On the Intra-Ovarian Egg of Some Osseous Fishes

The following is a list of the species of fishes whose ova or ovaries I examined:—Trigla gurnardus, Gadus virens, G. æglefinus, G. luscus, G. merlangus, Lophius piscatorius, Salmo salar, Anarrhichas lupus, Conger vulgaris, Blennius pholis, Hippoglossoides limandoides.

In several cases the investigation was carried out on fresh ovaries; others were only inspected in a preserved condition. I cut sections of all of them. They were hardened in weak chromic or in picrosulphuric acid.

With regard to the result of my researches, I may mention that there were two features which seemed to me of special interest. Firstly, the development of the yolk, and secondly, the origin of the egg-membranes and the follicle. I think I have been successful in tracing the first, and also answered part of the latter question. But the smallness of the objects presents great difficulties, and the ova, after they pass a certain size, become so opaque that their structure has to be studied entirely from sections of hardened specimens. I propose to divide this paper into five chapters, beginning with the nucleus and its changes, and finishing up with a general account of the development of the intra-ovarian egg. An abstract of this paper was read before the Royal Society at their meeting in the beginning of December last. It will be found in this year’s Proceedings of the Society.

The various stages of the growing ovum have all, or nearly all, been seen in the gurnard’s egg; but, in order to show that they also occur in the other forms, where it was possible, illustrations have been copied from sections of the different species examined.

I found the smallest ova, measuring 0·011 mm. in diameter, in the ovary of the Haddock. In these eggs the nucleus occupies almost the whole of the interior (fig. 1). A very narrow zone of protoplasm, which, as far as I could ascertain, was not bounded by a membrane, surrounded the nucleus. The wall-less ovum lies in the endothelial or connective tissue stroma of the ovary. The nucleoli (fig. 1, n′.)—there are a great number of them—rest as a rule on the inner surface of the nuclear wall, barring a few, which take up a central position. With an ordinary high power (Zeiss F⁴) nothing more of an internal structure can be detected.

The next size (fig. 2) shows a division of the protoplasm into two distinct layers or zones, an outer lighter and an inner darker or more denser one. The egg in this stage is still tolerably transparent. Fig. 2 represents a small gurnard’s egg (Trigla gurnardus) 0·030 mm. in diameter. The nucleoli show an inclination to gather still more towards the periphery of the nucleus, and the central portion rarely reveals a germinal spot, while there appears instead an intra-nuclear network which will be more minutely described in larger eggs. In ova of this bulk, and also in somewhat larger ones, one or more of the nucleoli become larger than the others, and in their interior highly refractive specks are visible which have sometimes been described as endonucleoli. Another peculiarity about the large germinal spots is that they are always surrounded by a light portion which does not stain with carmine.

In somewhat larger ova, of a diameter of 0·080 mm. (fig. 3), the dark zone round the nucleus is seen to have increased considerably while the light one remained stationary. The large nucleus which is represented in fig. 4 shows that in some cases the big nucleoli disappear almost completely, leaving an unstained part around them. Frequently one spot was seen in the centre of the nucleus among what is generally known as the “chromatic” substance. This consists of very minute granules distinguished from the rest of the nuclear substance by its greater consistence, a higher refractive power, as well as by its capability of assuming a strong tint in certain solutions of colouring matter which are used among microscopists for staining nuclei. The small granules are suspended in a network of threads, which has so often been described in both animal and vegetable cells, and which plays a conspicuous part in the karyokinetic figures of the dividing nucleus. It is specially well seen in fig. 9, n. f., representing a nucleus and its surrounding protoplasm of a middle-sized gurnard’s egg.

I agree with my friend Dr. Will in not attaching any morphological significance to the nucleoli. They must be regarded as large masses of chromatic substance. In some instances they are entirely absent; in others one or more may be present.

To return to the large nucleoli which, as has been mentioned, are occasionally present in the ova of the gurnard, they are never wanting in the eggs of the conger eel (Conger vulgaris). They stain slightly darker than the small ones. In several cases (fig. 5, n′) I noticed a small nucleolus being constricted off from a large one. In an egg of Gadus virens (fig. 6), measuring 0·105 mm. in diameter, the dark protoplasm (vr.¹) surrounding the nucleus in smaller ova had been separated in form of a ring, and internally to it another narrower ring (pr.²) of protoplasm was frequently present. The zone of light protoplasm externally had increased considerably meanwhile. In some instances, however, the dark zone had invested the whole of the ovum, and the light portion had entirely disappeared. Another feature which came under my notice now was that the dark zone contained the faint outlines of spots corresponding in size to nucleoli (fig. 7, sp.), none of which, however, were seen outside in the light protoplasm. Only in Hippoglossoides did I occasionally observe similar spots close to the surface of the egg.

There can be little doubt that these spots are nucleoli which have travelled through the nuclear membrane into the surrounding protoplasm, and are gradually dissolved there. I notice here incidentally that possibly some find their way to the surface of the egg to form the nuclei of the follicular epithelium. This is only a supposition; but it will be referred to again more fully in a subsequent chapter.

In a larger egg of Gadus virens, 0·132 mm. in diameter (fig. 7), we still find a granular protoplasmic ring round the nucleus; however, a great change has come over the nucleoli, which are now no more closely attached to the nuclear wall. They seem to have become broken up or dissolved in some way, assuming all sorts of shapes. Some are crescent-shaped, some are like rods, and others again are reduced to small specks or angular fragments. Now what is the significance of all this ?

To commence with the relation of the dark protoplasm towards the light one, I may previously state that this division of the contents of the ovum has been seen by several authors not only in fishes and amphibians, but also in invertebrate forms. Bambeke¹ observed a division of the protoplasm into two zones in the young ova of Leuciscus rutilus, Hippo-campus antiquorum, and Lota vulgaris. Eimer² believes in the idendity of His’ “Rindenschicht” with His’ “Zonoid layer;” his figures, however, show clearly that in young ova, at anyrate, it corresponds to the light protoplasm which I described, in distinction to the more granular part internally to it. The fact that the yolk is divided into zones has also been observed in Sepia by Lankester.³ Finally, my friend Dr. Will⁴ has noticed the same phenomenon in Orthoptera. No doubt the formation of protoplasmic rings round the nucleus of the growing egg has been seen in a still greater number of animal groups than I have just enumerated.

I think there can be little hesitation in regarding the dark central protoplasm as owing its origin to the nucleus, although there appear to be cases, such as recorded by Lankester,? in which the protoplasm is nourished entirely from without.

But a difficulty presents itself here. Has the dark part originated by a simple transformation of the light portion, or has another substance been added to the protoplasm from the nucleus causing this change? The latter seems the most probable of the two cases. This view is considerably strengthened by an observation which was published by Ransom¹ in 1867, in the ‘Philosophical Transactions? He says: “The action of the water is not the same on the free uninjured germinal vesicle in young ova as it is on those still within the egg. I found that the results were in a great measure due to the influence exerted by the constituents of the yolk, which were carried into the vesicle by osmose. When the ovum was acted upon by water, the germinal spots were gradually seen to become pale, and finally disappear. These facts strongly suggest the notion that the germinal spots are soluble in some of the constituents of the yolk, and we may thus explain their disappearance in ripe ova.

Ransom likewise observed that in the earlier ovum of Gasterosteus leiverus and pungitis—the two species which he examined—the germinal spots, which were embedded in the colloid matrix of the vesicle, were to be seen at the periphery of the vesicle only, so as to be in contact with the inner surface of the nuclear wall. The germinal spots were often “tailed and vacuolate.”

In an older stage, such as that represented by fig. 8, the dark ring encompassing the nucleus has evidently been absorbed, and the latter has dimished in size while the egg itself has continued to grow larger. Indications of the boundary of the old germinal vesicle are still seen (fig. 9, n’.), and the space between it and the reduced one is filled with dark granular protoplasm, which seems still to be produced by the action of the nucleus. A new process, however, begins at this stage, namely, the formation of the yolk spherules, which will be described in the next paragraph.

If we look at figure 9, which represents the nucleus of a large egg surrounded by a zone of protoplasm indicating the outlines of its predecessor, we are at once struck by the peculiar protuberances which make their appearance all over its outer surface. They were best seen in sections through the hardened ova of the gurnard. These measured somewhere about 0·130 mm. in diameter. These diverticula, buds or “stolons,” as they have been called by Balbiani, are pushed out by the nucleus. Part of the nucleoli resting upon the diverticula is drawn into them and carried away towards the exterior of the egg. They have the appearance now of minute vesicles or cells containing a nucleus. For such, indeed, they have been mistaken even by the most recent writer on the subject, Ovsi-annikov. The vesicle is either a portion of the nuclear wall which has become constricted off, or it may be a later formation. Sometimes the vesicles thus formed do not contain any nucleolar matter and remain unaffected by staining reagents. Like the others they travel towards, but they do not quite reach the surface of the egg, leaving a cortical layer of protoplasm which is the “Rindenschicht” of His. The vesicles with their nucleolar contents are the yolk-spherules. The solid mass in their interior soon breaks up into fine granules, and it is in this condition that the yolk-spherules are found in the largest intra-ovarian eggs. The granules, however, are at first of a dark colour, which they only lose on the ovum becoming ripe. In the mature egg the yolk is perfectly transparent.

The nuclear network which has been mentioned above is specially well seen at this stage (Fig. 9, n.f.). The threads connecting the minute granules in the interior of the nucleus seem to be made up of fine dots rather than solid fibres.

The vesicles with clear contents might possibly be what is known as “oil globules,” on the significance of which my friend Mr. Prince¹ has recently published an interesting paper. Their oily contents would thus originate from the clear nuclear substance. I merely throw this out as a suggestion, but if it should ultimately be proved correct, it would form another addition to these most interesting and instructive phenomena which the ovum of osseous fishes is undergoing during its growth. In the ripe gurnard’s egg a large oil-globule is to be found at the periphery of the yoke, and similar ones occur in many other marine Teleosteans. The largest yoke spherules in the gurnard (fig. 10) have a diameter of 0·015 mm. In the frog-fish (Lophius piscatorius), however, their size varies from 0·018 up to 0·090 mm. (fig. 11). The contents in the latter case consist, besides the small granules, of peculiar crescent-shaped bodies and vacuoles. In many of them dark crystal-line bodies were seen enclosed in a vesicle. Ovsiannikov¹ likewise makes mention of crystals and peculiar oval bodies as occurring in the yolk spherules of Osmerus eperlanus.

With regard to the process of budding from the nucleus which I just described as leading to the origin of the yolk-spherules, similar changes of the nucleus have been noticed by various observers; but only Will² derives the yolk-spherules from these buds. He investigated the intra-ovarian egg of Amphibia. No other writer seems to have seen anything corresponding to this process in Vertebrates. Roule,³ Fol,⁴ and Balbiani⁵ also describe the formation of diverticula from the surface of the nucleus. The first two studied the ova of Ascidians, the latter those of Myriapods. However, the ultimate fate of these diverticula containing nucleolar portions is to become cells of the follicular epithelium, which in Teleostean ova has been formed before those changes began. That the yolk-spherules originate within the egg has been rigorously maintained by some of the greatest authorities, such as Balfour and Gegenbaur, but neither of the two mentions the budding process of the nucleus. Balfour¹ writes on this subject: “Yolk-spherules arise as extremely minute, highly-refracting particles in a stratum of protoplasm some little way below the surface, and are always most numerous at the pole opposite the germinal vesicle.” “It deserves to be specially noted that when the yolk-spherules are first formed, the peripheral layer of the ovum is entirely free from them.” “Two points about the spherules appear clearly to point to their being developed in the protoplasm of the ovum, and not in the follicular epithelium. 1. That they do not make their appearance in the superficial stratum of the ovum. 2. That no yolk-spherules are present in the cells of the follicular epithelium, in which they could not fail to be detected.” Balfour’s opinion is that Gegenbaur’s² account of the formation of the yolk-spherules in birds is probably correct. “Protoplasmic molecules,” says Gegenbaur, grow into larger granules. These become larger again, and are transformed into vesicles, which increase continuously in volume. New solid products now take their origin in the interior, and dissolve into fine granules.” “The yolk-spherules originate in the interior of the egg, and not from the follicular epithelium.” These observations, which were made in birds’, agree with those in reptiles’ eggs.

On the other hand, yolk-spherules appear also sometimes to be formed from without. At any rate,³ Beddard regards it as highly probable. His view, which was deduced from the study of the ovarian ovum of Lepidosiren (Protopterus), is supported, among other facts, by their being no external membrane in this stage. The follicle lies immediately on the yolk, and masses of migrating cells were seen in course of being budded off from it.

To return to the nucleus again; it rapidly degenerates as the egg grows older. Whether the variously shaped particles of the remaining nucleoli, which we have seen in figure 7, subsequently congregate in the centre, in order to become round again, I have not been able to observe. Such a transformation, however, according to Iwakawa,¹ seems to obtain in the young ova of Triton. At any rate the nucleus becomes smaller and smaller, to the benefit of the yolk. In an almost ripe ovum very little of it is left (fig. 12). It then lies excentrically, and consists of clear contents, with the exception of a few round nucleoli. More of the latter are seen just outside the nucleus in the surrounding yolk. Large vacuoles (va.) have also made their appearance in the immediate neighbourhood of the nucleus, but their presence may be due to the fixing reagent. It is important to note that no trace of a membrane can be made out at this stage of the germinal vesicle.

The gradual decomposition of the nucleus in the growing egg has been seen by many authors, and we have descriptions not only from vertebrate, but also from invertebrate ova. Thus Hertwig,² in his researches on the formation of the egg in Toxopneustes lividus, says, “When the ovum becomes ripe, the nucleus undergoes regressive metamorphosis, and is driven by means of protoplasmic contractions to the surface of the yolk. Its membrane dissolves, and its contents break up and are reabsorbed by the yolk. The nucleolus, however, seems to remain unchanged, and appears to travel into the yolk mass, becoming the permanent nucleus of the ripe ovum.”

With regard to the question as to the presence or absence of a membrane in the nucleus, I could see a double contour distinctly in the section represented by figure 7. I believe it is now generally recognised that the nucleus is surrounded by a membrane, and most of the references I can find support this fact. Thus Hertwig, in the paper quoted above, speaks of the nuclear membrane as being of a distinct double contour and well defined from the surrounding part, as well as from the contents of the nucleus. I could cite many other authorities, but it would lead me too far.

At the recent meeting of the British Association at Birmingham, I submitted a paper on the egg membranes of osseous fishes. I pointed out that, in a section of the gurnard’s egg, I had seen a thin membrane internally to the egg capsule or “zona radiata,” and that it had been already noticed by Ransom and other observers.

I think the egg-membranes constitute that part of the Teleostean ovum on which most has been written. I shall attempt to throw some light on the discrepancies which we find among zoologists in regard to these membranes. All zoologists agree as to the existence of one more or less thick layer, which most of them believe to be pierced by minute pores. Now I find that this layer has no less than seven different names, as will be seen by the subjoined list:

1. Zona radiata.—Balfour, Beneden, Brook, McIntosh, Ovsiannikov, Reichert, Solger.

2. Vitell. membr.—Aubert, Beddard (?), Cunningham, Haeckel, Kölliker, Waldeyer.

3. Egg-capsule.—His, Müller, Prince.

4. Yolk-sac.—Ransom.

5. Zona pellucida.—Eimer.

6. Chorion.—Leuckart, Rathke.

7. Egg-shell.—Oellacher, Vogt.

Leaving out the follicular layer, or “granulosa” as it has been called especially by continental investigators, we have besides the above-mentioned membrane, descriptions of one or two or even three others. As I am here only dealing with the intra-ovarian egg of a few marine forms, some of which were not even ripe enough to possess egg-membranes, my observations will necessarily be somewhat incomplete in this respect. I will commence with a description of the “zona radiata,” following Balfour and others in adopting this name for the principal membrane. As it is probably in all cases pierced by radiating pores, the term “zona radiata” indicates its most prominent morphological character. The name “vitelline membrane” is misleading, additional membranes having been described, some of which no doubt owe their origin also to the vitellus. I believe one of the principal causes of the very great divergence of opinion as to the number of membranes, is that in some cases only intra-ovarian and in others ripe ova have been examined. Balfour¹ has shown in his careful researches on the intra-ovarian egg of Elasmobranchs, that in many cases an absorption of part or the whole of the membranes takes place. The disappearance of the “zonoid layer” in the ripe ovum of the gurnard, shows that such an absorption may also occur in Teleosteans. The ova of Lepidosiren, described by Beddard,² afford another example of an imbibition of the membrane taking place. I may mention here incidentally that I am inclined to look upon his zona radiata as the above-mentioned zonoid layer.

In Trigla gurnardus the zona radiata in section does not show a distinct striation, but in the fresh egg it is well visible. Its thickness (figs. 8, 13, 14, 15, z.) averages about 0·008 mm. It is often granular and stains darkly as a rule in carmine and haematoxylin. Internally to it is a much broader layer (figs. 8, 13, 14, 30), which in section appears to be the inner portion of the zona, the stripes being apparently continued through both. The width of the latter is about 0·025 mm. Both layers are striped, i. e. provided with minute radial pores. I was inclined at first to consider these two layers as belonging to one membrane, namely, the “zona radiata.”

However, its semi-fluid condition distinguishes it from the much firmer and elastic zona radiata. Hitherto it has always been looked upon as the outer portion of the yolk, and has been described by Gegenbaur in the ova of birds, reptiles, and Elasmobranchs as the “belle Randschicht,” and by His as the “zonoid layer.” It stains only slightly, and I have found it as a rule devoid of granules or vesicles. In the ripe ova this zonoid layer disappears entirely. I cannot agree with His, according to whom it is replaced by the egg-capsule or zona, as the latter is always formed before the zonoid layer.

Brock¹ thinks that this thick layer is a peculiarity, probably in some relation to the nourishment and growth of the egg.

According to Kupffer,² the membrane surrounding the yolk in the herring’s egg consists of two layers, viz. an inner finely striated and an outer one into which the striæ are not continued. The striæ, he says, might be radial pores, however, he must point out that, as the pores are not found in the external layer, they certainly do not open exteriorly. Eimer³ first noticed the stripes in the zonoid layer, which he believes are due to processes from the follicle. He saw the stripes in Alburnis lucidus, Salmo fario, and Perca fluviatilis, but they only occupied the outer half and are not so delicate as those in the zona. That this layer belongs to the yolk, he says, is seen by the fact that in separating it follows always the latter. My own observations are in direct opposition to this last statement. Eimer also speaks of a membrane externally to the zona. He believes that it originates from the follicular layer and therefore might be looked upon as a chorion.

The same author regards the radial striation in the zona radiata as being due to little rods between which are found pores. He likewise examined the ovum of Reptiles, in which he noticed besides the zona and the external “chorion,” an internal membrane, of which I shall have to speak presently.

Kölliker⁴ says “there is in all fishes” eggs an outer, more resistant, thinner layer externally to the zona which may even preserve the striation in some cases.

Leuckart¹ makes mention of an outer membrane-like boundary of the zona, through which canals are continued, and Aubert² as well as Remak³ speak to the same effect.

A few other observers such as His, Lereboullet,⁴ Solger,⁵ and Ransom,⁶ do not make any reference to a layer externally to the zona, but admitting at the same time the porous structure of the latter; while Haeckel,⁷ speaks of a structureless vitelline membrane only, having delicate dark spots. Ovsian-nikov⁸ again, the most recent writer on the ova of osseous fishes, mentions three layers as occurring in Perca fluviatili s. Prolongations from the follicular cells into the canals of the zona were seen distinctly. No doubt they serve for nourishing the egg. There is a distinct zona radiata externa in Osmerus eperlanus, while in Gasterosteus there is only one layer.

Lindgren,⁹ who has recently examined the structure of the mammalian ovum, found that the zona pellucida, which corresponds to our zona radiata, is pretty often completely homogeneous in ripe eggs. He believes that this is due to different physiological conditions of the egg. Author used the highest powers available.

Johann Müller¹⁰ was to my knowledge the first to identify the punctures on the surface of the zona radiata with pores piercing it. Since then, although we occasionally find statements to the contrary, the great bulk of zoologists maintain that pores exist in all cases. May not the fact that some of the recent observers have not noticed them be due to the peculiar physiological condition of the egg which Lindgren referred to ? Careful investigation of a large number of both intra-and extra-ovarian ova of marine fishes would no doubt help to clear up the matter.

In the zona radiata of the ripe egg of Gadus morrhua (spirit preparations) no trace of a striation could be made out with a magnifying power of about 800 diam. Cunningham,¹ however, has seen the stripes in ova of the same species which were prepared with Perenyi’s fluid, and I firmly believe they exist in all cases. As the same observer has recently pointed out, the pores may occasionally (Myxine glutinosa) be branched.

To return to His’ zonoid layer, I stated my reasons for considering it as one of the egg-membranes, although all previous observers looked upon it merely as the modified external part of the yolk.

The ova of Blennius pholis have one layer only. The zonoid layer is always absent.

In speaking of the zonoid layer, His² says “It is probable that it belongs to the zona radiata, but the detailed history of both formations has still to be done.”

A question which has occupied the attention of many modern observers on this subject is whether the pores of the zona radiata receive processes from the surrounding follicular cells. It seems to have first been suggested by Waldeyer³ that the egg-membranes are secretions from the follicular epithelium, and that the latter sends prolongations through the pores into the interior of the egg. Eimer¹ is quite positive in his statements of having seen prolongations from the follicular cells project into the pores of the zona. This, however, was only observed in the eggs of the ringed snake. Brock² again makes mention of similar processes in the ovum of the barbel and that of Servanus hepatus. Finally, the theory of the egg being nourished by means of prolongations from the follicular cells finds a strong supporter in Lindgren.³ He shows, moreover, that in the egg of the rabbit even follicular cells occasionally travel through the pores of the zona. Some were observed inside it, and others half way out. I myself have not been able to trace any processes from the follicular cells into the ovum, but it seems to me quite probable that such prolongations do exist. At any rate I believe that the ovum receives its raw material as it were through the radial pores from the follicle. It is then assimilated and transformed by the nucleus and the egg nourished in this manner.

Before I proceed to a description of the follicular layer, I must mention another membrane which has been described by some authors, while its existence has been denied by others, I am referring to an extremely delicate membrane covering the yolk internally to the zona radiata. I have seen such a membrane in some cases in the young gurnard’s egg (fig. 13, m. i.), that is to say, only in sections of hardened specimens, and not in the adult egg. It has been first described by Ransom, who calls it the inner yolk-sac,” in distinction to the outer yolk-sac (zona radiata). He also isolated it in Gasterosteus. Oellacher did the same in the trout, after treatment with chloride of gold. Allen Thomson, as well as Kölliker, Eimer. Lereboullet, and Aubert, describe an inner delicate membrane.

Although Ovsiannikov saw this layer in Perca fluviatilis, he found no trace of it in Lota vulgaris, and comes to the conclusion that it is an artificial product.

His, Waldeyer, Brock, and others, deny the existence of an inner membrane.

In theory, the presence of such a membrane would explain much which seems at present very difficult. If it really existed, we would naturally regard the zona as being secreted from it. It would also place the pores of the former into the same category as those occurring in cuticular formations, with which they indeed have much resemblance.

The follicular layer, or granulosa, surrounds the egg-membranes which I have just mentioned. In the next paragraph I shall speak about its development; hence I need only describe its appearance in the ripe intra-ovarian egg.

In the gurnard’s egg it consists of a layer of closely-set cells, which has an average thickness of 0·006 mm. Seen from above, the cells present hexagonal outlines with a central nucleus (fig. 16). The egg lies in a stroma of connective tissue.

In the shanny’s egg (Blennius pholis) the follicle is peculiarly modified (fig. 15,f.). The depth of the cells, which in one half of the egg is only about 0·007 mm., gradually increases until it reaches 0·032 mm. at the opposite side. The cells at that side become drawn out and taper towards the surface of the egg. The space between the cells is filled up with interstitial substance (i. s.). Another feature about the follicle in this case is that it touches the zona in all parts, except in a circular portion (c.p.), where it is not in immediate contact with it. This space is filled with an apparently viscid substance, which no doubt is secreted by the follicular cells.

Similar peculiar modifications of the granulosa have been observed by Eimer and Brock. McLeod¹ likewise mentions a granulosa composed of elongated cells as occurring in Gobius niger.

Although I have never seen prolongations from the follicular cells pass through the zona, as has been described by Eimer and Lindgren, I strongly believe that the follicle forms the most important source for the nourishment of the egg.

Lindgren¹ observed in the mammalian ovum that granulosa cells occasionally travelled into the egg by means of the pores of the zona. Some were seen inside the egg, and others half way out, and prolongations from the follicular cells were frequently traced to the interior. I may mention that nothing of the kind was noticed by me in the eggs of osseous fishes; indeed, the pores of the zona are much too narrow to allow of their being traversed by the follicular cells.

On account of my not being able to obtain all the different ovarian stages, I have made no observations on the origin of the egg. Whether the ovum originates from a simple transformation of an epithelial cell, or whether several unite, as in Elasmobranchs, has not been observed. I believe, however, that probably only one cell is concerned in the formation of the egg. This view is held by Brock² and Kolessnikov.³ The former makes the following statement:—“We often find immediately under the epithelium, and frequently in direct communication with it, numerous masses of cells. These masses are seldom utricular—more often they are round or wedge-shaped. The cells show every gradation from epithelial cells to the smallest ova. The whole process lasts only a very short time.”

Kolessnikov also mentions that the primary eggs are formed by the growth of some epithelial cells, one cell giving rise to one ovum. A division of the ovum, as described by Waldeyer, could not be found by the same author in either Amphibians or Teleosteans.

The smallest ova I saw were those of Gadus æglefinus, which measured only 00·11 mm. in diameter (fig. 1). By far the greater portion of the egg was taken up by the nucleus, and many of the numerous nucleoli were already ranged round the inner surface of the nuclear membrane. A small zone of protoplasm covered the nucleus outside. Apparently there was no cell-membrane. The follicular layer was like-wise absent.

In the next larger size, such as we find in fig. 2, which was taken from the gurnard’s ovary, we find that the protoplasm surrounding this nucleus is divided into a dark and light zone. The explanation of this peculiar feature was given in Chapter II. Among the nucleoli some are frequently of a very large size. In figures 6 and 7 we now notice a follicular layer surrounding the yolk and bulging somewhat into it, but the cells composing it are large and not numerous. In fact at first a few cells cover the whole of the ovum.

I have not arrived at any definite conclusion as to the origin of the follicular layer. There are three possible ways in which it might have arisen:

1. From outside the egg, by an aggregation of epithelial cells round the ovum, as in Elasmobranchs (Balfour).

2. From outside the egg by connective tissue or endothelial cells collecting at the periphery of the ovum. Teleosteans (His, Ovsiannikov). Cephalopoda (Lankester).

3. From inside the egg:

   • From the vitellus as in Ascidians (Sabatier).

   • From the nucleus as in Ascidians (Roule, Fol). Myriapods (Balbiani).. Orthoptera (Will).

Although some of my observations seem to show that the follicular epithelium takes its origin from the interior of the egg, others point the opposite way, and, on the whole, I think it would probably be more correct to assign its origin to the connective tissue. At any rate it is ready formed before an egg-membrane can be discovered. Brock can give no opinion as to the origin of the follicle. According to His the very young ova are often surrounded by a double membrane of endothelium, while there is no trace as yet of a granulosa.

In a young egg (fig. 6), as I mentioned before, a few large flat cells cover its whole surface. As the ovum increases in size they become more numerous and much smaller, until in the ripe eggs they have the appearance as seen in figures 6, 14, 15,f.

I have already spoken of the peculiar modification of the follicular layer in Blennius pholis, and need not refer to it again.

With regard to the origin of the egg-membranes, it was stated that they appear after the follicle. The first membrane is the zona radiata (figs. 8, 13, 14, 15, z). When it is fully developed the inner “zonoid” layer is formed in those eggs in which it is found. In Blennius pholis no zonoid layer ever appears. In the gurnard an inner layer was seen in sections of middle-sized eggs, but in later stages no such layer could be discovered. I have had occasion to refer to this layer in a previous paragraph. I myself have no doubt that the egg-membranes originate from the yolk. Ovsiannikov¹, however, holds that the zona is derived from the follicle; and Cunningham,² although he has not had an opportunity of investigating the subject more closely, comes to a similar conclusion.

According to Eimer an outer layer chorion, of which I saw nothing in the ova which I examined, originates from the follicular cells. He, as well as most other authors who have dealt with this subject, agree in the vitelline origin of the zona radiata and the zonoid layer. In the ripe egg the zonoid layer has disappeared completely.

Before concluding this short note on the intra-ovarian egg of osseous fishes, I must refer to a few points which have not been dealt with. An opening appears in the zona radiata before the extrusion of the ovum from the ovary. This “micropyle,” as it has been called by its discoverer “Doyère,” may represent an enlarged canal of the zona, but I have made no direct observation to prove this assertion. According to Cunningham the micropyle in Myxine glutinosa is formed by the growth of a cellular process from the follicular epithelium towards the vitelline while the vitelline membrane is being formed.

Another point which has still to be made out, is the ultimate fate of the nucleus and the appearance of a “discus proligerus “in the unfertilised egg. I hope, however, to make additional researches at some future period, in order to clear up these matters.

Illustrating Dr. R. Scharff’s paper “On the Intra-ovarian Egg of some Osseous Fishes.”

FIG. 1.—Intra-ovarian ova of Gadus æglefinus. (Zeiss D³.) n′. Nucleoli.

FIG. 2.—Intra-ovarian ovum of Trigla gurnardus. (Zeiss D⁴.) N. Nucleus. n′. Nucleoli.

FIG. 3.—Intra-ovarian ovum of Trigla gurnardus. (Zeiss D⁴.) N. Nucleus, n′ Nucleoli, pr. Dark protoplasmic ring. l.p. Light protoplasm.

FIG. 4.—Nucleus of intra-ovaria novum of Hippoglossides limandoides (Zeiss D⁴.) n′. Nucleoli.

FIG. 5.—Intra-ovarian ovum of Conger vulgaris. (Zeiss D³.) n′. Nucleoli.

FIG. 6.—Intra-ovarian ovum of Gadus virens. (Zeiss D⁴.) ri. Nucleoli. Pr¹ External protoplasmic ring. Pr². Internal protoplasmic ring. f. Follicle. I. p. Light protoplasm.

FIG. 7.—Intra-ovarian ovum of Gadus virens. (Zeiss F³.) Nucleoli. n.f. Nuclear figure. N. m. Nuclear membrane, pr. Protoplasmic ring. sp. Spots (nucleoli ?). f. Follicle.