Chapter XII · (continued 3)

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.

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].

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.

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].

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.

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.

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.

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.

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.

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.

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.

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].

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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].

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.

By the stage seven days after birth the ovary has reached a sufficiently advanced stage to answer the more important question I set myself to solve, nevertheless, partly to reconcile the apparent discrepancy between my account and that of Dr Foulis, and partly to bring my description up to a better known condition of the ovary, I shall make a few remarks about some of the succeeding stages.

In a young rabbit about four weeks old the ovary is a very beautiful object for the study of the nuclei, &c.

The pseudo-epithelium is now formed of a single layer of columnar cells, with comparatively scanty protoplasm. In it there are present a not inconsiderable number of developing ova.

A layer of connective tissue—the albuginea—is now present below the pseudo-epithelium, which contains a few small nests with very young permanent ova. The layer of medium sized nests internal to the albuginea forms a very pretty object in well stained sections, hardened in Kleinenberg's picric acid. The ova in it have all assumed the permanent form, and are provided with beautiful reticulate nuclei, with, as a rule, one more especially developed nucleolus, and smaller granular bodies. Their diameter varies from about 0.028 to 0.04 mm. and that of their nucleus from 0.016 to 0.02 mm. The majority of these ova are not provided with a follicular investment, but amongst them are numerous small cells, clearly derived from the germinal epithelium, which are destined to form the follicle (vide fig. 40Aand B). In a few cases the follicles are completed, and are then formed of very flattened spindle-shaped (in section) cells. In the majority of cases all the ova of each nest are quite distinct, and each provided with a delicate vitelline membrane (fig. 40A) In other instances, which, so far as I can judge, are more common than in the previous stages, the protoplasm of two or more ova is fused together.

Examples of this are represented in Pl. 26, fig. 40A. In some of these the nuclei in the undivided protoplasm are all of about the same size and distinctness, and probably the protoplasm eventually becomes divided up into as many ova as nuclei; in other cases, however, one or two nuclei clearly preponderate over the others, and the smaller nuclei are indistinct and hazy in outline. In these latter cases I have satisfied myself as completely as in the case of Elasmobranchii, that only one or two ova (according to the number of distinct nuclei) will develop out of the polynuclear mass, and that the other nuclei atrophy, and the material of which they were composed serves as the nutriment for the ova which complete their development. This does not, of course, imply that the ova so formed have a value other than that of a single cell, any more than the development of a single embryo out of the many in one egg capsule implies that the embryo so developing is a compound organism.

In the innermost layer of the germinal epithelium the outlines of the original large nests are still visible, but many of the follicles have been cut off by ingrowths of stroma. In the still intact nests the formation of the follicles out of the cells of the germinal epithelium may be followed with great advantage. The cells of the follicle, though less columnar than was the case at an earlier period, are more so than in the case of follicles formed in the succeeding stages. The previous inequality in the cells of the follicles is no longer present.

The tubuliferous tissue in the zona vasculosa appears to me to have rather increased in quantity than the reverse; and is formed of numerous solid columns or oval masses of cells, separated by strands of connective tissue, with typical spindle nuclei.

It is partially intelligible to me how Dr Foulis might from an examination of the stages similar to this, conclude that the follicle cells were derived from the stroma; but even at this stage the position of the cells which will form the follicular epithelium, their passage by a series of gradations into obvious cells of the germinal epithelium and the peculiarities of their nuclei, so different from those of the stroma cells, supply a sufficient series of characters to remove all doubt as to the derivation of the follicle cells. Apart from these more obvious points, an examination of the follicle cells from the surface, and not in section, demonstrates that the general resemblance in shape of follicle cells to the stroma cells is quite delusory. They are in fact flat, circular, or oval, plates not really spindle-shaped, but only apparently so in section. While I thus fundamentally differ from Foulis as to the nature of the follicle cells, I am on this point in complete accordance with Waldeyer, and my own results with reference to the follicle cannot be better stated than in his own words (pp. 43, 44).

At six weeks after birth the ovary of the rabbit corresponds very much more with the stages in the development of the ovary, which Foulis has more especially studied, for the formation of the follicular epithelium, than during the earlier stages. His figure (Quart. Journ. Mic. Sci., Vol. XVI., Pl. 17, fig. 6) of the ovary of a seven and a half months' human fœtus is about the corresponding age. Different animals vary greatly in respect to the relative development of the ovary. For example, the ovary of a lamb at birth about corresponds with that of a rabbit six weeks after birth. The points which may be noticed about the ovary at this age are first that the surface of the ovary begins to be somewhat folded. The appearances of these folds in section have given rise, as has already been pointed out by Foulis, to the erroneous view that the germinal epithelium (pseudo-epithelium) became involuted in the form of tubular open pits. The folds appear to me to have no connection with the formation of ova, but to be of the same nature as the somewhat similar folds in Elasmobranchii. A follicular epithelium is present around the majority of the ova of the middle layer, and around all those of the inner layer of the germinal epithelium. The nests are, moreover, much more cut up by connective tissue ingrowths than in the previous stages.

The follicle cells of the middle layers are very flat, and spindle-shaped in section, and though they stain more deeply than the stroma cells, and have other not easily characterised peculiarities, they nevertheless do undoubtedly closely resemble the stroma cells when viewed (as is ordinarily the case) in optical section.

In the innermost layer many of the follicles with the enclosed ova have advanced considerably in development and are formed of columnar cells. The somewhat heterodox view of these cells propounded by Foulis I cannot quite agree to. He says (Quart. J. Mic. Sci., Vol. XVI., p. 210): The protoplasm which surrounds the vesicular nuclei acts as a sort of cement substance, holding them together in the form of a capsular membrane round the young ovum. This capsular membrane is the first appearance of the membrana granulosa. I must admit that I find nothing similar to this, nor have I met with any special peculiarities (as Foulis would seem to indicate) in the cells of the germinal epithelium or other cells of the ovary.

Figure 41 is a representation of an advanced follicle of a six weeks' rabbit, containing two ova, which is obviously in the act of dividing into two. Follicles of this kind with more than one ovum are not very uncommon. It appears to me probable that follicles, such as that I have figured, were originally formed of a single mass of protoplasm with two nuclei; but that instead of one of the nuclei atrophying, both of them eventually developed and the protoplasm subsequently divided into two masses. In other cases it is quite possible that follicles with two ova should rather be regarded as two follicles not separated by a septum of stroma.

On the later stages of development of the ovary I have no complete series of observations. The yolk spherules I find to be first developed in a peripheral layer of the vitellus. I have not been able definitely to decide the relation of the zona radiata to the first formed vitelline membrane. Externally to the zona radiata there may generally be observed a somewhat granular structure, against which the follicle cells abut, and I cannot agree with Waldeyer (loc cit., p. 40) that this structure is continuous with the cells of the discus, or with the zona radiata. Is it the remains of the first formed vitelline membrane? I have obtained some evidence in favour of this view, but have not been successful in making observations to satisfy me on the point, and must leave open the question whether my vitelline membrane becomes the zona radiata or whether the zona is not a later and independent formation, but am inclined myself to adopt the latter view. The first formed membrane, whether or no it becomes the zona radiata, is very similar to the vitelline membrane of Elasmobranchii and arises at a corresponding stage.

Summary of observations on the mammalian ovary.—The general results of my observations on the mammalian ovary are the following:—

(1) The ovary in an eighteen days' embryo consists of a cylindrical ridge attached along the inner side of the Wolffian body, which is formed of two parts; (a) an external epithelium—two or three cells deep (the germinal epithelium); (b) a hilus or part forming in the adult the vascular zone, at this stage composed of branched masses of epithelial tissue (tubuliferous tissue) derived from the walls of the anterior Malpighian bodies, and numerous blood-vessels, and some stroma cells.

(2) The germinal epithelium gradually becomes thicker, and after a certain stage (twenty-three days) there grow into it numerous stroma ingrowths, accompanied by blood-vessels. The germinal epithelium thus becomes honeycombed by strands of stroma. Part of the stroma eventually forms a layer close below the surface, which becomes in the adult the tunica albuginea. The part of the germinal epithelium external to this layer becomes reduced to a single row of cells, and forms what has been spoken of in this paper as the pseudo-epithelium of the ovary. The greater part of the germinal epithelium is situated internal to the tunica albuginea, and this part is at first divided up by strands of stroma into smaller divisions externally, and larger ones internally. These masses of germinal epithelium (probably sections of branched trabeculæ) may be spoken of as nests. In the course of the development of the ova they are broken up by stroma ingrowths, and each follicle with its enclosed ovum is eventually isolated by a layer of stroma.

(3) The cells of the germinal epithelium give rise both to the permanent ova and to the cells of the follicular epithelium. For a long time, however, the cells remain indifferent, so that the stages, like those in Elasmobranchii, Osseous Fish, Birds, Reptiles, &c., with numerous primitive ova embedded amongst the small cells of the germinal epithelium, are not found.

(4) The conversion of the cells of the germinal epithelium into permanent ova commences in an embryo of about twenty-two days. All the cells of the germinal epithelium appear to be capable of becoming ova: the following are the stages in the process, which are almost identical with those in Elasmobranchii:—

(a) The nucleus of the cells loses its more or less distinct network, and becomes very granular, with a few specially large granules (nucleoli). The protoplasm around it becomes clear and abundant—primitive ovum stage. It may be noted that the largest primitive ova are very often situated in the pseudo-epithelium. (b) A segregation takes place in the contents of the nucleus within the membrane, and the granular contents pass to one side, where they form an irregular mass, while the remaining space within the membrane is perfectly clear. The granular mass gradually develops itself into a beautiful reticulum, with two or three highly refracting nucleoli, one of which eventually becomes the largest and forms the germinal spot par excellence. At the same time the body of the ovum becomes slightly granular. While the above changes, more especially those in the nucleus, have been taking place, the protoplasm of two or more ova may fuse together, and polynuclear masses be so formed. In some cases the whole of such a polynuclear mass gives rise to only a single ovum, owing to the atrophy of all the nuclei but one, in others it gives rise by subsequent division to two or more ova, each with a single germinal vesicle.

(5) All the cells of a nest do not undergo the above changes, but some of them become smaller (by division) than the indifferent cells of the germinal epithelium, arrange themselves round the ova, and form the follicular epithelium.

(6) The first membrane formed round the ovum arises in some cases even before the appearance of the follicular epithelium, and is of the nature of a vitelline membrane. It seems probable, although not definitely established by observation, that the zona radiata is formed internally to the vitelline membrane, and that the latter remains as a membrane, somewhat irregular on its outer border, against which the ends of the follicle cells abut.

General Observations on the Structure and Development of the Ovary.

In selecting Mammalia and Elasmobranchii as my two types for investigation, I had in view the consideration that what held good for such dissimilar forms might probably be accepted as true for all Vertebrata with the exception of Amphioxus.

The structure of the ovary.—From my study of these two types, I have been led to a view of the structure of the ovary, which differs to a not inconsiderable extent from that usually entertained. For both types the conclusion has been arrived at that the whole egg-containing part of the ovary is really the thickened germinal epithelium, and that it differs from the original thickened patch or layer of germinal epithelium, mainly in the fact that it is broken up into a kind of meshwork by growths of vascular stroma. If the above view be accepted for Elasmobranchii and Mammalia, it will hardly be disputed for the ovaries of Reptilia and Aves. In the case also of Osseous Fish and Amphibia, this view of the ovary appears to be very tenable, but the central core of stroma present in the other types is nearly or quite absent, and the ovary is entirely formed of the germinal epithelium with the usual strands of vascular stroma[413]. It is obvious that according to the above view Pflüger's egg-tubes are merely trabeculæ of germinal epithelium, and have no such importance as has been attributed to them. They are present in a more or less modified form in all types of ovaries. Even in the adult Amphibian ovary, columns of cells of the germinal epithelium, some indifferent, others already converted into ova, are present, and, as has been pointed out by Hertwig[414], represent Pflüger's egg-tubes.

The formation of the permanent ova.—The passage of primitive ova into permanent ova is the part of my investigation to which the greatest attention was paid, and the results arrived at for Mammalia and Elasmobranchii are almost identical. Although there are no investigations as to the changes undergone by the nucleus in other types, still it appears to me safe to conclude that the results arrived at hold good for Vertebrates generally[415]. As has already been pointed out the transformation which the so-called primitive ova undergo is sufficient to shew that they are not to be regarded as ova but merely as embryonic sexual cells. A feature in the transformation, which appears to be fairly constant in Scyllium, and not uncommon in the rabbit, is the fusion of the protoplasm of several ova into a syncytium, the subsequent increase in the number of nuclei in the syncytium, the atrophy and absorption of a portion of the nuclei, and the development of the remainder into the germinal vesicles of ova; the vitellus of each ovum being formed by a portion of the protoplasm of the syncytium.

As to the occurrence of similar phenomena in the Vertebrata generally, it has already been pointed out that Ed. van Beneden has described the polynuclear masses in Mammalia, though he does not appear to me to have given a complete account of their history. Götte[416] describes a fusion of primitive ova in Amphibia, but he believes that the nuclei fuse as well as the bodies of the ova, so that one ovum (according to his view no longer a cell) is formed by the fusion of several primitive ova with their nuclei. I have observed nothing which tends to support Götte's view about the fusion of the nuclei, and regard it as very improbable. As regards the interpretation to be placed upon the nests formed of fused primitive ova, Ed. van Beneden maintains that they are to be compared with the upper ends of the egg tubes of Insects, Nematodes, Trematodes, &c. There is no doubt a certain analogy between the two, in that in both cases certain nuclei of a polynuclear mass increase in size, and with the protoplasm around them become segmented off from the remainder of the mass as ova, but the analogy cannot be pressed. The primitive ova, or even the general germinal epithelium, rather than these nests, must be regarded as giving origin to the ova, and the nests should be looked on, in my opinion, as connected more with the nutrition than with the origin of the ova. In favour of this view is the fact that as a rule comparatively few ova are developed from the many nuclei of a nest; while against the comparison with the egg tubes of the Invertebrata it is to be borne in mind that many ova appear to develop independently of the nests.

In support of my view about the nests there may be cited many analogous instances from the Invertebrata. In none of them, however, are the phenomena exactly identical with those in Vertebrata. In the ovary of many Hydrozoa (e.g. Tubularia mesembryanthemum), out of a large number of ova which develop up to a certain point, a comparatively very small number survive, and these regularly feed upon the other ova. During this process the boundary between a large ovum and the smaller ova is indistinct: in the outermost layer of a large ovum a number of small ova are embedded, the outlines of the majority of which have become obscure, although they can still be distinguished. Just beyond the edge of a large ovum the small ova have begun to undergo retrogressive changes; while at a little distance from the ovum they are quite normal. An analogous phenomenon has been very fully described by Weismann[417] in the case of Leptodera, the ovary of which consists of a germogene, in which the ova develop in groups of four. Each group of four occupies a separate chamber of the ovary, but in summer only one of the four eggs (the third from the germogene) develops into an ovum, the other three are used as pabulum. In the case of the winter eggs the process is carried still further, in that the contents of the alternate chambers, instead of developing into ova, are entirely converted, by a series of remarkable changes, into nutritive reservoirs. Fundamentally similar occurrences to the above are also well known in Insects. Phenomena of this nature are obviously in no way opposed to the view of the ovum being a single cell.

With reference to the origin of the primitive ova, it appears to me that their mode of development in Mammals proves beyond a doubt that they are modified cells of the germinal epithelium. In Elasmobranchii their very early appearance, and the difficulty of finding transitional forms between them and ordinary cells of the germinal epithelium, caused me at one time to seek (unsuccessfully) for a different origin for them. Any such attempts appear to me, however, out of the question in the case of Mammals.

The egg membranes.—The homologies of the egg membranes in the Vertebrata are still involved in some obscurity. In Elasmobranchii there are undoubtedly two membranes present. (1) An outer and first formed membrane—the albuminous membrane of Gegenbaur—which, in opposition to previous observers, I have been led to regard as a vitelline membrane. (2) An inner radiately striated membrane, formed as a differentiation of the surface of the yolk at a later period. Both these membranes usually atrophy before the ovum leaves the follicle. In Reptilia[418] precisely the same arrangement is found as in Elasmobranchii, except that as a rule the zona radiata is relatively more important. The vitelline membrane external to this (or as it is usually named the chorion) is, as a rule, thin in Reptilia; but in Crocodilia is thick (Gegenbaur), and approaches the condition found in Scyllium and other Squalidæ. It appears, as in Elasmobranchii, to be formed before the zona radiata. A special internal differentiation of the zona radiata is apparently found (Eimer) in many Reptilia. No satisfactory observations appear to be recorded with reference to the behaviour of the two reptilian membranes as the egg approaches maturity. In Birds[419] the same two membranes are again found. The first formed and outer one is, according to Gegenbaur and E. van Beneden, a vitelline membrane; and from the analogy of Elasmobranchii I feel inclined to accept their view. The inner one is the zona radiata, which disappears comparatively early, leaving the ovum enclosed only by the vitelline membrane, when it leaves the follicle. All the large-yolked vertebrate ova appear then to agree very well with Elasmobranchii in presenting during some period of their development the two membranes above mentioned.

Osseous fish have almost always a zona radiata, which it seems best to assume to be equivalent to that in Elasmobranchii. Internal to this is a thin membrane, the equivalent, according to Eimer, of the membrane found by the same author within the zona in Reptilia. A membrane equivalent to the thick vitelline membrane of Elasmobranchii would seem to be absent in most instances, though a delicate membrane, external to the zona, has not infrequently been described; Eimer more especially asserts that such a membrane exists in the perch within the peculiar mucous covering of the egg of that fish.

In Petromyzon, a zona radiata appears to be present[420], which is divided in the adult into two layers, both of them perforated. The inner of the two perhaps corresponds with the membrane internal to the zona radiata in other types. In Amphibia the single late formed and radiately striated (Waldeyer) membrane would appear to be a zona radiata. If the suggestion on page 605 turns out to be correct the ova of Mammalia possess both a vitelline membrane and zona radiata. E. van Beneden[421] has, moreover, shewn that they are also provided at a certain period with a delicate membrane within the zona.

The reticulum of the germinal vesicle.—In the course of description of the ovary it has been necessary for me to enter with some detail into the structure of the nucleus, and I have had occasion to figure and describe a reticulum identical with that recently described by so many observers. The very interesting observations of Dr Klein in the last number of this Journal[422] have induced me to say one or two words in defence of some points in my description of the reticulum. Dr Klein says, on page 323, I have distinctly seen that when nucleoli are present—the instances are fewer than is generally supposed; they are accumulations of the fibrils of the network. I have no doubt that Klein is correct in asserting that nucleoli are fewer than is generally supposed; and that in many of these instances what are called nucleoli are accumulations, natural or artificial, of the fibrils of the network; but I cannot accept the universality of the latter statement, which appears to me most certainly not to hold good in the case of ova, in which nucleoli frequently exist in the absence of the network.

Again, I find that at the point of intersection of two or more fibrils there is, as a rule, a distinct thickening of the matter of the fibrils, and that many of the dots seen are not merely, as Dr Klein would maintain, optical sections of fibrils.

It appears to me probable that both the network and the nucleoli are composed of the same material—what Hertwig calls nuclear substance—and if Dr Klein merely wishes to assert this identity in the passage above quoted, I am at one with him.

Although a more or less distinct network is present in most nuclei (I have found it in almost all embryonic nuclei) it is not universally so. In the nuclei of primitive ova I have no doubt that it is absent, though present in the unmodified nuclei of the germinal epithelium; and it is present only in a very modified form in the nuclei of primitive ova undergoing a transformation into permanent ova. The absence of the reticulum does not, of course, mean that the substance capable of forming a reticulum is absent, but merely that it does not assume a particular arrangement.