Chapter XII: The Organs of Excretion (5)

The development of the yolk appears to me to present special difficulties, owing to the fact pointed out by His[391] that the conditions of development vary greatly according to whether the ovary is in a state of repose or of active development. I do not feel satisfied with my results on this subject, but believe there is still much to be made out. Observations on the yolk spherules may be made either in living ova, in ova hardened in osmic acid, or in ova hardened in picric or chromic acids. The two latter reagents, as well as alcohol, are however unfavourable for the purpose of this study, since by their action the yolk spherules appear frequently to be broken up and otherwise altered. This has to some extent occurred in Pl. 25, fig. 21, and the peculiar appearance of the yolk of this ovum is in part due to the action of the reagent. On the whole I have found osmic acid the most suitable reagent for the study of the yolk, since without breaking up the developing spherules, it stains them of a deep black colour. The yolk spherules commence to be formed in ova, of not more than 0.06 mm. in the ovaries of moderately old females. In young females they are apparently not formed in such small ova. They arise as extremely minute, highly refracting particles, in a stratum of protoplasm _some little way below the surface, and are always most numerous at the pole opposite the germinal vesicle_. Their general arrangement is very much that figured and described by Allen Thomson in Gasterosteus[392], and by Gegenbaur and Eimer in young Reptilian ova. In section they naturally appear as a ring, their general mode of distribution being fairly typically represented on Pl. 25, fig. 27. The ovum represented in fig. 27 was 0.5 mm. in diameter, and the yolk spherules were already largely developed; in smaller ova they are far less numerous, though arranged in a similar fashion. The developing yolk spherules are not uniformly distributed but are collected in peculiar little masses or aggregations (Pl. 25, fig. 21). These resemble the granular masses, figured by His (_loc. cit._ Pl. 4, fig. 33) in the Salmon, and may be compared with the aggregations figured by Götte in his monograph on _Bombinator igneus_ (Pl. 1, fig. 9). It deserves to be especially noted, that when the yolk spherules are first formed, the _peripheral layer of the ovum_ is entirely free from them, a feature which is however apt to be lost in ova hardened in picric acid (Pl. 25, fig. 21). Two points about the spherules appear clearly to point to their being developed in the protoplasm of the ovum, and not in the follicular epithelium. (1) That they do not make their appearance in the superficial stratum of the ovum. (2) That no yolk spherules are present in the cells of the follicular epithelium, in which they could not fail to be detected, owing to the deep colour they assume on being treated with osmic acid.

Footnote 391: _Das Ei bei Knochenfischen._

Footnote 392: "Ovum" in Todd's _Encyclopædia_, fig. 69.

It need scarcely be said that the yolk spherules at this stage are not cells, and have indeed no resemblance to cells. They would probably be regarded by His as spherules of fatty material, unrelated to the true food yolk.

As the ova become larger the granules of the peripheral layer before mentioned gradually assume the character of the yolk spheres of the adult, and at the same time spread towards the centre of the egg. Not having worked at fresh specimens, I cannot give a full account of the growth of the spherules; but am of opinion that Gegenbaur's account is probably correct, according to which the spheres at first present gradually grow and develop into vesicles, in the interior of which solid bodies (nuclei of His?) appear and form the permanent yolk spheres. When the yolk spheres are still very small they have the typical oblong form[393] of the ripe ovum, and this form is acquired while the centre of the ovum is still free from them.

Footnote 393: The peculiar oval, or at times slightly
rectangular and striated yolk spherules of Elasmobranchii are
mentioned by Leydig and Gegenbaur (Pl. 11, fig. 20), and
myself, _Preliminary Account of Development of Elasmobranch
Fishes_, and by Filippi and His in _Osseous Fishes_.

The growth of the yolk appears mainly due to the increase in size and number of the individual yolk spheres. Even when the ovum is quite filled with large yolk spheres, the granular protoplastic network of the earlier stages is still present, and serves to hold together the constituents of the yolk. In the cortical layer of nearly ripe ova, the yolk has a somewhat different character to that which it exhibits in the deeper layers, chiefly owing to the presence of certain delicate granular (in hardened specimens) bodies, whose nature I do not understand, and to special yolk spheres rather larger than the ordinary, provided with numerous smaller spherules in their interior, which are probably destined in the course of time to become free and to form ordinary yolk spheres.

The mode of formation of the yolk spheres above described appears to me to be the normal, and possibly the only one. Certain peculiar structures have, however, come under my notice, which may perhaps be connected with the formation of the yolk. One of these resembles the bodies described by Eimer[394] as "Dotterschorfe." I have only met these bodies in a single instance in ova of 0.6 mm., from the ovary (in active growth) of a specimen of _Scy. canicula_ 23 inches in length. In this instance they consisted of homogeneous clear bodies (not bounded by any membrane) of somewhat irregular shape, though usually more or less oval, and rarely more than 0.02 mm. in their longest diameter. They were very numerous in the peripheral layer of the ovum, but quite absent in the centre, and also not found outside the ovum (as they appear to be in Reptilia). Yolk granules formed in the normal way, and staining deeply by osmic acid, were present, but the "Dotterschorfe" presented a marked contrast to the remainder of the ovum, in being absolutely unstained by osmic acid, and indeed they appeared more like a modified form of vacuole than any definite body. Their general appearance in Scyllium may be gathered from Eimer's figure 8, Pl. 11, though they were much more numerous than represented in that figure, and confined to the periphery of the ovum.

Footnote 394: "Untersuchung über die Eier d. Reptilien,"
_Archiv f. mikros. Anat._ Vol. VIII.

Dr Eimer describes a much earlier condition of these structures, in which they form a clear shell enclosing a central dark nucleus. This stage I have not met with, nor can I see any grounds for connecting these bodies with the formation of the yolk, and the fact of their not staining with osmic acid is strongly opposed to this view of their function. Dr Eimer does not appear to me to bring forward any satisfactory proof that they are in any way related to the formation of the yolk, but wishes to connect them with the peculiar body, well known as the yolk nucleus, which is found in the Amphibian ovum[395].

Footnote 395: Vide Allen Thomson, article "Ovum," Todd's
_Encyclopædia_, p. 95.

Another peculiar body found in the ova may be mentioned here, though it more probably belongs to the germinal vesicle than to the yolk. It has only been met with in the vitellus of some of the medium sized ova of a young female. Examples of this body are represented on Pl. 25, fig. 25A, _x_. As a rule there is only one in each of the ova in which they are present, but there may be as many as four. They consist of small vesicles with a very thick doubly contoured membrane, which are filled with numerous deeply staining spherical granules. At times they contain a vacuole. Some of the larger of them are not very much smaller than the germinal vesicle of their ovum, while the smallest of them present a striking resemblance to the nucleoli (fig. 25B), which makes me think that they may possibly be nucleoli which have made their way out of the germinal vesicle. I have not found them in the late stages or large ova.

The following measurements shew the size of some of these bodies in relation to the germinal vesicle and ovum:--

Diameter of Germinal Diameter of Body in
Diameter of Ovum. Vesicle. Vitellus.

0.096 mm. 0.03 mm. 0.009 mm.
0.064 mm. 0.025 mm. 0.012 mm.
{0.019 mm.
0.096 mm. 0.03 mm {0.003 mm.

_Germinal vesicle._--Gegenbaur[396] finds the germinal vesicle completely homogeneous and without the trace of a germinal spot. In Raja granules or vesicles may appear as artificial products, and in Acanthias even in the fresh condition isolated vesicles or masses of such may be present. To these structures he attributes no importance.

Footnote 396: _Loc. cit._

Alexander Schultz[397] states that there is nothing remarkable in the germinal vesicle of the Torpedo egg, but that till the egg reaches 0.5 mm., a single germinal spot is always present (measuring about 0.01 mm.), which is absent in larger ova.

Footnote 397: _Loc. cit._

The bodies described by Gegenbaur are now generally recognised as germinal spots, and will be described as such in the sequel. I have very rarely met with the condition with the single nucleolus described by Schultz in Torpedo.

My own observations are confined to Scyllium. In very young females, with ova not larger than 0.09 mm., the germinal vesicle has the same characters as during the embryonic periods. The contents are clear but traversed by a very distinct and deeply staining reticulum of fibres connected with the several nucleoli which are usually present and situated close to the membrane.

In a somewhat older female in the largest ova of about 0.12 mm., the germinal vesicle measures about 0.06 mm., and usually occupies an eccentric position. It is provided with a distinct though delicate membrane. The network, so conspicuous during the embryonic period, is not so clear as it was, and has the appearance of being formed of lines of granules rather than of fibres. The fluid contents of the nucleus remain as a rule, even in the hardened specimens, perfectly clear, though they become in some instances slightly granular. There are usually two, three, or more nucleoli generally situated, as described by Eimer, close to the membrane of the vesicle, the largest of which may measure as much as 0.006 mm. They are highly refracting bodies, containing in most instances a vacuole, and very frequently a smaller spherical body of a similar nature to themselves[398]. Granules are sometimes also present in the germinal vesicle, but are probably only extremely minute nucleoli.

Footnote 398: Compare, with reference to several points, the
germinal vesicle at this stage with the germinal vesicle of the
frog's ovum figured by O. Hertwig, _Morphologisches Jahrbuch_,
Vol. III. pl. 4, fig. 1.

In ova of 0.5 mm. the germinal vesicle has a diameter of 0.12 mm. (Pl. 25, fig. 21). It is usually shrunk in hardened specimens though nearly spherical in the living ovum. Its contents are rendered granular by reagents though quite clear when fresh, and the reticulum of the earlier stages is sometimes with difficulty to be made out, though in other instances fairly clear. In all cases the fibres composing it are very granular. The membrane is thick. Peculiar highly refracting nucleoli, usually enclosing a large vacuole, are present in considerable numbers, and are either arranged in a circle round the periphery, or sometimes aggregated towards one side of the vesicle; and in addition, numerous deeply staining smaller granular aggregations, probably belonging to the same category as the nucleoli (from which in the living ovum they can only be distinguished by their size), are scattered close to the inner side of the membrane over the whole or only a part of the surface of the germinal vesicle. In a fair number of instances bodies like that figured on Pl. 25, fig. 27, are to be found in the germinal vesicle. They appear to be nucleoli in which a number of smaller nucleoli are originating by a process of endogenous growth, analogous perhaps to endogenous cell-formation. The nucleoli thus formed are, no doubt, destined to become free. The above mode of increase for the nucleoli appears to be exceptional. The ordinary mode is, no doubt, that by simple division into two, as was long ago shewn by Auerbach.

Of the later stages of the germinal vesicle and its final fate, I can give no account beyond the very fragmentary statements which have already appeared in my monograph on Elasmobranch Fishes.

_Formation of fresh ova and ovarian nests in the post-embryonic stages._--Ludwig[399] was the first to describe the formation of ova in the post-embryonic periods. His views will be best explained by quoting the following passage:--

"The follicle of Skates and Dog-fish, with the ovum it contains, is to
be considered as an aggregation of the cells of the single-layered
ovarian epithelium which have grown into the stroma, and of which one
cell has become the ovum and the others the follicular epithelium. The
follicle, however, draws in with it into the stroma a number of
additional epithelial cells in the form of a stalk connecting the
follicle with the superficial epithelium. At a later period the lower
part of the stalk at its junction with the follicle becomes
continuously narrowed, and at the same time a rupture takes place in
the cells which form it. In this manner the follicle becomes at last
constricted off from the stalk, and so from its place of origin in the
superficial epithelium, and subsequently lies freely in the stroma of
the ovary."

Footnote 399: _Loc. cit._

He further explains that the separation of the follicles from the epithelium takes place much earlier in Acanthias than in Raja, and that the sinkings of the epithelium into the stroma may have two or three branches each with a follicle.

Semper gives very little information with reference to the post-embryonic formation of ova. He expresses his agreement on the whole with Ludwig, but, amongst points not mentioned by Ludwig, calls attention to peculiar aggregations of primitive ova in the superficial epithelium, which he regards as either rudimentary testicular follicles or as nests similar to those in the embryo.

My observations on this subject do not agree very closely with those either of Ludwig or Semper. The differences between us partly, though not entirely, depend upon the fundamentally different views we hold about the constitution of the ovary and the nature of the epithelium covering it (vide pp. 555 and 556).

In very young ovaries (Pl. 24, fig. 8) nests of ova (in my sense of the term) are very numerous, but though usually superficial in position are also found in the deeper layers of the ovary. They are especially concentrated in their old position, close to the dorsal edge of the organ. In some instances they do not present quite the same appearance as in the embryo, owing to the outlines of the ova composing them being distinct, and to the presence between the ova of numerous interstitial cells derived from the germinal epithelium, and destined to become follicular epithelium. These latter cells at first form a much flatter follicular epithelium than in the embryonic periods, so that the smaller adult ova have a much less columnar investment than ova of the same size in the embryo. A few primitive ova may still be found in a very superficial position, but occasionally also in the deeper layers. I am inclined to agree with Semper that some of these are freshly formed from the cells of the germinal epithelium.

In the young female with ova of about 0.5 mm. nests of ova are still fairly numerous. The nests are characteristic, and present the various remarkable peculiarities already described in the embryo. In many instances they form polynuclear masses, not divided into separate cells, generally, however, the individual ova are distinct. The ova in these nests are on the average rather smaller than during the embryonic periods. The nests are frequently quite superficial and at times continuous with the pseudo-epithelium, and individual ova also occasionally occupy a position in the superficial epithelium. Some of the appearances presented by separate ova are not unlike the figures of Ludwig, but a growth such as he describes has, according to my observations, no existence. The columns which he believes to have grown into the stroma are merely trabeculæ connecting the deeper and more superficial parts of the germinal epithelium; and his whole view about the formation of the follicular epithelium round separate ova certainly does not apply, except in rare cases, to Scyllium. It is, indeed, very easy to see that most freshly formed ova are derived from nests, as in the embryo; and the formation of a follicular epithelium round these ova takes place as they become separated from the nests. A few solitary ova, which have never formed part of a nest, seem to be formed in this stage as in the embryo; but they do not grow into the stroma surrounded by the cells of the pseudo-epithelium, and only as they reach a not inconsiderable size is a definite follicular epithelium formed around them. The follicular epithelium, though not always formed from the pseudo-epithelium, is of course always composed of cells derived from the germinal epithelium.

In all the ova formed at this stage the nucleus would seem to pass through the same metamorphosis as in the embryo.

In the later stages, and even in the full-grown female of Scyllium, fresh ova seemed to be formed and nests also to be present. In Raja I have not found freshly formed ova or nests in the adult, and have had no opportunity of studying the young forms.

_Summary of observations on the development of the ovary in Scyllium and Raja._

(1) The ovary in the embryo is a ridge, triangular in section, attached along the base. It is formed of a core of stroma and a covering of epithelium. A special thickening of the epithelium on the outer side forms the true germinal epithelium, to which the ova are confined (Pl. 24, fig. 1). In the development of the ovary the stroma becomes differentiated into an external vascular layer, especially developed in the neighbourhood of the germinal epithelium, and an internal lymphatic portion, which forms the main mass of the ovarian ridge (Pl. 24, figs. 2, 3, and 6).

(2) At first the thickened germinal epithelium is sharply separated by a membrane from the subjacent stroma (Pl. 24, figs. 1, 2, and 3), but at about the time when the follicular epithelium commences to be formed round the ova, numerous strands of stroma grow into the epithelium, and form a regular network of vascular channels throughout it, and partially isolate individual ova (Pl. 24, figs. 7 and 8). At the same time the surface of the epithelium turned towards the stroma becomes irregular (Pl. 24, fig. 9), owing to the development of individual ova. In still later stages the stroma ingrowths form a more or less definite tunic close to the surface of the ovary. External to this tunic is the superficial layer of the germinal epithelium, which forms what has been spoken of as the pseudo-epithelium. In many instances the protoplasm of its cells is produced into peculiar fibrous tails which pass into the tunic below.

(3) _Primitive ova._--Certain cells in the epithelium lining the dorsal angle of the body-cavity become distinguished as primitive ova by their abundant protoplasm and granular nuclei, at a very early period in development, even before the formation of the genital ridges. Subsequently on the formation of the genital ridges these ova become confined to the thickened germinal epithelium on the outer aspect of the ridges (Pl. 24, fig. 1).

(4) _Conversion of primitive ova into permanent ova._--Primitive ova may in Scyllium become transformed into permanent ova in two ways--the difference between the two ways being, however, of secondary importance.

(_a_) A nest of primitive ova makes its appearance, either by continued division of a single primitive ovum or otherwise. The bodies of all the ova of the nest fuse together, and a polynuclear mass is formed, which increases in size concomitantly with the division of its nuclei. The nuclei, moreover, pass through a series of transformations. They increase in size and form delicate vesicles filled with a clear fluid, but contain close to one side a granular mass which stains very deeply with colouring reagents. The granular mass becomes somewhat stellate, and finally assumes a reticulate form with one more highly refracting nucleoli at the nodal points of the reticulum. When a nucleus has reached this condition the protoplasm around it has become slightly granular, and with the enclosed nucleus is segmented off from the nest as a special cell--a permanent ovum (figs. 13, 14, 15, 16). Not all the nuclei in a nest undergo the whole of the above changes; certain of them, on the contrary, stop short in their development, atrophy, and become employed as a kind of pabulum for the remainder. Thus it happens that out of a large nest perhaps only two or three permanent ova become developed.

(_b_) In the second mode of development of ova the nuclei and protoplasm undergo the same changes as in the first mode; but the ova either remain isolated and never form part of a nest, or form part of a nest in which no fusion of the protoplasm takes place, and all the primitive ova develop into permanent ova. Both the above modes of the formation continue through a great part of life.

(5) _The follicle._--The cells of the germinal epithelium arrange themselves as a layer around each ovum, almost immediately after its separation from a nest, and so constitute a follicle. They are at first flat, but soon become more columnar. In Scyllium they remain for a long time uniform, but in large eggs they become arranged in two or three layers, while at the same time some of them become large and flask-shaped, and others small and oval (fig. 29). The flask-shaped cells have probably an important function in the nutrition of the egg, and are arranged in a fairly regular order amongst the smaller cells. Before the egg is quite ripe both kinds of follicle cells undergo retrogressive changes (Pl. 25, fig. 23).

In Raja a great irregularity in the follicle cells is observable at an early stage, but as the ovum grows larger the cells gradually assume a regular arrangement more or less similar to that in Scyllium (Pl. 25, figs. 30-33).

(6) _The egg membranes._--Two membranes are probably always present in Elasmobranchii during some period of their growth. The first formed and outer of these arises in some instances before the formation of the follicular epithelium, and would seem to be of the nature of a vitelline membrane. The inner one is the zona radiata with a typical radiately striated structure. It is formed from the vitellus at a much later period than the proper vitelline membrane. It is more developed in Scyllium than in Raja, but atrophies early in both genera. By the time the ovum is nearly ripe both membranes are very much reduced, and when the egg (in Scyllium and Pristiurus) is laid, no trace of any membrane is visible.

(7) _The vitellus._--The vitellus is at first faintly granular, but at a later period exhibits a very distinct (protoplasmic) network of fibres, which is still present after the ovum has been laid.

The yolk arises, in the manner described by Gegenbaur, in ova of about 0.06 mm. as a layer of fine granules, which stain deeply with osmic acid. They are at first confined to a stratum of protoplasm slightly below the surface of the ovum, and are most numerous at the pole furthest removed from the germinal vesicle. They are not regularly distributed, but are aggregated in small masses. They gradually grow into vesicles, in the interior of which oval solid bodies are developed, which form the permanent yolk-spheres. These oval bodies in the later stages exhibit a remarkable segmentation into plates, which gives them a peculiar appearance of transverse striation.

Certain bodies of unknown function are occasionally met with in the vitellus, of which the most remarkable are those figured at _x_ on Pl. 25, fig. 25A.

(8) _The germinal vesicle._--A reticulum is very conspicuous in the germinal vesicle in the freshly formed ova, but becomes much less so in older ova, and assumes, moreover, a granular appearance. At first one to three nucleoli are present, but they gradually increase in number as the germinal vesicle grows older, and are frequently situated in close proximity to the membrane.

THE MAMMALIAN OVARY (Pl. 26).

The literature of the mammalian ovary has been so often dealt with that it may be passed over with only a few words. The papers which especially call for notice are those of Pflüger[400], Ed. van Beneden[401], and especially Waldeyer[402], as inaugurating the newer view on the nature of the ovary, and development of the ova; and of Foulis[403] and Kölliker[404], as representing the most recent utterances on the subject. There are, of course, many points in these papers which are touched on in the sequel, but I may more especially here call attention to the fact that I have been able to confirm van Beneden's statement as to the existence of polynuclear protoplasmic masses. I have found them, however, by no means universal or primitive; and I cannot agree in a general way with van Beneden's account of their occurrence. I have found no trace of a germogene (Keimfache) in the sense of Pflüger and Ed. van Beneden. My own results are most in accordance with those of Waldeyer, with whom I agree in the fundamental propositions that both ovum and follicular epithelium are derived from the germinal epithelium, but I cannot accept his views of the relation of the stroma to the germinal epithelium.

Footnote 400: _Die Eierstöcke d. Säugethiere u. d. Menschen_,
Leipzig, 1863.

Footnote 401: "Composition et Signification de l'oeuf," _Acad.
r. de Belgique_, 1868.

Footnote 402: _Eierstock u. Ei._ Leipzig, 1870.

Footnote 403: _Trans. of Royal Society, Edinburgh_, Vol. XXVII.
1875, and _Quarterly Journal of Microscopical Science_,
Vol. XVI.

Footnote 404: _Verhandlung d. Phys. Med. Gesellschaft_,
Würzburg, 1875, N. F. Bd. VIII.

In the very interesting paper of Foulis, the conclusion is arrived at, that while the ova are derived from the germinal epithelium, the cells of the follicle originate from the ordinary connective tissue cells of the stroma. Foulis regards the zona pellucida as a product of the ovum and not of the follicle. To both of these views I shall return, and hope to be able to shew that Foulis has not traced back the formation of the follicle through a sufficient number of the earlier stages. It thus comes about that though I fully recognise the accuracy of his figures, I am unable to admit his conclusions. Kölliker's statements are again very different from those of Foulis. He finds certain cords of cells in the hilus of the ovary, which he believes to be derived from the Wolffian body, and has satisfied himself that they are continuous with Pflüger's egg-tubes, and that they supply the follicular epithelium. To the general accuracy of Kölliker's statements with reference to the relations of these cords in the hilus of the ovary I can fully testify, but am of opinion that he is entirely mistaken as to their giving rise to the follicular epithelium, or having anything to do with the ova. I hope to be able to give a fuller account of their origin than he or other observers have done.

My investigations on the mammalian ovary have been made almost entirely on the rabbit--the type of which it is most easy to procure a continuous series of successive stages; but in a general way my conclusions have been controlled and confirmed by observations on the cat, the dog, and the sheep. My observations commence with an embryo of eighteen days. A transverse section, slightly magnified, through the ovary at this stage, is represented on Pl. 26, fig. 35, and a more highly magnified portion of the same in fig. 35A. The ovary is a cylindrical ridge on the inner side of the Wolffian body, composed of a superficial epithelium, the germinal epithelium (_g.e._), and of a tissue internal to this, which forms the main mass of it. In the latter two constituents have to be distinguished--(1) an epithelial-like tissue (_t_), coloured brown, which forms the most important element, and (2) vascular and stroma elements in this.

The germinal epithelium is a layer about 0.03 - 0.04 mm. in thickness. It is (vide fig. 35A, _g.e._) composed of two or three layers of cells, with granular nuclei, of which the outermost layer is more columnar than the remainder, and has elongated rather than rounded nuclei. Its cells, though they vary slightly in size, are all provided with a fair amount of protoplasm, and cannot be divided (as in the case of the germinal epithelium of Birds, Elasmobranchii, &c.), into primitive ova, and normal epithelial cells. Very occasionally, however, a specially large cell, which, perhaps, deserves the appellation primitive ovum, may be seen. From the subjacent tissue the germinal epithelium is in most parts separated by a membrane-like structure (fluid coagulum); but this is sometimes absent, and it is then very difficult to determine with exactness the inner border of the epithelium. The tissue (_t_), which forms the greater mass of the ovary at this stage, is formed of solid columns or trabeculæ of epithelial-like cells, which present a very striking resemblance in size and character to the cells of the germinal epithelium. The protoplasm of these cells stains slightly more deeply with osmic acid than does that of the cells of the germinal epithelium, so that it is rather easier to note a difference between the two tissues in osmic acid than in picric acid specimens. This tissue approaches very closely, and is in many parts in actual contact with the germinal epithelium. Between the columns of it are numerous vascular channels (shewn diagrammatically in my figures) and a few normal stroma cells. This remarkable tissue continues visible through the whole course of the development of the ovary, till comparatively late in life, and during all the earlier stages might easily be supposed to be about to play some part in the development of the ova, or even to be part of the germinal epithelium. It really, however, has nothing to do with the development of the ova, as is easily demonstrated when the true ova begin to be formed. In the later stages, as will be mentioned in the description of those stages, it is separated from the germinal epithelium by a layer of stroma; though at the two sides of the ovary it is, even in later stages, sometimes in contact with the germinal epithelium.

In most parts this tissue is definitely confined within the limits of the ovary, and does not extend into the mesentery by which the ovary is attached. It may, however, be traced _at the anterior end_ of the ovary into connection with the walls of the Malpighian bodies, which lie on the inner side of the Wolffian body (vide fig. 35B), and I have no doubt that it grows out from the walls of these bodies into the ovary. In the male it appears to me to assist in forming, together with cells derived from the germinal epithelium, the seminiferous tubules, the development of which is already fairly advanced by this stage. I shall speak of it in the sequel as tubuliferous tissue. The points of interest in connection with it concern the male sex, which I hope to deal with in a future paper, but I have no hesitation in identifying it with the segmental cords (_segmentalstränge_) discovered by Braun in Reptilia, and described at length in his valuable memoir on their urogenital system[405]. According to Braun the segmental cords in Reptilia are buds from the outer walls of the Malpighian bodies. The bud from each Malpighian body grows into the genital ridge before the period of sexual differentiation, and sends out processes backwards and forwards, which unite with the buds from the other Malpighian bodies. There is thus formed a kind of trabecular work of tissue in the stroma of the ovary, which in the Lacertilia comes into connection with the germinal epithelium in both sexes, but in Ophidia in the male only. In the female, in all cases, it gradually atrophies and finally vanishes, but in the male there pass into it the primitive ova, and it eventually forms, with the enclosed primitive ova, the tubuli seminiferi. From my own observations in Reptilia I can fully confirm Braun's statements as to the entrance of the primitive ova into this tissue in the male, and the conversion of it into the tubuli seminiferi. The chief difference between Reptilia and Mammalia, in reference to this tissue, appears to be that in Mammalia it arises only from a few of the Malpighian bodies at the anterior extremity of the ovary, but in Reptilia from all the Malpighian bodies adjoining the genital ridge. More extended observations on Mammalia will perhaps shew that even this difference does not hold good.

Footnote 405: _Arbeiten a. d. Zool.-zoot. Institut Würzburg_,
Bd. IV.

It is hardly to be supposed that this tissue, which is so conspicuous in all young ovaries, has not been noticed before; but the notices of it are not so numerous as I should have anticipated. His[406] states that the parenchyma of the sexual glands undoubtedly arises from the Wolffian canals, and adds that while the cortical layer (Hulle) represents the earlier covering of a part of the Wolffian body, the stroma of the hilus, with its vessels, arises from a Malpighian body. In spite of these statements of His, I still doubt very much whether he has really observed either the tissue I allude to or its mode of development. In any case he gives no recognisable description or figure of it.

Footnote 406: _Archiv f. mikros. Anat._ Vol. I. p. 160.

Waldeyer[407] notices this tissue in the dog, cat, and calf. The following is a free translation of what he says, (p. 141):--"In a full grown but young dog, with numerous ripe follicles, there were present in the vascular zone of the ovary numerous branched elongated small columns (Schläuche) of epithelial cells, between which ran blood-vessels. They were only separated from the egg columns of the cortical layer by a row of large follicles. There can be no doubt that we have here remains of the sexual part of the Wolffian body--the canals of the parovarium--which in the female sex have developed themselves to an extraordinary extent into the stroma of the sexual gland, and perhaps are even to be regarded as _homologues of the seminiferous tubules_ (the italics are my own). I have almost always found the above condition in the dog, only in old animals these seminiferous canals seem gradually to atrophy. Similar columns are present in the cat, only they do not appear to grow so far into the stroma." Identical structures are also described in the calf.

Footnote 407: _Loc. cit._

Romiti gives a very similar description to Waldeyer of these bodies in the dog[408]. Born also describes this tissue in young and embryonic ovaries of the horse as the _Keimlager_[409]. The columns described by Kölliker[410] and believed by him to furnish the follicular epithelium, are undoubtedly my tubuliferous tissue, and, as Kölliker himself points out, are formed of the same tissue as that described by Waldeyer.

Footnote 408: _Archiv f. mikr. Anat._ Vol. X.

Footnote 409: _Archiv f. Anatomie, Physiologie, u. Wiss.
Medicin._ 1874.

Footnote 410: _Loc. cit._

Egli gives a very clear and accurate description of this tissue, though he apparently denies its relation with the Wolffian body.

My own interpretation of the tissue accords with that of Waldeyer. In addition to the rabbit, I have observed it in the dog, cat, and sheep. In all these forms I find that close to the attachment of the ovary, and sometimes well within it, a fair number of distinct canals with a large lumen are present, which are probably to be distinguished from the solid epithelial columns. Such large canals are not as a rule present in the rabbit. In the dog solid columns are present in the embryo, but later they appear frequently to acquire a tubular form, and a lumen. Probably there are great variations in the development of the tissue, since in the cat (not as Waldeyer did in the dog) I have found it most developed.

In the very young embryonic ovary of the cat the columns are very small and much branched. In later embryonic stages they are frequently elongated, sometimes convoluted, and are very similar to the embryonic tubuli seminiferi. In the young stages these columns are so similar to the egg tubes (which agree more closely with Pflüger's type in the cat than in other forms I have worked at) that to any one who had not studied the development of the tissue an embryo cat's ovary at certain stages would be a very puzzling object. I have, however, met with nothing in the cat or any other form which supports Kölliker's views.

My next stage is that of a twenty-two days' embryo. Of this stage I have given two figures corresponding to those of the earlier stage (figs. 36 and 36A).

From these figures it is at once obvious that the germinal epithelium has very much increased in bulk. It has a thickness 0.1 - 0.09 mm. as compared to 0.03 mm. in the earlier stage. Its inner outline is somewhat irregular, and it is imperfectly divided into lobes, which form the commencement of structures nearly equivalent to the nests of the Elasmobranch ovary. The lobes _are not_ separated from each other by connective tissue prolongations; the epithelium being at this stage perfectly free from any ingrowths of stroma. The cells constituting the germinal epithelium have much the same character as in the previous stage. They form an outer row of columnar cells internal to which the cells are more rounded. Amongst them a few large cells with granular nuclei, which are clearly primitive ova, may now be seen, but by far the majority of the cells are fairly uniform in size, and measure from 0.01 - 0.02 mm. in diameter, and their nuclei from 0.004 - 0.006 mm. The nuclei of the columnar outer cells measure about 0.008 mm. They are what would ordinarily be called granular, though high powers shew that they have the usual nuclear network. There is no special nucleolus. The rapid growth of the germinal epithelium is due to the division of its cells, and great masses of these may frequently be seen to be undergoing division at the same time. Of the tissue of the ovary internal to the germinal epithelium, it may be noticed that the tubuliferous tissue derived from the Malpighian bodies is no longer in contact with the germinal epithelium, but that a layer of vascular stroma is to a great extent interposed between the two. The vascular stroma of the hilus has, moreover, greatly increased in quantity.

My next stage is that of a twenty-six days' embryo, but the characters of the ovary at this stage so closely correspond with those of the succeeding one at twenty-eight days that, for the sake of brevity, I pass over this stage in silence.

Figs. 37 and 37A are representative sections of the ovary of the twenty-eighth day corresponding with those of the earlier stages.

Great changes have become apparent in the constitution of the germinal epithelium. The vascular stroma of the ovary has grown into the germinal epithelium precisely as in Elasmobranchii. It appears to me clear that the change in the relations between the stroma and epithelium is not due to a mutual growth, but entirely to the stroma, so that, as in the case of Elasmobranchii, the result of the ingrowth is that the germinal epithelium is honeycombed by vascular stroma. The vascular growths generally take the paths of the lines which separated the nests in an earlier condition, and cause these nests to become the egg tubes of Pflüger. It is obvious in figure 37 that the vascular ingrowths are so arranged as imperfectly to divide the germinal epithelium into two layers separated by a space with connective tissue and blood-vessels. The outer part is relatively thin, and formed of a superficial row of columnar cells, and one or two rows of more rounded cells; the inner layer is much thicker, and formed of large masses of rounded cells. The two layers are connected together by numerous trabeculæ, the stroma between which eventually gives rise to the connective tissue capsule, or tunica albuginea, of the adult ovary.

The germinal epithelium is now about 0.19 to 0.22 mm. in thickness. Its cells have undergone considerable changes. A fair number of them (fig. 37A, _p.o._), especially in the outer layer of the epithelium, have become larger than the cells around them, from which they are distinguished, not only by their size, but by their granular nucleus and abundant protoplasm. They are in fact undoubted primitive ova with all the characters which primitive ova present in Elasmobranchii, Aves, &c. In a fairly typical primitive ovum of this stage the body measures 0.02 mm. and the nucleus 0.014 mm. In the inner part of the germinal epithelium there are very few or no cells which can be distinguished by their size as primitive ova, and the cells themselves are of a fairly uniform size, though in this respect there is perhaps a greater variation than might be gathered from fig. 37A. The cells are on the average about 0.016 mm. in diameter, and their nuclei about 0.008 to 0.001 mm., considerably larger, in fact, than in the earlier stage. The nuclei are moreover more granular, and make in this respect an approach to the character of the nuclei of primitive ova.

The germinal epithelium is still rapidly increasing by the division of its cells, and in fig. 37A there are shewn two or three nuclei in the act of dividing. I have represented fairly accurately the appearance they present when examined with a moderately high magnifying power. With reference to the stroma of the ovary, internal to the germinal epithelium, it is only necessary to refer to fig. 37 to observe that the tubuliferous tissue (_t_) forms a relatively smaller part of the stroma than in the previous stage, and is also further removed from the germinal epithelium.

My next stage is that of a young rabbit two days after birth, but to economise space I pass on at once to the following stage five days after birth. This stage is in many respects a critical one for the ovary, and therefore of great interest. Figure 38 represents a transverse section through the ovary (on rather a smaller scale than the previous figures) and shews the general relations of the tissues.

The germinal epithelium is very much thicker than before--about 0.38 mm. as compared with 0.22 mm. It is divided into three obvious layers: (1) an outer epithelial layer which corresponds with the pseudo-epithelial layer of the Elasmobranch ovary, average thickness 0.03 mm. (2) A middle layer of small nests, which corresponds with the middle vascular layer of the previous stage; average thickness 0.1 mm. (3) An inner layer of larger nests; average thickness 0.23 mm.

The general appearance of the germinal epithelium at this stage certainly appears to me to lend support to my view that the whole of it simply constitutes a thickened epithelium interpenetrated with ingrowths of stroma.

The cells of the germinal epithelium, which form the various layers, have undergone important modifications. In the first place a large number of the nuclei--at any rate of those cells which are about to become ova--have undergone a change identical with that which takes place in the conversion of the primitive into the permanent ova in Elasmobranchii. The greater part of the contents of the nucleus becomes clear. The remaining contents arrange themselves as a deeply staining granular mass on one side of the membrane, and later on as a somewhat stellate figure: the two stages forming what were spoken of as the granular and stellate varieties of nucleus. To avoid further circumlocution I shall speak of the nucleus undergoing the granular and the stellate modifications. At a still later period the granular contents form a beautiful network in the nucleus.

The pseudo-epithelium (fig. 38A) is formed of several tiers of cells, the outermost of which are very columnar and have less protoplasm than in an earlier stage. In the lower tiers of cells there are many primitive ova with granular nuclei, and others in which the nuclei have undergone the granular modification. The primitive ova are almost all of the same size as in the earlier stage. The pseudo-epithelium is separated from the middle layer by a more or less complete stratum of connective tissue, which, however, is traversed by trabeculæ connecting the two layers of the epithelium. In the middle layer there are comparatively few modified nuclei, and the cells still retain for the most part their earlier characters. The diameter of the cells is about 0.012 mm., and that of the nucleus about 0.008 mm. In the innermost layer (fig. 38B), which is not sharply separated from the middle layer, the majority of the cells, which in the previous stage were ordinary cells of the epithelium, have commenced to acquire modified nuclei. This change, which first became apparent to a small extent in the young two days after birth, is very conspicuous at this stage. In some of the cells the nucleus is modified in the granular manner, in others in the stellate, and in a certain number the nucleus has assumed a reticular structure characteristic of the young permanent ovum.

In addition, however, to the cells which are becoming converted into ova, a not inconsiderable number may be observed, if carefully looked for, which are for the most part smaller than the others, generally somewhat oval, and in which the nucleus retains its primitive characters. A fair number of such cells are represented in fig. 38B. In the larger ones the nucleus will perhaps eventually become modified; but the smaller cells clearly correspond with the interstitial cells of the Elasmobranch germinal epithelium, and are destined to become converted into the epithelium of the Graafian follicle. In some few instances indeed (at this stage very few), in the deeper part of the germinal epithelium, these cells commence to arrange themselves round the just formed permanent ova as a follicular epithelium. An instance of this kind is shewn in fig. 38B, _o_. The cells with modified nuclei, which are becoming permanent ova, usually present one point of contrast to the homologous cells in Elasmobranchii, in that they are quite distinct from each other, and not fused into a polynuclear mass. They have around them a dark contour line, which I can only interpret as the commencement of the membrane (zona radiata?), which afterwards becomes distinct, and which would thus seem, as Foulis has already insisted, to be of the nature of a vitelline membrane.

In a certain number of instances the protoplasm of the cells which are becoming permanent ova appears, however, actually to fuse, and polynuclear masses identical with those in Elasmobranchii are thus formed (cf. E. van Beneden[411]). These masses become slightly more numerous in the succeeding stages. Indications of a fusion of this kind are shewn in fig. 38B. That the polynuclear masses really arise from a fusion of primitively distinct cells is clear from the description of the previous stages. The ova in the deeper layers, with modified granular nuclei, measure about 0.016 - 0.02 mm., and their nuclei from 0.01 - 0.012 mm.

Footnote 411: _Loc. cit._

With reference to the tissue of the hilus of the ovary, it may be noticed that the tubuliferous tissue (_t_) is relatively reduced in quantity. Its cells retain precisely their previous characters.

The chief difference between the stage of five days and that of two days after birth consists in the fact that during the earlier stage comparatively few modified nuclei were present, but the nuclei then presented the character of the nuclei of primitive ova.

I have ovaries both of the dog and cat of an equivalent stage, and in both of these the cells of the nests or egg tubes may be divided into two categories, destined respectively to become ova and follicle cells. Nothing which has come under my notice tends to shew that the tubuliferous tissue is in any way concerned in supplying the latter form of cell.

In a stage, seven days after birth, the same layers in the germinal epithelium may be noticed as in the last described stage. The outermost layer or pseudo-epithelium contains numerous developing ova, for the most part with modified nuclei. It is separated by a well marked layer of connective tissue from the middle layer of the germinal epithelium. The outer part of the middle layer contains more connective tissue and smaller nests than in the earlier stage, and most of the cells of this layer contain modified nuclei. In a few nests the protoplasm of the developing ova forms a continuous mass, not divided into distinct cells, but in the majority of instances the outline of each ovum can be distinctly traced. In addition to the cells destined to become ova, there are present in these nests other cells, which will clearly form the follicular epithelium. A typical nest from the middle layer is represented on Pl. 26, fig. 39A.

The nests or masses of ova in the innermost layer are for the most part still very large, but, in addition to the nests, a few isolated ova, enclosed in follicles, are to be seen.

A fairly typical nest, selected to shew the formation of the follicle, is represented on Pl. 26, fig. 39B.

The nest contains (1) fully formed permanent ova, completely or wholly enclosed in a follicle. (2) Smaller ova, not enclosed in a follicle. (3) Smallish cells with modified nuclei of doubtful destination. (4) Small cells obviously about to form follicular epithelium.

The inspection of a single such nest is to my mind a satisfactory proof that the follicular epithelium takes its origin from the germinal epithelium and not from the stroma or tubuliferous tissue. The several categories of elements observable in such a nest deserve a careful description.

(1) _The large ova in their follicles._--These ova have precisely the character of the young ova in Elasmobranchii. They are provided with a granular body invested by a delicate, though distinct membrane. Their nucleus is large and clear, but traversed by the network so fully described for Elasmobranchii. The cells of their follicular epithelium have obviously the same character as many other small cells of the nest. Two points about them deserve notice--(_a_) that many of them are fairly columnar. This is characteristic only of the first formed follicles. In the later formed follicles the cells are always flat and spindle-shaped in section. In this difference between the early and late formed follicles Mammals agree with Elasmobranchii. (_b_) The cells of the follicle are much more columnar towards the inner side than towards the outer. This point also is common to Mammals and Elasmobranchii.

Round the completed follicle a very delicate membrana propria folliculi appears to be present[412].

Footnote 412: _Loc. cit._, Waldeyer, p. 23, denies the
existence of this membrane for Mammalia. It certainly is not so
conspicuous as in some other types, but appears to me
nevertheless to be always present.

The larger ova, with follicular epithelium, measure about 0.04 mm., and their nucleus about 0.02 mm., the smaller ones about 0.022 mm., and their nucleus about 0.014 mm.

(2) _Medium sized ova._--They are still without a trace of a follicular epithelium, and present no special peculiarities.

(3) _The smaller cells with modified nuclei._--I have great doubt as to what is the eventual fate of these cells. There appear to be three possibilities.

(_a_) That they become cells of the follicular epithelium; (_b_) that they develop into ova; (_c_) that they are absorbed as a kind of food by the developing ova. I am inclined to think that some of these cells may have each of the above-mentioned destinations.

(4) _The cells which form the follicle._--The only point to be noticed about these is that they are smaller than the indifferent cells of the germinal epithelium, from which they no doubt originate by division. This fact has already been noticed by Waldeyer.

The isolated follicles at this stage are formed by ingrowths of connective tissue cutting off fully formed follicles from a nest. They only occur at the very innermost border of the germinal epithelium. This is in accordance with what has so often been noticed about the mammalian ovary, viz. that the more advanced ova are to be met with in passing from without inwards.