Chapter XII: The Organs of Excretion (6)

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

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

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

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

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

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

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

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

At six weeks after birth the ovary of the rabbit corresponds very much more with the stages in the development of the ovary, which Foulis has more especially studied, for the formation of the follicular epithelium, than during the earlier stages. His figure (_Quart. Journ. Mic. Sci._, Vol. XVI., Pl. 17, fig. 6) of the ovary of a seven and a half months' human 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 Elasmobranchii. A follicular epithelium is present around the majority of the ova of the middle layer, and around all those of the inner layer of the germinal epithelium. The nests are, moreover, much more cut up by connective tissue ingrowths than in the previous stages.

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

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

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

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

_Summary of observations on the mammalian ovary._--The general results of my observations on the mammalian ovary are the following:--

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

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

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

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

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

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

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

GENERAL OBSERVATIONS ON THE STRUCTURE AND DEVELOPMENT OF THE OVARY.

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

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

Footnote 413: My view of the structure of the ovary would seem
to be that held by Götte, _Entwicklungsgeschichte d. Unke_, pp.
14 and 15.

Footnote 414: _Loc. cit._ 36.

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

Footnote 415: 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.

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

Footnote 416: _Entwicklungsgeschichte 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 mesembryanthemum_), out of a large number of ova which develop up to a certain point, a comparatively very small number survive, and these regularly feed upon the other ova. During this process the boundary between a large ovum and the smaller ova is indistinct: in the outermost layer of a large ovum a number of small ova are embedded, the outlines of the majority of which have become obscure, although they can still be distinguished. Just beyond the edge of a large ovum the small ova have begun to undergo retrogressive changes; while at a little distance from the ovum they are quite normal. An analogous phenomenon has been very fully described by Weismann[417] in the case of Leptodera, the ovary of which consists of a germogene, in which the ova develop in groups of four. Each group of four occupies a separate chamber of the ovary, but in summer only one of the four eggs (the third from the germogene) develops into an ovum, the other three are used as pabulum. In the case of the winter eggs the process is carried still further, in that the contents of the alternate chambers, instead of developing into ova, are entirely converted, by a series of remarkable changes, into nutritive reservoirs. Fundamentally similar occurrences to the above are also well known in Insects. Phenomena of this nature are obviously in no way opposed to the view of the ovum being a single cell.

Footnote 417: _Zeit. für wiss. Zool._ Bd. XXVII.

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

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

Footnote 418: Gegenbaur, _loc. cit._; Waldeyer, _loc. cit._;
Eimer, _loc. cit._; and Ludwig, _loc. cit._

Footnote 419: Gegenbaur, Waldeyer, E. van Beneden, Eimer.

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

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

Footnote 420: Carlberla, _Zeit. f. wiss. Zool._ Bd. XXX.

Footnote 421: _Loc. cit._

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

Footnote 422: [_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 sub-germinal nuclei in Elasmobranchii 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[423], 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.

Footnote 423: [This Edition, p. 252.]

EXPLANATION OF PLATES 24, 25, 26.

PLATE 24.

LIST OF REFERENCE LETTERS.

_dn._. Modified nucleus of primitive ovum. _do._. Permanent ovum in the act of being formed. _dv._. Developing blood-vessels. _dyk._ Developing yolk. _ep._ Non-ovarian epithelium of ovarian ridge. _fe._ Follicular epithelium. _gv._ Germinal vesicle. _lstr._ Lymphatic region of stroma. _nn._ Nests of nuclei of ovarian region. _o._ Permanent ovum. _ovr._ Ovarian portion of ovarian ridge. _po._ Primitive ovum. _pse._ Pseudo-epithelium of ovarian ridge. _str._ Stroma ingrowths into ovarian epithelium. _v._ Blood-vessel. _vstr._ Vascular region of stroma adjoining ovarian ridge. _vt._ Vitelline membrane. _x._ Modified nucleus. _yk._ Yolk. _zn._ Zona radiata.

Fig. 1. 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 _Scyllium 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 (_nn_), 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.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_, 10 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 vascular stroma. Zeiss A, ocul. 2. _Osmic 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. _Osmic 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 _f.e._ is seen a surface view of the follicular epithelium of an ovum. Zeiss C, ocul. 2. _Picric acid._

Fig. 11. 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, 16, 17, 18 [Figs. 17 and 18 are on Pl. 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._

PLATE 25.

LIST OF REFERENCE LETTERS.

_do._ Permanent ovum in the act of being formed. _dyk._ Developing yolk. _fe._ Follicular epithelium. _fe´._ Secondary follicular epithelium. _gv._ Germinal vesicle. _nn._ Nests of nuclei of ovarian region. _o._ Permanent ovum. _pse._ Pseudo-epithelium. _str._ Stroma ingrowths into ovarian epithelium. _vt._ Vitelline membrane. _x._ Modified nucleus. _yk._ Yolk (vitellus). _zn._ 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 (_f.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 _Scyllium stellare_ 16-1/2 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 canicul_a 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 D D, 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.

PLATE 26.

COMPLETE LIST OF REFERENCE LETTERS.

_do._ Developing ovum. _fc._ Cells which will form the follicular epithelium, _fe._ Follicular epithelium. _ge._ Germinal epithelium. _mg._ Malpighian body. _n._ Nest of cells of the germinal epithelium. _nd._ Nuclei in the act of dividing. _o._ Permanent ovum. _ov._ Ovary. _po._ Primitive ovum. _t._ 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 (_t._), 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. 35A . 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. 35B. 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. 1.

Fig. 36. Transverse section through the ovary of an embryo rabbit of twenty-two days, hardened in osmic acid. It is coloured in the same manner as fig. 35. Zeiss A, ocul. 2.

Fig. 36A. 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 37A. The same parts of an ovary of a twenty-eight days' embryo as figs. 36 and 36A 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. 38A. Vertical section through a small part of the surface of the same ovary as fig. 38. Zeiss D D, ocul. 2.

Fig. 38B. 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. 39A. 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. 39B. 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. 40A and 40B. 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.

XIII. ON THE EXISTENCE OF A HEAD-KIDNEY IN THE EMBRYO CHICK, AND ON
CERTAIN POINTS IN THE DEVELOPMENT OF THE MÜLLERIAN DUCT[424].
BY F. M. BALFOUR AND A. SEDGWICK.

Footnote 424: From the _Quarterly Journal of Microscopical
Science_, Vol. XIX. 1879.

(With Plates 27 and 28.)

The following paper is divided into three sections. The first of these records the existence of certain structures in the embryo chick, which eventually become in part the abdominal opening of the Müllerian duct, and which, we believe, correspond with the head-kidney, or "Vorniere" of German authors. The second deals with the growth and development of the Müllerian duct. With reference to this we have come to the conclusion that the Müllerian duct does not develop entirely independently of the Wolffian duct. The third section of our paper is of a more general character, and contains a discussion of the rectifications in the views of the homologies of the parts of the excretory system in Aves, necessitated by the results of our investigations.

We have, as far as possible, avoided entering into the extended literature of the excretory system, since this has been very fully given in three general papers which have recently appeared by Semper[425], Fürbinger[426], and by one of us[427].

Footnote 425: "Das Urogenital-System der Plagiostomen,"
_Arbeiten a. d. zool.-zoot. Institut. Würzburg_.

Footnote 426: "Zur vergl. Anat. u. Entwick. d.
Excretionsorgane d. Vertebraten," _Morphologisches Jahrbuch_,
Vol. IV.

Footnote 427: "On the Origin and History of the Urinogenital
Organs of Vertebrates," _Journal of Anat. and Phys._, Vol. X.
[This Edition No. VII.]

All recent observers, including Braun[428] for Reptilia, and Egli[429] for Mammalia, have stated that the Müllerian duct develops as a groove in the peritoneal epithelium, which is continued backward as a primitively solid rod in the space between the Wolffian duct and peritoneal epithelium.

Footnote 428: _Arbeiten a. d. zool.-zoot. Institut. Würzburg_,
Vol. IV.

Footnote 429: _Beitr. zur Anat. u. Entwick. d.
Geschlechtsorgane_, Inaug. Diss., Zürich, 1876.

In our preliminary account we stated[430], in accordance with the general view, that the Müllerian duct was formed as a groove, or elongated involution of the peritoneal epithelium adjoining the Wolffian duct. We have now reason to believe that this is not the case. In the earliest condition of the Müllerian duct which we have been able to observe, it consists of three successive open involutions of the peritoneal epithelium, connected together by more or less well-defined ridge-like thickenings of the epithelium. We believe, on grounds hereafter to be stated, that the whole of this formation is equivalent to the head-kidney of the Ichthyopsida. The head-kidney, as we shall continue to call it, takes its origin from the layer of thickened epithelium situated near the dorsal angle of the body-cavity, close to the Wolffian duct, which has been known since the publication of Waldeyer's important researches as the germinal epithelium. The anterior of the three open involutions or grooves is situated some little distance behind the front end of the Wolffian duct. It is simply a shallow groove in the thickest part of the germinal epithelium, and forms a corresponding projection into the adjacent stroma. In front the projection is separated by a considerable interval from the Wolffian duct; but near its hindermost part it almost comes into contact with the Wolffian duct. The groove extends in all for about five of our sections, and then terminates by its walls becoming gradually continued into a slight ridge-like thickening of the germinal epithelium. The groove arises as a simple depression in a linear area of thickened germinal epithelium. The linear area is, however, continued very considerably further forward than the groove, and sometimes exhibits a slight central depression, which might be regarded as a forward continuation of the groove. The passage from the groove to the ridge may best be conceived by supposing the groove to be suddenly filled up, so as to form a solid ridge pointing inwards towards the Wolffian duct.

Footnote 430: _Proceedings of Royal Society_, 1878.

The ridge succeeding the first groove is continued for about six sections, and is considerably more prominent at its posterior extremity than in front. It is replaced by groove number two, which appears as if formed by the reverse process to that by which the ridge arose, viz., by a hollowing out of the ridge on the side towards the body-cavity. The wall of the second groove is, after a few sections, continued into a second ridge or thickening of the germinal epithelium, which, however, is so faintly marked as to be hardly visible in its middle part. In its turn this ridge is replaced by the third and last groove. This vanishes after one or two sections, and behind the point of its disappearance we have failed to find any further traces of the head-kidney. The whole formation extends through about twenty-four of our sections and one and a half segments (muscle-plates).

We have represented (Plate 27, Series A, Nos. 1-10) a fairly complete series of sections through part of the head-kidney of an embryo slightly older than that last described, containing the second and third grooves and accessory parts. The connection between the grooves and the ridges is very well illustrated in Nos. 3, 4, and 5 of this series. In No. 3 we have a prominent ridge, in the interior of which there appears in No. 4 a groove, which becomes gradually wider in Nos. 5 and 6. Both the grooves and ridges are better marked in this than in the younger stage; but the chief difference between the two stages consists in the third groove no longer forming the hindermost limit of the head-kidney. Instead of this, the last groove (No. 7) terminates by the upper part of its walls becoming constricted off as a separate rod, which appears at first to contain a lumen continuous with the open groove. This rod (Nos. 7, 8, 9, 10) situated between the germinal epithelium and Wolffian duct is continued backward for some sections. It finally terminates by a pointed extremity, composed of not more than two cells abreast (Nos. 8-10).

Our third stage, sections of which are represented in series B (Plate 27), is considerably advanced beyond that last described. The most important change which has been effected concerns the ridges connecting the successive grooves. A lumen has appeared in each of these, which seems to open at both ends into the adjacent grooves. At the same time the cells, which previously constituted the ridge, have become (except where they are continuous with the walls of the grooves) partially constricted off from the germinal epithelium. The ridges, in fact, now form ducts situated in the stroma of the ovarian ridge, in the space between the Wolffian duct and the germinal epithelium. The duct continuous with the last groove is somewhat longer than before. In a general way, the head-kidney may now be described as a duct opening into the body-cavity by three groove-like apertures, and continuous behind with the rudiment of the true Müllerian duct. Although the general constitution of the head-kidney at this stage is fairly simple, there are a few features in our sections which we do not fully understand, and a few points about the organ which deserve a rather fuller description than we have given in this general sketch.

The anterior groove (Nos. 1-3, series B, Pl. 27) is at first somewhat separated from the Wolffian duct, but approaches close to it in No. 3. In Nos. 2 and 3 there appears a rod-like body on the outer side of the walls of the groove. In No. 2 this body is disconnected with the walls of the groove, and even appears as if formed by a second invagination of the germinal epithelium. In No. 3 this body becomes partially continuous with the walls of the groove, and finally in No. 4 it becomes completely continuous with the walls of the groove, and its lumen communicates freely with the groove[431].

Footnote 431: A deep focus of the rather thick section
represented in No. 3 shewed the body much more nearly in the
position it occupies in No. 4.

The last trace of this body is seen on the upper wall of the groove in No. 5. We believe that the body (_r_1) represents the ridge between the first and second grooves of the earlier stage; so that in passing from No. 3 to No. 5 we pass from the first to the second groove. The meaning of the features of the body _r_1 in No. 2 we do not fully understand, but cannot regard them as purely accidental, since we have met with more or less similar features in other series of sections. The second groove becomes gradually narrower, and finally is continued into the second ridge (No. 8). The ridge contains a lumen, and is only connected with the germinal epithelium by a narrow wall of cells. A narrow passage from the body-cavity leads into that wall for a short distance in No. 8, but it is probably merely the hinder end of the groove of No. 7. The third groove appears in No. 11, and opens into the lumen of the second ridge (_r_2) in No. 12. In No. 13 the groove is closed, and there is present in its place a duct (_r_3) connected with the germinal epithelium by a wall of cells. This duct is the further development of the third ridge of the last stage; its lumen opens into the body-cavity through the third and last groove (_gr_3). In the next section this duct (_r_3) is entirely separated from the germinal epithelium, and it may be traced backwards through several sections until it terminates by a solid point, very much as in the last stage.

In the figures of this series (B) there may be noticed on the outer side of the Müllerian duct a fold of the germinal epithelium (_x_) forming a second groove. It is especially conspicuous in the first six sections of the series. This fold sometimes becomes much deeper, and then forms a groove, the upper end of which is close to the grooves of the head-kidney. It is very often much deeper than these are, and without careful study might easily be mistaken for one of these grooves. Fig. C, taken from a series slightly younger than B, shews this groove (_x_) in its most exaggerated form.

The stage we have just described is that of the fullest development of the head-kidney. In it, as in all the previous stages, there appear to be only three main openings into the body-cavity; but we have met in some of our sections with indications of the possible presence of one or two extra rudimentary grooves.

In an embryo not very much older than the one last described the atrophy of the head-kidney is nearly completed, and there is present but a single groove opening into the body-cavity.

In series D (Pl. 28) are represented a number of sections from an embryo at this stage. Nos. 1 and 2 are sections through the hind end of the single groove now present. Its walls are widely separated from the Wolffian duct in front, but approach close to it at the hinder termination of the groove (No. 2). The features of the single groove present at this stage agree closely with those of the anterior groove of the previous stages. The groove is continued into a duct--the Müllerian duct (as it may now be called, but in a previous stage the hollow ridge connecting the first and second grooves of the head-kidney)--which, after becoming nearly separated from the germinal epithelium, is again connected to it by a mass of cells at two points (Nos. 5, 6, and 8). The germinal epithelium is slightly grooved and is much reduced in thickness at these points of contact (_gr_2 and _gr_3), and we believe that they are the remnants of the posterior grooves of the head-kidney present at an earlier stage.

The Müllerian duct has by this stage grown much further backwards, but the peculiarities of this part of it are treated in a subsequent section.

We consider that, taking into account the rudiments we have just described, as well as the fact that the features of the single groove at this stage correspond with those of the anterior groove at an earlier stage, we are fully justified in concluding that _the permanent abdominal opening of the Müllerian duct corresponds with the anterior of our three grooves_.

Although we have, on account of their indefiniteness, avoided giving the ages of the chicks in which the successive changes of the head-kidney may be observed, we may, perhaps, state that all the changes we have described are usually completed between the 90th and 120th hour of incubation.

_The Glomerulus of the Head-Kidney._

In connection with the head-kidney in Amphibians there is present, as is well known, a peculiar vascular body usually described as the glomerulus of the head-kidney. We have found in the chick a body so completely answering to this glomerulus that we have hardly any hesitation in identifying it as such.

In the chick the glomerulus is paired, and consists of a vascular outgrowth or ridge projecting into the body-cavity on each side at the root of the mesentery. It extends from the anterior end of the Wolffian body to the point where the foremost opening of the head-kidney commences. We have found it at a period slightly earlier than that of the first development of the head-kidney. It is represented in figs. E and F, Pl. 28, _gl_, and is seen to form a somewhat irregular projection into the body-cavity, covered by a continuation of the peritoneal epithelium, and attached by a narrow stalk to the insertion of the embryonic mesentery (_me_).

In the interior of this body is seen a stroma with numerous vascular channels and blood corpuscles, and a vascular connection is apparently becoming established, if it is not so already, between the glomerulus and the aorta. We have reason to think that the corpuscles and vascular channels in the glomerulus are developed _in situ_. The stalk connecting the glomerulus with the attachment of the mesentery varies in thickness in different sections, but we believe that the glomerulus is continued unbroken throughout the very considerable region through which it extends. This point is, however, difficult to make sure of owing to the facility with which the glomerulus breaks away.