Paper - Oogenesis in the white mouse (1917)
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Oogenesis in the White Mouse
H. M. Kingery
From the Dcpaitiitenl of Histology and Emhri/ologi/, Cornell University, Ithaca,
Material and methods 264
Morphogenesis of the ovary 265
Proliferations of cells from the germinal epitheliiun 265
The formation of 'primitive germ cells' 267
The formation of 'definitive germ cells' 268
The tunica albuginea, rete ovarii, and periovarian capsul or bursaovarica 269
Oogenesis. The development and differentiation of the germ cells 271
First proliferation : 'primitive oocytes' 272
Second proliferation: 'definitive oocytes' 276
Discussion. Literature and observations 281
The genuineness of a 'new formation' of egg-cells 282
The origin of the definitive ova '. 283
The 'new formation' of egg-cells 287
The question of the mitochondria 291
The prolonged potentiality of the germinal epithelium for germ cell
The question of synizesis and synapsis 294
The degeneration of the cells of the first and second proliferations. .... 300
Summary and conclusions * 301
In the last two decades or so there has been produced a vast amount of cytological work which has had as its object the description and explanation of the structure, behavior, and nature of the chromosomes. Particular attention has been paid to the maturation divisions in the two processes oogenesis and spermatogenesis and to the stages leading up to these. Two facts seem to stand out clearly enough: — that by these two rapidly succeeding divisions the amount of the chromatin and the number of the chromosomes in the mature germ cells is reduced to half that found in the somatic cells. There have arisen in the interpretation and explanation of these facts differences of opinion, some of which will be taken up later in a discussion of synizesis and synapsis.
Of the two processes, spermatogenesis has received more attention; it is completed in less time and in many forms a single adult testis will show all the stages of development from spermatogonium to spermatozoon. Oogenesis, on the other hand, is extended over a long period; in mammals at least and in many other forms as well, the process may start in the young embryo and continue into adult life. The formation of the deutoplasm — vitellogenesis — with the consequent enormous growth in size of the egg-cell, further comphcates this process. Material for the study of the development of the male germ cells is easier to obtain, then, and is more convenient to work with. Nevertheless, oogenesis has not been neglected.
In 1900 V. Winiwarter published his paper on oogenesis in the rabbit and man, and in 1908 there appeared his monographic work in collaboration with Sainmont on the organogenesis of the ovary and oogenesis in the cat. These two studies have bad an important influence on the work since done along similar lines. An instance which might be mentioned is that the terminology, first proposed by von Winiwarter and later slightly modified by both authors, has been adopted by a large number of those who have made a study of oogenesis and particularly, perhaps, of spermatogenesis.
Von Winiwarter and Sainmont state that they found the cat a more favorable form to work with than the rabbit because the period of gestation is longer (sixty days instead of twenty-eight) ; the development of the animal is slower and consequently that of the ovary is more gradual, which made it easier to obtain the various stages. On the other hand, it seemed to me that it might be desirable to make a study of a form which bad a short sexual cycle, which could be readily obtained in large numbers, and which could be easily bred. The white or albino mouse was selected for this work as it seemed to offer a number of advantages. The sexual cycle is completed in about sixtydays (Kirkham, '16) of which the period of gestation occupies twenty or so. For this reason it is possible to secure a complete series of ovaries from birth to sexual maturity, as well as during embryonic life, without involving an excessive amount of material. The small size of the ovaries of even adult mice reduces the manipulation, a consideration not to be despised when making a large number of preparations.
Von Winiwarter and Sainmont state — reserving a full description for a later chapter which, as yet, has not appeared — that the definitive ova are formed after birth in the young kitten about three and a half or four months old, shortly before sexual maturity. This new formation of egg-cells takes place by a differentiation of cells from the germinal epithelium of the ovarj\ If this post-natal formation is of more than an exceedingly limited and special significance, it will be found to occur in other forms. Accordingly, in a form like the mouse which has a short sexual cycle, making it fairly easy to secure a complete series of ovaries between birth and sexual maturity, it should be possible to confirm or disprove such a new formation of egg-cells.
In this study, the emphasis is laid on oogenesis, which is used to mean the development of the definitive germ cells or ova from undifferentiated cells into primary oocytes in mature or nearly mature Graafian follicles. Further development, that is, maturation, will not be discussed, being beyond the limits planned for this study. My results agree with those of von Winiwarter and Sainmont in that, in the mouse, this process of oogenesis or differentiation of the definitive ova takes place after birth in the period before sexual maturity. Furthermore, in the mouse, as they found in the cat, the germ cells formed before birth during embryonic and foetal life all degenerate and have nothing to do with the development of the definitive ova. The development of these cells, however, and the morphogenesis of the ovary will be briefly described in order that the process of oogenesis proper may receive the setting necessary for its appreciation and understanding.
=Material and Method
The material on which this study is based consists of a rather complete series, serially sectioned, of ovaries of white mice ranging from embryos of 10 mm. length to adults — ^in all nearly 300 ovaries. From birth to two days afterwards, the. ovaries were fixed at intervals of two to three hours; from two to forty days, at intervals of one day; and from forty to sixty days, at intervals of five days. A number of ovaries from adult mice, pregnant and not pregnant, were also fixed. The series of embryonic and foetal ovaries ranges from those of embryos 10 mm. in length, at intervals of one millimeter, to foetuses 25 mm. long, or practically full term. These embryos, according to Kirkham's table ('16 a) range from about fourteen to twenty or twenty one days post coitum.
For fixation, Hermann's, Flemming's, Carnoy's (6-3-1, with and without the addition of mercuric chloride), picro-acetoformol (Bouin's), and sublimate-acetic were used, each ovary of a mouse being treated in a different manner. It was found that Hermann's and Flemming's fluids preserved the nuclear structure excellently in the interior of the younger ovaries, but that the outer two or three layers of cells were over-fixed, presenting the glassy, homogeneous appearance with lack of detail characteristic of cells over-fixed with osmic acid. Carnoy's fluids gave excellent preservation of the nuclei, but in a number of cases the sections, when stained with iron hematoxylin, showed a solid black band or border, one to three cells deep around the ovaries, completely obscuring detail. Picro-acetoformol was the most useful and successful fixing fluid for preserving the outer layers of cells — particularly the germinal epithelium. I might add that where there was any tissue in contact with the ovaries (a bit of the peritoneal wall was sometimes snipped off with the smaller ovaries) "or where the periovarian capsule was thicker than usual, due to its penetration by the oviduct, Flemming's fluid preserved the outer cells very well, the extraneous tissue becoming over-fixed and saving the ovary itself from that fate. The figures from Flemming and Hermann material are of cells thus protected.
Iron hematoxylin (Heidenhain's) was used for staining most of the sections, and for bringing out the details of the chromatin it can hardh' be improved. Flemming's triple, usually one of the shorter methods, was used with Plemming fixed material in a number of cases, and proved ver}^ useful in bringing out the nucleoli and the idiosome.
A large number of ovaries of different ages were also fixed and stained for mitochondria. Benda's own method was used, with varying success. More reliable and certain, and better in several waj^s, is the following method: the tissue is fixed in Benda's fluid (Flemming's, with about one drop of glacial acetic acid to each 10 cc.) or Helly's, or Zenker's fluid (with the acetic acid reduced as above) and then mordanted in 2-2.5 per cent potassium dichromate for two to four weeks, the longer time being perhaps preferable. Sections are stained with Weigert's copper hematoxylin. Tissues fixed in Benda's fluid with no subsequent mordantage, and stained with copper hematoxylin also gave very good results. By this method the mitochondria are stained black or blue-black and stand out very clearly against the yellow background of the cytoplasm.
Morphogenesis of the Ovary
In the development of the cat's ovary, von Winiwarter and Sainmont ('08) describe two proliferations of cells from the germinal epithelium before birth, forming medullary and cortical cords respectively, and lay some emphasis on these as separate down-growths. The germ cells and follicle cells of the first proliferation and the germ cells of the second all degenerate by the time the kitten is a few months old. At the age of about three and a half or four months, by a renewal of the activity of the germinal epithelium, a third proliferation of cells occurs, from which develop the definitive egg-cells or ova, making up the definitive cortex of the ovary. Kingsbury ('13) does not distinguish between a first and a second proliferation of cells in the embryo, considering them parts of one continuous process. From the cells which arise first from the germinal epithelium are formed the 'medullary cords,' and the 'cortical cords' or tubes of
Pfltjger are formed later, outside of or peripheral to these; and medullary are not to be sharply distinguished from cortical cords. Kingsbury also found no evidence of a third or post foetal proliferation of cells shortly before sexual maturity.
In the mouse there is a single continuous proliferation of cells from the germinal epithelium which extends up to about birth. This proliferation of cells does not take the form of tubular Mown-growths/ as in the cat and other forms, but the cells are in irregular masses just beneath the epithelium. Later, as the ovary grows in size and the stroma cells increase in number, these latter tend to wander in and break these masses of cells into short, thick structures which are, however, imperfectly and incompletely separated from each other (fig. 9). The small size of the ovary may be responsible for this pattern in the development; there is not enough room for the formation of the pronounced cords or egg-tubes found in other larger forms. These groups of cells are not at first separated from the germinal epithelium, but with the development of the tunica albuginea these cell-masses become cut off about the time of parturition. The development of the tunica will be taken up later. As was stated above, this process of cell-formation is a continuous process in the mouse; there is no distinction to be made between a first or medullary and a second or cortical proliferation. The inner regions of these cell-masses or groups might be termed 'medullary,' and what might be called 'medullary follicles' are probably formed by the separation of some of the germ cells with their follicle cells from these masses. These 'medullary follicles' would contain cells formed first from the germinal epithelium, which consequently are further along in development than those more peripherally located.
There is a small amount of stroma in the ovary at this time, formed by the differentiation and multiplication of mesenchymal cells which have wandered in at the hilum. Blood vessels are present as a few capillary loops. The stroma cells, as mentioned above, grow peripherally in strands and partially separate the cell-masses from each other (fig. 9). These stroma cells, on reaching the base of the germinal epithelium, begin to grow around under this as the developing tunica albuginea, partially separating these masses from the epithelium. These groups of cells retain a partial connection with the epithelium until about birth when the tunica is practically complete, although not very thick. At this time, the tunica is made up of one or two layers of spindle-shaped 6r fusiform cells. Even in the ovary of the adult mouse the tunica is not a very dense structure, consisting of a few layers (3 to 6) of flattened or fusiform cells. It seems to be continuous with the stroma in places where the latter is radially arranged, and hence appears thicker in some regions than in others. As will be seen later, the tunica has a part to play in the development of the definitive ova.
The cell-masses are made up of two kinds of cells — those with large round nuclei and a relatively small amount of cytoplasm, the 'primitive germ cells or oocytes, and smaller cells with smaller round or oval nuclei, the 'indifferent cells,' which are to be regarded as the future folHcle cells. In embryos or foetuses of 23 mm. length (ca. nineteen days post coitum) a few egg-cells in the central part of the ovary possess follicles made up of a single layer of these cells. As they surround an oocyte at first, they are somewhat flattened, perhaps as a result of an increase in size on the part of the germ cell: the folhcle cells may be stretched out a little before dividing to keep pace with the oocyte in its growth. This follicle-formation proceeds toward the periphery, and is quite rapid from this time up to and after birth (fig. 10). By three days after birth all the germ cells in the ovary proper are surrounded by follicles, A\hich, in the central part may be two-layered, of cuboidal cells, and peripherally just under the epitheUum are made up of a single layer of flattened cells. This condition persists for a few days, the centrally located follicles gradually growing in size (fig. 11). In young mice of 14 days, the central follicles have from three to five layers of cuboidal cells and fill up the interior of the ovary, constituting the bulk of it (fig. 12). Clearly marked off from these is a superficial layer of egg-cells two to five deep, in primary follicles, made up of a single layer of flattened cells. The large central egg-cells with their follicles are those which have arisen by the proliferation of cells from the germinal epithelium before birth, as described above; the oocytes in the primary follicles have been formed from the epithelium by a proliferation beginning about birth or shortly after — a process to be described below. Cavities begin to appear in the larger follicles between fifteen and eighteen days post partum, and a degeneration of the eggcells sets in at about the same time. The evidence shows that all these germ cells formed before birth degenerate and are resorbed, none of them developing into definitive ova. This degeneration takes the form of atrophy and resorbtion in some cases, but in others there may occur atresia folliculi, accompanied by the formation of a first polar body and a degenerative fragmentation of the egg-cell, simulating more or less closely a parthenogenetic cleavage (Kingery, '14).
- 1 In this paper I tise the terms 'primitive oocytes or germ cells' to refer to the cells formed from the germinal epithelium in the first or embryonic proliferation; 'definitive oocytes or germ cells' will be applied to the egg-cells arising from the germinal epithelium in the proliferation of cells between birth and sexual maturity The terms 'primordial geiTn cells' and 'primary gonocytes' have been used by authors in another sense and would not be appropriate here.
During the early development of the ovary the cells of the germinal epithelium vary in shape from rather tall cuboidal to somewhat flattened cells, a large number being more or less rounded. The nuclei are large, nearly filling the cell, and the cytoplasm is scant in amount. After birth certain of these cells begin their development into oocytes. They begin to grow and enlarge in situ in the epithehum. At first more or less spherical, as they become larger than the other cells of the epithelium they project from the surface of the ovary. These small protuberances are very noticeable between three and thirty days after birth (figs. 35, 36, 38, 1 to 3.) These cells soon become oval, however, with their long axes tangential to the surface of the ovary. As they enlarge, the adjacent epithelial cells are crowded to either side (or end) and are flattened against and around the egg-cells (figs. 35, 36.) As the egg-cells grow still larger, the bulging on the surface becomes more marked. In the course of further development some of these flattened cells extend up over (outside of) and under (inside of) the oocyte so that the latter is cornpletelj^ surrounded by a layer of flattened cells, a primary follicle, while still in the germinal epithelium (figs. 2, 3, 38). As growth proceeds, the other cells of the germinal epithelium extend up over this oocyte in its primary follicle which is in this manner 'left behind' in the tunica albuginea under the epithelium. The cells of the epithelium then unite over the egg-cell in its follicle, closing over it (cf. figs. 1 to 6, which are designed to show this) .
These primary follicles remain in the tunica albuginea at first, but as development goes on, they come to lie in the stroma
Text-fig. 1 to 6 These are to show the development of tlie follicle of a definitive germ cell and the way in which the oocyte migrates from the germinal epithelium into and through the tunica albuginea. In the figures, the free surface of the epithelium is up and the tunica and ovary down. Figure 3 is from an ovary of a mouse three da3's old, while the others are from the same ovary of a mouse twenty days after birth. X 625.
beneath it. The cells of the tunica, not a very dense layer of tissue, are apparently active in this migration of the follicles. They separate underneath (central to) and close up over (peripheral to) the follicles, and by a continuation of this process, these gradually pass through the tunica and reach the stroma beneath (figs. 3 to 6). New egg-cells in follicles are being continually added outside these, the later formed being, of course, younger than those more deeply located, which, in turn, are more peripherally situated than the cells originating during embryonic life, the primitive germ cells. These latter, as mentioned above, make up the bulk of the ovary for the first twenty or twenty-five days after birth (figs. 11,12). As they degenerate, they are gradually replaced by the definitive ova whose follicles have by this time begun to enlarge.
In the course of the degeneration of the primitive oocytes (those of embryonic origin) the space they occupied in the center of the ovary becomes filled with stroma and blood vessels which, in their growth, form the definitive medulla (figs. 11 to 13). This is well formed by about twenty-eight days after birth. What may now be termed the definitive cortex is filled with follicles containing oocytes of postnatal formation (definitive germ cells) and follicles of embryonic origin (primitive germ cells) which project into it from the medulla. Thus, the definitive medulla of the adult ovary includes the embryonic medulla and a large part if not all of the embryonic cortex. The definitive cortex is exclusively of post-partum formation.
The origin of the rete ovarii is still an open question. Von Winiwarter and Sainmont ('08) describe the rete of the cat as arising from the organ of Mihalkowics which forms a net-like structure of solid cords of cells which later acquire lumina. This net-work of tubules retains its connection with the uriniferous tubules (epoophoron) and forms a new connection with the inner ends of the medullary cords. The organ of Mihalkowics is described as arising from the capsules, of the glomeruli in the cephalic part of the mesonephros.
Felix (T2) states for man that, in the degeneration of the mesonephros the glomeruli, corpuscles, and secretory parts of the uriniferous tubules are the first to go, and the collecting tubules persist. The 'rete blastema,' a product of the epithelial nucleus which itself has arisen from the germinal epithelium, forms a net- work of cords. These cords, which are solid at first, but acquire lumina about birth, form the rete ovarii. They connect up with the collecting tubules of the mesonephros.
Kingsbury ('13) suggests that, in the cat, the rete may arise partly from the mesothelium at the cephalic end of the ovary and partly from ingrowths from the mesonephros.
In the mouse the mesonephros has a relatively slight development and degeneration sets in early. I did not make a special study of the origin and development of the rete ovarii in the mouse, reserving it for a possible future work. The suggestion is ventured that it apparently arises by ingrowths from the mesonephros. The rete is found as a constant structure in the adult ovary.
An interesting feature of the ovary of the mouse is the marked development of a peritoneal fold, forming a more or less complete covering or capsule for the organ (figs. 8, 9, 10, 12, 13). Huber ('15) mentions, but does not describe, a similar 'periovarian capsule' or 'bursa ovarica' in the rat. Van Beneden ('80) described in the bat a periovarian capsule which he said was closed, with no communication with the peritoneal cavity. Sobotta ('95) found in the mouse a connective tissue capsule which completely surrounds the ovary. He states that the space between capsule and ovary is filled with a clear serous liquor and that during heat the capsule becomes enlarged. Similar structures have been described for other mammals (Schmaltz, '11).
My material shows that this membrane arises as a fold of the peritoneum which grows over and encloses the ovary. The oviduct is in the fold as it develops, so that when the capsule is completely formed (embryo of 23 mm. length) the fimbriated end is inside, opening toward the ovary. This capsule is a delicate membrane made up of two layers of peritoneal epithelium between which is a small amount of connective tissue in which a few small blood vessels run. This was found to be apparently a complete capsule in rather more than half the cases examined; in the others there was an opening into the peritoneal cavity close to the hilum of the ovary, near where the oviduct penetrates the capsule.
As has been described above, there are in the ovary of the mouse, two proliferations of cells from the germinal epithelium, one during embryonic and foetal life, and the second extending from birth, or a day or so after, nearly to sexual maturity. The second proliferation constitutes oogenesis proper; but it is advisable to take up the development of the first or 'primitive germ cells' because of the part they play in the morphogenesis of the ovary, and because von Winiwarter and Sainmont have described the history of these cells in the cat with so much attention to detail, as 'oogenesis.'
It was stated above that this first proliferation of cells from the germinal epithelium is a continuous process, but it should be understood that the rate of proliferation is not uniform. It is well marked in embryos from 12 to 22 mm. length (ca. fifteen to nineteen days post coitum), when it becomes slower. From this time on it is rather slow and at about three days post partum it has ceased altogether and the second proliferation has begun. This second state of activity on the part of the germinal epithelium is manifested rather slowly, and it is over shortly before sexual maturity. It may be that the two proliferations in the mouse are parts of one process, the second (post partum) being a continuation of the first; the discussion of this will be taken up later.
The earlier stages of the nuclear development of these germ cells of the first or embryonic proliferation, the 'primitive oocytes,' are usually passed through by the cells while still in the germinal epithelium. After a number of cell-divisions a cell begins to differentiate. The resting stage after each mitosis von Winiwarter calls 'protobroque.' After the last oogonial mitosis in the mouse, the cell returns to this stage (fig. 14). The nucleus shows a delicate network of chromatin on a reticulum of linin. At the intersections the chromatin is massed in larger granules, among which are two or three nucleoli, one of which is usually larger than the others. With Flemming's fluid and triple stain, the red nucleoli stand out distinctly from the blue- violet reticulum of chromatin. No idiosome is seen at this stage, and the mitochondrial content is made up of granules sparsely scattered through the cytoplasm. Cells in this phase are found in the germinal epithelium, in embryos from 13 mm. length up to birth.
There is no indication of the stages 'poussieroide' or 'deutobroque' of von Winiwarter and Sainmont ('08). In the case of the former, the suggestion might be ventured that this appearance (to judge from their figures) is an artifact, due to overfixation with the osmic acid fixer used (Flemming's). The cells are described as being in the germinal epithelium, and I have found that over-fixation is regularly the fate of the cells in the outer laj^ers of the ovary.
In the mouse, the cells pass from the 'protobroque' to the ieptotene' stage', which is of short duration. The chromatin net-work gradually becomes heavier in places and breaks down in others, so that the result is the formation of long slender bands or threads, connected by delicate cross-bars (figs. 15, 16). These cross-bars disappear later and the chromatin is then arranged as a tangle of long slender threads (fig. 17). At least one nucleolus is present, frequently hidden in the knot of chromatin bands. An idiosome is not yet distinguishable. It is difficult to say whether the chromatin threads have any definite arrangement; in some cells they seem to have more or less the form of a horse-shoe, with their ends all toward the same side of the nucleus. This disposition, however, does not seem to be universal. Cells in this phase are usually found in the germinal epithelium, but many are also encountered beneath this in the ovary.
This stage soon passes into the following in which the chromatin threads undergo a contraction to one side of the nucleus — synizesis (fig. 18). This contraction is not extreme in the mouse and the chromatin bands can usually be distinguished, a few extending out of the tangle. The nucleoli are hidden among the threads and it is difficult to make them out; one at least, however, is present at this time. No idiosome was distinguished, so it is hard to say with certainty whether the contraction of the chromatin is toward that side of the cell. The mitochondria have increased in number and are massed around the nucleus. Cells in this stage are most frequently found in the ovary under the germinal epithelium.
The next stage, 'pachytene,' is to a certain extent synchronous with the preceding. During synizesis the long slender threads of chromatin begin to shorten and thicken. As this progresses, they assume, at the same time, a moniliforin appearance, as if made up of granules strung together (figs. 18, 19). At first a fairly regular arrangement as loops is seen in some of the cells, a persistence of the disposition in the preceding stage. This definite arrangement of the chromatin threads, if indeed it be of constant occurrence, is soon lost, and with the further shortening and thickening of the bands, they become irregularly arranged in the nucleus. At least one nucleolus is visible, at first among the ends of the chromatin threads and later almost anywhere in the nucleus. I was unable to distinguish an idiosome at this stage, but the mitochondrial granules, sometimes with a few rods among them, have begun to accumulate in the still rather scant cytoplasm at one side of the nucleus, forming a well-marked crescent-shaped mass. Later, when theidiosome is clearly distinguishable, it occurs in the center of this group of mitochondria, so it is safe to assume that this crescent marks its location. When a definite arrangement of the chromatin threads can be made out, their ends are directed toward this accumulation of mitochondria.
This pachytene stage is of rather long duration — in fact, cells in this phase are found in the ovary up to a couple of days after birth. It passes into the 'diplotene' stage of von Winiwarter. That is, some of the thick monliform threads of chromatin begin to split longitudinally (fig. 20). Y-shaped and ring-shaped chromatin segments are formed as a result, depending on whether the bands split at one end first or in the middle. In threads split at one end, the arms of the Y occasionally become twisted around each other, in the 'chiasmatypie' of Jansens, to which some have attached such importance in the explanation of the behavior of certain characters in inheritance — in 'crossing over,' for example. One or two nucleoli are visible during this stage, which is relatively short and blends with that following. Cells in this phase are found in ovaries of embryos about 22 mm . long, and as late as ovaries of mice a day or so after birth. Follicle formation begins at about the time the cells enter this stage. Practically all the early diplotene cells are in primary follicles, made up of a single layer of flattened cells. Later diplotene germ cells, and those in the following stage (dictye or dictyate) are in follicles more advanced in development.
The idiosome is clearly distinguishable for the first time in the diplotene stage. A mitochondrial technique is apparently the best method for bringing this out, but it is visible after Flemming's or Hermann's fluids. It appears as a deeply staining body in the cytoplasm, close to the nucleus. At first it is hidden in the crescent-shaped mass of mitochondrial granules, but when these become scattered through the cytoplasm, as happens shortly, the idiosome stands out clearly. Only occasionally are centrosomes seen in it.
There is a certain overlapping of this stage and the next, the 'dictye' or 'dictyate.' In many cells, while some chromatin threads are splitting, others begin to thin out and lose their character of threads or bands. The chromatin becomes arranged in irregular masses and granules at the intersections of a network or reticulum made up, partly of chromatin and partly of Unin (figs. 21 to 23). One large nucleolus and two or more smaller ones are usually present. The chromatin and linin are frequently in close relation with the large nucleolus which stains much less intensely as it enlarges. The idiosome is plainly visible in the cell near the nucleus, and the initochondria, mostly granules, but with beaded rods and threads appearing in increasing numbers, are evenly scattered throughout the cytoplasm.
Von Winiwarter and Sainmont ('08) consider this stage, dictye described and named by the former in his work on the rabbit a form of degeneration and not a normal step in development. R. Van der Stricht ('11) who employs von Winiwarter's terminology, states that this is not always a stage marking degeneration, but is frequently normal, appearing in maturing Graafian follicles. In the mouse, this stage bears a resemblance to the later phases of the development of the definitive oocytes (figs. 44 to 46), and is to be correlated with the marked growth in size of both nucleus and cell. This growth, together with the fact that these cells degenerate without forming ova, undoubtedly are important factors in producing these nuclear changes. This degeneration and its bearing on the origin of the definitive ova will be discussed later.
Oogenesis proper, the second or post-foetal proliferation of cells from the germinal epithelium, begins within two or three days after birth and extends nearly to sexual maturity; that is, the process lasts about thirty-five or forty days. At birth the first proliferation has become so slow that the cells are differentiating in the epithelium instead of in the ovary underneath, and pachytene and diplotene cells are seen in the epithelium itself. This condition does not last, however, as the process apparently stops entirely within three days after birth. That is to say, the second proliferation is an indication of a renewed activity on the part of the germinal epithelium, and may be considered as beginning where the first* left off. Whether the two are to be considered as parts of one continuous process is a question which will be discussed later.
Since the developing definitive oocytes pass through the early stages of their differentiation in the germinal epithelium, it is somewhat difficult to determine the correct seriation of stages. Criteria employed are the relative sizes of the nuclei and of the whole egg-cell in a single ovary, and the size and appearance of the cells and nuclei in ovaries of different ages.
The formation of egg-cells from the epithelium is most rapid during the time from three to twenty-five days post partum, and in a single ovary of this period practically all the early stages may be seen. Advantage has been taken of this fact in making the drawings : a number have been drawn from one ovary (eleven days post partum). It has been possible thus to show relative size very clearly, and the cells have all had the same fixation and stain. However, cells from a number of other ovaries, differently fixed and stained, have been drawn; accordingly there is no ground for a possible contention that my results are due to any one special method. This process goes on practically up to sexual maturity although more slowly in the latter part of this period, and is completed forty or forty-five days after birth. At this age the majority of female mice are sexually mature, and the ovaries contain all the oocytes which will be differentiated.
The development of these germ cells, the definitive oocytes, is marked chiefly, perhaps, by the utter absence of the complicated chromosomal history which is usually associated with this process. In the description to follow, it seems convenient to speak of three stages in the development, 'a,' 'b,' and 'c;' these are, of course, more or less arbitrary, and are not sharply distinguished, one passing almost insensibly into the next.
Apparently any cell in the germinal epithelium is a potential egg-cell. From the cells of this epithelium develop oocytes, follicle cells, and residual germinal epithelial cells; and at first there is no w^ay of distinguishing the different kinds of cells or their potentialities. At first neither the mitochondrial content nor the nuclear structure is distinctive or characteristic of any one of these possible lines of development. It is not until one of these cells, in the course of its differentiation, begins to grow in size that a germ cell can be distinguished from other cells in the epithelium. It would be difficult to determine just what the factors are which determine the line of differentiation any cell of the epithelium will take.
To digress for a moment: Jenkinson ('13) states that the "oocytes of the outermost layer often lie practically in the epithelium," but he thinks that they have developed from the 'primordial germ cells' which have migrated in at the hilum of the ovary. There can be no doubt whatever that in the mouse the oocytes start their development in the epithelium. Figures 35, 36 and 38 have been drawn with the adjacent -cells of the epithelium to bring out this point. In figure 35, in which, through shrinkage, the epithelium has been slightly torn away from the ovary, it is show^n conclusively, I think, that the egg-cells are actually in and a part of the epithelium and not merely crowded against the basal side of it.
Stage 'a.' The cells of the germinal epithelium have, of course, been dividing during the growth of the ovary, keeping pace with its increase in size. This might be termed the "multiplication period' (figs. 25 to 27). Beginning about three days after birth, certain of these epithelial cells commence to grow in size, and from then on they may be considered oocytes (primary) since they develop into the definitive ova. These cells of the germinal epithelium, after each mitosis, return to a condition, the resting stage between divisions, which resembles that described as 'protobroque' by von Winiwarter (fig. 24). It is from this point, after the last 'oogonial division' that the egg-cells in the mouse begin their further development. The cells in this stage are scarcely distinguishable from those in the resting condition between mitoses, and also resemble the 'protobroque' cells of the embryonic proliferation already described, although they may be considerably smaller (figs. 24 and 14). The nucleus is made up of a delicate network or reticulum of chromatin on a Unin frame-work; small clumps or granules of chromatin are located here and there at the intersections of the net-work.The reticulum may be poorly defined and the granules of chromatin appear more or less isolated in the nucleus. Two to five nucleoli may be present, although the more usual number is two or three. FlemiTiing's fixation and triple stain, or some similar technique, is necessary to distinguish these nucleoli from the larger clumps of chromatin, with which they may be confused in iron hematoxylin preparations. One nucleolus is usually larger than the others and is sometimes oval or elongated (figs. 28, d and 29, d).
Stage '6,' (figs. 29 to 33). This stage is marked by a slight increase in size of both oocyte and nucleus. The chromatin granules may increase in size, and the reticulum becomes heavier. There seems to be.an actual increase in the amount of chromatin : as the whole chromatin network is heavier or coarser (figs. 29 to 34) from two to five nucleoli may be present, although, as in the preceding stage, two or three seems to be the more usual number.
Stage 'c,' (figs. 34 to 46). The next phase is marked by a gradual change in the nuclear structure. The whole cell is growing markedly in size and, as the nucleus enlarges, the reticulum of chromatin becomes attenuated and loses to a certain degree its character as a network. Apparently the cross-bars break and the chromatin 'flows back' to the longer strands, forming threads or strands with granules of chromatin on them (figs. 36 to 40). The chromatin loses more and more its staining reaction and the nucleus appears filled with more or less isolated strands and granules of faintly staining chromatin. These strands are eventually arranged as smaller masses and threads of irregular form, with granules scattered here and there, and with a faint, irregular, incomplete reticulum of linin. Two or three nucleoli are usually \'isible, one frequently larger and less intensely stained than the others (figs. 41 to 46).
This condition of the nucleus, with the chromatin widely scattered in irregular granules and strands and with two or three nucleoli, resembles to a certain extent the 'dictyate' stage described above for the primitive germ cells. This is not surprising, perhaps, when it is considered that the 'dictyate' stage of the primitive germ cell and stage 'c' of the definitive oocyte are each correlated with the enormous growth of the germ cell and its nucleus. Compare, for example, figure 3() with 46; the latter oocyte is so large that only the nucleus is shown in the drawing. This oocyte (fig. 46) is in a nearly mature Graafian follicle (text fig. 7) and is almost ready for maturation.
Text fig. 7 A definitive oocyte in a mature or nearly mature Graafian follicle. This is a sketch of the oocyte whose nucleus is shown in figure 46. The follicles of the mouse do not attain the large size nor the marked vesicular character of those of man}- other mammals. X 44.
As the oocytes enter stage 'b,' they begin to grow in size and, although at first more or less rounded, they become more oval, with their long axes tangential to the surface of the ovary; but their shape is apparently dependant on pressure and the effect of the adjacent cells. Oocytes in stages 'a' and 'b' are found in the germinal epithelium, and occasionally cells of stage 'c' occur here also (figs. 35, 36, 38). Usually however, oocytes in stage 'c' are in primary follicles or in Graafian follicles of various stages of development, up to the nearly mature primary oocyte in the adult or sexually mature ovary (figs. 4, 5, 6, 40, and 7). The manner in which the oocytes penetrate the tunica albuginea in leaving the germinal epithelium and the formation of the follicles have already been discussed (p. 384-385).
An idiosome is not visible in stage 'a,' and becomes well marked only in cells of stage 'b.' Centrosomes are not infrequently clearly distinguishable in the idiosomes (figs. 32, 38). The mitochondrial content of the cells of the germinal epithelium is very scanty, and consists of a few granules close around the nucleus (fig. 47). As the oocyte begins to differentiate and grow in size, the mitochondria increase in number and become located chiefly, perhaps, in the two ends of the cell, now somewhat oval in shape (fig. 48). They are arranged near the nucleus and, as differentiation proceeds, they tend to accumulate more in one end or side of the cell, forming a more or less crescent-shaped mass which marks the place where the idiosome develops (figs. 48 to 50). At about the beginning of stage 'c' the mitochondrial mass becomes dispersed and the granules are scattered quite uniformly through the cytoplasm, accompanying the growth of the cell (figs. 50, 51). There is no indication of a peripheral condensation of the mitochondria such as described for the cat's egg by R. Van der Stricht ('11).
The oocytes remain in this stage 'c,' which somewhat resembles von Winiwarter's 'dictye,' until ready for maturation. There can be no doubt of this. Egg-cells have been followed in their development up into mature or nearly mature Graafian follicles in sexually mature and adult ovaries, and the nuclei are all in this phase (figs. 44 to 46, 7). The further development of the oocytes, the maturation divisions and the formation of the mature ova, was not investigated, as beyond the scope of this work.
Discussion: Literature and Observations
The monographic work by von Winiwarter and Sainmont ('08) on the organogenesis of the ovary and oogenesis in the cat has had its influence on studies since made along similar lines, and as has been mentioned above, their revision of von Winiwarter's terminology has been adopted by a large number of authors. But so far, no one, apparently, has called attention to a feature of their work which seems to me to be rather inconsistent and illogical. Their account of oogenesis is presented with great care and attention to detail, but, as they themselves state, all of the cells which go through this process degenerate, and not one develops into a definitive ovum.^ It is not quite clear just how the course of development of such cells, all of which degenerate, constitutes oogenesis. If oogenesis be used in a broad sense to mean the course of development of egg-cells, with no regard to their ultimate fate, then perhaps, the application of the term is justified here. However, if oogenesis is taken to mean the development of the definitive ova, von Winiwarter and Sainmont have not described such a process.
They state that the definitive ova are formed by a renewed activity of the germinal epithelium shortly before sexual maturity.^ They merely mention this and say a full description will follow later. In a note appearing in 1908 at about the same as their memoir (von Winiwarter and Sainmont 'OS*"), they discuss very briefly (four pages) the post-foetal formation of ova in the cat. They state here that the question of a new formation
"Au second chapitre du present memoire nous avons demontre que les cordons corticaux ou tubes de Pfliiger ainsi que toutes les formations auxquelles ils donnent naissance (ovules, foUicuIes primordiaux, follicules de de Graaf developpes) ne sont que productions transitoires, au m6me titre que les cordons meduUaires." (Von Winiwater and Sainmont, '08, p. 165.)
' "Pendant que ces dernieres modifications se deroulent I'activite de I'assise epitheliale entre une derniere fois en jeu pour aboutir aux invaginations epithe liales Ces invaginations, jointes aux cellules folliculcuses de zone
corticale primitive, aboutissent k la formation de la zone corticale definitive de I'ovaire, h laquelle, seule, sera reservee la production des oeufs definitifs. Son histoire appartient a un chapitre ulterieur." (Von Winiwarter and Sainmont, '08, p. 259-260.) of ova has not been settled one way or the other for two reasons : — no one has taken the time and pains to examine a complete ('liiekenlose') series of ovaries, and second, there have not been any definite characteristics by which a young egg-cell might be recognized. Concerning this second point these authors state further :
Nun ist es von einem von uns (v. Winiwarter, '00) nachgewiesen worden, dass das Ei der Saugetiere im Laufe der Wachstumsperiode eine Reihe von Kernveranderungen durchmacht, welche so characteristisch sind, dass sie mit Sicherheit erlauben, einen jungen Oocyten von alien iibrigen epithelialen Zellen des Ovarium s zu unterscheiden. Von diesem Prinzip ausgehend, hat sich schon damals einer von uns (v. Winiwarter) dahin ausgesprochen, dass eine Neubildung von Eiern nur dann als bewiesen gelten konne, wenn die als neugebildete Eier angesehenen Elemente die characteristischen Kernmetamorphosen der ersten Entwicklungsstadien des Ovariums erkennen lassen ("que les pretendus ovules de nouvelle formation montreraient les metamorphoses nucleaires caracteristiques des premiers stades de developpement de Fovaire"). ('08 a, p. 613-614.)
It seems to me that this is rather an unwarranted assumption, made by von Wlniw^arter himself in 1900 and repeated by von Winiwarter and Sainmont in 1908. The account given by these authors of the development of these egg-cells, is very carefully worked out. The egg-cells, in their development, pass through certain stages which have been very carefully characterized, and then every one undergoes degeneration. It is not quite clear just what justification there is for the dictum that any cells of new formation, in order to be considered egg-cells, must pass through the same stages in their development as the earlier formed cells which degenerate and disappear. It would seem that the ultimate fate of any cells of new formation should have some bearing on the question of whether they are to be considered definitive egg-cells. If these cells, formed after birth, can be shown to develop into the definitive ova, if they can be traced through all the stages up to eggs in mature Graafian follicles, it would seem that the question of whether or not their earlier development was like that of the cells formed in the embryo, w^as a matter of slight importance.
To return to the two points raised by them concerning a new formation of egg-cells; — I think my series of ovaries is complete enough to satisfy any requirements. As for their second consideration, it seems to be rather an unwarranted assumption; the ultimate fate of the cells under discussion is what should decide their status, and not the nuclear metamorphoses characteristic of the first stages of the development of the ovary." Perhaps their promised later chapter will throw some light on this question.
The problem of the origin of the definitive ova is one which has been of interest to many workers, but is not yet definitely settled. I shall take up this point only briefly, referring to the papers by Swift ('14 and '15) and Firket ('14) for a more detailed discussion.
The two more important views as to the origin of the definitive germ cells are: (1) that they develop by a process of differentiation from the mesothelial cells of the germinal epithelium covering the ovary; and (2) that they develop from 'primordial germ cells' or 'Ureier,' which themselves have had their origin elsewhere (entoderm) and have migrated to the epithelium of the genital ridge, there to become differentiated into the ova. The first is the older view, and, presented by Waldeyer in 1870, is today held probably by a majority of those who have considered the question. The second conception is comparatively recent and probably grew out of Hoffmann's work in 1893. He showed ('93) that the 'primordial germ cells' or 'Ureier' which Waldeyer and his school believed were differentiated from cells of the germinal epithelium, were, in birds, present in the embryo, long before the germinal epithelium or gonad had appeared, and that they were found far from the site of the future reproductive organ. They were found in entoderm, in splanchnic mesenchyme and between the two, and later migrated to the mesothelial covering of the gonad when it started its development. He believed that the definitive ova were the direct descendants of these 'primordial germ cells.'
'Primordial germ cells' have been described in a number of forms, including fishes, reptiles, birds, and mammals. For a more detailed discussion and bibliography reference is again made to Swift ('14 and '15) and Firket ('14). To mention a few cases among the mammals, Jenkinson ('13) states that in the rabbit the 'primordial germ cells,' originating in the splanchnopleure of the yolk-stalk, migrate to the gonad and there develop into the definitive ova; he believes that the definitive germ cells of the mouse have a similar origin. He adds, however: — "This mode of origin of the germ cells does not, of course, preclude the formation of others from the cells of the sex-cords, that is from the germinal epithelium, and it is indeed quite possible that this occurs." Kirkham ('16) says that the primordial germ cells in the mouse give rise to the definitive ova, and Rubaschkin ('12) holds that they persist and form the definitive ova in the guinea pig it is evident, then, that this second view is becoming more generally accepted. There is, however, a fatal weakness in the evidence heretofore presented in its support. Those who hold this conception have, for the most part, been content to trace these 'primordial germ cells' in or to the germinal epithelium, and then assume that they proceed there to develop into the definitive ova. The case has been considered proven when these cells had been followed into the embryonic ovary. Very few have studied the further history of these cells. Swift (loc. cit.), who is very positive that the primordial germ cells form the definitive ova, carried his investigations only as far as an embryo of fourteen days incubation, stating that d' Hollander ('04) and Sonnenbrodt ('08) describe the development of these cells into definitive ova. As a matter of fact, d' Hollander did not Swift states ('15, p. 450) that v. Berenberg-Gossler "has confirmed the findings of Swift in all the essential points." This is rather misleading, for the fact is that while von Berenberg-Gossler does confirm the actual facts — that large cells, 'primordial germ cells', are present in duck embryos of 24 to 32 somites, he does not accept Swift's interpretation of this. He states:— Alles in allem, bin ich der Ansicht, dass das ganze verhalten dieser zellen in hohem grade davor warnt, siefiir keimbahnzellen zu halten, und dass ihre Genese iiberhaupt ihre Geschlechtszellennatur sehr zweifelhaft macht. . . . Als Hauptergebnis meiner bisherigen Untersuchungen sehe ich die Erkenntnis an, dass man von einer Keimbahn bei Sauropsiden nicht mehr reden kann." (Von Berenberg-Gossler, '14, p. 261-262. The emphasis is Jiia.) study material from chicks more than twenty days after hatching (v. p. 161), and his evidence on this point is therefore not conclusive. Sonnenbrodt studied material from chicks just hatched to hens several years old; he states that the egg-cells in the ovary at hatching are oocytes, and that they develop into the definitive ova. He apparently accepts d'Hollander's views as to the origin of these oocytes. Although neither of these works alone is conclusive on this point, together they serve to support Swift's view.
Nevertheless, it has not been conclusively demonstrated that these 'primordial germ cells' develop into the definitive ova. In fact, there is weighty evidence to show that they play no part in the formation of the latter. Firket ('14) states that Dustin, Kuchekewitsch, and Allen admit that these 'primordial germ cells' degenerate totally or partly in Amphibians and Sauropsids; Allen and Popoff admit a degeneration in the testis and Skrobansky in the ovary of mammals. Kirkham ('16) states that in the male mouse the 'primordial germ cells' all degenerate, while in the female they form the definitive ova. Von Winiwarter and Sainmont ('08) hold that these 'primordial germ cells' in the cat are temporarily hypertrophied cells and have nothing to do with the process of oogenesis.
Firket ('14) studied material from chick embryos from eightytwo hours incubation to hatching, and in addition, the ovaries from chicks just hatched up to young hens of six months, an age when the hens of most breeds are sexually mature and have begun to lay. He believes that his work proves the sexual or genital nature of these 'primordial germ cells,' or 'primary gonocytes,' as he prefers to call them. His studies show that the gonocytes of the medullary zone (formed by the first of the two embryonic proliferations frorn the germinal epithelium, as von Winiwarter described for the cat) develop into oocytes which pass through the first stages of the growth period and then degenerate. They have all disappeared in the chick fourteen days after hatching. The oocytes of the cortical zone (second embryonic proliferation) practically aU degenerate, although he states that he can not be sure that they all do. There is a new formation of germ cells in the cortical region, from cells derived from the germinal epithelium, and from these the definitive oocytes develop; but it is not improbable, at least, that a small number of the 'primordial germ cells' as well, are differentiated into definitive ova. One of his conclusions may be pertinent here: —
II faut, done, morphologiquement parlant, considerer les gonocytes primaires (primordial germ cells) des Vertebres comme etant un rappel phylogenique des gonocytes definitifs des classes inferieurs, notament des Cyclostomes et des Acraniens. L'epuisement graduel, dans la serie phylogenique des elements de cette lignee a necessite I'apparition, au cours de I'ontogenese, d'une seconde lignee de gonocytes, moins precoces. ('14, p. 330-331 )
In the mouse, the evidence here presented shows that the definitive ova originate from cells of the germinal epithelium by a process of differentiation, and that this process takes place between birth and sexual maturity. The primitive germ cells also arise from the germinal epithelium, in the embryo. But the relations of the 'primordial germ cells' are not clear. Jenkinson ('13) and Kirkham ('16) both think that they form the definitive ova in the mouse. But Kirkham's paper is merely an abstract, unaccompanied by figures, and therefore can not be considered conclusive. Jenkinson devotes a few pages in his book to this question, but is hardly convincing.
It would seem that, in the mouse, there are three possible courses of development open for the 'primordial germ cells': — 1) they may persist and form the primitive germ cells or oocytes and so eventually degenerate; 2) they may, after reaching the germinal epithelium, develop into the definitive germ cells; or, 3), they may degenerate completely. The evidence presented, I think, shows that neither the first nor thp second is the course followed. The objection mentioned above, that no one has traced these cells through to mature ova, also disposes of the second possibility. There is, indeed, the further possibility that the 'primordial germ cells,' after entering the germinal epithelium, lose their size and characteristics and become indistinguishable from the mesothelial cells. It would be extremely difficult to prove or disprove this, but the burden of the proof would, it seems to me, rest upon any one who supported such a view. The last course, degeneration, seems to be the most probable fate of these cells.
There are not many examples in the literature of a new formation of epg-cells after birth. Van Beneden ('80) described in the adult bat a formation of egg-cells from the germinal epithelium. From his description and figures, the process resembles the formation of oocytes in the mouse before sexual maturity; but, as he did not connect this process with that in the embryo, it is difficult to say whether it is strictly comparable with the condition in the mouse.
Lane-Claypon ('05), in a study of the ovary of the rabbit, concludes that from the germinal epithelium are formed definitive ova, follicle cells, and interstitial cells. These interstitial cells, from their origin, are potential egg-cells, and, under the proper stimulus, are capable of developing into ova. In this case, the proper stimulus is provided in some way by pregnancy, and a number of these cells become differentiated into oocytes. Apparently these conclusions have not been confirmed by other authors, and hence lose something of their force.
Von Winiwarter and Sainmont ('08) state that in the cat, at about the age of three and one-half or four months, a renewal of the activity of the germinal epithelium provides a new supply of germ cells which develop into the definitive ova, when all the egg-cells of the first and second proliferations have degenerated. In a note appearing about the same time ('08"^), they state that these definitive ova come either entirely from this third proliferation, or partly from it and partly from undifferentiated cells left over from the second.^
^ "Es tauchen nun jetzt in den Epithelhaufen und Striingcn der Cortdcalis Kleine Gruppen von Zellen auf , deren Kerne im Staubfonnigen oder deutobrochen Stadium sind. Diese Fonnen waren schon seit langer Zeit nicht mehr vorhanden, und da sie den ersten Stufen des Wachstuins des Oocyten entsprechen, ist es augenscheinlich, dass sie niit einer Neubildung von Eiern zusammenhangen. . . Wir glauben bewiesen -zu haben, dass in Saugetierovarium nicht nur samtliche Markstrjinge, sondern auch alle Eier und Follikel der primitiven Corticalis dem Untergang anheimfallen. Die definitiven Eier entstammen entweder von undifferenzierten Zellen der zweiten Proliferation (Pflugersche Schlauchej oder von Zellen der dritten Wucherung oder invaginations 6pithelials. Es ist uns nicht moglich, wenigstens morphologisch, die Elemente der einen und anderen zu unterscheiden." (v. Winiwarter and Sainmont, '08 a, p. 616.) Kingsbury ('13), while admitting that his material was too scant to permit a conclusive statement, inclined to the opinion that there was no evidence of a new formation of ova by a third proliferation from the germinal epithelium of the cat's ovary.
R. Van der Stricht ('11) apparently does not consider this question at all, as, in his work on the vitellogenesis of the cat's egg, he makes no mention of such a new formation of ova. But, as he accepts von Winiwarter's terminology and course of development for the egg-cells, and states, further, that oocytes with diplotene or dictye nuclei are found in the adult ovary, it may be assumed that he derives the definitive ova from the second proliferation.
Rubaschkin ('12) states that he can corroborate the conclusions of von Winiwarter and Sainmont that the cells of the first and second proliferations in the cat degenerate. He says nothing, however, about a third proliferation, but one is led to infer that in his opinion one does occur. He further states that Waldeyer had described a proliferation of cells in the post partum development of the ovaries of several animals, but I think Rubaschkin has been misled. In the reference mentioned by him, Waldeyer ('70) says that he believes there is no new formation of egg-cells from the surface epithelium, but that any epithelial down-growths present in post partum ovaries of dogs and rabbits are left over from the embryonic proliferation. Moreover, he states: —
Auch bei Katzen, von welchen ich mehr.ere zur Zeit der Frlihjahrsbrunst untersuchte, fand ich, wie gesagt, Nichts von einer derartigen Neubildiing. Meine Untersuchungen sind so zahlreich, dass, wenn sie wirklich vorkommen sollte, wir eine sehr scltene Aiisnahme imd am allerwenigsten eine Regel vor uns hatten. ('70, p. 45).
Rubaschkin also says that he found a third proliferation of cells from the germinal epithelium of the ovary of the guinea pig which occurs before birth and which he considers the source of the definitive ova.
Firket ('14) describes in the embryo chick a third proliferation of cells from the germinal epithelium which he states forms the most if not all of the definitive ova. As mentioned above, however, he is not able to state definitely that all the mature ova come from this source. In both these last cases however, it may be questioned whether such a formation of egg-cells before birth is strictly comparable to a proliferation of cells after birth and before sexual maturity.
Felix ('12) in describing the development of the ovary in man says that there is an early proliferation of cells from the germinal epithelium, forming the 'epithelial nucleus.' Later, a 'young cortical zone' becomes differentiated, whether from the outer part of the epithelial nucleus, or by a renewed activity on the part of the epithelium resulting in a proliferation of cells, he is unable to say with certainty, although inclining toward the former view. With the exception of this possible proliferation from the germinal epithelium, there is no addition of epithelial cells to the ovary. After the tunica albuginea is formed (in embryos of 180 mm. length) no cells can be added to the interior of the ovary. Apparently there is no possibility of a new formation of ova from the germinal epithelium.
From the foregoing, it is clear that a new formation of germ cells after birth is not of very general occurrence. It may be that, as von Winiwarter and Sainmont ('08 a) suggest, such a formation of ova has been overlooked because a careful study has not been made of a complete series of ovaries. Certainly, the study of a complete series of ovaries between birth and sexual maturity and into adult life, has shown that in the mouse there is such a proliferation of germ cells after birth.
It is possible that the criticism may be made that the egg-cells described above, in the germinal epithelium of postnatal ovaries, as developing primary oocytes, are, in reality, cells which have already passed through the earlier stages of development in the embryonic ovary, and are, perhaps in von Winiwarter's diplotene or dictye stages. I have gone over this carefully and am confident that such is not the case. In the first place, these eggcells differ in many particulars from the cells described as diplotene or dictye, and are not to be confused with them. Secondly, these post natal oocytes are actually smaller than the primitive oocytes of the later stages (cf. figs. 21 and 22 with 28 to 31). Thirdly, intermediate forms can be observed between these cells and ordinary cells of the germinal epithelium on the one hand, and between these cells and oocytes in mature Graafian follicles on the other. The evidence, then, presented by a study of a series of mouse ovaries between birth and sexual maturity shows rather conclusively that there is a new formation of germ cells after birth, and that the definitive ova come from these cells. • Rubaschkin ('12) from his work on the guinea pig states that developing egg-cells may be distinguished from other cells in the ovary by a difference in the mitochondrial content. Mitochondria in the germ cells appear exclusively in the form of granules, while in other cells, including those of the germinal epithelium rods or threads are found. He thus distinguishes sharply between germ cells and epithelial cells, in accordance with his theory of the 'Keimbahn.' The granular form is the type found in the embryonic cells; as differentiation of the cells proceeds the mitochondria become transformed into rods and threads. The germ cells, accordingly, show their embryonic or undifferentiated condition by the granular type of their mitochondria.
Firket ('14) finds that, in the chick, the type of mitochondria is not constant in the germ cells. Swift ('14) states that the mitochondria in the 'primordial germ cells' of the chick are usually rods, although granules are frequently present also. This is directly opposed to the results of Tschaschin ('10), a student of Rubaschkin, who describes the mitochondria of the 'primordial germ cells' of the chick as exclusively granular.
In this connection the work of Lewis and Robertson ('16) is of interest. In tissue cultures of the testicular follicles of the grasshopper, Chorthippus curtipennis, the mitochondria were observed in the living germ cells in the course of their development. Granular in the primary spermatagonia, the mitochondria become granular threads in the secondary sperniatogonia and assume a long thread-like appearance during mitosis. In the growth period of the primary spermatocytes, the mitochondria are again of the granular form. This would show that in this form the shape of the, mitochondria is not constant in the germ cells during their development.
In the mouse, the mitochondria in the developing definitive oocytes are almost entirely of the granular type. The cells of the germinal epithelimn have a small amount of cytoplasm in which are a few granular mitochondria (fig. 47, upper cell). In the course of the growth of one of these cells as a primary oocyte, the number of mitochondria increases, keeping pace with the development of the cell (fig. 47, lower cell.) The mitochondria are of the granular type until the cell is quite large, in a follicle of cuboidal cells, single-layered or even stratified, when rods and threads begin to appear (figs. 50, 51). Thus it is evident that the granular type of mitochondria prevails in the earlier stages of the developing definitive oocytes.
But in the mouse the mitochondria of the cells of the germinal epithelium are also of the granular form. And further, in the folhcle cells surrounding the oocytes, the mitochondria, distributed chiefly in the part of the cells toward the oocyte, are granular, rod-like, or thread-hke. In the cells of some follicles granules appear, and in the cells of others rods or threads are found; in the same follicle, some cells may have granules and others rods or threads, and it is not at all rare to find all kinds of mitochondria in the same cell (fig. 52). It is seen, then, that, in the mouse, while the granular type of mitochondria is predominent, perhaps, in the developing definitive oocytes, this is not a distinctive feature, for epithelial cells and follicle cells as well have a similar mitochondrial content.
This might be expected from the work of Lewis and Lewis ('15) on mitochondria in tissue cultures. They find that the mitochondria can be observed in the living cell, unstained, and that these mitochondria are not constant in form, but change their shape repeatedly. Rods or threads may be seen to break up into granules, and granules fuse to form larger granules. Accordingly, one would not expect to find the mitochondria of any one shape constant in or peculiar to any particular kind of cell.
Schaxel ('11) states that the shape of the mitochondria varies according to the method of fixation and staining : — that with the Benda technique, rods or threads predominate, while after Altmann's or similar methods, granules are found. While I have not made a special study of this point, my results seem to bear out the conclusions of Schaxel; however, although mitochondria of one type or the other may predominate in preparations made by one or the other of these methods, both granules and rods or threads are found in both kinds of preparations. Lewis and Lewis think it quite probable that the mitochondria, 'malleable' or plastic as they appear to be, may have their shape affected by different methods of treatment.
Moreover, it is possible that the mitochondria represent the structural expression of the reducing substances concerned in cellular respiration (Kingsbury, '12). As Lewis and Lewis suggest, it is possible that they are continually being formed and as continually destroyed (oxidized) in the cytoplasm in the course of the metabolic activity of the cell. It is evident that a pronounced change in the metabolism of the egg-cell is correlated with its marked growth in size, from a small cell in the germinal epithelium to a mature ovum. Furthermore, the part played by the follicle cells in the growth of the oocyte must also affect the metabolic activity of those cells. It would seem reasonable, then, to conclude that it is only natural that the mitochondria should not be constant in shape in any one particular kind of cell, but should vary in different cells, or even in the same cell at different times.
In returning now to the question of the post partum proliferation of germ cells, the present discussion will take up only the w^ork of von Winiwarter and Sainmont on the cat in connection with my results in the mouse. From the little these authors have said about this new formation of egg-cells, the process is limited to a brief period of time shortly before sexual maturity (three and one-half to four months). As a result of a multiplication of cells in the germinal epithelium, masses or strands of cells grow down into the tunica albuginea, retaining a connection with the epithelium. In these strands, certain of the cells appear with their nuclei in the 'deutobroque' stage. The germ cells of embryonic origin have largely degenerated by this time, so there is room in the ovary for these new egg-cells which, together with their folhcle cells — which are apparently left over from the first proliferation (v, footnote 3, p. 281) — will make up the definitive cortex. Von Winiwarter and Sainmont do not say so in so many words, but, since they repeat (cf. p. 281 to 283) the assumption made by the senior author in 1900, that cells of a new formation must pass through the nuclear transformations seen in the germ cells of embryonic origin, the inference is that these cells of this post partum proliferation do possess such a nuclear history. The promised later chapter, dealing with this particularly, will doubtless clear up a number of the questions arising from their preliminary account.
In the mouse the period during which egg-cells are formed from the germinal epithehum is prolonged, from birth or shortly after, nearly to sexual maturity, instead of being limited to a small part of that time. The process is much more marked during the first half of this period, and becomes gradually slower until it stops shortly before sexual maturity. Correlated with this prolonging of the period during which germ cells are formed from the germinal epithelium is the entire absence of anything resembling 'cords' or tubular down-growths in the ovary after birth. As has been described above (p. 268 and 269), the germ cells arise singly and make their way individually out of the germinal epithelium into and through the tunica albuginea. It might be considered that the 'tubular' or 'cord-like' down-growths, such as those found in the cat, are here, shallowed out and retarded, reduced to single cells. It is entirely probable that this prolonging of the period, during which definitive oocytes arise from the germinal epithelium, and the 'down-growth' of individual cells, instead of groups of cells, are to be correlated with the small size of the ovary in the mouse. There is not enough room in the organ to contain all the definitive ova, as well as the degenerating primitive oocytes, and, as a result, the process of formation of the former is retarded and prolonged over a long period, and the germ cells arise singly instead of in groups. These definitive egg-cells are added outside the earlier formed primitive oocytes, and room for their growth is provided for b}^ the degeneration and resorbtion of these latter and their folUcles, as well as by the growth of the whole ovary.
From this it is apparent that the potentiality of the germinal epithelium for germ-cell formation lasts for a relatively long time in the mouse. At birth, the cells of the germinal epithelium seem equally capable of developing into oocytes, follicle cells, or epithelial cells, and it is not evident just what the factors are which determine their eventual fate. As the ovary becomes more mature and the cells more differentiated, this potentiality of the cells of the germinal epithelium is lost and after sexual maturity no more egg-cells or follicle cells are derived from the epithelium.
The question of synizesis and synapsis'^ has been given marked attention during the last few years. Whether synizesis represents a real condition in the development of the germ cells (egg or spermatozoon) has been discussed quite thoroughly and will be taken up only briefly here. Reference is made to the works of von Winiwarter and Sainmont ('08), Duesberg ('08), Meves ('07), and others who discuss the question and the literature. Here it will be enough to say that Meves, Duesberg, McClung, and many others consider synizesis an artifact due to faulty fixation. The first two authors, however, admit that there is at a definite period in the development of the germ cells, a tendency, more or less marked in different forms, on the part of the chromatin to contract when brought into contact with the fixative. On the other hand, von Winiwarter and Sainmont, and probably a majority of those who have given the matter consideration, believe that synizesis is a normal stage in the development of the germ cells, of rather marked theoretical importance.
The existence of synapsis or a conjugation of chromatin threads is affirmed and denied; and those who believe it takes place are not agreed on the manner of its occurrence. Union side by side (parasynapsis) and end to end (telosynapsis) have been described in different forms; and in some of these same forms, union of any sort has been denied. For example, von Winiwarter and Sainmont describe a parallel or side by side conjugation of chromatin threads in the cat, and R. Van der Stricht ('11), while accepting the results of these authors on many points, states that he finds no evidence whatever for such an occurrence.
- There is some confusion in regard to the usage of these terms. Von Winiwarter and Sainmont use 'synapsis' to mean a contraction of the chromatin to one side of the nucleus. The better usage seems to be, however, to restrict this term to the conjugation of the chromatin threads or chromosomes, and to employ 'synizesis' to apply to the stage where the chromatin is contracted in the nucleus, as McClung suggested in 1905. Accordingly, the terms will be used here in this sense.
In the mouse the primitive germ cells, as has been described, pass through in the course of their development, a stage which may be termed synizesis. The contraction is more marked, perhaps, in ovaries whose preservation was not the best, but the condition is present, nevertheless,' in well-fixed material. Probably, therefore, its occurrence here is not to be considered an artifact.
In the development of the definitive germ cells, however, which are formed after birth, there is not the slightest indication of synizesis. There is no period in the differentiation of these oocytes when there is the least appearance of a contraction of the chromatin (figs. 24 to 46). In the mouse, synapsis does not occur, either in the development of the primitive germ cells, or in the differentiation of the definitive oocytes. In the former, there is no evidence of a union, side by side or end to end, of the chromatin threads. These bands of chromatin exhibit no parallelism whatever until they begin to split lengthwise in what is termed the diplotene stage, rather late in development. In the definitive oocytes there are no definite, well-defined chromatin threads in the whole course of early development, from the cell in the germinal epithelium to the nearly mature oocyte in its Graafian follicle, and accordingly, there can be no question of a union of chromatin bands at all.
There can be no doubt about the facts in regard to the absence of synizesis and synapsis in the development of the definitive oocytes in the mouse. A careful search was made of the growing egg-cells in the germinal epithelium of ovaries from birth to sexual maturity for just these stages. At first it was thought that they would be encountered, and their absence was doubted. But further study showed convincingly that these stages were not part of the developmental history of these definitive ova. This agrees with the results Duesberg ('08) reports for the spermatogenesis in the rat; he finds no indication of synapsis. I might say here that the early part of the development of the definitive oocytes in the mouse bears a noticeable resemblance to the growth period of the spermatocytes in the rat, according to Duesberg ('08). Compare his figures 4 to 14 with my figures 28 to 40.
It is evident, then, that there is no general agreement as to the facts of synizesis and synapsis, that is, whether there is or is not a union, side by side or end to end, of chromatin threads during or after a period when the chromatin is more or less contracted in the nucleus. Naturally, then, the interpretations placed on these phenomena do not agree. Three views may be mentioned and briefly discussed here.
The first is the one held, perhaps, by most workers at present. This is that, during synapsis in oocyte or spermatocyte, chromosomes of maternal and paternal origin unite side by side, and that there may occur an interchange of materials during the more or less complete fusion. In one of the maturation divisions following, maternal chromosomes are separated from paternal; thus, since whole chromosomes pass into daughter cells, a reduction in the number of chromosomes is brought about. This is the view of those who hold what have been termed 'ultimate particle' theories of development and inheritance, based on Weismann's theories and the hypothesis of the individuality of the chromosomes. These bodies are made up of elements or particles — 'factors' (which may be ultramicroscopic) — Hnearly arranged, and derived from each parent. These 'factors' are the 'determiners' of the characters ('unit characters') in the new individual. During synapsis, when maternal chromosomes conjugate with paternal, and later, when these chromosomes separate, there is a segregation and redistribution of these 'factors,' so the mature ovum or each spermatozoon has a set of 'factors' differing from those of oocyte or spermatocyte, the character of which has been determined in all probability by chance. The characters of the new individual are determined by the factors brought in by each germ cell in fertilization. Those who hold this view have used the behavior of the chromosomes to explain certain modes of inheritance and have constructed compHcated theories to account for the facts. It would seem, however, that they have confused the explanation of the phenomena with the teleological significance they have attached thereto.
Another school (Meves, Duesberg, et al.) avoids the difficulty of explanation or interpretation by denying the facts. For them, synizesis is merely a tendency on the part of the chromatin to contract, at a certain period in the development of the germ cells, and the contraction figure is due to imperfect fixation in a faulty technique. Synapsis does not exist; those who describe this condition have confused the seriation of stages, and the apparent conjugation is really a splitting and separation of chromatin threads, a precocious preparation for the following division (first maturation division).
A third view is that attributed to R. Hertwig by von Winiwarter and Sainmont ('08), by Kingsbury and Hirsch ('12) and by Levy ('15), although I have been unable to find the reference where he discusses just this point. This view is that synizesis and synapsis represent a suppressed or abortive mitosis.
According to this view, on the one hand, synizesis represents 'an attempt on the part of the spermatogonia to divide again — which fails; while on the other hand, the reputed conjugation of chromosomes occurring at about this time is but the imperfect fission and subsequent fusion of daughter chromosomes of such abortive division (Kingsbury and Hirsch, '12).
In discussing the degeneration of secondary spermatogonia in Desmognathus, Kingsbury and Hirsch point out a resemblance between the degeneration stages and synizesis, and make the suggestion that synizesis may represent or be an expression of a "running out of the sper'matogonial stock." It may be the checking or terminating of the period of multiplication. Synapsis is apparently absent in Desmognathus.
If synizesis and synapsis represent an abortive mitosis, it would be expected that somatic cells would show similar phenomena under the proper conditions. And, indeed, the conclusions of Marcus ('07) as reported by Popoff ('08) demonstrate this.
In the development of the thymus, Marcus found that the cells, after a period of mitosis, enter on a phase somewhat similar to the growth period of the germ cells, which leads through suppressed or abortive mitosis to degeneration. Marcus found, just before the abortive mitosis, cells in stages which he considered true synizesis. Popoff ('08), in a discussion of this, considers that this finding of synizesis in somatic cells puts an end to the special significance this stage has been given in the development of the germ cells. The significance of synapsis depends on, first, the hypothesis of the individuality of the chromosomes, and, second, on the occurrence of synizesis and synapsis exclusively in the germ cells. Popoff, rejecting the first and considering the second proved not to be true, denies any special significance to synizesis and synapsis.
Metz ('16) finds that in the Diptera, although nothing resembling synizesis is evident, there occurs in the somatic cells a pairing of chromosomes which is very similar to synapsis (conjugation of chromosomes). He states: — The similarity between the figures in the somatic cells of flies and those in the germ cells of many animals (including flies) makes it seem very probable that essentially the same cause is operative in both cases. If this be true it would seem that in the development of a fly each cell-division is preceeded by an attempt at synapsis. Or, in other words, the tendency to undergo synapsis is so marked as to bring about a close approximation of homologous chromosomes during each cell generation. ('16, p. 255).
In the mouse, synizesis occurs only in the development of the primitive germ cells, all of which degenerate, as has been described above. It may be that, even in this early stage in the development of these cells, degeneration has set in, manifesting itself as yet, however, only in this manner. The splitting of the chromatin threads later (diplotene stage) may be a precocious longitudinal division in preparation for a mitosis which never occurs. This may be due (R. Hertwig, Kingsbury and Hirsch, et al.) to a derangement of the nucleo-cytoplasmic rel^-tionship, brought about by a setting in of degenerative processes. Certain of the forces governing this relationship may become inoperative and others abnormally strong so that the attempt on the part of the cell" to perform its usual functions results in such conditions as synizesis and, in some forms, of synapsis.
This would explain the occurrence of synapsis in the second proliferation of germ cells in the cat as described by von Winiwarter and Sainmont. In the third proliferation described by them, it is to be inferred that synizesis and synapsis occur, but the disturbance in the 'play of forces' governing the nucleocytoplasmic relationship is not great enough to bring about their suppression; the cells after a 'checking/ recover and go through the maturation process. In the case of the somatic cells described by Metz, it might be that the disturbance of this relationship is not great enough to interfere markedly with cell-de vision. In the mouse, the 'disturbance in the play of forces' may be greater in the young (embryonic) ovary, perhaps because of its immature condition, and bring about the complete degeneration of the cells, while in the adult or sexually mature mouse this may not be marked enough to manifest itself at all.
The results of Wodsadelek ('16) on the spermatogenesis of the mule may be of interest here. He finds that the course of spermatogenesis is apparently normal up to the beginning of the growth period of the primary spermatocytes, at which time the germ cells begin to degenerate. The author states that there is no evidence of synizesis, but that there is a more or less marked attempt at synapsis or conjugation on the part of a. varying number of chromosomes He says:
Cells of this nature, m which a great deal of fusion had apparently taken place, invariably show more or less pronounced indications of decay, and the question arises as to whether this unusual amount of fusion is due to the condition of decay or whether the degeneration sets in because of the unusual amount of fusion. It appears, however, that the great amount of fusion is caused by the existing degenerate condition of the cell in general, for invariably masses of chromatin material bearing no resemblance to normal chromosomes or threads are present in these cells. ('16, p. 20).
In these spermatocytes, the attempt at synapsis is apparently an indication of the degenerate condition of the cells. The more of the chromosomes which 'pair,' the more marked the degeneration. It might not be going too far to conclude that in cases where synizesis and synapsis occur as undoubted facts, the cells are in a more or less marked condition of degeneration. In some cases, such as the primitive germ cells of the mouse, the cells of the second proliferation of the cat (von Winiwarter and Sainmont), etc., the degeneration is so pronounced that the cells never recover. In other cases, for example synizesis and synapsis, as described in the germ cells of an increasingly large number of forms, the degenerate condition is so slight that although it brings about synizesis and, in many instances, synapsis, the cells recover and proceed to maturation.
It was stated in the earlier part of this paper that the eggcells of the first or embryonic proliferation in the mouse all degenerate and disappear. The evidence for this is both direct and circumstantial. In ovaries of mice, from about seventeen days post partum up to those sexually mature and adult, eggcells in their follicles may be seen in various stages of degeneration and atresia. Usually the atretic follicles are large, and at first are located near the center (future medulla) of the ovary, but smaller degenerating follicles are in the primitive cortex as well. As stated above, some of these degenerating egg-cells undergo a degenerative fragmentation and may form a first polar body and second polar spindle, and may even break up into fragments, with or without nuclei, so that the whole process resembles parthenogenesis (Kingery, '14). This is evidence, of course, that a large number of these germ cells of embryonic origin degenerate and are resorbed. Furthermore, in ovaries from a few days after birth to sexual maturitj^ there is a peripheral zone of egg-cells in primary follicles made up of a single layer of flattened cells. This zone, which may be from three to six or eight cells deep, is composed of definitive oocytes I, formed from the germinal epithelium after birth and located in and under the tunica albuginea. A study of ovaries between birth and sexual maturity shows that, in the mouse as in other forms, there is formed an over-supply of oocytes, and a vast number of these egg-cells must tail to reach maturity. The development and differentiation of the ovary has had a centrifugal direction all along, and most probably this is adhered to in this instance. The primitive germ cells, centrally located, are the first to start on their course of degeneration, a process which later involves many of the definitive oocytes, probably beginning with those more deeply situated. This degeneration of definitive eggcells, which in all likelihood sets in before sexual maturity, is continued through the whole sexual life of the individual, as is, of course, too well known to need emphasis. Since, then, a large number of definitive oocytes degenerate, and since these are situated more superficially than the primitive germ cells, of which an extremely large number certainly degenerate, it is not unreasonable to conclude that all these primitive oocytes degenerate and are resorbed, and that the definitive ova are all of a later, that is, post natal, origin from the germinal epithelium.
Summary and Conclusions
1. In the development of the ovary of the mouse there are two proliferations of cells from the germinal epithelium. The first, occurring before birth, gives rise to germ cells, the 'primitive germ cells,' all of which degenerate and are resorbed; the second, extending from birth or a few days after nearly to sexual maturity, forms the definitive ova.
2. The evidence here presented, while not conclusive, indicates the possibility that the rete ovarii is formed by in-growths from the mesonephros.
3. A periovarian capsule (bursa ovarica), which develops as a fold of the peritoneum, encloses the ovary more or less completely. The oviduct penetrates this capsule, which is a closed sac with no evident opening into the peritoneal cavity in about 50 per cent of the cases examined.
4. The follicle cells of the primitive oocytes arise from 'indifferent cells' which, also originating from the germinal epithelium, grow down into, or are left behind in, the ovary along with the germ cells. In the course of development, they come to surround the egg-cells, forming thus the primitive follicles.
5. The follicles of the definitive oocytes arise also from the germinal epithelium. While the egg-cell is developing in the epithelium, the cells adjacent to it extend up around and down under it, enclosing it while it is still in the epithelium. Thus, when the oocyte is left behind, or under the epithelium in the tunica albuginea, it is surrounded by a primary follicle made up of a few flattened cells.
6. In the mouse the primitive germ cells have a course of development different from that of the definitive oocytes. The former, of the first or embryonic proliferation, undergo synizesis; then they pass through the stages pachytene, diplotene, dictyate or dictye (of von Winiwarter) and are then overtaken by degeneration, which may be atrophy or a degenerative fragmentation.
The cells formed from the germinal epithelium after birth, the definitive oocytes, have a different history; their chromatin is in the form of an irregular network, clumps of chromatin at the intersections, with from two to five nucleoli in the meshes. With the growth of the oocyte, the chromatin becomes attenuated and stains more faintly until there is the appearance of irregular granules of chromatin connected by very slender chromatin strands and the remnants of the linin reticulum, and one large faintly s.tainir^g nucleolus with frequently one or two others more deeply stained. This condition persists until the egg is in a mature Graafian follicle, ready for maturation.
7. The evidence from a study of the mouse ovary, while not, perhaps, conclusive on this point, tends to show that the 'primordial germ cells' ('Ureier') probably degenerate, playing no part in the development of the definitive ova.
Oocytes originating from cells of the germinal epithelium by a process of differentiation, the 'primitive germ cells,' develop ,to a certain point and then degenerate. None of these cells take part in the formation of the definitive ova.
8. The definitive germ cells develop by a process of differentiation from cells of the germinal epithelium. Stages can be observed, transitional between mesothelial cells on the one hand and primary oocytes in Graafian follicles on the other. This differentiation takes place after birth and before sexual maturity, constituting a 'new formation' of germ cells.
9. IVIitochondria, which, although granular in the developing definitive oocytes, are also found as rods or threads in the germ cells and frequently appear in all forms in the follicle cells, are not to be considered characteristic of the germ cells. Certainly no one particular form is constant in the germ cells, a condition which is not surprising when one considers the probable nature of the mitochondj-ia and the different methods for demonstrating them.
10. The new formation of germ cells from the germinal epithelium is prolonged and extends from birth or shortly afterwards to approximately sexual maturity. This prolonging of the potentiality of the germinal epithehum for germ cell production and the absence of 'cell-cords' are probably to be correlated with the small size of the ovary of the mouse.
11. Synizesis is found constantly in the development of the primitive oocytes; while in the differentiation of the definitive ova, neither synizesis nor synapsis occur. In the case of the primitive germ cells, which are fated to degenerate, the suggestion is repeated that synizesis may represent a stage in degeneration, wherein the normal relations of nucleus and cytoplasm, and thp forces governing them are disturbed. The fact that similar stages have been found in somatic cells militates against the attributing of any special genetic significance to these conditions. •
12. The evidence shows that all the primitive oocytes (embryonic proliferation) develop to a certain extent and then degenerate. This is accomplished by the time the mouse is sexually mature.
I wish to acknowledge my indebtedness to the Department of Histology and Embryology for materials and facilities for work. I also desire to express my appreciation of the kindness and encouragement of Dr. B. F. Kingsbury whose interest and criticism have been of great assistance to me in this study.
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Explanation of Figures
Figures 8 to 12 were drawn in outline with a projection lantern and the details filled in from the specimens. The other figures were all drawn with a camera lucida, at table level. Text figures 1 to 6 were made with the aid of a Zeiss apochromatic objective, 2 mm., 1.4 N. A., and compensating ocular X 8, at a magnification of 1875 diameters; the cuts, reduced to one third, are X 625. Figure 7 was drawn at a magnification of 133 and reduced to one third (X 44). The other figures, with the exception of 51, were made with a Zeiss apochromatic objective, 2 mm., 1.4 N. A., and compensating ocular X 12, at a magnification of 2667 diameters; reduced one fourth off, they are X 2000 in the plates. Figure 51 was drawn with compensating ocular X 6, and is half this magnification.
I wish to express my appreciation of the careful and painstaking work of Mr. R. S. Outsell who made all my drawings for me.
Cu H., Weigert's copper hematoxylin P.A.F., Picro-aceto-formol (Bouin's)
FL, Flemming's fluid P-P-, Post-partum
Herm., Hermann's fluid Tri., Flemming's triple stain (safranI.H., Heidenhain's iron hematoxylin in, gentian violet, and orange G.)
EXPLANATION OF FIGURES
8 Transection of ovary of embryo 11 mm. long. The oviduct (Miillerian duct) is at the left in a fold which later fomis the perioan capsule. X 67.
9 Transection of ovary of embryo 17 mm. X 67.
10 Transection of ovary of mouse between 9 and 19 hours after birth. The capsule completely surrounds the ovary. X 67.
11 Transection of ovary of mouse 10 days after birth. The capsule was left out of this drawing. X 67.
12 Transection of ovary of mouse 15 days post partum. The center is filled with large follicles containing primitive oocytes. X 33.
13 Transection of ovary of mouse 39 days old — practically sexually mature. The definitive cortex filled with definitive oocytes in their follicles is shown, and the medulla with its blood vessels, stroma, etc. X 33.
EXPLANATION OF FIGURES
14 Cell of germinal epithelium, 'protobroque.' 16 mm. embryo. Herm., I.H.
15 Primitive oocyte in the epithelium. Transition from protobroque to leptotene. Same ovary as 14.
16 Primitive oocyte in leptotene stage. Two nucleoli are visible. 14 mm. embryo. P.A.F., I.H.
17 Beginning of synizesis, primitive oocyte. 17 mm. embryo. Carnoy's I.H.
18 Synizesis, primitive oocyte. 16 mm. embryo. Fl., Tri.
• 19 Pachytene stage, primitive oocyte. 19 mm. embryo. Herm., I.H.
20 Diplotene stage, primitive oocyte. Embryo near term. Herm., I.H.
21 Primitive oocyte, transition from diplotene to dictyate (dictye). Same ovary as figure 20.
22 Dictyate stage. Primitive oocyte in follicle of a single layer of flattened cells. The idiosome is visible. Mouse three days p.p. Fl., Tri.
23 Dictyate stage. The nucleus only of a primitive oocyte in a follicle of 4 or more layers of cells, with a cavity (antrum) just forming. Thirteen days p.p. Fl. Tri.
24 Cell of the germinal epithelium, resembling 'protobroque' stage. Twentyeight days p.p. Fl.,Tri.
25, 26, 27 Cells of the germinal epithelium in mitosis. Mouse eleven days p.p. P.A.F., I.H.
EXPLANATION OF FIGURES
28 Three cells from the germinal epithelimn (the free surface is up), 'd' is a definitive oocyte in stage 'a' ('protobroque'?), while the others are undifferentiated epithelial cells. Mouse eleven days p.p. P.A.F., I.H.
29 Germinal epithelium, 'd' is a definitive oocyte in stage 'a'; 'e' and 'f are oocytes in stage 'b'; 'g' is an ordinary epithelial cell, and 'h' is a cell of the tunica albuginea. Same ovary as figure 28.
30 to 33 Stage 'b'. 30 and 33 are from the same ovary as figure 28; 31 is from a mouse twelve days p.p., Herm., I.H. and 32 is from a mouse twelve days p.p., FL, Tri.
34 Transition, 'b' to 'c'. Same ovary as figure 28.
35 Transition, 'b' to 'c'. The germinal epithelium has been torn away from the ovary, probably through shrinkage, carrying with it a developing oocyte. Same ovary as figure 28.
36 Definitive oocyte, stage 'c'. Early stage of follicle formation. The free surface of the epithelium is up. Mouse ten days p.p., FL, I.H.
EXPLANATION OF FIGURES
37 Stage 'c'. This oocyte was surrounded by a follicle of flattened cells and was partly in the tunica albuginea beneath. Same ovary as figure 28. (Eleven days p.p., P.A.F., I.H.)
38 Stage 'c'. Follicle formation. The free surface of the epithelium is toward the left. Same ovary as figure 37.
39 Stage 'c'. This oocyte was in a follicle of flattened cells, under the epithelium and surrounded by the connective tissue cells of the tunica albuginea. Same ovary as figure 37.
40 Stage 'c'. Primary oocyte in follicle just under the germinal epithelium. Mouse twenty-eight days p.p. FL, Tri.
41 Oocyte in primary follicle of flattened cells. Mouse sexually mature (ninety days p.p.). Carnoy's, I.H.
42 Stage 'c'. Oocyte in primary follicle of cuboidal cells. Mouse sexually mature (sixty-seven days p.p.). FL, I.H.
43 Stage 'c'. Oocyte in follicle of tall cuboidal cells which are in two layers in places. Same ovary as figure 42.
EXPLANATION OF FIGURES
44 Stage 'c'. Nucleus of oocyte in follicle of 2 to 3 layers of cuboidal cells. Mouse sexually mature (sixty-seven days p.p.)- F^-, 1-H. (Same ovary as figure 42)
45 Stage 'c'. Nucleus of definitive oocyte in follicle of 3 to 5 layers of cells, A slight vacuolation in^the follicle indicates the beginning of the formation of a cavity (antrum folliculi). Adult mouse, pregnant. P.A.F., I.H.
46 Stage 'c'. Nucleus of definitive oocyte in mature Graafian follicle (this follicle is shown in text figure 7). Adult mouse, Herm., I.H.
47 Two cells from the germinal epithelium of an ovary twenty-eight days p.p. The upper cell is an indifferent epithelial cell and the lower is a definitive oocyte in stage 'a'. A mitochondrial technique was used (Benda's fluid, Cu H).
48 Definitive oocyte in germinal epitheliiun, stage 'b'. To show mitochondria. Zenker's + 2 drops of acetic acid; potassium dichromate, fourteen days; Cu H. Mouse seventeen days p.p.
49 Oocyte in the germinal epithelium, with cells flattened around each end (figs. 28, 29). Mitochondria and idiosome shoM'n. Same ovary as figure 48.
50 Oocyte in the germinal epithelimn, about the same stage as figure 28 or 29. Mitochondria and idiosome are shown. The former are mostly granules, but there is an indication of the formation of granular threads. Mouse eleven days p.p. Same technique as ovary shown in figure 48.
51 Oocyte in follicle of 3 to 4 layers of cells, showing the uniform distribution of the mitochondria. The idiosome is also shown. Mouse twenty-six days old. Zenker's + 2 drops of acetic acid; potassium dichromate, three weeks; Cu H. X 1000.
52 Part of an oocyte and adjacent follicle cells. Note that the mitochondria in the egg-cell are all granular, while both granules and threads appear in the follicle cells. Same ovary as figure 47.
Cite this page: Hill, M.A. (2024, March 4) Embryology Paper - Oogenesis in the white mouse (1917). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Paper_-_Oogenesis_in_the_white_mouse_(1917)
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