Paper - Origin of the sex-cords and definitive spermatogonia in the male chick (1916)
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Swift CH. Origin of the sex-cords and definitive spermatogonia in the male chick. (1916) Amer. J Anat. 20(3): 375-410.
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Origin Of The Sex-Cords And Definitive Spermatogonia In The Male Chick
Charles H. Swift
From the Hull Laboratory of Anatomy, University of Chicago
Introduction and review of literature 375
Materials and method of study 383
The indifferent gonad 385
Differentiation of sex and origin of the sexual cords 387
The evolution of the sexual or seminiferous cords of the embryonic testis... 390 The interstitial cells and the mitochondrial crescent of the spermatogoniimi. 403
Introduction and Review of Literature
The year 1870, in which appeared Waldeyer's famous article, Eierstock und Ei, really marks the beginning of modern research relative to the sex glands, their origin and products.
According to a majority of the investigators who have studied the question since 1870, there are two possible sources of origin of the definitive male cells — either from cells of the germinal epithelium or from primordial germ-cells.
These terms require some elucidation and wdll be explained before taking up the more important articles which bear on the subject.
In the early stages of all vertebrate embryos, the epithelium, or mesothelium, lining the coelomic cavity is flat and a single layer of cells in thickness. After the appearance of the Wolffian body the coelomic epithelium on its ventro-medial surface begins to change. The cells begin to elongate and become columnar or cylindrical and they may finally be several layers in thickness. This change is strictly limited; viewed in section the elongated cells protrude out from the general level of coelomic epitheUum like a .small narrow hillock and if looked at from above, the area has the appearance of a light narrow streak or stria on the medial and anterior face of the pink mesonephros. This region of differentiated coelomic epithelium is the germinal streak of Kolliker ('61) and the germinal epithelium of Waldeyer (70).
The primordial germ-cells (ureier, keimzellen, gonocytes, ovules males, ovules primordiaux, urgeschlechtszellen, and germcells of the various authors) are found among the cells of the germinal epithelium from its very commencement. They are easily recognized, for they are quite different from the surrounding cylindrical cells. They are large, oval or round, have a large excentric nucleus, which appears vesicular and does not have much chromatin. The cytoplasm also stains faintly. These cells were discerned by Bornhaupt ('67) and were clearly described by Waldeyer ('70), both investigators noticing them in the germinal epithelium of the chick. Since Bornhaupt many investigators in every vertebrate phylum have seen the cells, and yet there is great uncertainty, even at the present time, in regard to their origin and fate.
The earlier students, as a rule, believed that they arose in situ, in the germinal epithelium from the epithelial cells by a process of differentiation (Waldeyer, 70; Balfour, '78; and Semon, '87). Of late, however, there is an increasing belief that they antedate the germinal epithelium; that they originate in some region of the embryo at a distance from the site of the future gonads, and only migrate into the Wolffian region at about the time of the appearance of the germinal epithelium (Niissbaum, '80; Hoffman, '92; Eigenmann, '92; Beard, '04; Rubaschkin, '07; and Swift, '14).
Having given these short explanations of terms which will be frequently employed we are now in a position to review the more important papers which relate to the origin of the definite sex-cells in the male.
Waldeyer ('70), although he described so exactly the germinal epithelium and primordial germ-cells, did not believe that either played any role in the histogenesis of the male sex gland. He believed that the sex cords arose in the middle portion of the Wolffian body and that they grew into the stroma under the germinal epithelium. The primordial germ-cells degenerated and the definitive sex elements arose from the Wolffian tissue.
Braun (77), like Waldeyer (70) and Bornhaupt ('67), described two kinds of cells in the germinal epithelium, the cylindrical, differentiated, peritoneal or coelomic cells and the large primordial germ-cells. Braun found in reptiles (Lacerta) that the walls of the Wolffian tubules produced cell buds, which grew in height, anastomosed one with the other, and ended by sending up cord-like, cellular processes which fused with the germinal epithelium. He called these processes segmental cords, and believed that they gave rise to the seminiferous tubules of the testis.
In reality, Braun did not describe the origin of the true sexual cords, but rete cords, or cords of uro-genital union. Up to the time of Mihalkowics ('85) the true sex cords were constantly confused, both as to origin and function with the rete cords. The rete cords, or cords of uro-genital union, arise prior to the sex cords in the male and female. The rete cords, according to Mihalkowics ('85) and Sainmont ('05), are derived from the Wolffian tubules and the epithelium of Bo^\Tiian's capsule. According to Allen ('04, '05 and '06), they appear in reptiles and mammals as ingrowths of the germinal epithelium. Bouin ('00), in the frog, Firket ('14), and Swift ('15), in the chick, beheved them to arise as condensations of the mesenchyme between the germinal epithelium and the Wolffian body. Whatever their origin they do not produce the definitive sex-cells, but are developed into the tubuli recti and rete testis; interposed between the tubuli contorti, which are of true sex cord origin, which produce the definitive sex-cells, and the vasa efferentia, which are modified Wolffian tubules.
Mihalkowics ('85) described the origin of the true sex cords from the germinal epithelium and replaced Braun's name segmental cord with sex cord. He did not get the details exactly correct — that was left for Janosik — but his idea was correct.
He believed that cells of the germinal epithelium migrated into the subjacent mesenchyme and grouped themselves into cords which later united with the overlying epithelium, while Janosik described the sex cords arising as invaginations or processes of the germinal epithelium. For a very complete* account of the origin of rete cords and the two series of sex cords in the female and the single series in the male, the reader is referred to Allen ('04) and Firket ('14).
Ntissbaum ('80) in Rana and in Teleosts, agreed with most of investigators of the period prior to Mihalkowics, that the segmental or medullary cords arose as outgrowths of the Malphigian capsules. However, he disagreed with them as to the origin of the large clear, primordial germ-cells which were present in the cords and germinal epithelium. He did not believe them to be differentiated coelmic cells because he found them away from the site of the gonads, in the mesentery, because there were no transition forms and because they contained a great deal of vitellus, which was not present in the neighboring cells of the germinal epithelium. He believed them to be primitive cells and the direct ancestors of the adult sexual cells.
In passing it should be said that Ntissbaum was the first to hint at a distant site of origin and migration for the primordial germ-cells. He also formulated the hypothesis that the sexual cells do not come from any cells that have given up their embryonic character or have gone into building any part of the body, nor do sexual cells ever go into body formation." Here, then, is a suggestion of the geim-plasm idea.
Laulanie ('86), in the chick, thought that the primordial sexual gland was bi-sexual; that the germinal epithelium produced the female elements and that the male elements or cells were present in the network of cords, which were developed from the stroma in the midst of the gonad. The cortical ovules, in the germinal epithelium were female and the medullary ovules were male. According to his idea, the gonad was not indifferent but hermaphroditic. Later on in development one of the sexual elements degenerated and the gland became male or female as the case might be. This idea is of interest only because of its novelty and historic interest.
We now come to two important papers, which will always stand out as landmarks, and which, together with those of Waldeyer, Niissbaum and Mihalkowics, will always be studied in connection with any work on the origin of the sexual cells. I refer to the work of Semon ('87) and Hoffman ('92).
Semon studied the indifferent gonad and the testis of the chick. Like Balfour ('78), Braun ('77), and Nussbaum ('80), he believed the sex cords to be outgrowths of the capsules of the Wolffian bodies. He saw, as Waldeyer did, two kinds of cells in the germinal epithelium, the columnar epithelial coelomic cells, and the large clear primordial germ-cells. He described the various stages which the columnar cells passed through in becoming the primordial germ-cells. In fact, his opinion' in regard to the primordial germ-cells is exactly that of Waldeyer. He found that the sexual cords, growing out from the Malphigian bodies, reached the germinal epithelium about the sixth day and that the primordial germ-cells passed into them. The cords became separated from the epithelium by a connective tissue layer, differentiated from the stroma, called the albuginea. The cords anastomosed freely with one another, became tubular and in this way developed the tubuli contorti seminiferi. The cavity in the cords began to appear during the third week of incubation and at hatching had reached a great size. These tubes contained two kinds of cells, like the germinal epithelium, the small peritoneal cells and the large clear cells which resembled and were descendants of the primordial germ-cells. In regard to the products of these two varieties of cells it is better to quote Semon: Es kann natiirlich keinen Zweifel unterliegen, das die kleinen Zellen der Segmentalstrange die sogenannten Stiitzzellen der Samenkanalchen, die Ureier aber die grossen, runden Hodenzellen reprasentiernen."
Semon and Nussbaum ('80) were the first to show a continuity between the primordial germ-cells and the male sexual cells of the adult. It must be remembered, however, that Semon obtained his ureier from the cells of the germinal epithelium by a process of differentiation, while Niissbaum noticed the primordial germ-cells at a distance from the sexual primordium.
Hoffman ('92) employed in his researches about a dozen species of birds, mainly taken from the group of waders, and in three of them, Haematopus ostralegus, Sterna paradisea, and Gallinula chloropus, there was sufficient evidence brought out to show that some, if not all the primordial germ-cells, did not arise in the modified coelomic epithelium. In the three species mentioned above, he found at the proper time, numbers of the primordial ova in the germinal epithelium, but, in addition, he found cells, supposedly primordial ova, because of their resemblance to those found later in the germinal epithelium in embryos of 23 somites. An embryo of 23 somites does not possess the so-called germinal epithelium, the coelomic epithelium over the Wolffian body not having been modified at this age, yet in these he found primordial germ-cells far removed from the site of the future sex gland, in the splanchnic plate of mesoderm, in the region between splanchnic mesoderm and entoderm and in the entoderm itself.
To quote Hoffman :
Maintenant qu'il est evident que les ovules primitifs ne derivent pas des cellules peritoneales previlegiees, mais qu'ils se rencontrent deja dans tres jeunes periodes de developpement, bien que leur premiere ebauche chez les Vertebres, soit encore entierement inconnue il sera necessaire de laisser tomber le mot "epithelium genuinatif." Voila pourquoi j'appellerai desormais, cette partie de I'epithelium peritoneal, qui se transforme en un assise, dont les cellules sont disposees en pousieurs rangees et entre lesquelles sont placees les ovules primitifs, couche des ovules primitifs.
This couche des ovules primitifs" gave origin to the true sexual cords in the male, and the medullary cords and cortical cords in the female.
The true sexual cords in the male and the medullary cords in the female are homologous — the cords of first proliferation according to Firket ('14). The cortical cords — cords of second proliferation — occur only in the female and appear later. The true sexual cords in the male produce the sexual elements of the male. The medullary cords in the female — their equivalents — go partly into the formation of ovarian stroma, but in greater part disappear, while the second crop in the female — the cortical cords — give rise to the sexual elements in the female and the follicular epithelium (Swift, '15).
Hoffman found in Totanus calidrus, Vanellus cristatus, and Limosa ergocephala that the true sexual cords, just after hatching, developed a lumen and became the seminiferous tubule.
To quote Hoffman again :
Les tubes seminiferes renferment deux especes de cellules; des cellules grandes et rondes et des cellules plus petites d'une forme conique ou cylindrique qui les premiere enveloppent. Les grandes cellules ont un noyau arrondi et volumineux, qui ne contient que peu de chromatine et qui ne se colore que tres faiblement par les reactifs; son diametre est de 9-11 /x. Le corps protoplasmatique est egalement tres pale, les contours sont ordinairement tres indistincts. Le noyau des petites cellules est ovale, il a une longueur de 6-7 /z, une largeur de 4-5 fj, et est tiente tres fortement. Les deux especes de cellules se trouvent melees sans aucun ordre, ne formant ordinairement qu'une seule assise, comme cela se voit dans les sections, qui ont coupees le tube ou exactement dans son axe longitudinale ou trans versale. Entre les grandes cellules des tubes seminiferes et les ovules primordiaux de la couche des ovules primitifs, il n'y a pas de moindre difference. II n'est pas douteux que les grandes cellules soient des ovules primordiaux emigres et qu'elles representent les propres cellules du testicule — les spermatogonies — tandis que les petites cellules forment les cellules de soutient (Stiitzzellen des auteurs allemands).
Hoffman's work is of extreme importance because of the continuity which he established. The primordial germ-cell was first seen away from the germinal epithelium; it migrated into that structure, then passed into the sexual cord, which the germinal epithelium produced, became a spermatogonium, and later developed into the mature sexual cell.
According to Semon the peritoneal mesoblastic cell is the ancestor of the sperm; according to Hoffman the primordial germ-cell of whose ancestry he is ignorant, is the direct forebear of the sperm.
Allen ('04) made a very thorough study of the development of the testis and ovary of the pig and rabbit and found the sexand rete-cords to be invaginations of the peritoneum.
In regard to the primordial germ-cells he said:
Primitive sex cells are being formed in the rete cords from the syncitial cells that have retained the primitive character exhibited by the peritoneal cells, from which these cords arise. This development of undifferentiated peritoneal derivatives to form primitive sex cells is probably homologous with the process by which germinative cells (descendants of the peritoneal cells of the germinal epithelium) of the seminiferous tubules of the testis and the cells of the cords of Pfliiger of the ovary are being transformed into primitive sex-cells.
And again :
Clear transition forms are found to connect the primitive sex-cells with the germinative cells of the seminiferous tubules. Transition forms connecting the germinative cells with the sex cells occur in the pig embryos between 2.5 cm. and 13 cm. leng-th; later states show no transition forms.
As regards the formation of the definitive sexual cells he said :
It has been shown in the pig and rabbit, however, that these sex cells, appearing in the indifferent stages, do not contribute to the formation of functional sex products in the ovary. The same is probably true of the testis, it being at least certain that the great bulk of the spermatogonia are formed from the germinative cells — cells derived originall}' from the peritoneum and maintaining, first, their indifferent character, at least so far as our technique is able to show.
Dustin ('07, '10) studied the question of germ-cells origin, migration and evolution in Amphibia — Triton and Rana— and in Reptilia^ — Chrysemys.
In the first paper, dealing with the Amphibia, he reported that the primordial germ-cells were of mesoblastic origin ; from a part of the primitive coelom which he called gonocle. He called the germ cells gonocytes of the first line and found that they reached the sites of the definitive sex glands by active migration. He found that a second line of gonocytes was produced by transformation of small germinative cells, which were in their turn the result of peritoneal epitheliimi proliferation. Both lines of gonocytes were able to produce definitive sexual cells.
In his second paper relating to Chrysemys, he found that there were also two lines of gonocytes, with this exception, that in the reptile the primary gonocyte came from the entoderm.
Deux especes de cellules sexuelles se succedent done au cours de I'ontogenese ; Tune privient de I'endoderme; elle est extraordinairement precoce et subit line serie de migrations qui liii assurcnt trois localisations succcssives (gl. paires primaries, glande impaire, gl. paires definitives); I'autre est d'origine mesodermique et se developpe in situ, au niveau des glandes definitives.
Chez Chrysemys marginata, les gonocytes de la premiere espece ne presentent de karyokineses qu'au debut de leur differenciation ; ulterieurement ils ne se multiplient plus; beaucoup d'entre eux deg^nerent meme.
He believed that more of the primary gonocytes functioned, in producing definitive sexual cells, in Amphibia than in Reptilia.
Rubaschkin ('12), working with the guinea-pig, stated that the definitive sex cells in both testis and ovary are descendants of the primordial germ-cells. Rubaschkin used as his criterion for distinguishing the primordial germ-cells the granular type of mitochondria. He believed that the primitive cells, or blastomeres, possessed this type of mitochondria, and that as the somatic tissues were differentiated from them, the cells of the tissues acquired the rod-shaped mitochondria. In this way he was able to recognize the germ-cell at any stage in the young embryo and differentiate it from the ordinary somatic cell. By this means he came to the conclusion that the definitive sex cells were descended from the primordial germ-cells.
For a more detailed account of the literature on the subject of the origin of the definitive sexual cells, the reader is referred to the ample bibliographies of Bouin ('00), Allen ('04), and Firket ('14).
Materials and Method of Study
In the course of this investigation two fixatives were employednamely, Meves' modification of Flemming's fluid and the acetic, osmic-bichromate mixture. In a previous investigation on the primordial germ-cells of the chick ('14) the osmic acid content of these fluids was found to be a serious disadvantage owing to their staining of the numerous vitellus granules, which, when blackened, obscured the cytological details. However, that drawback was not present in the embryos and young chicks used in this research for the germ-cells had lost their vitellus content, and the preservation of the mitochondrial granules and attractions sphere was a decided asset.
In this work only the testes were fixed and not the whole embryo, as was the custom, when dealing with the younger stages.
In the case of the youngest stages used in this investigation, the testes were allowed to remain in the killing fluid 24 hours but as there were signs of over-fixation in the sections, this time was reduced to 10 hours with much better results.
All the sections were cut 4/z in thickness, and following the acetic-osmic-bichromate fluid stained by Bensley's anilin-acid fuchsin — Wright's blood stain method and the anilin-acid-fuchsin-methyl green method. There is no need of entering into detail in regard to these staining methods since they have been described by Bensley ('11), Cowdry ('12), and Swift ('15).
Following Meves' fluid the iron hematoxylin stain was employed.
The following table will show at a glance the number of stages employed, their age, and the methods used in fixation and staining.
Meves' fixation and iron-hematoxylin stain
Bensley's anilin acid fuchsin and methylereen stain
osmic-bichromate ' fixation
Bensley's anilin acid fuchsin and Wright's blood stain
The Indifferent Gonad
If the viscera are removed from the abdominal cavity of a 5-day chick embryo, the Wolffian bodies will be seen on the posterior wall on either side of the mid-line. On the ventromedial surface of each mesonephros, beginning at the cephalad extremity and extending caudad about two-thirds of the length of each Wolffian body, a narrow, white, rounded ridge will be observed. These narrow elongated elevations are the indifferent gonads. At this time they have a length of about 2 mm. and there is a noficeable difference in size in favor of the left gonad.
On examining a section through the anterior portion of the Wolffian body, the gonads will be seen to resemble two small abrupt hillocks on the surface of the mesonephros turned towards the mesentery, and, it will be noticed also that they have a broad connection with the mesonephros.
The microscope reveals, farther, that there are "three distinct tissues in the gonads.
Clothing the free surface of each genital anlagen, and extending for some distance over the root of the mesentery, is an epithelium made up of two to three layers of tall columnar cells. This tissue is the germinal epithelium of Bornhaupt ('67) and Waldeyer ('70) and all later investigators. This epithelium is made up of columnar or rather cylindrical cells, which have oval or round deep staining nuclei and possesses a distinct basement membrane. This last structure is very noticeable and at this stage — 5 days — can be followed over the germinal hillock as a regular curved line without any irregularities of any kind.
Under the germinal epithelium, which is thicker and more extensive over the left gonad, is another tissue — the stroma of the gonad. This tissue is nothing but embryonic mesenchyme, and as such is directly continuous with the same tissue in the Wolffian body and in the root of the developing mesentery. The cells of this tissue are loosely packed and have faintly staining nuclei. The cytoplasmic part of the cells appears as indistinctly bounded processes which anastomose with the processes of other neighboring cells. The cells, then, are apt to be stellate in appearance and the whole tissue to resemble a loose syncytium. This stroma, more voluminous in the case of the left gonad, fills up the region between the concave germinal epithelium and the Wolffian body and, in fact, forms most of the area of the gonad.
This stroma is denser just under the epithelium, and from this region, if the sections happens to be right, a narrow cord may be seen to extend obliquely towards the Wolffian body. This cord, the result of a condensation of naesenchyme tissue, is known as a rete cord or cord of uro-genital union and must not be confused with the sexual cord, which arises later and iri a different way. According to Firket ('14) there are 16 of these cords. It is not my intention to enter into the origin of these cords or to engage in the controversy as to their origin, for an excellent review of all the facts and literature will be found in Firket's ('14)- article.
The third tissue present in the gonad is made up of primordial germ-cells.
The cells are easily seen, for their great size, large nucleus, and clear cytoplasm make them conspicuous in the chick as in all the other forms in which they have been observed. They merit, however, a more extended description.
The primordial germ-cells do not form a compact or continuous tissue in the indifferent gonad of 5 days, but are present, usually, as isolated cells or groups of several cells in the germinal epithelium and subjacent stroma (Swift, '15). They may be even seen in the root of the mesentery subject to the same arrangement.
These primordial germ-cells are, at a glance, seen to be different from the other tissues of the gonad. Although present in the epithelium and stroma of the genital hillock yet I have never seen a stage which could be called a transition between them and the surrounding cells, for in addition to their large size, great nucleus and clear cytoplasm, they possess even at this late date (5 days) many droplets of vitellus in the cytoplasm and a conspicuous attraction-sphere (Swift '14 and '15). The cells of the germiiuil epithelium and stroma have none of the vitellus and inconspicuous spheres.
In fact the primordial germ-cells do not originate in the germinal epithelium as was believed by many of the older investigators, but at a distance from the site of the gonad. They arise in the germ wall entoderm (Swift, '14) and after an extended migration through the blood vessels (Swift '14 and von Berenberg-Gossler, '14) they reach the splanchnopleure entering into the formation of the mesentery, where they leave the vessels and then migrate in an amoeboid manner into the forming germinal epithelium.
During all their migration period and up to this time they divide very infrequently. That they do divide is proven by the increase of numbers from stage to stage (Firket, '14, and Swift, '14 and '15) and by the fact that they are frequently seen in small groups, which are probably the result of several successive divisions.
As was indicated in the short historical review of this article, it is still an open question as to the role played by the primordial germ-cells in the formation of the definitive sex-cells in the. male.
DIFFERENTIATION OF SEX AND ORIGIN OF THE SEXUAL CORDS
The period of embryonic development in the chick from 5| to 6^ days is of extreme interest and importance, for during that period the sexual cords appear and at its termination it is possible to tell the sex of the individual.
The sexual cords, which begin to appear at about the 132d hour of development, are the true sexual cords in the male or seminiferous cords, and in the female the medullary cords or cords of first proliferation. As the names imply, there is only one series of cords in the male while in the female there are two — the last series being known as the cortical cords or cords of second proliferation (Mihalkowics, '85, Hoffmann, '92, Firket, '14 and Swift, '15).
The cords of first proliferation are first noticed as buds or protrusions of the germinal epithelium into the underlying stroma. The basement membrane appears wavy bat is continuous around the swelling. The buds enlarge and elongate but remain attached to the germinal epithelium for some time (fig. 5, Swift, '15). These cords, which are at first small, are evidently of germinal epithelium origin, for the basement membrane of the latter structure is, for some time, continuous around the cord. Neither are they formed by infolding or invagination of the germinal epithelium, for in that case a tubular lumen would be present in each cord, and in some cases could be seen connected with the coelomic cavity. They are simply the result of increased local mitotic activity in the germinal epithelium. The primordial germ-cells which are present in the cords do not play any evident role in their formation, for none are ever found dividing just at the time of cord formation, while the peritoneal cells show signs of increased activity.
Although the cords begin to appear at 5| days, yet it is not until 12 hours later that the greatest activity of the germinal epithelium in their production occurs. At 6§ days they have about ceased to appear.
At the end of the period of cord production the interior of the gonad seems to be made up nearly entirely of sexual cords. They are close together, separated from one another by a little stroma; a majority of the cords are no longer attached to the germinal epithelium but are still surrounded by a definite basement membrane.
The cords have become autonomous, for, although no longer attached to the germinal epithelium, they are growing rapidly as is indicated by the rapid increase in diameter of the cord and size of the gonads.
The cords have a definite orientation — they are straight and extend from the germinal epithelium down towards the Wolffian body.
The primordial germ-cells are during this period found in three situations in the gonad, in the germinal epithelium, in the sexual cord, and in the small amount of stroma between the cords.
Wlien the embryo chick has reached the 156th hour of development (6^ days), the formation of cords of first proUferation ceases rather abruptly, and about this time it is possible to determine the sex of the individual.
In determining the sex of the embryo there are several criteria which are of value.
1. The relative size of the gonads. It has been known for a long time that the left gonad in the female grows rapidly while the right does not, so that in a short time after sexual cord formation, the left gonad or ovary has far outgrown the right. In the case of the male individual both gonads continue to grow. This criterion is of value, but too much reliance can be placed upon it especially during the 6th day, for in all cases, male as well as female, the left gonad is the larger. The left gonad is the larger during the indifferent stage and in the male is the larger even in the adult (Etzold, '91).
2. Thickness of the germinal epithelium. This and the following criterion I believe to be the most important. In the male the germinal epithelium is thin at the end of sexual cord formation and continues so, while in the female the germinal epithelium over the left gonad or ovary still consists of the several layers of columnar cells. In the male the epithelial cells become very quickly a single layer in thickness and the individual cells cuboid in character. This is an excellent test.
3. Number of primordial germ-cells in the germinal epithelium. In the germinal epithelium of the left female gonad the number of primordial germ-cells appears undiminished, while in the epitheliimi covering the male gonads there is hardly a single one to be seen (Hoffman, '92 and Swift, '15). In the case of the male they all appear to have gone into the sexual cords, and in the female to be remaining quiescent until the formation of the cortical cords, in the evolution of which they play so important a role (Swift, '15).
4. Attachment and growth of the sexual cords. The cords remain thin and attached to the germinal epithelium for a long time in the male gonad, while in the female they increase rapidly in diameter and become detached early in their evolution.
This rapid increase in size of the cords accounts for the more rapid growth of the embr\'onic ovan'.
This last criterion is of less weight than the preceding, and is of importance only when used in connection with the others.
The Evolution Of The Sexual Or Semixiferous Cords Of The Embryonic Testis
The next three embn.-onic stages studied, namely, 7, 8; and 9 days, may be described together, since they are characterized in common by the same facts, and the changes although occurring, are not abrupt or great.
During this period the sex of the indi\'idual can be easily ascertained, either with or without the aid of the microscope. In the latter case the first criterion mentioned above, relative size of the gonads, is amply sufficient, and with the microscope a single glance at the germinal epithelium, the sexual cords, and the number of primordial germ-cells in the epithelium is all that is needed.
Both testes are slowly increasing in size but in all cases the left is found to be the larger. They are becoming rounder and in the process the broad connection with the Wolffian body is being narrowed: in other words, the}' are being constricted or pinched off of the Wolffian body.
The germinal epitheUum is reduced to a single laj'er of cuboidal cells, and by the end of this period of 7 to 9 days of development, all connection with seminiferous cords is severed. This is brought about by a condensation of the mesenchj-me imder the epithelium to form the tunica albuginea.
The seminiferous cords, which will in the future claim most of our attention, make up the greater part of the testis. They have a definite orientation from the epithehimi obliquely toward the narrowing attachment of the testis with the Wolffian body.' Each cord is, of course, not entirety straight, but wav}'. They are separated one from another by a verj' thin layer, one might almost sa3' fihn, of stroma and a number can be followed at one time across the field of the microscope.
The cells of the seminiferous cords are of two kiiid? — the ordman' peritoneal cells, issue of the germinal epithelium, and the primordial germ-cells. The fomier are the more numerous and, at this period, can be easily seen to have definite membranes. They still preser^'e their cylindrical form, acquired in the formation of the germinal epithelium, and the dark staining round or oval nucleus.
The primordial germ-cells, although not numerous, are as conspicuous as when obser^'ed in the capillar}' of a 21 somite chick or the root of the forming mesentery- (Swift, '14). They are a Uttle smaller and have lost the characteristic ^'itellus but in all other ways are unchanged. They are still large, have the same clear cytoplasm, large round nucleus and conspicuous attraction sphere. From 2 to 8 of the primordial germ-cells are present in a single cord and there are no signs of division. In 9 embryos of this age studied I was not able to find a single primordial germ-cell in mitosis. This last fact is of interest when a comparison is made with the primordial germ-cells in the germinal epithelimn of the oyaiy of the same age. It will be recalled (Swift, '15) that during the 8th and 9th da^' in the female the gemi-cells are in an extremely active condition and that the formation of the cortical cords is well under way.
As regards the stroma only a few lines are necessars'. Only under the gemiinal epithehum is the connective tissue development considerable. In that region there is a broad band of tissue, the albuginea. In the rest of the gonad, between the seminiferous cords, the small anioimt of stroma still remains embr^'onic in nature, forming a kind of mesenchATual sjTicytium. It resembles the mesenchjTne of the mesonephros from which it was originally derived and with which it is still connected.
Four embrj'os aged 11 days were studied. Considerable change has taken place. The testes have increased greatly in size, are rounder, but still have a narrow attachment to the Wolffian body. On examining a section through the testis it will be noticed immediately that the stroma has increased in amount between the seminiferous cords (fig. 1), and that the layer under the epithelium has thickened. In fact most of the increase in the size of the testes is due to the increase in amount of connective tissue. The cells of this tissue are still mesenchymal in character; cell boundaries are hard to determine and the cytoplasmic processes of the cells unite forming a kind of syncytium. The nuclei are round or oval (fig. 1).
The seminiferous cords no longer run in a regular way as described above, but form a kind of network. On studying them one receives the impression that they have grown considerably in length and as a result have had to become convoluted in order to adapt themselves to a slower growing space. In all four embryos studied several peculiarities were found to be common in regard to the seminiferous cords. In the first place a cord runs only a short distance across the field before being lost. This indicates folding (fig. 1). In the second place there are more cords present in that part of the testis next the Wolffian body, and here the net formed by the cords is more compact. In the region towards the germinal epithelium there is always a single cord which tends to run parallel to the germinal epithelium separated from it by the developing albuginea and from the other cords by a thick layer of stroma.
The seminiferous cords are surrounded by a definite basement membrane (fig. 1), which they acquired when given off from the germinal epithelium.
The cells present in the cords are principally of the peritoneal t3^pe, although an occasional primordial germ-cell can be seen (fig. 1). The cell walls of the peritoneal cells can be seen but are not so distinct as formerly. Peritoneal cells are occasionally seen dividing but the primordial germ-cells are still quiescent. In this stage the increase in the cords is one of length but not of diameter.
In the 13 day embryo several important changes must be recorded.
There has been a great increase in the amount of stroma (fig. 2) and as a result of this, in the size of the testis, which is now almost separated from the mesonephros. In an embryo four days younger the stroma was present simply as a thin film between the seminiferous cords; two days later there had been some increase in the connective tissue, especially in the region under the germinal epithelium, but in these embryos of 13 days development the area of the mesenchyme far exceeds that of the cords (fig. 2). In addition a few interstitial cells are present in the stroma between the cords. These interstitial cells are very numerous in later stages, so that an account of them and their development will be deferred until those are described.
Fig. 1 Portion of a transverse section through the left testis of an 11 day chick embryo. This section shows the seminiferous cords, composed of peritoneal cells and containing several primordial germ-cells. The cords are separated from one another by a mesenchyme-like connective tissue.
The figures illustrating this article Avere drawn by Mr. A. B. Streedain. Zeiss apochromatic objective 1.5 mm., and compensating ocular 6 were employed for all the figures. The camei-a lucida was used in making all the drawings and magnification calculated at table level in all cases. The figures were reduced by one-fourth in reproduction giving a magnification of 1125 diameters for all the illustrations. The figures were drawn from preparations fixed in Bensley's acetic-osmic bichromate mixture and stained with Bensley's anilin acid fuchsia — Wright's blood stain. All the sections were cut 4 n in thickness.
int.C, interstitial cells
L., lumen of seminiferous cord
M.C., mitochondrial crescent
p.c, peritoneal cell
pr.o.. primordial germ-cell
-pr'.o'., primordial germ-cell in mitosis.
S., cell of support
S.C., seminiferous cord
Sp'., spermatogonium in mitosis
The seminiferous cords have a larger diameter, and now fomi an open net with large interstices (fig. 2). The cords anastomose with each other in eveiy conceivable plane and direction, so that their original orientation is completely lost. This tangle of cords now occupies the entire testis, the area of mesenchyme under the genninal epithelium, described in a previous stage,
Fig. 2 Portion of a transverse section through the left testis of a 13 day chick embryo. This section shows a seminiferous cord containing many primordial germ-cells. Two of the germ -cells are dividing.
having disappeared. The cords stain more intensely now, stand out more sharply against the light connective-tissue background and contain peritoneal cells and i)rimordial genn-cells. There has been an important change, however, for the latter are much more nimierous than in any preceding age (fig. 2). This increase in number of the germ-cells is evident even when examining the section under the low power microscope, for they are seen as clear spots thickly sprinkled throughout the cords. On using the higher powers this is confirmed, and, in addition, it is seen that the prmiordial genn-cells are actively dividing (fig. 2). In a single section through the testis from 4-10 are foimd in mitosis. The increase in the size of the seminiferous cord is due to the increase in the number of the germcells for the peritoneal cells are quiescent.
Semon ('87) in the chick and Popoff ('09) in the chick and a number of other fonns, described the 'ureier' and 'les o\ailes males' (primordial germ-cells) as having a definite position near the center of the seminiferous cords, while the peritoneal cells occupied the periphery. I have not found that to be the case (figs. 2 and 3). In all stages up to and including the 15 daj^ stages examined, I find them to be scattered evenly throughout the cord — some at its center and some at the periphery. Later on they are all found at the periphery of the cord.
A few words are necessary in regard to the structure of the primordial germ-cells. As will be seen from all the figures illustrating this article there is little change in the primordial germcells or their tissue. In fact, there is little change to record in all their history from origin until they become oogonia or spermatogonia, unless it is a gradual loss of vitellus and a progressive slight decrease in size (Swift, '14 and '15). However, with the beginning of division in the female line of germ-cells, which resulted in the oogonia, there was a change in the arrangement of the mitochondria (Swift, '15). While the primordial germcells were quiescent in the germinal epithelium of the embryonic ovary, up to the 8 day stage, the mitochondria were evenly scattered in the cytoplasm (Swift, '15). At 8 days the germ cells began to divide, and in all the later stages the mitochondria were found grouped around the attraction-sphere in a characteristic manner. It is a striking and suggestive fact that the same change occurs in the male line. In all the primordial germ-cells in the sexual cords of the male chick, up to and including the 1 1 day stage, the rod shaped and granular mitochondria are evenly distributed (fig. 1). This arrangement also holds for the germ-cells of the 13 day embryo and for the few present in the 15 day stage.
Fig. 3 Portion of a, transverse section through tlie right testis of a 15 day chick embryo. This section shows a massive seminiferous cord, composed of primordial germ-cells, spermatogonia and peritoneal cells. One of the germcells is dividing. The spermatogonia, at the left, contain the characteristic mitochondrial crescent which serves to identify them.
Thus, beginning with the 13 da}^ embryo, a new mitochondrial arrangement is noticed; the mitochondria are grouped around the attraction-sphere, so that they form a granular crescent or cap on the nucleus, depending upon the plane of the section (figs. 3, 4, 5 and 6). As was stated above and in a former article (Swift, '15) this same arrangement was observed in the oogonia of the chick and it is my beUef that any cell in the male which has this character is a spennatogonium. Certainly all the spermatogonia of much later periods possess this distinguishing character. I believe, then, that spermatogonia foniiation begins in the male chick embryo at 13 days as a result of primordial germ-cell division, and that the peculiar arrangement of mitochondria around the attraction-sphere, which will be taken up at greater length in a later chapter, distinguishes the spermatogonium from the primordial germ-cell.
Fig. 4 Portion of a transverse section through the left testis of a 17 day chick embryo. This section of a massive seminiferous cord contains peritoneal cells and spermatogonia. All the spermatogonia contain the mitochondrial crescent. At this age practically all the primordial germ-cells have given way to the spermatogonia.
At this time, also, it is necessary to say something about the continuity of the mitochondria in the germ line.
In this regard, I can say that they are always present in the primordial genn-cells, oogonia and spermatogonia, either as short rods or granules, depending somewhat upon the fixation. About the same amount seems to be present in the germ-cells at all stages, from origin in the primitive streak stage to oogonia and spermatogonia formation in the 8 and 13 day embryo respectively. Even in the oogonia and spermatogonia the number of mitochondria remains about as in the germ-cells, for the mass around the attraction-sphere is formed at the expense of the rest of the cytoplasm (figs. 4, 5 and 6).
In the 15 day chick embryo there are an immense number of interstitial cells in the stroma. They appear grouped in masses and cords in the stroma between the seminiferous cords. So numerous are they that it is possible to identify them in the section before removal of the paraffin. This can be easily done since they appear black, owing to the staining of their many fatty granules by the osmic acid.
The seminiferous cords branch and anastomose to form a net as described under the 13 day embryo. They contain, however, relatively and absolutely, many more spermatogonia, for the increase has been in that element owing to continued division of the germ-cells (fig. 3). The number of spermatogonia, compared with the preceding stage, is enormous. They are seen several hundred in a field, when the low power microscope is used. Numbers of mitoses in the primordial germ-cells and spermatogonia are present (fig. 3), and this is the stage in which they are found dividing most actively.
In regard to the 17 day chick embryo not a great deal need be said, for, in it, the findings closely resemble those described in the 15 day embryo.
The interstitial cells are present in greater quantity, and there has been some increase in the size of the cords ; the amount of cord tissue, too, as compared with the stroma, has increased (fig. 4). Practically all the primordial germ-cells have been changed by division, giving rise to the spermatogonia (fig. 4). There is no evidence of division in the peritoneal cells but there number has not decreased. In certain regions of the seminiferous cord complex, it is evident that the spermatogonia are beginning to assume a position at the periphery of the cord, next to the basement membrane. This is very evident in the next stage. There is no evidence of a lumen beginning to appear in the cords. They still are solid, although it was at this age that Semon ('87), in the chick, described one as being present.
In the next embryo, that of 20 days development, there are some important changes to record.
The seminiferous cords are very massive and seem to be growing at the expense of the stroma, which is considerably reduced in quantity (fig. 5). The interstitial cells (fig. 5), too, are not as numerous as in the two preceding stages. The greatest changes, however, have taken place in the structure and orientation of the cords.
The spermatogonia, which hitherto have had no definite arrangement, being scattered helter-skelter throughout the cord, now begin to be arranged in a definite manner. They are placed against the basement membrane of the seminiferous cord in such a way that their long axes are at right angles to the long axis of the cord (fig. 5). Another striking fact is to be noted, that the attraction-sphere, with its mitochondrial body, is next to the basement membrane, while the nucleus is turned toward the axis of the cord (fig. 5). That is to say, the vegetative pole of the spermatogonium is next the basement membrane. The nucleus is excentrically placed, and always the greatest mass of cytoplasm, in which is placed the attractionsphere, is towards the basement membrane (fig. 5).
The cords, also, are beginning to have a cavity. This cavity is far from being continuous and can be seen here and there only, in the central axis of the seminiferous cord (fig. 5). This lumen is formed, not as one would suppose, by a fissure appearing between cells and then enlarging, but by a liquefaction of the cells in the central axis of the cord. When this destruction which involves chiefly the peritoneal cells, is complete, a slit appears in the debris and the lumen is formed (fig. 5). The peri
Fig. 5 Portion of a transverse section through the right testis of a 20 daychick embryo. This section shows a seminiferous cord in which a lumen is beginning to develop. The spermatogonia are next to the basement membrane, with elongating peritoneal cells between them. Notice that the mitochondrial crescents of the spermatogonia are next to the basement membrane of the cord. A small mass of interstitial cells is present in the stroma.
toneal cells are the principal sufferers because the spermatogonia have placed themselves along the wall. ..:J do not know positively through what agency they are .Enabled to place themselves against the basement membrane, • byt suppose that it is through their ability to mo\'e like an amoeba. They or rather their ancestors, the primordial germcells, had this power in their early history, and it may ha\'e b(HMi in abeyance during their sojourn in the germinal epithelimn and sex cords, only to be assumed again at this time. Color is lent to this theory by the fact that frequently, at this stage, the end of the spermatogonium turned toward the basement mem
Fig 6 Portion of a transverse section through the left testis of a chick 3 days old. This section shows a seminiferous tubule containing spermatogonia and supporting cells. The spermatogonia are next the basement membrane and have a definite polarity — nucleus near the central portion of the tubule and mitochondrial crescent at the basement membrane. The supporting cells are descendants of the peritoneal cells.
brane is pointed, as if it were forcing its way in that direction between the intervening cells (fig. 5).
The cords, although forming a network, are again beginning to have a definite orientation, that is, they run obliquely from the germinal epithelium towards the remains of the Wolffian body.
In this, and the next stage to be described, the position of the peritoneal cells is also of great interest. In general there are one to three of them between adjacent spermatogonia and their long axes, too, are at right angles to the long axis of the cord (figs. 5 and 6). Their nuclei are placed next the basement membrane, while most of the cytoplasm is towards the central axis of the cord or tubule (figs. 5 and 6). This is the reverse of the condition in the spermatogonia.
In this stage and in the next we begin to have an idea of what obtains in the adults which have been studied. The spermatogonia at the basement membrane of the tube and between them the supporting cells — cells of Setroli — which are derived from the peritoneal cells.
In the next stage — a 3 day chick — the stroma is greatly reduced in amount as are the interstitial cells also. Most of the testis is taken up by the seminiferous cords and tubules, which are arranged in the way described in the 20 day chick embryo.
Most of the seminiferous cords have by this time acquired a ca\dty and may now be called tubules. In these seminiferous tubules nearly all the speniiatogonia are against the basement membranes, as described before (fig. 6). The peritoneal cells have lengthened, or been pressed out, so that they begin to look like the supporting cells of an adult seminiferous tubule (fig. 6). In this as well as in the last stage, the spermatogonia are occasionally seen dividmg (fig. 6). In these rare cases the mitotic figure is arranged, not like that which gives origin to the spermatocyte, but so that the line of cleavage between the daughter cells is at right angles to the long axis of the seminiferous tubule (fig. 6). In this way both daughter spermatogonia are kept in their proper position, next the membrane, and the number of peritoneal cells, or cells of support, which is still too large, is reduced by pressure (fig. 6).
In the 6 and 10 day chicks there are no changes of any moment to record, except that there is a progressive increase in the size of the lumen of the seminiferous cords and a reduction in the amount of stroma and in the number of interstitial cells. The cavity in the seminiferous cord, even at 10 days is slit-Uke
iii(l is very far from resembling the large cavity pictm-ed by Semon ('87) in stages of about this age.
THE INTERSTITIAL CELLS AND THE MITOCHONDRIAL CRESCENT OF THE SPERMATOGONIUM
It is not possible to enter into any account of the various opinions which have been advanced and held as regards the origin of the interstitial cells of the testis. For the various theories on this subject the reader is referred to Sainmont's article which appeared in 1905. However, to give a slight historical background a few leading articles will be cited.
Tourneux (79) identified the interstitial cells in the testes of various animals and described them as being differentiated connective tissue cells.
Niissbaum ('80) thought that the interstitial cells were derived from cell columns, which were given off by the germinal epithelium in early embryonic history.
Plato ('97) described the different stages which exist between the connective tissue cells and the differentiated interstitial cells. He studied the question in the testes of the cat and various other animals. In connection with this work he brought forward a rather novel theory which ascribed a nourishing function to the interstitial cells. He beheved that they manufactured fat, which was passed through the basement membrane of the seminiferous tubule into the cells of Sertoli and used as food in spermatogenesis.
Allen ('03) and Whitehead ('04) believed that the connective tissue elements of both testis and ovary of the pig and rabbit, from which the interstitial cells were differentiated were derived from the peritoneum. Allen says: "In early stages they (the connective tissue elements) are not distinguishable from the cells which make up the sex-cords, except that the latter are marked off from the stroma by their membrana propria."
Whitehead "found himself in accord with the conclusion of Allen, that the mterstitial tissue of the testis is derived from the peritoneum, meaning thereby the mesothelium of the genital ridge."
There are, then, two main opinions held as to the origin of the interstitial cells; the one school, including Kolliker, Tourneux (79) and Plato ('97), hold that they arise from the connective tissue cells; the other group, to which belong Niissbaum ('80) and von Bardeleben, believe that they come from the general epithelium. Allen ('03) and Whitehead ('04) must be classed as holding the latter, and Sainmont ('05) the former opinion.
In the male chick embryo the interstitial cells are first evident in the 13 day testis. They are not numerous and are evenly scattered throughout the testis. Usually they appear as single cells but at times 2-6 may be found together. The cells themselves have various forms, sometimes cubical, sometimes polyhedral and frequently fusiform. The nucleus is round or oval, stains deeply and usually has a central position (fig. 5). The cytoplasm of these cells also stains strongly and appears dense (fig. 5). The interstitial cells are remarkable, however, because of the immense amoimt of fat which they contain. This fat in the stained specimen has been dissolved out, but the vacuoles, which contained it are present, and occupy no small part of the cytoplasm (fig. 5). If the section be examined carefully intermediate stages between the connective tissue cells and the interstitial cells can be seen. That is, certain of the connective tissue cells will be seen containing small vacuoles and arranged at times in small groups.
In the 15 and 17 day embryonic testes there is an immense amount of interstitial cell tissue. It is arranged in masses and cords of vaiying sizes between the seminiferous cords. The interstitial cells of these and later stages can be easily seen before the section is stained and while it is still in paraffin. The interstitial cell cords and masses, owing to the contained fat, stain black with osmic acid and hence show up dark against the lighter background. Sainmont ('05) described a rich plexus of capillaries in relation to these masses of interstitial cells in the cat, but in the chick there seems to be no increase in vascularity around them.
Beginning with hatching and continuhig until the chick is 10 days old there is a progressive decrease in the amount of interstitial tissue as compared witli tlie true connective tissue and the seminiferous cords. I do not know whether there is an actual decrease, or only apparent, due to the great increase in the size of the testis. In any event I was not able to find any signs of degeneration in the interstitial cells.
It is an interesting fact that the first appearance of the interstitial cells in the testis of the 13 day embryo is synchronous with the appearance of the spermatogonia, and that they only reach their maximum development when all the primordial germ-cells have become spermatogonia.
There is no doubt, in the chick, that they arise from the connective tissue of the testis, which in its turn is derived from the general mesenchyme of the mesonephros. That they arise from the connective tissue elements by simple differentiation is proven, first, by the presence of transitional forms at the time of their origin, and secondly, by the very rapid increase in the numbers of the interstitial cells in the absence of any evidence of division.
In a previous paper (Swift, '15), and in a preceding page of this article, a short account was given of the arrangement of the mitochondria around the attraction-sphere of the oogonia and spermatogonia respectively. I shall hereafter call this body, made up of sphere and mitochondria, the mitochondrial crescent.
As has been previously stated, the mitochondrial crescent consists of attraction-sphere and mitochondria, and occupies that part of the cell in which the cytoplasm is most voluminous ; in other words, it occupies a part of the vegetative pole of the cell. The whole body in one plane, appears like a crescent fixed on, or capping the nucleus, the attraction-sphere in the middle enclosing the centrosomes, around it a thin clear area, and extending down on either side the arms of the crescent composed of mitochondria (figs. 3, 4, 5 and 6). In case the knife does not section the nucleus, but passes through the sphere, then the mitochondrial crescent appears as a circular mass, composed of attraction-sphere, clear area and surrounding circle of mitochondria (fig. 4). In nearly all cases the cytoplasm which suspends the mitochondrial portion of the mitochondrial crescent appears denser and stains darker than that of the rest of the cell (figs. 4, 5 and 6).
This mitochondrial crescent appears in cells which are the result of primordial germ-cell division, in other words, in the oogonia and spermatogonia. It appears in a few cells in the female at 8 days and in the male at 13 days and this marks the ending of the primordial germ-cell line and the beginning of the oogonial and spermatogonia! generations. By 17 days in the male (fig. 4) eveiy cell of primordial germ-cell lineage possesses it, thus indicating that all are spermatogonia. When the spermatogonia place themselves against the basement membrane, when the cavity begins to appear in the cords, they do so in such a manner that the mitochondrial crescent is against the basement membrane (figs. 5 and 6).
This mitochondrial crescent, whose appearance and formation I have just described, is the same structure which D'HoUander ('04) described in the oocytes of the female chick. He, however, called the central sphere and its surrounding clear area the yolk nucleus of Balbiani and the mitochondrial portion the 'couche vitellogene' or 'couche palleale.' His methods did not demonstrate the mitochondria, but in the region of the mitochondrial crescent which they occupy his text described, and his figures showed a denser zone of cytoplasm. This denser zone may be dissolved mitochondria or it may be more condensed cytoplasm, which seems also to be present in mv preparations (fig- 4).
There is, of course, a great deal of dispute as to the nature of the yolk nucleus — its cytological structure and its role in the cell.
Mertens ('94), in his study of the yolk nucleus of Balbiani in the oocytes of birds and mammals, came to the conclusion that two very different entities are described under the one name, first certain elements found in the cytoplasm which originate in the nucleus and, secondly, the attraction-sphere, which van Bambeke called the 'couche palleale' and Van der Stricht the 'couche vitellogene.' At the present moment I am not prepared to say am^thing positively as to the origin of the denser material in the mitochondrial portion of the mitochondrial crescent, but I am very certain that the attraction-sphere and its surrounding body of mitochondria ought not to be called the yolk nucleus of Balbiani for it does not have a nuclear origin.
The denser ground substance in the mitochondrial portion of the mitochondrial crescent may be derived from the nucleus and fall into the class of a true yolk nucleus or a chromidial substance in the sense of R. Hertwig.
It may be that the mitochondrial portion of the mitochondrial crescent plays a role in the oocyte in the formation of vitellus, as has been suggested, acts as a true 'couche vitellogene; ' but as to its function in the male, I cannot even surmise.
1. In the male chick the true sexual cords or seminiferous cords originate from the germinal epithelium during the sixth and seventh days of development and are the result of localized activity of the epithelium. Nearly all the primordial germ-cells present in the germinal epithehum are carried down into the seminiferous cords but they play only a passive role for at this time they show no evidences of cell division.
2. The sexual cords remain attached to the germinal epithelium for only a short time, and continue to grow, after formation of the albuginea, as a result of division of the peritoneal cells.
3. At the end of the seventh day of development the sex of the individual can be easily told, for in the male the gonads are of nearly equal size, while in the female the left gonad is much the larger. In the male the germinal epithelium remains thin after the formation of the sexual cords and contains very fewprimordial germ-cells, while in the female the epithelium of the left gonad or ovary continues to be thick and contains many primordial germ-cells.
4. During the eighth and ninth days of development the gonads increase slowly in size and the thin sexual cords make up most of the volume of the testes. They are separated from one another by a thin layer of stroma and have a definite orientation from germinal epithelium obliquely down towards the Wolffian body. When the embryo is 11 days old the stroma begins to increase in quantity and the seminiferous cords commence to meander and to anastomose with each other.
5. Up to the thirteenth day of development the primordial germ-cells in the sexual cords do not divide, their numbers remaining about the same as when they left the germinal epithelium. Beginning at this stage they divide actively and continue to do so for the next four daj'^s. The primordial germ-cells give rise to the spermatogonia, which are from now on very numerous in the sexual cords. The spermatogonia differ from the primordial germ-cells in possessing the mitochondrial crescent. This occupies part of the vegetative pole and consists of attraction-sphere containing centrosomes, and mitochondria, which in section are seen to extend over the nuclear membrane, capping the nucleus. The arms of the mitochondrial body may embrace more than one-half of the nucleus. This mitochondrial body is suspended in dense cytoplasm and is exactly comparable to the yolk nucleus as described by D'Hollander and Van der Stricht in the oocyte. I have also described it in the young oogonia of the chick. The interstitial cells appear in the stroma on the thirteenth day but reach their greatest development during the seventeenth day. They are simply differentiated stroma cells, since they do not divide and transitional forms can be seen.
6. Cavities begin to appear in the network of seminiferous cords during the twentieth day. These cavities are formed, not by fissures between the cells, but by Uquefaction of the cells in the central axis of the cord. At the twentieth day the spermatogonia are found against the basement membrane, with the nucleus towards the central axis of the cord and the mitochondrial crescent near the basement membrane. They probably reach this position by amoeboid migration. The elongated cells between the spermatogonia are derived from the peritoneal cells of the seminiferous cords.
7. The primordial germ-cells give rise to the spermatogonia and the coelomic cells of the germinal epithelium produce the supporting cells of the seminiferous tubule.
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