Chapter XII: The Organs of Excretion (4)

If the series of ovaries which I have represented be examined, it will not, I think, be denied that the general appearances are very much in favour of my view. The thickened patch of ovarian epithelium can apparently be traced through the whole series of sections, and no indications of its sudden reduction to the thin pseudo-epithelium are apparent. The most careful examination that I have been able to make brings to light nothing tending to shew that the general appearances are delusive. The important difference between us refers to _our views of the nature of the tissue subjacent to the pseudo-epithelium_. If my results be accepted, it is clear that the whole ovarian region is an epithelium interpenetrated by connective tissue ingrowths, so that the region below the pseudo-epithelium is a kind of honeycomb or trabecular net-work of germinal epithelium, developing ova of all stages and sizes, and composed of cells capable of forming follicular epithelium for developing ova. Ludwig figures what he regards as the formation of the follicular epithelium round primitive ova during their passage into the stroma. It is quite clear to me, that his figures of the later stages, 33 and 34, represent fully formed permanent ova surrounded by a follicular epithelium, and that their situation in contact with the pseudo-epithelium is, so to speak, an accident, and it is quite possible that his figures 31 and 32 also represent fully formed ova; but I have little hesitation in asserting that he has not understood the mode of formation of the follicular epithelium, and that, though his statement that it is derived from the germinal epithelium is quite correct, his account of the process is completely misleading. The same criticism does not exactly apply to Semper's statements. Semper has really observed the formation of the follicular epithelium round young ova; but, nevertheless, he appears to me to give an entirely wrong account of the relation of the stroma to the germinal epithelium. The extent of the difference between Semper's and my view may perhaps best be shewn by a quotation from Semper, _loc. cit._, 465:--"In females the nests of primitive ova sink in groups into the stroma. In these groups one cell enlarges till it becomes the ovum, the neighbouring cells increase and arrange themselves around the ova as follicle cells."

Although the histological changes which take place in the succeeding stages are not inconsiderable, they do not involve any fundamental change in the constitution of the ovarian region, and may be described with greater brevity than has been so far possible.

In a half-grown female, with an ovarian region of 3mm. in breadth, and 0.8mm. in thickness, the stroma of the ovarian region has assumed a far more formed aspect than before. It consists (Pl. 24, fig. 10) of a basis in most parts fibrous, but in some nearly homogeneous, with a fair number of scattered cells. Immediately below the pseudo-epithelium, there is an imperfectly developed fibrous layer, forming a kind of tunic, in which are imbedded the relatively reduced epithelial trabeculæ of the previous stages. They appear in sections as columns, either continuous with or independent of the pseudo-epithelium, formed of normal cells of the germinal epithelium, nests of ova, and permanent ova in various stages of development. Below this there comes a layer of larger ova which are very closely packed. A not inconsiderable number of the larger ova have, however, a superficial situation, and lie in immediate contact with the pseudo-epithelium. Some of the younger ova, enclosed amongst epithelial cells continuous with the pseudo-epithelium, are very similar to those figured by Ludwig. It is scarcely necessary to insist that this fact does not afford any argument in favour of his interpretations. The ovarian region is honeycombed by large vascular channels with distinct walls, and other channels which are perhaps lymphatic.

The surface of the ovarian region is somewhat irregular and especially marked by deep oblique transverse furrows. It is covered by a distinct, though still irregular pseudo-epithelium, which is fairly columnar in the furrows but flattened along the ridges. The cells of the pseudo-epithelium have one peculiarity very unlike that of ordinary epithelial cells. Their inner extremities (vide fig. 10) are prolonged into fibrous processes which enter the subjacent tissue, and bending nearly parallel to the surface of the ovary, assist in forming the tunic spoken of above. This peculiarity of the pseudo-epithelial cells seems to indicate that they do not essentially differ from cells which have the character of undoubted connective tissue cells, and renders it possible that the greater part of the tunic, which has apparently the structure of ordinary connective tissue, is in reality derived from the original germinal epithelium, a view which tallies with the fact that in some instances the cells of the tunic appear as if about to assist in forming the follicular epithelium of some of the developing ova. In Raja, the similarity of the pseudo-epithelium to the subjacent tissue is very much more marked than in Scyllium. The pseudo-epithelium appears merely as the superficial layer of the ovarian tunic somewhat modified by its position on the surface. It is formed of columnar cells with vertically arranged fibres which pass into the subjacent layers, and chiefly differ from the ordinary fibres in that they still form parts of the cell-protoplasm enclosing the nucleus. In Pl. 25, fig. 34, an attempt is made to represent the relations of the pseudo-epithelium to the subjacent tissue in Raja. Ludwig's figures of the pseudo-epithelium of the ovary, in the regular form of its constituent cells, and its sharp separation by a basement membrane from the tissue below, are quite unlike anything which I have met with in my sections either of Raja or Scyllium.

Close to the dorsal border of the ovary the epithelial cells of the non-ovarian region have very conspicuous tails, extending into a more or less homogeneous substance below, which constitutes a peculiar form of tunic for this part of the ovarian ridge.

In the full-grown female the stroma of the ovarian region is denser and has a more fibrous aspect than in the younger animal. Below the pseudo-epithelium it is arranged in two or three more or less definite layers, in which the fibres run at right angles. It forms a definite ovarian tunic. The pseudo-epithelium is much more distinct, and the tails of its cells, so conspicuous in previous stages, can no longer be made out.

_Formation of the permanent ova and the follicular epithelium._--In my monograph on the development of Elasmobranch Fishes an account was given of the earliest stages in the development of the primitive ova, and I now take up their development from the point at which it was left off in that work. From their first formation till the stage spoken of in my monograph as P, their size remains fairly constant. The larger examples have a diameter of about 0.035 mm., and the medium-sized examples of about 0.03 mm. The larger nuclei have a diameter of about 0.16 mm., but their variations in size are considerable. If the above figures be compared with those on page 350 of my monograph on Elasmobranch Fishes, it will be seen that the size of the primitive ova during these stages is not greater than it was at the period of their very first appearance.

The ova (Pl. 24, fig. 1) are usually aggregated in masses, which might have resulted from division of a single ovum. The outlines of the individual ova _are always distinct_. Their protoplasm is clear, and their nuclei, which are somewhat passive towards staining reagents, are granular, with one to three nucleoli. I have noticed, up to stage P, the occasional presence of highly refractive spherules in the protoplasm of the primitive ova already described in my monograph (pp. 353, 354, Pl. 12, fig. 15). They seem to occur up to a later period than I at first imagined. Their want of constancy probably indicates that they have no special importance. Professor Semper has described similar appearances in the male primitive ova of a later period.

As to the distribution of the primitive ova in the germinal epithelium, Professor Semper's statement that the larger primitive ova are found in masses in the centre, and that the smaller ova are more peripherally situated is on the whole true, though I do not find this distribution sufficiently constant to lay so much stress on it as he does.

The passive condition of the primitive ova becomes suddenly broken during stage Q, and is succeeded by a period of remarkable changes. It has only been by the expenditure of much care and trouble that I have been able to elucidate to my own satisfaction what takes place, and there are still points which I do not understand.

Very shortly after stage Q, in addition to primitive ova with a perfectly normal nucleus, others may be seen in which the nucleus is apparently replaced by a deeply stained irregular body, smaller than the ordinary nuclei (Pl. 24, fig. 11, _d.n._). This body, by the use of high objectives, is seen to be composed of a number of deeply stained granules, and around it may be noticed a clear space, bounded by a very delicate membrane. The granular body usually lies close to one side of this membrane, and occasionally sends a few fine processes to the opposite side.

The whole body, _i.e._ all within the delicate membrane is, according to my view, a modified nucleus; as appears to me very clearly to be shewn by the fact that it occupies the normal position of a nucleus within a cell body. Semper, on the other hand, regards the contained granular body as the nucleus, which he compares with the spindles of Bütschli, Auerbach, &c.[374]. This interpretation appears to me, however, to be negatived by the position of these bodies. The manner in which Semper may, perhaps, have been led to his views will be obvious when the later changes of the primitive ova are described. The formation of these nuclei would seem to be due to a segregation of the constituents of the original nuclei; the solid parts becoming separated from the more fluid. As a rule, the modified nuclei are slightly larger than the original ones. In stage Q the following two tables shew the dimensions of the parts of three unmodified and of three modified nuclei taken at random.

Footnote 374: _Loc. cit._ p. 361.

_Primitive ova with unmodified nuclei_--

Nuclei.

0.014 mm.
0.012 mm.
0.01 mm.

_Primitive ova with modified nuclei_--

Granular
Nuclei. Bodies in nuclei.

0.018 mm. 0.006 mm.
0.018 mm. 0.006 mm.
0.012 mm. 0.009 mm.

For a slightly older stage than Q, the two annexed tables also shew the comparative size of the modified and unmodified nuclei:

_Unmodified nuclei of normal primitive ova--_

0.014 mm.
0.016 mm.
0.014 mm.
0.016 mm.
0.016 mm.

_Nuclei of primitive ova with modified nuclei--_

Granular
Nuclei. Bodies in Nuclei.

0.018 mm. 0.008 mm.
0.016 mm. 0.008 mm.
0.016 mm. 0.01 mm.
0.016 mm.
0.018 mm.

These figures bring out with clearness the following points: (1) that the modified nuclei are slightly but decidedly larger on the average than the unmodified nuclei; (2) that the contained granular bodies _are very considerably_ smaller than ordinary nuclei.

Soon after the appearance of the modified nuclei, remarkable changes take place in the cells containing them. Up to the time such nuclei first make their appearance the outlines of the individual ova are very clearly defined, but subsequently, although numerous ova with but slightly modified nuclei are still to be seen, yet on the whole the outlines of all the primitive ova are much less distinct than before; and this is especially the case with the primitive ova containing modified nuclei.

From cases in which three or four ova are found in a mass with modified nuclei, but in which the outline of each ovum is fairly distinct, it is possible to pass by insensible gradations to other cases in which two or three or more modified nuclei are found embedded in a mass of protoplasm in which no division into separate cells can be made out (fig. 14). For these masses I propose to employ the term nests. They correspond in part with the _Ureiernester_ of Professor Semper.

Frequently they are found in hardened specimens to be enclosed in a membrane-like tunic which appears to be of the nature of coagulated fluid. These membranes closely resemble and sometimes are even continuous with trabeculæ which traverse the germinal epithelium. Ovaries differ considerably as to the time and completeness of the disappearance of the outlines marking the separate cells, and although, so far as can be gathered from my specimens, the rule is that the outlines of the primitive ova with modified nuclei soon become indistinct, yet in one of my best preserved ovaries very large nests with modified nuclei are present in which the outline of each ovum is as distinct as during the period before the nuclei undergo these peculiar changes (Pl. 24, fig. 12). In the same ovary other nests are present in which the outlines of the individual ova are no longer visible. The section represented on Pl. 24, fig. 2, is fairly average as to the disappearance of the outlines of the individual ova.

It is clear from the above statements, that in the first instance the nests are produced by the coalescence of several primitive ova into a single mass or syncytium; though of course, the several separate ova of a nest may originally, as Semper believes, have arisen from the division of a single ovum. In any case there can be no doubt that the nests of separate ova increase in size as development proceeds; a phenomenon which is more reasonably explained on the view that the ova divide, than on the view that they continue to be freshly formed. The same holds true for the nests of nuclei and this, as well as other facts, appears to me to render it probable that the nests grow by division of the nuclei without corresponding division of the protoplasmic matrix. I cannot, however, definitely prove this point owing to my having found nests, with distinct outlines to the ova, as large as any without such outlines.

The nests are situated for the most part near the surface of the germinal epithelium. The smaller ones are frequently spherical, but the larger are irregular in form. The former are about 0.05 mm. in diameter; the latter reach 0.1 mm. Scattered generally, and especially in the deeper layers, and at the edges of the germinal epithelium, are still unmodified or only slightly modified primitive ova. These unmodified primitive ova are aggregated in masses, but in these masses the outlines of each ovum, though perhaps less clear than in the earlier period, are still distinct.

When the embryo reaches a length of seven centimètres, and even in still younger embryos, further changes are observable. In the first place many of the modified nuclei acquire fresh characters, and it becomes necessary to divide the modified nuclei into two categories. In both of these the outer boundary of the nucleus is formed by a very delicate membrane, the space within which is perfectly clear except for the granular body. In the variety which now appears in considerable numbers the granular body has an irregular star-like form. The rays of the star are formed of fibres frequently knobbed at their extremities, and the centre of the star usually occupies an eccentric position. Typical examples of this form of modified nucleus, which may be spoken of as the stellate variety, are represented on Pl. 25, fig. 17; between it and the older granular variety there is an infinite series of gradations, many of which are represented on Pl. 24, figs. 12, 14, 15, 16. Certain of the stellate nuclei exhibit two centres instead of one, and in some cases, like that represented on Pl. 25, fig. 19, the stellate body of two nuclei is found united. Both of these forms are possibly modifications of the spindle-like form assumed by nuclei in the act of dividing, and may be used in proving that the nests increase in size by the division of the contained nuclei. In addition to the normal primitive ova, a few of which are still present, there are to be found, chiefly in the deeper layers of the germinal epithelium, larger ova differing considerably from the primitive ova. They form the permanent ova (Pl. 24, fig. 3, _o_). Their average diameter is 0.04 mm., compared with 0.03 mm., the diameter of original primitive ova. The protoplasm of which they are composed is granular, but at first a membrane can hardly be distinguished around them; their nucleus is relatively large, 0.02 - 0.027 mm. in diameter. It presents the characters ascribed by Eimer[375], and many other recent authors[376], to typical nuclei (vide Pl. 24, fig. 3, and Pl. 24, 25, figs. 13, 14, 15, 16, 17, 18). It is bounded by a distinct membrane, within which is a more or less central nucleolus from which a number of radial fibres which stain very deeply pass to the surface; here they form immediately internal to the membrane a network with granules at the nodal points. In some instances the regularity of the arrangement of these fibres is very great, in other instances two central nucleoli are present, in which case the regularity is considerably interfered with. The points in which the youngest permanent ova differ from the primitive may be summed up as follows:--

(1) The permanent ova are larger, the smallest of them being larger than the average primitive ova in the proportion of four to three. (2) They have less protoplasm as compared to the size of the nucleus. (3) Their protoplasm is granular instead of being clear. (4) Their nucleus is clear with exception of a network of fibres instead of being granular as in the primitive ova. It thus appears that the primitive ova and permanent ova are very different in constitution, though genetically related in a way to be directly narrated.

Footnote 375: _Archiv f. micr. Anat._ Vol. XIV.

Footnote 376: Vide especially Klein, _Quart. Journ. of Mic.
Sci._ July 1878.

The formation of permanent ova is at its height in embryos of about seven centimètres or slightly larger. The nests at this stage are for the most part of a very considerable size and contain a large number of nuclei, which have probably, as before insisted, originated from a division of the smaller number of nuclei present in the nests at an earlier stage. Figs. 14-18 are representations of nests at this period. The diameter of the nuclei is, on the whole, slightly greater than at an earlier stage. A series of measurements gave the following results:--

0.016 mm.
0.016 mm.
0.018 mm.
0.02 mm.
0.02 mm.

Both varieties of modified nuclei are common enough, though the stellate variety predominates. The nuclei are sometimes in very close contact, and sometimes separated by protoplasm, which in many instances is very slightly granular. In a large number of the nests nothing further is apparent than what has just been described, but in a very considerable number one or more nuclei are present, which exhibit a transitional character between the ordinary stellate nuclei of my second category, and the nuclei of permanent ova as above described; and in these nests the formation of permanent ova is taking place. Permanent ova in the act of development are indicated in my figures by the letters _do_. Many of the intermediate nuclei are more definitely surrounded by granular protoplasm than the other nuclei of the nests, and accordingly have their outlines more sharply defined. Between nuclei of this kind, and others as large as those of the permanent ova, there are numerous transitional forms. The larger ones frequently lie in a mass of granular protoplasm projecting from the nest, and only united with it by a neck (Pl. 24, figs. 14 and 16). For prominences of this kind to become independent ova, it is only necessary for the neck to become broken through. Nests in which such changes are taking place present various characters. In some cases several nuclei belonging to a nest appear to be undergoing conversion into permanent ova at the same time. Such a case is figured on Pl. 25, figs. 17 and 18. In these cases the amount of granular protoplasm in the nest and around each freshly formed ovum is small. In the more usual cases only one or two permanent ova at the utmost are formed at the same time, and in these instances a considerable amount of granular protoplasm is present around the nucleus of the developing permanent ovum. In such instances it frequently happens several of the nuclei not undergoing conversion appear to be in the process of absorption, and give to the part of the nest in which they are contained a very hazy and indistinct aspect (Pl. 24, fig. 15). Their appearance leads me to adopt the view _that while some of the nuclei of each nest are converted into the nuclei of the permanent ova, others break down and are used as the pabulum, at the expense of which the protoplasm of the young ovum grows_.

It should, however, be stated, that after the outlines of the permanent ova have become definitely established, I have only observed in a single instance the inclusion of a nucleus within an ovum (Pl. 25, fig. 24). In many instances normal nuclei of the germinal epithelium may be so observed within the ovum.

The nuclei which are becoming converted into the nuclei of permanent ova gradually increase in size. The following table gives the diameter of four such nuclei:--

0.022 mm.
0.022 mm.
0.024 mm.
0.032 mm.

These figures should be compared with those of the table on page 564.

The ova when first formed are situated either at the surface or in the deeper layers of the germinal epithelium. Though to a great extent surrounded by the ordinary cells of the germinal epithelium, they are not at first enclosed in a definite follicular epithelium. The follicle is, however, very early formed.

My observations lead me then to the conclusion that in a general way the permanent ova are formed by the increase of protoplasm round some of the nuclei of a nest, and the subsequent separation of the nuclei with their protoplasm from the nest as distinct cells--a mode of formation exactly comparable with that which so often takes place in invertebrate egg tubes.

Besides the mode of formation of permanent ova just described, a second one also seems probably to occur. In ovaries just younger than those in which permanent ova are distinctly formed, there are present primitive ova, with modified nuclei of the stellate variety, or nuclei sometimes even approaching in character those of permanent ova, which are quite isolated and not enclosed in a definite nest. The body of these ova is formed of granular protoplasm, but their outlines are very indistinct. Such ova are considerably larger than the normal primitive ova. They may measure 0.04 mm. In a slightly later stage, when fully formed permanent ova are present, isolated ones are not infrequent, and it seems natural to conclude that these isolated ova are the direct descendants of the primitive ova of the earlier stage. It seems a fair deduction that in some cases primitive ova undergo a direct metamorphosis into permanent ova by a modification of their nucleus, and the assumption of a granular character in their protoplasm, without ever forming the constituent part of a nest.

It is not quite clear to me that in all nests the coalescence of the protoplasm of the ova necessarily takes place, since some nests are to be found at all stages in which the ova are distinct. Nevertheless, I am inclined to believe that the fusion of the ova is the normal occurrence.

The mode of formation of the permanent ova may then, according to my observations, take place in two ways:--1. By the formation of granular protoplasm round the nucleus in a nest, and the separation of the nucleus with its protoplasm as a distinct ovum. 2. By the direct metamorphosis of an isolated primitive ovum into a permanent ovum. The difference between these two modes of formation does not, from a morphological point of view, appear to be of great importance.

The above results appear clearly to shew that _the primitive ova in the female are not to be regarded as true ova, but as the parent sexual cells which give rise to the ova_: a conclusion which completely fits in with the fact that cells exactly similar to the primitive ova in the female give rise to the spermatic cells in the male.

Slightly after the period of their first formation the permanent ova become invested by a very distinct and well-marked, somewhat flattened, follicular epithelium (Pl. 24, fig. 3). Where the ova lie in the deeper layers of the germinal epithelium, the follicular epithelium soon becomes far more columnar on the side turned inwards, than on that towards the surface, especially when the inner side is in contact with the stroma (Pl. 24, fig. 7, and Pl. 25, figs. 24 and 26). This is probably a special provision for the growth and nutrition of the ovum.

There cannot be the smallest doubt that the follicular epithelium is derived from the general cells of the germinal epithelium--a point on which my results fully bear out the conclusions of Ludwig and Semper.

The larger ova themselves have a diameter of about 0.06 mm., and their nucleus of about 0.04 mm. The vitellus is granular, and provided with a distinct, though delicate membrane, which has every appearance of being a product of the ovum itself rather than of the follicular epithelium. The membrane would seem indeed to be formed in some instances even before the ovum has a definite investment of follicle cells. The vitellus is frequently vacuolated, but occasionally the vacuoles appear to be caused by a shrinking due to the hardening reagent. The nucleus has the same peculiar reticulate character as at first. Its large size, as compared with the ovum, is very noticeable.

With this stage the embryonic development of the ova comes to a close, though the formation of fresh ova continues till comparatively late in life. I have, however, two series of sections of ovaries preserved in osmic acid, from slightly larger embryos than the one last described, about which it may be well to say a few words before proceeding to the further development of the permanent ova.

The younger of these ovaries was from a Scyllium embryo 10 centimètres long, preserved in osmic acid.

A considerable number of nests were present (Pl. 24, fig. 13), exhibiting, on the whole, similar characters to those just described.

A series of measurements of the nuclei in them were made, leading to the following results:--

0.014 mm.
0.014 mm.
0.016 mm.
0.016 mm.
0.018 mm.
0.018 mm.

Thus, if anything, the nuclei were slightly smaller than in the younger embryo. It is very difficult in the osmic specimens to make out clearly the exact outlines of the various structures, the nuclei in many instances being hardly more deeply stained than in the protoplasm around them. The network in the nuclei is also far less obvious than after treatment with picric acid. The permanent ova were hardly so numerous as in the younger ovary before described. A number of these were measured with the following results:--

Ovum. Nucleus.

0.03 mm. 0.014 mm.
0.034 mm. 0.018 mm.
0.028 mm. 0.016 mm.
0.03 mm. 0.02 mm.
0.04 mm. 0.02 mm.
0.04 mm. 0.02 mm.
0.048 mm. 0.02 mm.

These figures shew that the nuclei of the permanent ova are smaller than in the younger embryo, and it may therefore be safely concluded that, in spite of the greater size of the embryo from which it is taken, the ovary now being described is in a more embryonic condition than the one last dealt with.

Though the permanent ova appeared to be formed from the nests in the manner already described, it was fairly clear from the sections of this ovary that many of the original primitive ova, after a metamorphosis of the nucleus and without coalescing with other primitive ova to form nests, become converted directly into the permanent ova. Many large masses of primitive ova, or at least of ova with the individual outlines of each ovum distinct, were present. The average size of ova composing these was however small, the body measuring about 0.016 mm., and the nucleus 0.012 mm. Isolated ova with metamorphosed nuclei could also be found measuring 0.022, and their nuclei about 0.014 mm.

The second of the two ovaries, hardened in osmic acid, was somewhat more advanced than the ovary in which the formation of permanent ova was at its height. Fewer permanent ova were in the act of being formed, and many of these present had reached a considerable size, measuring as much as 0.07 mm. Nests of the typical forms were present as before, but the nuclei in them were more granular than at the earlier period, and on the average slightly smaller. A series measured had the following diameters:--

0.01 mm.
0.012 mm.
0.014 mm.
0.016 mm.

One of these nests is represented on Pl. 25, fig. 20. Many nests with the outlines of the individual ova distinct were also present.

On the whole it appeared to me, that the second mode of formation of permanent ova, viz. that in which the nest does not come into the cycle of development, preponderated to a greater extent than in the earlier embryonic period.

POST-EMBRYONIC DEVELOPMENT OF THE OVA.--My investigations upon the post-embryonic growth and development of the ova, have for the most part been conducted upon preserved ova, and it has been impossible for me, on this account, to work out, as completely as I should have wished, certain points, more especially those connected with the development of the yolk.

Although my ovaries have been carefully preserved in a large number of reagents, including osmic acid, picric acid, chromic acid, spirit, bichromate of potash, and Müller's fluid, none of these have proved universally successful, and bichromate of potash and Müller's fluid are useless. Great difficulties have been experienced in distinguishing the artificial products of these reagents. My investigations have led me to the result, that in the gradual growth of the ova with the age of the individual the changes are not quite identical with those during the rapid growth which takes place at periods of sexual activity, after the adult condition has been reached--a result to which His has also arrived, with reference to the ova of Osseous Fish. I propose dealing separately with the several constituents of the egg-follicle.

_Egg membranes._--A vitelline membrane has been described by Leydig[377] in Raja, and an albuminous layer of the nature of a chorion[378] by Gegenbaur[379] in Acanthias--the membranes described in these two ways being no doubt equivalent.

Footnote 377: _Rochen u. Haie._

Footnote 378: By _chorion_ I mean, following E. van Beneden's
nomenclature, a membrane formed by the follicular epithelium,
and, by _vitelline membrane_, one formed by the vitellus or
body of the ovum.

Footnote 379: "Bau und Entwicklung d. Wirbelthiereier," &c.,
_Müll. Archiv_, 1861.

Dr Alex. Schultz[380] has more recently investigated a considerable variety of genera and finds three conditions of the egg membranes. (1) In Torpedo, a homogeneous membrane, which is of the nature of a chorion. (2) In Raja, a homogeneous membrane which is, however, perforated. (3) In Squalidæ, a thick homogeneous membrane, internal to which is a thinner perforated membrane. He apparently regards the perforated inner membrane as a specialised part of the simple membrane found in Torpedo, and states that this membrane is of the nature of a chorion.

Footnote 380: "Zur Entwicklungsgeschichte d. Selachier,"
_Arch. f. mikr. Anat._ Vol. XI.

My own investigations have led me to the conclusion that though the egg-membranes can probably be reduced to single type for Elasmobranchii, yet that they vary with the stage of development of the ovum. Scyllium (stellare and canicula) and Raja have formed the objects of my investigation. I commence with the two former.

It has already been stated that in Scyllium, even before the follicular epithelium becomes formed, a delicate membrane round the ovum can be demonstrated, which appears to me to be derived from the vitellus or body of the ovum, and is therefore of the nature of a vitelline membrane. It becomes the vitelline membrane of Leydig, the albuminous membrane of Gegenbaur, and homogeneous membrane of Schultz.

In a young fish (not long hatched) with ova of not more than 0.12 mm., this membrane, though considerably thicker than in the embryo, is not thick enough to be accurately measured. In ova of 0.5 mm. from a young female (Pl. 25, fig. 21) the vitelline membrane has a thickness of 0.002 mm. and is quite homogeneous[381]. Internally to it may be observed very faint indications of the differentiation of the outermost layer of the vitellus into the perforated or radially striated membrane of Schultz, which will be spoken of as _zona radiata_.

Footnote 381: The apparent structure in the vitelline membrane
in my figure is merely intended to represent the dark colour
assumed by it on being stained. The zona radiata has been made
rather too thick by the artist.

In an ovum of 1 mm. from the nearly full grown though not sexually mature female, the zona radiata has increased in thickness and definiteness, and may measure as much as 0.004 mm. It is always very sharply separated from the vitelline membrane, but appears to be more or less continuous on its inner border with the body of the ovum, at the expense of which it no doubt grows in thickness.

In ova above 1 mm. in diameter, both vitelline membrane and zona radiata, but especially the latter, increase in thickness. The zona becomes marked off from the yolk, and its radial striæ become easy to see even with comparatively low powers. In many specimens it appears to be formed of a number of small columns, as described by Gegenbaur and others. The stage of about the greatest development of both the vitelline membrane and zona radiata is represented on Pl. 25, fig. 22.

At this time the vitelline membrane appears frequently to exhibit a distinct stratification, dividing it into two or more successive layers. It is not, however, acted on in the same manner by all reagents, and with absolute alcohol appears at times longitudinally striated.

From this stage onwards, both vitelline membrane and zona gradually atrophy, simultaneously with a series of remarkable changes which take place in the follicular epithelium. The zona is the first to disappear, and the vitelline membrane next becomes gradually thinner. Finally, when the egg is nearly ripe, the follicular epithelium is separated from the yolk by an immeasurably thin membrane--the remnant of the vitelline membrane--only visible in the most favourable sections (Pl. 25, fig. 23, _vt._). When the egg becomes detached from the ovary even this membrane is no longer to be seen.

Both the vitelline membrane and the zona radiata are found in Raja, but in a much less developed condition than in Scyllium. The vitelline membrane is for a long time the only membrane present, but is never very thick (Pl. 25, fig. 31). The zona is not formed till a relatively much later period than in Scyllium, and is always delicate and difficult to see (Pl. 25, fig. 32). Both membranes atrophy before the egg is quite ripe; and an apparently fluid layer between the follicular epithelium and the vitellus, which coagulates in hardened specimens, is probably the last remnant of the vitelline membrane. It is, however, much thicker than the corresponding remnant in Scyllium.

Though I find the same membranes in Scyllium as Alexander Schultz did in other Squalidæ, my results do not agree with his as to Raja. Torpedo I have not investigated.

It appears to me probable that the ova in all Elasmobranch Fishes have at some period of their development the two membranes described at length for Scyllium. Of these the inner one, or zona radiata, will probably be admitted on all hands to be a product of the peripheral protoplasm of the egg.

The outer one corresponds with the membrane usually regarded in other Vertebrates as a chorion or product of the follicular epithelium, but, by tracing it back to its first origin, I have been led to reject this view of its nature.

_The follicular epithelium._--The follicular epithelium in the eggs of Raja and Acanthias has been described by Gegenbaur[382]. He finds it flat in young eggs, but in the larger eggs of Acanthias more columnar, and with the cells wedged in so as to form a double layer. These observations are confirmed by Ludwig[383].

Footnote 382: _Loc. cit._

Footnote 383: _Loc. cit._

Alexander Schultz[384] states that in Torpedo, the eggs are at first enclosed in a simple epithelium, but that in follicles of .008 mm. there appear between the original large cells of the follicle (which he describes as granulosa cells and derives from the germinal epithelium) a number of peculiar small cells. He states that these are of the same nature as the general stroma cells of the ovary, and believes that they originate in the stroma. When the eggs have reached 0.1 - 0.15 mm., he finds that the small and large cells have a very regular alternating arrangement.

Footnote 384: _Loc. cit._

Semper records but few observations on the follicular epithelium, but describes in Raja the presence of a certain number of large cells amongst smaller cells. He believes that they may develop into ova, and considers them identical with the larger cells described by Schultz, whose interpretations he does not, however, accept.

My own results accord to a great extent with those of Dr Schultz, as far as the structure of the follicular epithelium is concerned, but I am at one with Semper in rejecting Schultz's interpretations.

In Scyllium, as has already been mentioned, the follicular epithelium is at first flat and formed of a single layer of uniform cells, each with a considerable amount of clear protoplasm and a granular nucleus. It is bounded externally by a delicate membrane--the membrana propria folliculi of Waldeyer--and internally by the vitelline membrane. In the ovaries of very young animals the cells of the follicular epithelium are more columnar on the side towards the stroma than on the opposite side, but this irregularity soon ceases to exist.

In many cases the nuclei of the cells of the follicular epithelium exhibit a spindle modification, which shews that the growth of the follicular epithelium takes place by the division of its cells. No changes of importance are observable in the follicular epithelium till the egg has reached a diameter of more than 1 mm.

It should here be stated that I have some doubts respecting the completeness of the history of the epithelium recorded in the sequel. Difficulties have been met with in completely elucidating the chronological order of the occurrences, and it is possible that some points have escaped my observation.

The first important change is the assumption of a palisade-like character by the follicle cells, each cell becoming very narrow and columnar and the nucleus oval (Pl. 25, fig. 28). In this condition the thickness of the epithelium is about 0.025 mm. The epithelium does not, however, become uniformly thick over the whole ovum, but in the neighbourhood of the germinal vesicle it is very flat and formed of granular cells with indistinct outlines, rather like the hypodermis cells of many Annelida. Coincidently with this change in the follicular epithelium the commencement of the atrophy of the membranes of the ovum, described in the last section, becomes apparent.

The original membrana propria folliculi is still present round the follicular epithelium, but is closely associated with a fibrous layer with elongated nuclei. Outside this there is now a layer of cells, very much like an ordinary epithelial layer, which may possibly be formed of cells of the true germinal epithelium (fig. 28, _fe´_). This layer, which will be spoken of as the secondary follicle layer, might easily be mistaken for the follicular epithelium, and it is possible that it has actually been so mistaken by Eimer, Clark, and Klebs, in Reptilia, and that the true follicular epithelium (in a flattened condition) has been then spoken of as the _Binnenepithel_.

In slightly older eggs the epithelial cells are no longer uniform or arranged as a single layer. The general arrangement of these cells is shewn in Pl. 25, fig. 29. A considerable number of them are more or less flask-shaped, with bulky protoplasm prolonged into a thin stem directed towards the vitelline membrane, with which, in many instances if not all, it comes in contact. These larger cells are arranged in several tiers. Intercalated between them are a number of elongated small cells with scanty protoplasm and a deeply staining nucleus, not very dissimilar to, though somewhat smaller than, the columnar cells of the previous stage. There is present a complete series of cells intermediate between the larger cells and those with a deeply stained nucleus, and were it not for the condition of the epithelium in Raja, to be spoken of directly, I should not sharply divide the cells into two categories. In surface views of the epithelium the division into two kinds of cells would not be suspected. There can, it appears to me, be no question that both varieties of cell are derived from the primitive uniform follicle cells.

The fibrous layer bounding the membrana propria folliculi is thicker than in the last stage, and the epithelial-like layer (_fe´_) which bounds it externally is more conspicuous than before. Immediately adjoining it are vascular and lymph sinuses. The thickness of the follicular epithelium at this stage may reach as much as 0.04 mm., though I have found it sometimes considerably flatter. The cells composing it are, however, so delicate that it is not easy to feel certain that the peculiarities of any individual ovum are not due to handling. The absence of the peculiar columnar epithelium on the part of the surface adjoining the germinal vesicle is as marked a feature as in the earlier stage. When the egg is nearly ripe, and the vitelline membrane has been reduced to a mere remnant, the follicular epithelium is still very columnar (Pl. 25, fig. 23). The thickness is greater than in the last stage, being now about 0.045 mm., but the cells appear only to form a single definite layer. From the character of their nuclei, I feel inclined to regard them as belonging to the category of the smaller cells of the previous stage, and feel confirmed in this view by finding certain bodies in the epithelium, which have the appearance of degenerating cells with granular nuclei, which I take to be the flask-shaped cells which were present in the earlier stage.

I have not investigated the character of the follicular epithelium in the perfectly ripe ovum ready to become detached from the ovary. Nor can I state for the last-described stage anything about the character of the follicular epithelium in the neighbourhood of the germinal vesicle.

As to the relation of the follicular epithelium to the vitelline membrane, and the possible processes of its cells continued into the yolk, I can say very little. I find in specimens teased out after treatment with osmic acid, that the cells of the follicular epithelium are occasionally provided with short processes, which might possibly have perforated the vitelline membrane, but have met with nothing so clear as the teased out specimens figured by Eimer. Nothing resembling the cells within the vitelline membrane, as described by His[385] in Osseous Fish, and Lindgren in Mammalia, has been met with[386].

Footnote 385: _Das Ei bei Knochenfischen._

Footnote 386: _Arch. f. Anat. Phys._ 1877.

My observations in Raja are not so full as those upon Scyllium, but they serve to complete and reconcile the observations of Semper and Schultz, and also to shew that the general mode of growth of the follicular epithelium is fundamentally the same in my representatives of the two divisions of the Elasmobranchii. In very young eggs, in conformity with the results of all previous observers, I find the follicular epithelium approximately uniform. The cells are flat, but extended so as to appear of an unexpected size in views of the surface of the follicle. This condition does not, however, last very long. A certain number of the cells enlarge considerably, others remaining smaller and flat. The differences between the larger and the smaller cells are more conspicuous in sections than in surface views, and though the distribution of the cells is somewhat irregular, it may still be predicted as an almost invariable rule that the smaller cells of the follicle will line that part of the surface of the ovum, near to which the germinal vesicle is situated. On Pl. 25, fig. 30, is shewn in section a fairly average arrangement of the follicle cells. Semper considers the larger cells of such a follicle to be probably primitive ova destined to become permanent ova. This view I cannot accept: firstly, because these cells only agree with primitive ova in being exceptionally large--the character of their nucleus, with its large nucleolus, being not very like that of a primitive ovum. Secondly, because they shade into ordinary cells of the follicle; and thirdly, because no evidence of their becoming ova has come before me, but rather the reverse, in that it seems probable that they have a definite function connected with the nutrition of the egg. To this point I shall return.

In the next stage the small cells have become still smaller. They are columnar, and are wedged in between the larger ones. No great regularity in distribution is as yet attained (Pl. 25, fig. 31). Such a regularity appears in a later stage (Pl. 25, fig. 32), which clearly corresponds with fig. 8 on Pl. 34 of Schultz's paper, and also with the stage of Scyllium in Pl. 25, fig. 29, though the distinction between the two kinds of cells is here far better marked than in Scyllium. The big cells have now become flask-shaped like those in Scyllium, and send a process down to the vitelline membrane. The smaller cells are arranged in two or three tiers, but the larger cells in a single layer. The distribution of the larger and smaller cells is in some instances very regular, as shewn in the surface view on Pl. 25, fig. 33. There can, it appears to me, be no doubt that Schultz's view of the smaller cells being lymph-cells which have migrated into the follicle cannot be maintained.

The thickness of the epithelium at this stage is about 0.04 mm. In the succeeding stages, during which the egg is rapidly growing to the colossal size which it eventually attains, the follicular epithelium does not to any great extent alter in constitution. It grows thicker on the whole, and as the vitelline membrane gradually atrophies, its lower surface becomes irregular, exhibiting somewhat flattened prominences, which project into the yolk. At the greatest height of the prominences the epithelium may reach a thickness of 0.06 mm., or even more. The arrangement of the tissues external to the follicular epithelium is the same in Raja as in Scyllium.

The most interesting point connected with the follicle, both in Scyllium and Raja and presumably in other Elasmobranchii is that its epithelium at the time when the egg is rapidly approaching maturity is composed with more or less of distinctness of two forms of cells. One of these is large flask-shaped and rich in protoplasm, the other is small, consisting of a mere film of protoplasm round a nucleus. Considering that the larger cells appear at the time of rapid growth, it is natural to interpret their presence as connected with the nutrition of the ovum. This view is supported by the observations of Eimer and Braun, on the development of Reptilian ova. In many Reptilian ova it appears from Eimer's[387] observations, that the follicular epithelium becomes several layers thick, and that a differentiation of the cells, similar to that in Elasmobranchii, takes place. The flask-shaped cells eventually undergo peculiar changes, becoming converted into a kind of beaker-cell, with prolongations through the egg membranes, which take the place of canals leading to the interior of the egg. Braun also expresses himself strongly in favour of the flask-shaped cells functioning in the nutrition of the egg[388]. That these cells in the Reptilian ova really correspond with those in Elasmobranchii appears to me clear from Eimer's figures, but I have not myself studied any Reptilian ovum. My reasons for dissenting from both Semper's and Schultz's views on the nature of the two forms of follicular cells have already been stated.

Footnote 387: _Archiv f. mikr. Anat._ Vol. VIII.

Footnote 388: Braun, "Urogenitalsystem d. Amphibien,"
_Arbeiten a. d. zool.-zoot. Institut Würzburg_, Bd. IV. He
says, in reference to the flask-shaped cell, p. 166, "Höchstens
würde ich die Funktion der grossen Follikelzellen als
_einzellige Drüsen_ mehr betonen."

_The Vitellus and the development of the yolk spherules._--Leydig, Gegenbaur, and Schultz, have recorded important observations on this head. Leydig[389] chiefly describes the peculiar characters of the yolk spherules.

Footnote 389: _Loc. cit._

Gegenbaur[390] finds in the youngest eggs fine granules, which subsequently develop into vesicles, in the interior of which the solid oval spheres, so characteristic of Elasmobranchii, are developed.

Footnote 390: _Loc. cit._

Schultz describes in the youngest ova of Torpedo the minute yolk spherules arranged in a semilunar form around the eccentric germinal vesicle. In older ova they spread through the whole. He also gives a description of their arrangement in the ripe ovum. Dr Schultz further finds in the body of the ovum peculiar protoplastic striæ, arranged as a series of pyramids, with the bases directed outwards. In the periphery of the ovum a protoplastic network is also present, which is continuous with the above-mentioned pyramidal structures.

My observations do not very greatly extend those of Gegenbaur and Schultz with reference to the development of the yolk, and closely agree with what Gegenbaur has given in the paper above quoted more fully for Aves and Reptilia than for Elasmobranchii.

In very young ova the body of the ovum is simply granular, but when it has reached about 0.5 mm. the granules are seen to be arranged in a kind of network, or sponge-work (Pl. 25, fig. 21), already spoken of in my monograph on Elasmobranch Fishes.

This network becomes more distinct in succeeding stages, especially in chromic acid specimens (Pl. 25, fig. 22), probably in part owing to a granular precipitation of the protoplasm. In the late stages, when the yolk spherules are fully developed, it is difficult to observe this network, but, as has been shewn in my monograph above quoted, it is still present after the commencement of embryonic development. An arrangement of the protoplasmic striæ like that described by Schultz has not come under my notice.