Nuclei of primitive ova with modified nuclei—
These figures bring out with clearness the following points: (1) that the modified nuclei are slightly but decidedly larger on the average than the unmodified nuclei; (2) that the contained granular bodiesare very considerablysmaller than ordinary nuclei.
Soon after the appearance of the modified nuclei, remarkable changes take place in the cells containing them. Up to the time such nuclei first make their appearance the outlines of the individual ova are very clearly defined, but subsequently, although numerous ova with but slightly modified nuclei are still to be seen, yet on the whole the outlines of all the primitive ova are much less distinct than before; and this is especially the case with the primitive ova containing modified nuclei.
From cases in which three or four ova are found in a mass with modified nuclei, but in which the outline of each ovum is fairly distinct, it is possible to pass by insensible gradations to other cases in which two or three or more modified nuclei are found embedded in a mass of protoplasm in which no division into separate cells can be made out (fig. 14). For these masses I propose to employ the term nests. They correspond in part with theUreiernesterof Professor Semper.
Frequently they are found in hardened specimens to be enclosed in a membrane-like tunic which appears to be of the nature of coagulated fluid. These membranes closely resemble and sometimes are even continuous with trabeculæ which traverse the germinal epithelium. Ovaries differ considerably asto the time and completeness of the disappearance of the outlines marking the separate cells, and although, so far as can be gathered from my specimens, the rule is that the outlines of the primitive ova with modified nuclei soon become indistinct, yet in one of my best preserved ovaries very large nests with modified nuclei are present in which the outline of each ovum is as distinct as during the period before the nuclei undergo these peculiar changes (Pl.24, fig. 12). In the same ovary other nests are present in which the outlines of the individual ova are no longer visible. The section represented onPl.24, fig. 2, is fairly average as to the disappearance of the outlines of the individual ova.
It is clear from the above statements, that in the first instance the nests are produced by the coalescence of several primitive ova into a single mass or syncytium; though of course, the several separate ova of a nest may originally, as Semper believes, have arisen from the division of a single ovum. In any case there can be no doubt that the nests of separate ova increase in size as development proceeds; a phenomenon which is more reasonably explained on the view that the ova divide, than on the view that they continue to be freshly formed. The same holds true for the nests of nuclei and this, as well as other facts, appears to me to render it probable that the nests grow by division of the nuclei without corresponding division of the protoplasmic matrix. I cannot, however, definitely prove this point owing to my having found nests, with distinct outlines to the ova, as large as any without such outlines.
The nests are situated for the most part near the surface of the germinal epithelium. The smaller ones are frequently spherical, but the larger are irregular in form. The former are about 0.05mm.in diameter; the latter reach 0.1mm.Scattered generally, and especially in the deeper layers, and at the edges of the germinal epithelium, are still unmodified or only slightly modified primitive ova. These unmodified primitive ova are aggregated in masses, but in these masses the outlines of each ovum, though perhaps less clear than in the earlier period, are still distinct.
When the embryo reaches a length of seven centimètres, and even in still younger embryos, further changes are observable.In the first place many of the modified nuclei acquire fresh characters, and it becomes necessary to divide the modified nuclei into two categories. In both of these the outer boundary of the nucleus is formed by a very delicate membrane, the space within which is perfectly clear except for the granular body. In the variety which now appears in considerable numbers the granular body has an irregular star-like form. The rays of the star are formed of fibres frequently knobbed at their extremities, and the centre of the star usually occupies an eccentric position. Typical examples of this form of modified nucleus, which may be spoken of as the stellate variety, are represented onPl.25, fig. 17; between it and the older granular variety there is an infinite series of gradations, many of which are represented onPl.24, figs. 12, 14, 15, 16. Certain of the stellate nuclei exhibit two centres instead of one, and in some cases, like that represented onPl.25, fig. 19, the stellate body of two nuclei is found united. Both of these forms are possibly modifications of the spindle-like form assumed by nuclei in the act of dividing, and may be used in proving that the nests increase in size by the division of the contained nuclei. In addition to the normal primitive ova, a few of which are still present, there are to be found, chiefly in the deeper layers of the germinal epithelium, larger ova differing considerably from the primitive ova. They form the permanent ova (Pl.24, fig. 3,o). Their average diameter is 0.04mm., compared with 0.03mm., the diameter of original primitive ova. The protoplasm of which they are composed is granular, but at first a membrane can hardly be distinguished around them; their nucleus is relatively large, 0.02 - 0.027mm.in diameter. It presents the characters ascribed by Eimer[375], and many other recent authors[376], to typical nuclei (videPl.24, fig. 3, andPl.24, 25, figs. 13, 14, 15, 16, 17, 18). It is bounded by a distinct membrane, within which is a more or less central nucleolus from which a number of radial fibres which stain very deeply pass to the surface; here they form immediately internal to the membrane a network with granules at the nodal points. In some instances the regularity of the arrangement of these fibres is very great, in other instancestwo central nucleoli are present, in which case the regularity is considerably interfered with. The points in which the youngest permanent ova differ from the primitive may be summed up as follows:—
(1) The permanent ova are larger, the smallest of them being larger than the average primitive ova in the proportion of four to three. (2) They have less protoplasm as compared to the size of the nucleus. (3) Their protoplasm is granular instead of being clear. (4) Their nucleus is clear with exception of a network of fibres instead of being granular as in the primitive ova. It thus appears that the primitive ova and permanent ova are very different in constitution, though genetically related in a way to be directly narrated.
The formation of permanent ova is at its height in embryos of about seven centimètres or slightly larger. The nests at this stage are for the most part of a very considerable size and contain a large number of nuclei, which have probably, as before insisted, originated from a division of the smaller number of nuclei present in the nests at an earlier stage. Figs. 14-18 are representations of nests at this period. The diameter of the nuclei is, on the whole, slightly greater than at an earlier stage. A series of measurements gave the following results:—
0.016mm.0.016mm.0.018mm.0.020mm.0.020mm.
Both varieties of modified nuclei are common enough, though the stellate variety predominates. The nuclei are sometimes in very close contact, and sometimes separated by protoplasm, which in many instances is very slightly granular. In a large number of the nests nothing further is apparent than what has just been described, but in a very considerable number one or more nuclei are present, which exhibit a transitional character between the ordinary stellate nuclei of my second category, and the nuclei of permanent ova as above described; and in these nests the formation of permanent ova is taking place. Permanent ova in the act of development are indicated in my figures by the lettersdo. Many of the intermediate nuclei are moredefinitely surrounded by granular protoplasm than the other nuclei of the nests, and accordingly have their outlines more sharply defined. Between nuclei of this kind, and others as large as those of the permanent ova, there are numerous transitional forms. The larger ones frequently lie in a mass of granular protoplasm projecting from the nest, and only united with it by a neck (Pl.24, figs. 14 and 16). For prominences of this kind to become independent ova, it is only necessary for the neck to become broken through. Nests in which such changes are taking place present various characters. In some cases several nuclei belonging to a nest appear to be undergoing conversion into permanent ova at the same time. Such a case is figured onPl.25, figs. 17 and 18. In these cases the amount of granular protoplasm in the nest and around each freshly formed ovum is small. In the more usual cases only one or two permanent ova at the utmost are formed at the same time, and in these instances a considerable amount of granular protoplasm is present around the nucleus of the developing permanent ovum. In such instances it frequently happens several of the nuclei not undergoing conversion appear to be in the process of absorption, and give to the part of the nest in which they are contained a very hazy and indistinct aspect (Pl.24, fig. 15). Their appearance leads me to adopt the viewthat while some of the nuclei of each nest are converted into the nuclei of the permanent ova, others break down and are used as the pabulum, at the expense of which the protoplasm of the young ovum grows.
It should, however, be stated, that after the outlines of the permanent ova have become definitely established, I have only observed in a single instance the inclusion of a nucleus within an ovum (Pl.25, fig. 24). In many instances normal nuclei of the germinal epithelium may be so observed within the ovum.
The nuclei which are becoming converted into the nuclei of permanent ova gradually increase in size. The following table gives the diameter of four such nuclei:—
0.022mm.0.022mm.0.024mm.0.032mm.
These figures should be compared with those of the table on page564.
The ova when first formed are situated either at the surface or in the deeper layers of the germinal epithelium. Though to a great extent surrounded by the ordinary cells of the germinal epithelium, they are not at first enclosed in a definite follicular epithelium. The follicle is, however, very early formed.
My observations lead me then to the conclusion that in a general way the permanent ova are formed by the increase of protoplasm round some of the nuclei of a nest, and the subsequent separation of the nuclei with their protoplasm from the nest as distinct cells—a mode of formation exactly comparable with that which so often takes place in invertebrate egg tubes.
Besides the mode of formation of permanent ova just described, a second one also seems probably to occur. In ovaries just younger than those in which permanent ova are distinctly formed, there are present primitive ova, with modified nuclei of the stellate variety, or nuclei sometimes even approaching in character those of permanent ova, which are quite isolated and not enclosed in a definite nest. The body of these ova is formed of granular protoplasm, but their outlines are very indistinct. Such ova are considerably larger than the normal primitive ova. They may measure 0.04mm.In a slightly later stage, when fully formed permanent ova are present, isolated ones are not infrequent, and it seems natural to conclude that these isolated ova are the direct descendants of the primitive ova of the earlier stage. It seems a fair deduction that in some cases primitive ova undergo a direct metamorphosis into permanent ova by a modification of their nucleus, and the assumption of a granular character in their protoplasm, without ever forming the constituent part of a nest.
It is not quite clear to me that in all nests the coalescence of the protoplasm of the ova necessarily takes place, since some nests are to be found at all stages in which the ova are distinct. Nevertheless, I am inclined to believe that the fusion of the ova is the normal occurrence.
The mode of formation of the permanent ova may then, according to my observations, take place in two ways:—1. By the formation of granular protoplasm round the nucleus in anest, and the separation of the nucleus with its protoplasm as a distinct ovum. 2. By the direct metamorphosis of an isolated primitive ovum into a permanent ovum. The difference between these two modes of formation does not, from a morphological point of view, appear to be of great importance.
The above results appear clearly to shew thatthe primitive ova in the female are not to be regarded as true ova, but as the parent sexual cells which give rise to the ova: a conclusion which completely fits in with the fact that cells exactly similar to the primitive ova in the female give rise to the spermatic cells in the male.
Slightly after the period of their first formation the permanent ova become invested by a very distinct and well-marked, somewhat flattened, follicular epithelium (Pl.24, fig. 3). Where the ova lie in the deeper layers of the germinal epithelium, the follicular epithelium soon becomes far more columnar on the side turned inwards, than on that towards the surface, especially when the inner side is in contact with the stroma (Pl.24, fig. 7, andPl.25, figs. 24 and 26). This is probably a special provision for the growth and nutrition of the ovum.
There cannot be the smallest doubt that the follicular epithelium is derived from the general cells of the germinal epithelium—a point on which my results fully bear out the conclusions of Ludwig and Semper.
The larger ova themselves have a diameter of about 0.06mm., and their nucleus of about 0.04mm.The vitellus is granular, and provided with a distinct, though delicate membrane, which has every appearance of being a product of the ovum itself rather than of the follicular epithelium. The membrane would seem indeed to be formed in some instances even before the ovum has a definite investment of follicle cells. The vitellus is frequently vacuolated, but occasionally the vacuoles appear to be caused by a shrinking due to the hardening reagent. The nucleus has the same peculiar reticulate character as at first. Its large size, as compared with the ovum, is very noticeable.
With this stage the embryonic development of the ova comes to a close, though the formation of fresh ova continues till comparatively late in life. I have, however, two series of sections of ovaries preserved in osmic acid, from slightly larger embryosthan the one last described, about which it may be well to say a few words before proceeding to the further development of the permanent ova.
The younger of these ovaries was from a Scyllium embryo 10 centimètres long, preserved in osmic acid.
A considerable number of nests were present (Pl.24, fig. 13), exhibiting, on the whole, similar characters to those just described.
A series of measurements of the nuclei in them were made, leading to the following results:—
0.014mm.0.014mm.0.016mm.0.016mm.0.018mm.0.018mm.
Thus, if anything, the nuclei were slightly smaller than in the younger embryo. It is very difficult in the osmic specimens to make out clearly the exact outlines of the various structures, the nuclei in many instances being hardly more deeply stained than in the protoplasm around them. The network in the nuclei is also far less obvious than after treatment with picric acid. The permanent ova were hardly so numerous as in the younger ovary before described. A number of these were measured with the following results:—
These figures shew that the nuclei of the permanent ova are smaller than in the younger embryo, and it may therefore be safely concluded that, in spite of the greater size of the embryo from which it is taken, the ovary now being described is in a more embryonic condition than the one last dealt with.
Though the permanent ova appeared to be formed from the nests in the manner already described, it was fairly clear fromthe sections of this ovary that many of the original primitive ova, after a metamorphosis of the nucleus and without coalescing with other primitive ova to form nests, become converted directly into the permanent ova. Many large masses of primitive ova, or at least of ova with the individual outlines of each ovum distinct, were present. The average size of ova composing these was however small, the body measuring about 0.016mm., and the nucleus 0.012mm.Isolated ova with metamorphosed nuclei could also be found measuring 0.022, and their nuclei about 0.014mm.
The second of the two ovaries, hardened in osmic acid, was somewhat more advanced than the ovary in which the formation of permanent ova was at its height. Fewer permanent ova were in the act of being formed, and many of these present had reached a considerable size, measuring as much as 0.07mm.Nests of the typical forms were present as before, but the nuclei in them were more granular than at the earlier period, and on the average slightly smaller. A series measured had the following diameters:—
0.010mm.0.012mm.0.014mm.0.016mm.
One of these nests is represented onPl.25, fig. 20. Many nests with the outlines of the individual ova distinct were also present.
On the whole it appeared to me, that the second mode of formation of permanent ova,viz.that in which the nest does not come into the cycle of development, preponderated to a greater extent than in the earlier embryonic period.
Post-embryonic Development of the Ova.—My investigations upon the post-embryonic growth and development of the ova, have for the most part been conducted upon preserved ova, and it has been impossible for me, on this account, to work out, as completely as I should have wished, certain points, more especially those connected with the development of the yolk.
Although my ovaries have been carefully preserved in a large number of reagents, including osmic acid, picric acid, chromic acid, spirit, bichromate of potash, and Müller's fluid, none of these have proved universally successful, and bichromate of potashand Müller's fluid are useless. Great difficulties have been experienced in distinguishing the artificial products of these reagents. My investigations have led me to the result, that in the gradual growth of the ova with the age of the individual the changes are not quite identical with those during the rapid growth which takes place at periods of sexual activity, after the adult condition has been reached—a result to which His has also arrived, with reference to the ova of Osseous Fish. I propose dealing separately with the several constituents of the egg-follicle.
Egg membranes.—A vitelline membrane has been described by Leydig[377]in Raja, and an albuminous layer of the nature of a chorion[378]by Gegenbaur[379]in Acanthias—the membranes described in these two ways being no doubt equivalent.
Dr Alex. Schultz[380]has more recently investigated a considerable variety of genera and finds three conditions of the egg membranes. (1) In Torpedo, a homogeneous membrane, which is of the nature of a chorion. (2) In Raja, a homogeneous membrane which is, however, perforated. (3) In Squalidæ, a thick homogeneous membrane, internal to which is a thinner perforated membrane. He apparently regards the perforated inner membrane as a specialised part of the simple membrane found in Torpedo, and states that this membrane is of the nature of a chorion.
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 roundthe 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 0.12mm., this membrane, though considerably thicker than in the embryo, is not thick enough to be accurately measured. In ova of 0.5mm.from a young female (Pl.25, fig. 21) the vitelline membrane has a thickness of 0.002mm.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 aszona radiata.
In an ovum of 1mm.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 0.004mm.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 1mm.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 onPl.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 remarkablechanges 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.008mm.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 - 0.15mm., 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 1mm.
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 narrowand columnar and the nucleus oval (Pl.25, fig. 28). In this condition the thickness of the epithelium is about 0.025mm.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 theBinnenepithel.
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 inPl.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 0.04mm., 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 0.045mm., 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. OnPl.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 onPl.34 of Schultz's paper, and also with the stage of Scyllium inPl.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 arrangedin 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 onPl.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 0.04mm.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 0.06mm., 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 correspondwith 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 0.5mm.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. Inthe 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 inPl.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 0.06mm.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 protoplasmsome 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 onPl.25, fig. 27. The ovum represented in fig. 27 was 0.5mm.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 distributedbut 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 onBombinator igneus(Pl.1, fig. 9). It deserves to be especially noted, that when the yolk spherules are first formed, theperipheral layer of the ovumis 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 granularprotoplastic 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 0.6mm., from the ovary (in active growth) of a specimen ofScy.canicula23 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 0.02mm.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 formationof 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 onPl.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:—
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 eggreaches 0.5mm., a single germinal spot is always present (measuring about 0.01mm.), 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 0.09mm., 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 0.12mm., the germinal vesicle measures about 0.06mm., 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 0.006mm.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 0.5mm.the germinal vesicle has a diameter of 0.12mm.(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 membraneis 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 onPl.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 constrictedoff 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 (videpp.555and556).
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 0.5mm.nests of ova are still fairly numerous. The nests are characteristic, and present the various remarkable peculiarities already describedin 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 epitheliumon 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 delicatevesicles 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 theirgrowth. 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 0.06mm.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 atxonPl.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.