The Works of Francis Balfour 1-12
|Embryology - 20 Nov 2019 Expand to Translate|
|Google Translate - select your language from the list shown below (this will open a new external page)|
العربية | català | 中文 | 中國傳統的 | français | Deutsche | עִברִית | हिंदी | bahasa Indonesia | italiano | 日本語 | 한국어 | မြန်မာ | Pilipino | Polskie | português | ਪੰਜਾਬੀ ਦੇ | Română | русский | Español | Swahili | Svensk | ไทย | Türkçe | اردو | ייִדיש | Tiếng Việt These external translations are automated and may not be accurate. (More? About Translations)
Foster M. and Sedgwick A. The Works of Francis Balfour Vol. I. Separate Memoirs (1885) MacMillan and Co., London.
Foster M. and Sedgwick A. The Works of Francis Balfour Vol. II. A Treatise on Comparative Embryology 1. (1885) MacMillan and Co., London.
Foster M. and Sedgwick A. The Works of Francis Balfour Vol. III. A Treatise on Comparative Embryology 2 (1885) MacMillan and Co., London.
Foster M. and Sedgwick A. The Works of Francis Balfour Vol. IV. Plates (1885) MacMillan and Co., London.
|Historic Disclaimer - information about historic embryology pages|
|Embryology History | Historic Embryology Papers)|
Draft Version - Notice removed when completed.
Vol I. Separate Memoirs (1885)
XII. On the Structure and Development of the Vertebrate Ovary
(With Plates 24, 25, 26.)
THE present paper records observations on the ovaries of but two types, viz., Mammalia and Elasmobranchii. The main points dealt with are three : I. The relation of the germinal epithelium to the stroma. 2. The connection between primitive ova in Waldeyer's sense and the permanent ova. 3. The homologies of the egg membranes.
The second of these points seems to call for special attention after Semper's discovery that the primitive ova ought really to be regarded as primitive sexual cells, in that they give rise to the generative elements of both sexes.
The Development of the Elasmobranch Ovary
The development of the Elasmobranch ovary has recently formed the subject of three investigations. The earliest of them, by H. Ludwig, is contained in his important work, on the ' Formation of the Ovum in the Animal Kingdom V Ludwig arrives at the conclusion that the ovum and the follicular epithelium are both derived from the germinal epithelium, and enters into some detail as to their formation. Schultz 3 , without apparently being acquainted with Ludwig's observations, has come to very similar results for Torpedo.
1 From the Quarterly Journal of Microscopical Science, Vol. 18, 1878.
2 Arbeilen a. d. zooL-zoot. Institut Wurzburg, Bd. I.
3 Archivf. micr. Anat. Vol. XI.
Semper 1 , in his elaborate memoir on the urogenital system of Elasmobranchs, has added very greatly to our knowledge on this subject. In a general way he confirms Ludwig's statements, though he shews that the formation of the ova is somewhat more complicated than Ludwig had imagined. He more especially lays stress on the existence of nests of ova (Ureierernester), derived from the division of a single primitive ovum, and of certain peculiarly modified nuclei, which he compares to spindle nuclei in the act of division.
My own results agree with those of previous investigators, in attributing to the germinal epithelium the origin both of the follicular epithelium and ova, but include a number of points which I believe to be new, and, perhaps, of some little interest ; they differ, moreover, in many important particulars, both as to the structure and development of the ovary, from the accounts of my predecessors.
The history of the female generative organs may conveniently be treated under two heads, viz. (i) the history of the ovarian ridge itself, and (2) the history of the ova situated in it. I propose dealing in the first place with the ovarian ridge.
The Ovarian ridge in Scy ilium. At the stage spoken of in my monograph on Elasmobranch Fishes as stage L, the ovarian ridge has a very small development, and its maximum height is about O'l mm. It exhibits in section a somewhat rounded form, and is slightly constricted along the line of attachment. It presents two surfaces, which are respectively outer and inner, and is formed of a layer of somewhat thickened germinal epithelium separated by a basement membrane from a central core of stroma. The epithelium is far thicker on the outer surface than on the inner, and the primitive ova are entirely confined to the former. The cells of the germinal epithelium are irregularly scattered around the primitive ova, and have not the definite arrangement usually characteristic of epithelial cells. Each of them has a large nucleus, with a deeply staining small nucleolus, and a very scanty protoplasm. In stage N the ovarian ridge has a pointed edge and narrower attachment than in stage L. Its greatest height is about O'l/ mm. There is more stroma, and the basement membrane is more distinct than before ; in other respects no changes worth recording have taken place. By stage P a distinction is observable between the right and left ovarian ridges ; the right one has, in fact, grown more rapidly than the left, and the difference in size between the two ridges becomes more and more conspicuous during the succeeding stages, till the left one ceases to grow any larger, though it remains for a great part of life as a small rudiment.
1 Arbeitcn a. d. zool.-zoot. Institut IVurzburg, Bd. n.
The right ovarian ridge, which will henceforth alone engage our attention, has grown very considerably. Its height is now about O'4 mm. It has in section (vide PI. 24, fig. i) a triangular form with constricted base, and is covered by a flat epithelium, except for an area on the outer surface, in length co-extensive with the ovarian ridge, and with a maximum breadth of about O'25 mm. This area will be spoken of as the ovarian area or region, since the primitive ova are confined to it. The epithelium covering it has a maximum thickness of about 0^05 mm., and thins off rather rapidly on both borders, to become continuous with the general epithelium of the ovarian ridge. Its cells have the same character as before, and are several layers deep. Scattered irregularly amongst them are the primitive ova. The germinal epithelium in the ovarian region is separated by a basement membrane from the adjacent stroma.
In succeeding stages, till the embryo reaches a length of 7 centimetres, no very important changes take place. The ovarian region grows somewhat in breadth, though in this respect different embryos vary considerably. In two embryos of nearly the same age, the breadth of the ovarian epithelium was 0*3 mm. in the one and 0^35 mm. in the other. In the former of these embryos, the thickness of the epithelium was slightly greater than in the latter, viz. o'OQ mm. as compared with o - o8. In both the epithelium was sharply separated from the subjacent stroma. There were relatively more epithelial cells in proportion to primitive ova than at the earlier date, and the individual cells exhibited great variations in shape, some being oval, some angular, others very elongated, and many of them applied to part of an ovum and accommodating themselves to its shape. In some of the more elongated cells very deeply stained nuclei were present, which (in a favourable light and with high powers) exhibited the spindle modification of Strasburger with great clearness, and must therefore be regarded as undergoing division. The ovarian region is' at this stage bounded on each side by a groove.
In an embryo of seven centimetres (PL 24, fig. 2) the breadth of the ovarian epithelium was o - 5, but its height only 0*06 mm. It was still sharply separated from the subjacent stroma, though a membrane could only be demonstrated in certain parts. The amount of stroma in the ovarian ridge varies greatly in different individuals, and no reliance can be placed on its amount as a test of the age of the embryo. In the base of the ovarian ridge the cells were closely packed, elsewhere they were still embryonic.
My next stage (PL 24, fig. 3, and fig. 4), shortly before the time of the hatching of the embry/o, exhibits in many respects an advance on the previous one. It is the stage during which a follicular covering derived from the germinal epithelium is first distinctly formed round the ova, in a manner which will be more particularly spoken of in the section devoted to the development of the ovum itself. The breadth of the ovarian region is 0^56 mm., and its greatest height close to the central border, O'I2 mm. a great advance on the previous stage, mainly, however, due to the larger size of the ova.
The ovarian epithelium is still in part separated from the subjacent stroma by a membrane close to its dorsal and ventral borders, but elsewhere the separation is not so distinct, it being occasionally difficult within a cell or so to be sure of the boundary of the epithelium. The want of a clear line between the stroma and the epithelium is rendered more obvious by the fact that the surface of the latter is somewhat irregular, owing to projections formed by specially large ova, into the bays between which are processes of the stroma. In an ovary about this stage, hardened in osmic acid, the epithelium stains very differently from the subjacent stroma, and the line of separation between the two is quite sharp. A figure of the whole ovarian ridge, shewing the relation between the two parts, is represented on PL 24, fig. 5.
The layer of stroma in immediate contact with the epithelium is very different from the remainder, and appears to be destined to accompany the vascular growths into the epithelium, which will appear in the next stage. The protoplasm of the cells composing it forms a loose reticulum with a fair number of oval or rounded nuclei, with their long axis for the most part parallel to the lower surface of the epithelium. It contains, even at this stage, fully developed vascular channels.
The remainder of the stroma of the ovarian ridge has now acquired a definite structure, which remains constant through life, and is eminently characteristic of the genital ridge of both sexes. The bulk of it (PI. 24, fig. 3, str) consists of closely packed polygonal cells, of about 0^014 mm. with large nuclei of about oxxx). These cells appear to be supported by a delicate reticulum. The whole tissue is highly vascular, with the numerous capillaries ; the nuclei in the walls of which stand out in some preparations with great clearness.
In the next oldest ovary, of which I have sections, the breadth of the ovarian epithelium is 07 mm. and its thickness CTO96. The ovary of this age was preserved in osmic acid, which is the most favourable reagent, so far as I have seen, for observing the relation of the stroma and epithelium. On PI. 24, fig. 6, is represented a transverse section through the whole breadth of the ovary, slightly magnified to shew the general relations of the parts, and on PI. 24, fig. 7, a small portion of a section more highly magnified. The inner surface of the ovarian epithelium is more irregular than in the previous stage, and it may be observed that the subjacent stroma is growing in amongst the ova. From the relation of the two tissues it is fairly clear that the growth which is taking place is a definite growth of the stroma into the epithelium, and not a mutual intergrowth of the two tissues. The ingrowths of the stroma are, moreover, directed towards individual ova, around which, outside the follicular epithelium, they form a special vascular investment in the succeeding stages. They are formed of a reticular tissue with comparatively few nuclei.
By the next stage, in my series of ovaries of Scy. camcula, important changes have taken place in the constitution of ovarian epithelium. Fig. 8, PI. 24, represents a portion of ths ovarian epithelium, on the same scale as figs. I, 2, 3, &c., and fig. 9 a section through the whole ovarian ridge slightly magnified. Its breadth is now 1*3 mm., and its thickness O'3 mm. B. 36
The ova have grown very greatly, and it appears to me to be mainly owing to their growth that the greater thickness of the epithelium is due, as well as the irregularity of its inner surface (vide fig. 9).
The general relation of the epithelium to the surrounding parts is much the same as in the earlier stage, but two new features have appeared (i) The outermost cells of the ovarian region have more or less clearly arranged themselves as a kind of epithelial covering for the organ ; and (2) the stroma ingrowths of the previous stage have become definitely vascular, and have penetrated through all parts of the epithelium.
The external layer of epithelium is by no means a very marked structure, the character of its cells varies greatly in different regions, and it is very imperfectly separated from the subjacent layer. I shall speak of it for convenience as pseudoepitlielium.
The greater part of the germinal epithelium forms anastomosing columns, separated by very thin tracts of stroma. The columns are, in the majority of instances, continuous with the pseudo-epithelium at the surface, and contain ova in all stages of development. Many of the cells composing them naturally form the follicular epithelium for the separate ova; but the majority have no such relation. They have in many instances assumed an appearance somewhat different from that which they presented in the last stage, mainly owing to the individual nuclei being more widely separated. A careful examination with a high power shews that this is owing to an increase in the amount of protoplasm of the individual cells, and it may be noted that a similar increase in the size of the bodies of the cells has taken place in the pseudo-epithelium and in the follicular epithelium of the individual ova.
The stroma ingrowths form the most important feature of the stage. In most instances they are very thin and delicate, and might easily be overlooked, especially as many of the cells in them are hardly to be distinguished, taken separately, from those of the germinal epithelium. These features render the investigation of the exact relation of the stroma and epithelium a matter of some difficulty. I have, however, been greatly assisted by the investigation of the ovary of a young example of Scyllium stellare, i6i centimetres in length, a section of which is represented in PI. 25, fig. 26. In this ovary, although no other abnormalities were observable, the stroma ingrowths were exceptionally wide ; indeed, quite without a parallel in my series of ovaries in this respect. The stroma most clearly divides up the epithelium of the ovary into separate masses, or more probably anastomosing columns, the equivalents of the egg-tubes of Pfluger. These columns are formed of normal cells of the germinal epithelium, which enclose ovarian nests and ova in all stages of development. A comparison of the section I have represented, with those from previous stages, appears to me to demonstrate that the relation of the epithelium and stroma has been caused by an ingrowth or penetration of the stroma into the epithelium, and not by a mutual intergrovvth of the two tissues. Although the ovary, of which fig. 26 represents a section was from Scy. stellare, and the previous ovaries have been from Scy. canicula, yet the thickness of the epithelium may still be appealed to in confirmation of this view. In the previous stage the thickness was about O'og6 mm., in the present one it is about O'i6mm., a difference of thickness which can be easily accounted for by the growth of the individual ova and the additional tracts of stroma. A pseudo-epithelium is more or less clearly formed, but it is continuous with the columns of epithelium. In the stroma many isolated cells are present, which appear to me, from a careful comparison of a series of sections, to belong to the germinal epithelium.
The thickness of the follicular epithelium on the inner side of the larger ova deserves to be noted. Its meaning is discussed on p. 567.
Quite a different interpretation to that which I have given has been put by Ludwig and Semper upon the parts of the ovary at this stage. My pseudo-epithelium is regarded by them as forming, together with the follicular epithelium of the ova, the sole remnant of the original germinal epithelium; and the masses of cells below the pseudo-epithelium, which I have attempted to shew are derived from the original germinal epithelium, aie regarded as parts of the ingrowths of the adjacent stroma.
Ludwig has assumed this interpretation without having had an opportunity of working out the development of the parts, but Semper attempts to bring forward embryological proofs in support of this position.
If the series of ovaries which I have represented be examined, it will not, I think, be denied that the general appearances are very much in favour of my view. The thickened patch of ovarian epithelium can apparently be traced through the whole series of sections, and no indications of its sudden reduction to the thin pseudo-epithelium are apparent. The most careful examination that I have been able to make brings to light nothing tending to shew that the general appearances are delusive. The important difference between us refers to our views of the nature of the tissue subjacent to the pseudoepithelium. If my results be accepted, it is clear that the whole ovarian region is an epithelium interpenetrated by connective tissue ingrowths, so that the region below the pseudo-epithelium is a kind of honeycomb or trabecular net-work of germinal epithelium, developing ova of all stages and sizes, and composed of cells capable of forming follicular epithelium for developing ova. Ludwig figures what he regards as the formation of the follicular epithelium round primitive ova during their passage into the stroma. It is' quite clear to me, that his figures of the later stages, 33 and 34, represent fully formed permanent ova surrounded by a follicular epithelium, and that their situation in contact with the pseudo-epithelium is, so to speak, an accident, and it is quite possible that his figures 31 and 32 also represent fully formed ova ; but I have little hesitation in asserting that he has not understood the mode of formation of the follicular epithelium, and that, though his statement that it is derived from the germinal epithelium is quite correct, his account of the process is completely misleading. The same criticism does not exactly apply to Semper's statements. Semper has really observed the formation of the follicular epithelium round young ova ; but, nevertheless, he appears to me to give an entirely wrong account of the relation of the stroma to the germinal epithelium. The extent of the difference between Semper's and my view may perhaps best be shewn by a quotation from Semper, loc. '/., 465: " In females the nests of primitive ova sink in groups into the stroma. In these groups one cell enlarges till it becomes the ovum, the neighbouring cells increase and arrange themselves around the ova as follicle cells."
Although the histological changes which take place in the succeeding stages are not inconsiderable, they do not involve any fundamental change in the constitution of the ovarian region, and may be described with greater brevity than has been so far possible.
In a half-grown female, with an ovarian region of 3 mm. in breadth, and O'8 mm. in thickness, the stroma of the ovarian region has assumed a far more formed aspect than before. It consists (PL 24, fig. 10) of a basis in most parts fibrous, but in some nearly homogeneous, with a fair number of scattered cells. Immediately below the pseudo-epithelium, there is an imperfectly developed fibrous layer, forming a kind of tunic, in which are imbedded the relatively reduced epithelial trabeculae of the previous stages. They appear in sections as columns, either continuous with or independent of the pseudo-epithelium, formed of normal cells of the germinal epithelium, nests of ova, and permanent ova in various stages of development. Below this there comes a layer of larger ova which are very closely packed. A not inconsiderable number of the larger ova have, however, a superficial situation, and lie in immediate contact with the pseudo-epithelium. Some of the younger ova, enclosed amongst epithelial cells continuous with the pseudp-epithelium, are very similar to those figured by Ludwig. It is scarcely necessary to insist that this fact does not afford any argument in favour of his interpretations. The ovarian region is honeycombed by large vascular channels with distinct walls, and other channels which are perhaps lymphatic.
The surface of the ovarian region is somewhat irregular and especially marked by deep oblique transverse furrows. It is covered by a distinct, though still irregular pseudo-epithelium, which is fairly columnar in the furrows but flattened along the ridges. The cells of the pseudo-epithelium have one peculiarity very unlike that of ordinary epithelial cells. Their inner extremities (vide fig. 10) are prolonged into fibrous processes which enter the subjacent tissue, and bending nearly parallel to the surface of the ovary, assist in forming the tunic spoken of above. This peculiarity of the pseudo-epithelial cells seems to indicate that they do not essentially differ from cells which have the character of undoubted connective tissue cells, and renders it possible that the greater part of the tunic, which has apparently the structure of ordinary connective tissue, is in reality derived from the original germinal epithelium, a view which tallies with the fact that in some instances the cells of the tunic appear as if about to assist in forming the follicular epithelium of some of the developing ova. In Raja, the similarity of the pseudo-epithelium to the subjacent tissue is very much more marked than in Scyllium. The pseudoepithelium appears merely as the superficial layer of the ovarian tunic somewhat modified by its position on the surface. It is formed of columnar cells with vertically arranged fibres which pass into the subjacent layers, and chiefly differ from the ordinary fibres in that they still form parts of the cell-protoplasm enclosing the nucleus. In PL 25, fig. 34, an attempt is made to represent the relations of the pseudo-epithelium to the subjacent tissue in Raja. Ludwig's figures of the pseudoepithelium of the ovary, in the regular form of its constituent cells, and its sharp separation by a basement membrane from the tissue below, are quite unlike anything which I have met with in my sections either of Raja or Scyllium.
Close to the dorsal border of the ovary the epithelial cells of the non-ovarian region have very conspicuous tails, extending into a more or less homogeneous substance below, which constitutes a peculiar form of tunic for this part of the ovarian ridge.
In the full-grown fpmale the stroma of the ovarian region is denser and has a more fibrous aspect than in the younger animal. Below the pseudo-epithelium it is arranged in two or three more or less definite layers, in which the fibres run at right angles. It forms a definite ovarian tunic. The pseudoepithelium is much more distinct, and the tails of its cells, so conspicuous in previous stages, can no longer be made out.
Formation of the permanent ova and tlie follicular epithelium. In my monograph on the development of Elasmobranch Fishes an account was given of the earliest stages in the development of the primitive ova, and I now take up their development from the point at which it was left off in that work. From their first formation till the stage spoken of in my monograph as P, their size remains fairly constant. The larger examples have a diameter of about O'O35 mm., and the medium-sized examples of about O'O3 mm. The larger nuclei have a diameter of about O'i6 mm., but their variations in size are considerable. If the above figures be compared with those on page 350 of v my monograph on Elasmobranch Fishes, it will be seen that the size of the primitive ova during these stages is not greater than it was at the period of their very first appearance.
The ova (PL 24, fig. i) are usually aggregated in masses, which might have resulted from division of a single ovum. The outlines of the individual ova are always distinct. Their protoplasm is clear, and their nuclei, which are somewhat passive towards staining reagents, are granular, with one to three nucleoli. I have noticed, up to stage P, the occasional presence of highly refractive spherules in the protoplasm of the primitive ova already described in my monograph (pp. 353, 354, PL 12, fig. 15). They seem to occur up to a later period than I at first imagined. Their want of constancy probably indicates that they have no special importance. Professor Semper has described similar appearances in the male primitive ova of a later period.
As to the distribution of the primitive ova in the germinal epithelium, Professor Semper's statement that the larger primitive ova are found in masses in the centre, and that the smaller ova are more peripherally situated is on the whole true, though I do not find this distribution sufficiently constant to lay so much stress on it as he does.
The passive condition of the primitive ova becomes suddenly broken during stage Q, and is succeeded by a period of remarkable changes. It has only been by the expenditure of much care and trouble that I have been able to elucidate to my own satisfaction what takes place, and there are still points which I do not understand.
Very shortly after stage O, in addition to primitive ova with a perfectly normal nucleus, others may be seen in which the nucleus is apparently replaced by a deeply stained irregular body, smaller than the ordinary nuclei (PL 24, fig. 11, d. //.).
This body, by the use of high objectives, is seen to be composed of a number of deeply stained granules, and around it may be noticed a clear space, bounded by a very delicate membrane. The granular body usually lies close to one side of this membrane, and occasionally sends a few fine processes to the opposite side.
The whole body, i.e. all within the delicate membrane is, according to my view, a modified nucleus ; as appears to me very clearly to be shewn by the fact that it occupies the normal position of a nucleus within a cell body. Semper, on the other hand, regards the contained granular body as the nucleus, which he compares with the spindles of BUtschli, Auerbach, &c.\ This interpretation appears to me, however, to be negatived by the position of these bodies. The manner in which Semper may, perhaps, have been led to his views will be obvious when the later changes of the primitive ova are described. The formation of these nuclei would seem to be due to a segregation of the constituents of the original nuclei ; the solid parts becoming separated from the more fluid. As a rule, the modified nuclei are slightly larger than the original ones. In stage Q the following two tables shew the dimensions of the parts of three unmodified and of three modified nuclei taken at random.
Primitive ova with unmodified nuclei
0-014 mm. O'Oi2 mm. 0*01 mm.
Primitive ova with modified nuclei
Granular Nuclei. Bodies in Nuclei.
O'oiS mm o - oo6 mm.
o'olS mm 0*006 mm.
o'oi2 mm 0-009 mm.
For a slightly older stage than Q, the two annexed tables also shew the comparative size of the modified and unmodified nuclei :
1 Loc. cit. p. 361.
Unmodified nuclei of normal primitive ova
0x114 mm. o - oi6 mm. o - oi4 mm. o'oi6 mm. ox>i6 mm.
Nuclei of primitive ova with modified nuclei
Granular Nuclei. Bodies in Nuclei.
o'oiS mm crooS mm.
o - oi6 mm o'ooS mm.
o'oi6 mm o'oi mm.
o'oi6 mm. ......
These figures bring out with clearness the following points : (i) that the modified nuclei are slightly but decidedly larger on the average than the unmodified nuclei ; (2) that the contained granular bodies are very considerably smaller than ordinary nuclei.
Soon after the appearance of the modified nuclei, remarkable changes take place in the cells containing them. Up to the time such nuclei first make their appearance the outlines of the individual ova are very clearly defined, but subsequently, although numerous ova with but slightly modified nuclei are still to be seen, yet on the whole the outlines of all the primitive ova are much less distinct than before ; and this is especially the case with the primitive ova containing modified nuclei.
From cases in which three or four ova are found in a mass with modified nuclei, but in which the outline of each ovum is fairly distinct, it is possible to pass by insensible gradations to other cases in which two or three or more modified nuclei are found embedded in a mass of protoplasm in which no division into separate cells can be made out (fig. 14). For these masses I propose to employ the term nests. They correspond in part with the Ureiernester of Professor Semper.
Frequently they are found in hardened specimens to be enclosed in a membrane-like tunic which appears to be of the nature of coagulated fluid. These membranes closely resemble and sometimes are even continuous with trabeculae which traverse the germinal epithelium. Ovaries differ considerably as to the time and completeness of the disappearance of the outlines marking the separate cells, and although, so far as can be gathered from my specimens, the rule is that the outlines of the primitive ova with modified nuclei soon become indistinct, yet in one of my best preserved ovaries very large nests with modified nuclei are present in which the outline of each ovum is as distinct as during the period before the nuclei undergo these peculiar changes (PI. 24, fig. 12). In the same ovary other nests are present in which the outlines of the individual ova are no longer visible. The section represented on PL 24, fig. 2, is fairly average as to the disappearance of the outlines of the individual ova.
It is clear from the above statements, that in the first instance the nests are produced by the coalescence of several primitive ova into a single mass or syncytium ; though of course, the several separate ova of a nest may originally, as Semper believes, have arisen from the division of a single ovum. In any case there can be no doubt that the nests of separate ova increase in size as development proceeds ; a phenomenon which is more reasonably explained on the view that the ova divide, than on the view that they continue to be freshly formed. The same holds true for the nests of nuclei and this, as well as other facts, appears to me to render it probable that the nests grow by division of the nuclei without corresponding division of the protoplasmic matrix. 1 cannot, however, definitely prove this point owing to my having found nests, with distinct outlines to the ova, as large as any without such outlines.
The nests are situated for the most part near the surface of the germinal epithelium. The smaller ones are frequently spherical, but the larger are irregular in form. The former are about 0*05 mm. in diameter; the latter reach 0*1 mm. Scattered generally, and especially in the deeper layers, and at the edges of the germinal epithelium, are still unmodified or only slightly modified primitive ova. These unmodified primitive ova are aggregated in masses, but in these masses the outlines of each ovum, though perhaps less clear than in the earlier period, are still distinct.
When the embryo reaches a length of seven centimetres, and even in still younger embryos, further changes are observable.
In the first place many of the modified nuclei acquire fresh characters, and it becomes necessary to divide the modified nuclei into two categories. In both of these the outer boundary of the nucleus is formed by a very delicate membrane, the space within which is perfectly clear except for the granular body. In the variety which now appears in considerable numbers the granular body has an irregular star-like form. The rays of the star are formed of fibres frequently knobbed at their extremities, and the centre of the star usually occupies an eccentric position. Typical examples of this form of modified nucleus, which may be spoken of as the stellate variety, are represented on PI. 25, fig. 17 ; between it and the older granular variety there is an infinite series of gradations, many of which are represented on PL 24, figs. 12, 14, 15, 1 6. Certain of the stellate nuclei exhibit two centres instead of one, and in some cases, like that represented on PL 25, fig. 19, the stellate body of two nuclei is found united. Both of these forms are possibly modifications of the spindle-like form assumed by nuclei in the act of dividing, and may be used in proving that the nests increase in size by the division of the contained nuclei. In addition to the normal primitive ova, a few of which are still present, there are to be found, chiefly in the deeper layers of the germinal epithelium, larger ova differing considerably from the primitive ova. They form the permanent ova (PL 24, fig. 3 o). Their average diameter is 0^04 mm., compared with 003 mm., the diameter of original primitive ova. The protoplasm of which they are composed is granular, but at first a membrane can hardly be distinguished around them ; their nucleus is relatively large, O'O2 0^027 mm. in diameter. It presents the characters ascribed by Eimer 1 , and many other recent authors*, to typical nuclei (vide PL 24, fig. 3, and PL 24, 25, figs. 13, 14, 15, 1 6, 17, 1 8). It is bounded by a distinct membrane, within which is a more or less central nucleolus from which a number of radial fibres which stain very deeply pass to the surface ; here they form immediately internal to the membrane a network with granules at the nodal points. In some instances the regularity of the arrangement of these fibres is very great, in other instances two central nucleoli are present, in which case the regularity is considerably interfered with. The points in which the youngest permanent ova differ from the primitive may be summed up as follows :
1 Archiv f. inter. Anat. Vol. xiv.
" Vide especially Klein, Quart. Jouni. of Mic. Sci. July 1878.
(i) 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 centimetres 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 :
o - oi6 mm. O'oi6 mm. 0*018 mm. O'O2 mm. o'O2 mm.
Both varieties of modified nuclei are common enough, though the stellate variety predominates. The nuclei are sometimes in very close contact, and sometimes separated by protoplasm, which in many instances is very slightly granular. In a large number of the nests nothing further is apparent than what has just been described, but in a very considerable number one or more nuclei are present, which exhibit a transitional character between the ordinary stellate nuclei of my second category, and the nuclei of permanent ova as above described ; and in these nests the formation of permanent ova is taking place. Permanent ova in the act of development are indicated in my figures by the letters d o. Many of the intermediate nuclei are more definitely surrounded by granular protoplasm than the other nuclei of the nests, and accordingly have their outlines more sharply defined. Between nuclei of this kind, and others as large as those of the permanent ova, there are numerous transitional forms. The larger ones frequently lie in a mass of granular protoplasm projecting from the nest, and only united with it by a neck (PI. 24, figs. 14 and 16). For prominences of this kind to become independent ova, it is only necessary for the neck to become broken through. Nests in which such changes are taking place present various characters. In some cases several nuclei belonging to a nest appear to be undergoing conversion into permanent ova at the same time. Such a case is figured on PL 25, figs. 17 and 18. In these cases the amount, of granular protoplasm in the nest and around each freshly formed ovum is small. In the more usual cases only one or two permanent ova at the utmost are formed at the same time, and in these instances a considerable amount of granular protoplasm is present around the nucleus of the developing permanent ovum. In such instances it frequently happens several of the nuclei not undergoing conversion appear to be in the process of absorption, and give to the part of the nest in which they are contained a very hazy and indistinct aspect (PI. 24, fig. 15). Their appearance leads me to adopt the view that while some of the nuclei of each nest are converted into the nuclei of the permanent ova, others break down and are iised 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 (PI. 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 :
o'O22 mm. ox>22 mm. 0x124 mm. 0x132 mm.
These figures should be compared with those of the table on page 564.
The ova when first formed are situated either at the surface or in the deeper layers of the germinal epithelium. Though to a great extent surrounded by the ordinary cells of the germinal epithelium, they are not at first enclosed in a definite follicular epithelium. The follicle is, however, very early formed.
My observations lead me then to the conclusion that in a general way the permanent ova are formed by the increase of protoplasm round some of the nuclei of a nest, and the subsequent separation of the nuclei with their protoplasm from the nest as distinct cells a mode of formation exactly comparable with that which so often takes place in invertebrate egg tubes.
Besides the mode of formation of permanent ova just described, a second one also seems probably to occur. In ovaries just younger than those in which permanent ova are distinctly formed, there are present primitive ova, with modified nuclei of the stellate variety, or nuclei sometimes even approaching in character those of permanent ova, which are quite isolated and not enclosed in a definite nest. The body of these ova is formed of granular protoplasm, but their outlines are very indistinct. Such ova are considerably larger than the normal primitive ova. They may measure 0^04 mm. In a slightly later stage, when fully formed permanent ova are present, isolated ones are not infrequent, and it seems natural to conclude that these isolated ova are the direct descendants of the primitive ova of the earlier stage. It seems a fair deduction that in some cases primitive ova undergo a direct metamorphosis into permanent ova by a modification of their nucleus, and the assumption of a granular character in their protoplasm, without ever forming the constituent part of a nest.
It is not quite clear to me that in all nests the coalescence of the protoplasm of the ova necessarily takes place, since some nests are to be found at all stages in which the ova are distinct. Nevertheless, I am inclined to believe that the fusion of the ova is the normal occurrence.
The mode of formation of the permanent ova may then, according to my observations, take place in two ways : i. By the formation of granular protoplasm round the nucleus in a nest, and the separation of the nucleus with its protoplasm as a distinct ovum. 2. By the direct metamorphosis of an isolated primitive ovum into a permanent ovum. The difference between these two modes of formation does not, from a morphological point of view, appear to be of great importance.
The above results appear clearly to shew that the primitive ova in the female are not to be regarded as true ova, but as the parent sexual cells ivJiich give rise to tlie 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 (PI. 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 (PI. 24, fig. 7, and PI. 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 O'o6 mm., and their nucleus of about 0^04 mm. The vitellus is granular, and provided with a distinct, though delicate membrane, which has every appearance of being a product of the ovum itself rather than of the follicular epithelium. The membrane would seem indeed to be formed in some instances even before the ovum has a definite investment of follicle cells. The vitellus is frequently vacuolated, but occasionally the vacuoles appear to be caused by a shrinking due to the hardening reagent. The nucleus has the same peculiar reticulate character as at first. Its large size, as compared with the ovum, is very noticeable.
With this stage the embryonic development of the ova comes to a close, though the formation of fresh ova continues till comparatively late in life. I have, however, two series of sections of ovaries preserved in osmic acid, from slightly larger embryos than the one last described, about which it may be well to say a few words before proceeding to the further development of the permanent ova.
The younger of these ovaries was from a Scyllium embryo 10 centimetres long, preserved in osmic acid.
A considerable number of nests were present (PI. 24, fig. 13), exhibiting, on the whole, similar characters to those just described.
A series of measurements of the nuclei in them were made, leading to the following results :
0*014 mm. o'oi4 mm. O'oi6 mm. O'oi6 mm. o'oiS mm. 0*018 mm.
Thus, if anything, the nuclei were slightly smaller than in the younger embryo. It is very difficult in the osmic specimens to make out clearly the exact outlines of the various structures, the nuclei in many instances being hardly more deeply stained than in the protoplasm around them. The network in the nuclei is also far less obvious than after treatment with picric acid. The permanent ova were hardly so numerous as in the younger ovary before described. A number of these were measured with the following results :
0*03 mm 0-014 mm.
0-034 mm 0-018 mm.
ox>28 mm. ...... o'oi6 mm.
0x13 mm O'O2 mm.
ox>4 mm O'O2 mm.
ox>4 mm. ...... ox>2 mm.
0^048 mm ox>2 mm.
These figures shew that the nuclei of the permanent ova are smaller than in the younger embryo, and it may therefore be safely concluded that, in spite of the greater size of the embryo from which it is taken, the ovary now being described is in a more embryonic condition than the one last dealt with.
Though the permanent ova appeared to be formed from the nests in the manner already described, it was fairly clear from the sections of this ovary that many of the original primitive ova, after a metamorphosis of the nucleus and without coalescing with other primitive ova to form nests, become converted directly into the permanent ova. Many large masses of primitive ova, or at least of ova with the individual outlines of each ovum distinct, were present. The average size of ova composing these was however small, the body measuring about o'Oi6'mm., and the nucleus O'OI2 mm. Isolated ova with metamorphosed nuclei could also be found measuring O'O22, and their nuclei about 0*014 mm. The second of the two ovaries, hardened in osmic acid, was somewhat more advanced than the ovary in which the formation of permanent ova was at its height. Fewer permanent ova were in the act of being formed, and many of these present had reached a considerable size, measuring as much as O'O/ mm. Nests of the typical forms were present as before, but the nuclei in them were more granular than at the earlier period, and on the average slightly smaller. A series measured had the following diameters :
o'oi mm. o - oi2 mm. o'oi4 mm. 0*016 mm.
One of these nests is represented on PI. 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 Miiller's fluid, none of these have proved universally successful, and bichromate of potash and Muller'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 1 in Raja, and an albuminous layer of the nature of a chorion 51 by Gegenbaur 3 in Acanthias the membranes described in these two ways being no doubt equivalent.
Dr Alex. Schultz 4 has more recently investigated a considerable variety of genera and finds three conditions of the egg membranes, (i) 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 Squalidae, 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 Elasmobranchs, 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.
1 Rochen u. ffaie.
8 By chorion I mean, following E. van Beneden's nomenclature, a membrane formed by the follicular epithelium, and, by vitelline membrane, one formed by the vitellus or body of the ovum.
8 "Bau und Entwicklung d. Wirbelthiereier," &c., Mull. Archiv, 1861.
4 "Zur Entwicklungsgeschichte d. Selachier," ArrJi.f. mikr. Anat. Vol. XI.
In a young fish (not long hatched) with ova of not more than O'i2 mm., this membrane, though considerably thicker than in the embryo, is not thick enough to be accurately measured. In ova of O'5 mm. from a young female (PI. 25, fig. 21) the vitelline membrane has a thickness of O'OO2 mm. and is quite homogeneous 1 . 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 I 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 O'OO4 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 I 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 striae 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 PI. 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.
1 The apparent structure in the vitelline membrane in my figure is merely intended to represent the dark colour assumed by it on being stained. The zona radiata has been made rather too thick by the artist.
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 v /.). 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 Squalidae, 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 1 . 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 8 .
Alexander Schultz 3 states that in Torpedo, the eggs are at first enclosed in a simple epithelium, but that in follicles of 1 Loc. fit. Lof. tit. Loc. cii. 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 O'l 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 develope 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 I 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 palisadelike character by the follicle cells, each cell becoming very narrow and columnar and the nucleus oval (PI. 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 PI. 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 v-itelline 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 ceils 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 1 in Osseous Fish, and Lindgren in Mammalia, has been met with 2 .
1 Das Ei bei Knochenfischen.
2 Arch.f. Anat. Phys. 1877.
My observations in Raja are not so full as those upon Scyllium, but they serve to complete and reconcile the observations of Semper and Schultz, and also to shew that the general mode of growth of the follicular epithelium is fundamentally the same in my representatives of the two divisions of the Elasmobranchii. In very young eggs, in conformity with the results of all previous observers, I find the follicular epithelium approximately uniform. The cells are flat, but extended so as to appear of an unexpected size in views of the surface of the follicle. This condition does not, however, last very long. A certain number of the cells enlarge considerably, others remaining smaller and flat. The differences between the larger and the smaller cells are more conspicuous in sections than in surface views, and though the distribution of the cells is somewhat irregular, it may still be predicted as an almost invariable rule that the smaller cells of the follicle will line that part of the surface of the ovum, near to which the germinal vesicle is situated. On PI. 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 (PI. 25, fig. 31). Such a regularity appears in a later stage (PI. 25, fig. 32), which clearly corresponds with fig. 8 on PI. 34 of Schultz's paper, and also with the stage of Scyllium in PI. 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 PI. 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 O'o6 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 Elasmobranchs 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 1 observations, that the follicular epithelium becomes several layers thick, and that a differentiation of the cells, similar to that in Elasmobranchs, 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 s . That these cells in the Reptilian ova really correspond with those in Elasmobranchs 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.
1 Archiv f. mikr. Anat. Vol. vin.
z Braun, " Urogeuitalsystem d. Amphibien," Arbeiten a. d. zool.-zoot. Institut
The Vitellus and the development of the yolk spherules. Leydig, Gegenbaur, and Schultz, have recorded important observations on this head. Leydig 1 chiefly describes the peculiar characters of the yolk spherules.
Gegenbaur 2 finds in the youngest eggs fine granules; which subsequently develop into vesicles, in the interior of which the solid oval spheres, so characteristic of Elasmobranchs, are developed.
Schultz describes in the youngest ova of Torpedo the minute yolk spherules arranged in a semi-lunar 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 striae, 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 spongework (PI. 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 (PI. 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 striae like that described by Schultz has not come under my notice.
U'urzburg, Bd. iv. He says, in reference to the flask -shaped cell, p. 166, "Hochstens wiirde ich die Funktion der grossen Follikelzellen als einselligt Dritsen mehr betonen."
1 Loc. (it. '* l.oc. cit.
The development of the yolk appears to me to present special difficulties, owing to the fact pointed out by His 1 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 othenvise altered. This has to some extent occurred in PI. 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 o - o6 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 akvays most numerous at the pole opposite the germinal vesicle. Their general arrangement is very much that figured and described by Allen Thomson in Gasterosteus 2 , 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 PI. 25, fig. 27. The ovum represented in fig. 27 was O'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. PI. 4, fig. 33) in the Salmon, and may be compared with the aggregations figured by Gotte in his monograph on Bombinator igneus (PI. I, 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 (PI. 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, (i) 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.
1 Das Ei bei Knochenfischen.
- " Ovum" in Todd's Encyclopedia, fig. 69.
It need scarcely be said that the yolk spherules at this stage are not cells, and have indeed no resemblance to cells. They would probably be regarded by His as spherules of fatty material, unrelated to the true food yolk.
As the ova become larger the granules of the peripheral layer before mentioned gradually assume the character of the yolk spheres of the adult, and at the same time spread towards the centre of the egg. Not having worked at fresh specimens, I cannot give a full account of the growth of the spherules ; but am of opinion that Gegenbaur's account is probably correct, according to which the spheres at first present gradually grow and develop into vesicles, in the interior of which solid bodies (nuclei of His ?) appear and form the permanent yolk spheres. When the yolk spheres are still very small they have the typical oblong form * 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.
1 The peculiar oval, or at times slightly rectangular and striated yolk spherules of Elasmobranchs are mentioned by Leydig and Gegenbaur (PI. n, fig. 20), and myself, Preliminary Account of Development of Elasmobranch Fis/us, and by Filippi and His in Osseous Fishes.
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 1 as " Dotterschorfe." I have only met these bodies in a single instance in ova of O'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 O'O2 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, PI. 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 1 .
1 " Untersuchung iiber die Eier d. Reptilian," Archiv f. mikros. Anat. Vol. VIII.
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 PI. 25, fig. 25 A, 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. 25 B), which makes me think that they may possibly be nucleoli which have made their way out of the germinal vesicle. I have not found them in the late stages or large ova.
The following measurements shew the size of some of these bodies in relation to the germinal vesicle and ovum :
Diameter of Germinal Diameter of Body in
Diameter of Ovum. Vesicle. Vitellus.
0^096 mm. . . 0*03 mm. . . o'oog mm. 0*064 mm. . . o - o25 mm. . . o'oi2 mm.
0-096 mm. 0-03 mm. J' 19 mm '
Germinal vesicle. Gegenbaur 2 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 3 states that there is nothing remarkable in the germinal vesicle of the Torpedo egg, but that till the egg reaches O'5 mm., a single germinal spot is always present (measuring about O'oi mm.), which is absent in larger ova.
1 Vide Allen Thomson, article "Ovum," Todd's Encyclopedia , p. 95. 2 Loc. cit. s l^oc. cil.
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 ccoo, 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 O'I2 mm., the germinal vesicle measures about O'o6 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 1 . Granules are sometimes also present in the germinal vesicle, but are probably only extremely minute nucleoli.
In ova of O'5 mm. the germinal vesicle has a diameter of O'I2 mm. (PI. 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.
1 Compare, with reference to several points, the germinal vesicle at this stage with the germinal vesicle of the frog's ovum figured by O. Hertwig, Morphologisches Jahrbuch, Vol. in. pi. 4, fig. r.
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 1 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."
1 Lot. fif.
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 viewi^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 (PI. 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 O'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 trabeculae 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.
(i) 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. i). 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 (PI. 24, figs. 2, 3, and 6).
(2) At first the thickened germinal epithelium is sharply separated by a membrane from the subjacent stroma (PI. 24, figs, i, 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 (PI. 24, figs. 7 and 8). At the same time the surface of the epithelium turned towards the stroma becomes irregular (PI. 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. i).
(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, 1 6). 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 (PI. 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 (PI. 25, figs. 30 33).
(6) The egg membranes. -Two membranes are probably always present in Klasmobranchs 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 O'o6 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. 25 A.
(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 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 PflUger 1 , Ed. van Beneden 2 , and especially Waldeyer 3 , as inaugurating the newer view on the nature of the ovary, and development of the ova ; and of Foulis 4 and Kolliker 5 , 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 Pfliiger 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. Kolliker'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 Pfliiger's egg-tubes, and that they supply the follicular epithelium. To the general accuracy of Kolliker'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.
1 Die Eierstocke d. Saugethiere it. d. Menschen, Leipzig, 1863.
a "Composition et Signification de 1'cEuf," Acad. r. dc Be^i<jtie, 1868.
3 Eierslock u. Ei. Leipzig, 1870.
4 Trans, of Riyal Society, Edinburgh, Vol. XXVii. 1875, and Quarterly Journal of Microscopical Science ) Vol. xvi.
6 Verhandlung d. P/iys. AM. Gcsdhchaft, Wiirzl.urg, 1875, N. F. Bd. vin.
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. 35 A. 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 (i) 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. 35 A, 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 (/), which forms the greater mass of the ovary at this stage, is formed of solid columns or trabeculae 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. 35 B), 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 (segmentalstrdnge] discovered by Braun in Reptilia, and described at length in his valuable memoir on their urogenital system 1 . 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 2 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.
1 Arbeiten a. d. Zool.-zoot. Institut Wiirzburg, Bd. iv.
2 Archiv f. mikros. Anat. Vol. I. p. 160.
Waldeyer 1 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 (Schlauche) 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 VVolffian 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 tnbnles (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 8 . Born also describes this tissue in young and embryonic ovaries of the horse as the Keimlager*. The columns described by Kolliker 4 and believed by him to furnish the follicular epithelium, are undoubtedly my tubuliferous tissue, and, as Kolliker 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.
1 Loc. dt.
2 Archiv f. inikr. Anat. Vol. x.
3 Archil'/. Anatomic, Physiologic, u. Ifiss. Maiiein. 1874.
4 Lot. tit.
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 Pfliiger'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 Kolliker'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 36 A).
From these figures it is at once obvious that the germinal epithelium has very much increased in bulk. It has a thickness o - i O'O9 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 arc 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 o - oi O'O2 mm. in diameter, and their nuclei from 0004 croo6 mm. The nuclei of the columnar outer cells measure about crooS 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.
Fi& s - 37 an d 37 A 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 Elasmobranchs. 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 Elasmobranchs, 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 Pfluger. 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 trabecuLne, 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 o - 22 mm. in thickness. Its cells have undergone considerable changes. A fair number of them (fig. 37 A,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 Elasmobranchs, Aves, &c. In a fairly typical primitive ovum of this stage the body measures O'O2 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. 3/A. The cells are on the average about O'Oi6 mm. in diameter, and their nuclei about O'OoS 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 37 A 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 (f) 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 O'22 mm. It is divided into three obvious layers: (i) 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 O'i 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 Elasmobranchs. 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. 38 A) 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 trabeculae 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 O'Oi2 mm., and that of the nucleus about O'OOS mm. In the innermost layer (fig. 38 B), 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. 38 B. 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. 38 B, o. The cells with modified nuclei, which are becoming permanent ova, usually present one point of contrast to the homologous cells in Elasmobranchs, 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 Elasmobranchs are thus formed (cf. E. van Beneden 1 ). These masses become slightly more numerous in the succeeding stages. Indications of a fusion of this kind are shewn in fig. 38 B. 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 O'Oi6 C'O2 mm., and their nuclei from O'Oi O'OI2 mm.
With reference to the tissue of the hilus of the ovary, it may be noticed that the tubuliferous tissue (/) is relatively reduced in quantity. Its cells retain precisely their previous characters.
1 Loc. cit.
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 PI. 26, fig. 39 A.
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 PI. 26, fig. 39 B.
The nest contains (i) 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 Elasmobranchs. 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 Elasmobranchs. 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 Elasmobranchs. (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 Elasmobranchs.
Round the completed follicle a very delicate membrana propria folliculi appears to be present 1 .
The larger ova, with follicular epithelium, measure about O'O4 mm., and their nucleus about 0*02 mm., the smaller ones about 0*022 mm., and their nucleus about OX)I4 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. 1 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.
1 Loc. cit., Waldeyer, p. 23, denies the existence of this membrane for Mammalia. It certainly is not so conspicuous as in some other types, but appears to me nevertheless to be always present.
The 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 O'O28 to 0*04 mm. and that of their nucleus from O'Oi6 to o - O2 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 fo.llicle (vide fig. 40 A and 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. 40 A).
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 PI. 26, fig. 40 A. 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 Elasmobranchs, 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., PI. 17, fig. 6) of the ovary of a seven and a half months' human foetus 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 Elasmobranchs. 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. y. Mic. Set., 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 Elasmobranchs and arises at a corresponding stage.
Summary of observations an tfie 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 trabeculae) 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 Elasmobranchs, 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 twentytwo 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 Elasmobranchs :
(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 pseudoepithelium, (b) A segregation takes place in the contents of the nucleus within the membrane, ar^d 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 tfie 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 epitJielium, 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 1 . It is obvious that according to the above view Pfluger's eggtubes are merely trabeculae 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 2 , represent Pfluger's egg-tubes.
The formation of tlie 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 1 . 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 Scy Ilium, 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.
1 My view of the structure of the ovary would seem to be that held by Gotte, Entwicklungsgeschichte d. Unke, pp. 14 and 15. 1 Loc. cit. 36.
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. Gotte 2 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 Gotte'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.
1 Since writing the above I have made out that in the Reptilia the formation of the permanent ova takes place in the same fashion as in Elasmobranchii and Mammalia.
2 Entwickiungsgeschichte d. Unke.
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 mesembryanthemuni), 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 1 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.
1 Zeit. fur wiss. Zool. Bd. xxvil.
TJie 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, (i) 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 1 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 Squalidae. It appears, as in Elasmobranchs, 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 2 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 Elasmobranchs 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 Elasmobranchs.
1 Gegenbaur, loc. cit.; Waldeyer,./^. cit.; Eimer, loc. cit.; and Ludwig, loc. cit. - Gegenbaur, Waldeyer, E. van Beneden, Eimer.
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 1 , 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 2 has, moreover, shewn that they are also provided at a certain period with a delicate membrane within the zona.
TJte 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 3 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.
1 Carlberla, Zrit. /. loiss. Zool. Bd. xxx. 3 Loc, cit.
3 [Quarterly Journal Microscopical Science, July 1878.]
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.
One of the most interesting points in Klein's paper, as well as in those of Heitzmann and Eimer, is the demonstration of a connection between the reticulum of the nucleus and fibres in the body of the cell. Such a connection I have not found in ova, but may point out that it appears to exist between the subgerminal nuclei in Elasmobranchs and the protoplasmic network in the yolk in which they lie. This point is called attention to in my Monograph on Elasmobranch Fishes, page 39 *, where it is stated that " the network in favourable cases may be observed to be in connection with the nuclei just described. Its meshes are finer in the vicinity of the nuclei, and the fibres in some cases appear almost to start from them." The nuclei in the yolk are knobbed bodies divided by a sponge work of septa into a number of areas each with a nucleolar body.
1 [This Edition, p. 252.]
EXPLANATION OF PLATES 24, 25, 26.
LIST OF REFERENCE LETTERS.
d n. Modified nucleus of primitive ovum, d o. Permanent ovum in the act of being formed, dv. Developing blood-vessels, d yk. Developing yolk, e p. Nonovarian epithelium of ovarian ridge, f e. Follicular epithelium, g v. Germinal vesicle. / str. Lymphatic region of stroma. n n. Nests of nuclei of ovarian region. o. Permanent ovum. ov r. Ovarian portion of ovarian ridge, p o. Primitive ovum. ps e. Pseudo-epithelium of ovarian ridge, str. Stroma ingrowths into ovarian epithelium, v. Blood-vessel, v str. Vascular region of stroma adjoining ovarian ridge. v t. Vitelline membrane, x. Modified nucleus, yk. Yolk, z n. Zona radiata.
Fig. i. Transverse section of the ovarian ridge of an embryo of Scy. canicula, belonging to stage P, shewing the ovarian region with thickened epithelium and numerous primitive ova. Zeiss C, ocul. 2. Picric acid.
Fig. 2. Transverse section of the ovarian ridge of an embryo of Scyttitim canicula, considerably older than stage Q. Zeiss C, ocul. 2. Picric acid. Several nests, some with distinct ova, and others with the ova fused together, are present in the section (. .), and several examples of modified nuclei in still distinct ova are also represented. One of these is marked x. The stroma of the ovarian ridge is exceptionally scanty.
Fig. 3. Transverse section through part of the ovarian ridge, including the ovarian region of an almost ripe embryo of Scyllium canicula. Zeiss C, ocul. 2. Picric acid. Nuclear nests (n. .), developing ova (d. o.), and ova (o.), with completely formed follicular epithelium, are now present. The ovarian region is still well separated from the subjacent stroma, and does not appear to contain any cells except those of the original germinal epithelium.
Fig. 4. Section through ovarian ridge of the same embryo as fig. 3, to illustrate the relation of the stroma (str.) and ovarian region. Zeiss a a, ocul. 2. Picric acid.
Fig. 5. Section through the ovarian ridge of an embryo of Scyllium canicula, ro cm. long, in which the ovary was slightly less advanced than in fig. 3. To illustrate the relation of the ovarian epithelium to the subjacent vascula stroma. Zeiss A, ocul. 2. Ostnic acid. y. points to a small separated portion of the germinal epithelium.
Fig. 6. Section through the ovarian ridge of an embryo of Scyllium canicula, slightly older than fig. 5. To illustrate the relation of the ovarian epithelium to the subjacent vascular stroma. Zeiss A, ocul. 2. Ostnic acid.
Fig. 7. More highly magnified portion of the same ovary as" fig. 6. To illustrate the same points. Zeiss C, ocul. 2 . Osmic acid.
Fig. 8. Section through the ovarian region (close to one extremity, where it is very small) from a young female of Scy. canicula. Zeiss C, ocul. 2. Picric acid. It shews the vascular ingrowths amongst the original epithelial cells of the ovarian region.
Fig. 9. Section through the ovarian region of the same embryo as fig. 8, at its point of maximum development. Zeiss A, ocul. 2. Picric acid.
Fig. 10. Section through superficial part of the ovary of an embryo, shewing the pseudo-epithelium ; the cells of which are provided with tails prolonged into the general tissue of the ovary. At/, e. is seen a surface view of the follicular epithelium of an ovum. Zeiss C, ocul. 2. Picric acid.
Fig. 1 1 . Section through part of an ovary of Scyllium canicula of stage Q, with three primitive ova, the most superficial one containing a modified nucleus.
Fig. 12. Section through part of an ovary of an example of Scyllium canicula, 8 cm. long. The section passes through a nest of ova with modified nuclei, in which the outlines of the individual ova are quite distinct. Zeiss E, ocul. 2. Picric acid.
Fig. 13. Section through part of ovary of the same embryo as in fig. 5. The section passes through a nest of nuclei, with at the least two developing ova, and also through one already formed permanent ovum. Zeiss E, ocul. 2. Osmic acid.
Figs. 14, 15, 1 6, 17, 1 8 [Figs. 17 and 18 are on PI. 25]. Sections through parts of the ovary of the same embryo as fig. 3, with nests of nuclei and a permanent ova in the act of formation. Fig. 14 is drawn with Zeiss D D, ocul. 2. Figs. 15, 16, 17, with Zeiss E, ocul. 2. Picric acid.
LIST OF REFERENCE LETTERS.
do. Permanent ovum in the act of being formed, dyk. Developing yolk. J e. Follicular epithelium, f e'. Secondary follicular epithelium, g v. Germinal vesicle. nn. Nests of nuclei of ovarian region, o. Permanent ovum. pse. Pseudo epithelium. sir. Stroma ingrowths into ovarian epithelium, vt. Vitelline membrane, x. Modified nucleus, yk. Yolk (vitellus). z n. Zona radiata.
[Figs. 17 and 18. Vide description of Plate 24.] .
Fig. 19. Two nuclei from a nest which appear to be in the act of division. From ovary of the same embryo as fig. 3.
Fig. 20. Section through part of an ovary of the same embryo as fig. 6, containing a nest of nuclei. Zeiss F, ocul. 2. Osmic acid.
Fig. 21. Ovum from the ovary of a half-grown female, containing isolated deeply stained patches of developing yolk granules. Zeiss B, ocul. 2. Picric acid.
Fig. 22. Section through a small part of the ovum of an immature female of Scyllium canicula, to shew the constitution of the yolk, the follicular epithelium, and the egg membranes. Zeiss E, ocul. 2. Chromic acid.
Fig. 23. Section through part of the periphery of a nearly ripe ovum of Scy. canicula. Zeiss C, ocul. 2. It shews the remnant of the vitelline membrane (v. t.) separating the columnar but delicate cells of the follicular epithelium (/ e.) from the yolk (yk.). In the yolk are seen yolk-spherules in a protoplasmic network. The transverse markings in the yolk-spherules have been made oblique by the artist.
Fig. 24. Fully formed ovum containing a second nucleus (x), probably about to be employed as pabulum ; from the same ovary as fig. 5. The follicular epithelium is much thicker on the side adjoining the stroma than on the upper side of the ovum. Zeiss F, ocul. 2. Osmic acid.
Fig. 25. A. Ovum from the same ovary as fig. 21, containing in the yolk three peculiar bodies, similar in appearance to the two small bodies in the germinal vesicle. B. Germinal vesicle of a large ovum from the same ovary, containing a body of a strikingly similar appearance to those in the body of the ovum in A. Zeiss E, ocul. 2. Picric acid.
Fig. 26. Section of the ovary of a young female of Scy Ilium stellare i6 centimetres in length. The ovary is exceptional, on account of the large size of the stroma ingrowths into the epithelium. Zeiss C, ocul. 2. Osmic acid.
Fig. 27. Ovum of Scyllium canicula, 5 mm. in diameter, treated with osmic acid. The figure illustrates the development of the yolk and a peculiar mode of proliferation of the germinal spots. Zeiss A, ocul. 2.
Fig. 28. Small part of the follicular epithelium and egg membranes of a somewhat larger ovum of Scyllium canicula than fig. 22. Zeiss D D, ocul. 2.
Fig. 29. The same parts as in fig. 28, from a still larger ovum. Zeiss D D, ocul. 2.
Fig. 30. Ovum of Raja with follicular epithelium. Zeiss C, ocul. 2.
Fig. 31. Small portion of a larger ovum of Raja than fig. 30. Zeiss DD, ocul. 2.
Fig. 32. Follicular epithelium, &c., from an ovum of Raja still larger than fig. 31. Zeiss D D, ocul. 2.
Fig- 33- Surface view of follicular epithelium from an ovum of Raja of about the same age as fig. 33.
Fig- 34- Vertical section through the superficial part of an ovary of an adult Raja to shew the relation of the pseudo-epithelium to the subjacent stroma. Zeiss D D, ocul. 2.
COMPLETE LIST OF REFERENCE LETTERS.
d o. Developing ovum. fc. Cells which will form the follicular epithelium, f e. Follicular epithelium, g e. Germinal epithelium, mg. Malpighian body. n. Nest of cells of the germinal epithelium, n d. Nuclei in the act of dividing, o. Permanent ovum, o v. Ovary. / o. Primitive ovum. /. Tubuliferous tissue, derived from Malpighian bodies.
Fig. 35. Transverse section through the ovary of an embryo rabbit of eighteen days, hardened in osmic acid. The colours employed are intended to render clear the distinction between the germinal epithelium (ge.) and the tubuliferous tissue (tf, which has grown in from the Wolffian body, and which gives rise in the male to parts of the tubuli seminiferi. Zeiss A, ocul. 2.
Fig. 35 A. Transverse section through a small part of the ovary of an embryo from the same female as fig. 35, hardened in picric acid, shewing the relation of the germinal epithelium to the subjacent tissue. Zeiss D D, ocul. 2.
Fig- 35 B. Longitudinal section through part of the Wolffian body and the anterior end of the ovary of an eighteen days' embryo, to shew the derivation of tubuliferous tissue (t.) from the Malpighian bodies, close to the anterior extremity of the ovary. Zeiss A, ocul. i.
Fig- 36. Transverse section through the ovary of an embryo rabbit of twentytwo days, hardened in osmic acid. It is coloured in the same manner as fig. 35. Zeiss A, ocul. 2.
Fig. 36 A. Transverse section through a small part of the ovary of an embryo, from the same female as fig. 36, hardened in picric acid, shewing the relation of the germinal epithelium to the stroma of the ovary. Zeiss D D, ocul. 2.
Figs. 37 and 37 A. The same parts of an ovary of a twenty-eight days' embryo as 'figs. 36 and 36 A of a twenty-two days' embryo.
Fig. 38. Ovary of a rabbit five days after birth, coloured in the same manner as figs. 35, 36 and 37, but represented on a somewhat smaller scale. Picric acid.
Fig. 38 A. Vertical section through a small part of the surface of the same ovary as fig. 38. Zeiss D D, ocul. 2.
Fig. 38 B. Small portion of the deeper layer of the germinal epithelium of the same ovary as fig. 38. The figure shews the commencing differentiation of the cells of the germinal epithelium into true ova and follicle cells. Zeiss D D, ocul. 2.
Fig. 39 A. Section through a small part of the middle region of the germinal . epithelium of a rabbit seven days after birth. Zeiss D D, ocul. 2.
Fig. 39 B. Section through a small part of the innermost layer of the germinal epithelium of a rabbit seven days after birth, shewing the formation of Graafian follicles. Zeiss D D, ocul. 2.
Figs. 40 A and 40 B. Small portions of the middle region of the germinal epithelium of a rabbit four weeks after birth. Zeiss D D, ocul. 2.
Fig. 41. Graafian follicle with two ova, about to divide into two follicles, from a rabbit six weeks after birth. Zeiss D D, ocul. 2.