

? 1932 Anat. Bee. 54, 87-98.




Champy, Ch. 1920 Arch. Zool. exp. gen. 60, 461-500. ? 1926 C.B. Soc. Biol., Paris, 94, 1082. ? 1927 C.B. Soc. Biol., Paris, 96, 597.

Champy, Ch. and Morita, J. 1928 Arch. exp. Zellforsch. 5, 308-40. Dantchakoff, W. 1932 Z. Zellforsch. mikr. Anat. 15, 581-644.

? 1936 Bull. Biol. 70, 241-307. Esaki, S. 1928 Z. mikr. anat. Forsch. 15, 368-404. Fano, G. and Garofolini, L. 1927 J. Physiol. 63, vi-vii.

? ? 1928 Arch. sci. biol. 12. Fell, H. B. and Robison, R. 1929 Biochem. J. 23, 767-84. Firket, J. 1920 Anat. Bee. 18, 309-21.

Hargitt, Geo. T. 1930 J. Morph. Physiol. 49, 277-321. ? 1930 a J. Morph. Physiol. 49, 333-47.

Kingery, H. M. 1917 J. Morph. Physiol. 30, 261-305. Maccabruni, F. 1913 Ann. ostet. ginec. 1, 57-65. Martinovitch, P. N". 1937 Nature, Lond., 139, 413. Mihailoff, W. 1937 Z. Zellforsch. 26, 174-201.

Mjassojedoff, S. W. 1925 Arch. mikr. Anat. 104, 1-24. Muratori, G. 1935 Arch. ital. anat. embriol. 35, 397-412.

?? 1937 Contr. Embryol. 154, Carnegie Inst. Wash., Publ. 479, 59-69. Olivo, M. O. 1934 Arch. ital. anat. embriol. 33, 718-25. Pincus, G. 1936 "The Eggs of Mammals." New York: The Macmillan Co. Pratt, B. H. and Long, J. A. 1917 J. Morph. Physiol. 29, 441-56. Rauh, W. 1926 Z. Anat. Entwicklungsm. 78, 637-68. Robson, J. M. 1934 "Recent advances in Sex and Reproductive Physiology.'1

London: J. and A. Churchill, Ltd. Wermel, E. M. 1931 Z. Zellforsch. 13, 545-65.

? 1933 Arch. exp. Zellforsch. 14, 554-73. Willier, B. H., Gallagher, T. F. and Koch, F. C. 1937 Physiol. Zool. 10, 101-22. Wolff, E. K. and Zondek, B. K. 1925 Virchows Arch. 254, 1-16.

Description of plates

All figures are photographs of stained preparations

Plate 9

Fig. 1. Normal ovary of a 16-day rat embryo. Control to figs. 2, 3, 4, 5, 6, 7, 8, 9, 10. All the germ cells are of the oogonial type. Bouin-Allen and Feulgen. x 1200.

Fig. 2. Section through a 16-day embryonic rat ovary cultivated in vitro for 42 hr., showing the leptotene stage of meiosis. Note the fine strands of the chromatin. Bouin-Allen and Feulgen. x 1000.

Fig. 3. Section through the same ovary as that shown in fig. 2. The leptotene and

synaptene stages of the meiotic prophase are seen, x 1000.

Fig. 4. Dividing oogonia in a 16-day embryonic rat ovary fixed after 42 hr. in vitro. Bouin-Allen and Feulgen. x 1000.

Fig. 5. Section through a 16-day embryonic rat ovary after 50 hr. cultivation showing nests and cords of cells which have developed in vitro. Note the healthy appearance of the ovary. Bouin-Allen and Feulgen. x 140.



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Fig. 6. The abnormal division of two adjacent germ cells (a) is seen (lower clumped metaphase figure in a different plane of visibility), and the normal division of a somatic cell (b) in the germinal epithelium. From a 16-day embryonic rat ovary fixed after 50 hr. in vitro. Bouin-Allen and Feulgen. x 1000.

Plate 10

Fig. 7. Section through a 16-day embryonic rat ovary after 71 hr. cultivation

showing the oocytes in the deep contraction stage (synizesis) of the meiotic

prophase. Bouin-Allen and Feulgen. x 1000.

Fig. 8. Section through a 15-16-day embryonic rat ovary after 6-16 days in vitro. Numerous oocytes in the pachytene phase of the meiosis. Bouin-Allen and

Feulgen. x 1200.

Fig. 11. Section through a 16-day mouse ovary after 19 days' cultivation. Nearly full grown ova are closely packed in the region of the original nests and cords. An incomplete single layer of cells surrounds some of the ova; the other ova are naked. Bouin-Allen and Meyer's haematoxylin. x 400.

Plate 11

Fig. 9. Section through a 15-16-day embryonic rat ovary after 7-2 days in vitro. Oocytes in the diplotene phase of the meiosis are shown. Bouin-Allen and Feulgen. x 1200.

Fig. 10. A large ring-like ovum in a 16-day embryonic rat ovary grown in vitro for 24 days. Bouin-Allen and Feulgen. x 1000.

Fig. 12. Section through the ovary of a 19-20-day embryonic rat after 21 days in vitro. Typical Graafian follicles have developed during cultivation and the nuclei of the oocytes have passed into the resting stage. Bouin-Allen and Meyer's haematoxylin. x 180. See fig. 13.

Fig. 13. Normal 19-20-day embryonic rat ovary. Control to fig. 12. x 290.

Fig. 14. Section through the same explant as that shown in fig. 12. Numerous residual ova are seen, x 100.

Fig. 15. Section through the ovary of a 4-day (post-embryonic) rat ovary after 9 days' cultivation in vitro. Bouin-Allen and Meyer's haematoxylin. x 640.



the development in vitro of the mammalian gonad. ovary and ovogenesis

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