CHAPTER I.

THE RIPE OVARIAN OVUM.

The ripe ovum is nearly spherical, and, after the removal of its capsule, is found to be unprovided with any form of protecting membrane.

My investigations on the histology of the ripe ovarian ovum have been made with the ova of the Gray Skate (_Raja batis_) only, and owing to a deficiency of material are somewhat imperfect.

The bulk of the ovum is composed of yolk spherules, imbedded in a protoplasmic matrix. Dr Alexander Schultz[70], who has studied with great care the constitution of the yolk, finds, near the centre of the ovum, a kernel of small yolk spherules, which is succeeded by a zone of spherules which gradually increase in size as they approach the surface. But, near the surface, he finds a layer in which they again diminish in size and exhibit numerous transitional forms on the way to molecular yolk granules. These Dr Schultz regards as in a retrogressive condition.

Footnote 70: _Archiv für Micro. Anat._ Vol. XI. 1875.

Another interesting feature about the yolk is the presence in it of a protoplasmic network. Dr Schultz has completely confirmed, and on some points enlarged, my previous observations on this subject[71]. Dr Schultz's confirmation is the more important, since he appears to be unacquainted with my previous investigations. In my paper (_loc. cit._), after giving a description of the network I make the following statement as to its distribution.

"A specimen of this kind is represented in Plate 14, fig. 2, _n.y_, where the meshes of the network are seen to be finer immediately around the nuclei, and coarser in the intervals. The specimen further shews, in the clearest manner, that this network is not divided into areas, each representing a cell and each containing a nucleus. I do not know to what extent this network extends into the yolk. I have never yet seen the limits of it, though it is very common to see the coarsest yolk-granules lying in its meshes. Some of these are shewn in Plate 14, fig. 2, _y.k._" [This edition, p. 65.]

Footnote 71: _Quart. Journ. Micro. Science_, Oct. 1874. [This edition, No. V.]

Dr Schultz, by employing special methods of hardening and cutting sections of the whole egg, has been able to shew that this network extends, in the form of fine radial lines, from the centre to the circumference; and he rightly states, that it exhibits no cell-like structures. I have detected this network extending throughout the whole yolk in young eggs, but have failed to see it with the distinctness which Dr Schultz attributes to it in the ripe ovum. Since it is my intention to enter fully both into the structure and meaning of this network in my account of a later stage, I say no more about it here.

At one pole of the ripe ovum a slight examination demonstrates the presence of a small circular spot, sharply distinguished from the remainder of the yolk by its lighter colour. Around this spot is an area which is also of a lighter colour than the yolk, and the outer border of which gradually shades into the normal tint of the yolk. If a section be made through this part (vide Pl. 6, fig. 1) the circular spot will be found to be the germinal vesicle, and the area around it a disc of yolk containing smaller spherules than the surrounding parts. The germinal vesicle possessed the same structure in both the ripe eggs examined by me; and, in both, it was situated quite on the external surface of the yolk.

In one of my specimens it was flat above, but convex below; in the other and, on the whole, the better preserved of the two, it had the somewhat quadrangular but rather irregular section represented in Pl. 6, fig. 1. It consisted of a thickish membrane and its primitive contents. The membrane surrounded the upper part of the contents and exhibited numerous folds and creases (vide fig. 1). As it extended downwards it became thinner, and completely disappeared at some little distance from the lower end of the contents. These, therefore, rested below on the yolk. At its circumference the membrane of the disc was produced into a kind of fold, forming a rim which rested on the surface of the yolk.

In neither of my specimens is the cavity in the upper part of the membrane filled by the contents; and the upper part of the membrane is so folded and creased that sections through almost any portion of it pass through the folds. The regularity of the surface of the yolk is not broken by the germinal vesicle, and the yolk around exhibits not the slightest signs of displacement. In the germinal vesicle figured the contents are somewhat irregular in shape; but in my other specimen they form a regular mass concave above and convex below. In both cases they rest on the yolk, and the floor of the yolk is exactly moulded to suit the surface of the contents of the germinal vesicle. The contents have a granular aspect, but differ in constitution from the surrounding yolk. Each germinal vesicle measured about one-fiftieth of an inch in diameter.

It does not appear to me possible to suppose that the peculiar appearances which I have drawn and described are to be looked upon as artificial products either of the chromic acid, in which the ova were hardened, or of the instrument with which sections of them were made. It is hardly conceivable that chromic acid could cause a rupture of the membrane and the ejection of the contents of the vesicle. At the same time the uniformity of the appearances in the different sections, the regularity of the whole outline of the egg, and the absence of any signs of disturbance in the yolk, render it impossible to believe that the structures described are due to faults of manipulation during or before the cutting of the sections.

We can only therefore conclude that they represent the real state of the germinal vesicle at this period. No doubt they alone do not supply a sufficient basis for any firm conclusions as to the fate of the germinal vesicle. Still, if they cannot sustain, they unquestionably support certain views. The natural interpretation of them is that the membrane of the germinal vesicle is in the act of commencing to atrophy, preparatory to being extruded from the egg, while the contents of the germinal vesicle are about to be absorbed.

In favour of the extrusion of the membrane rather than its absorption are the following features:

(1) The thickness of its upper surface. (2) The extension of its edge over the yolk. (3) Its position external to the yolk.

In favour of the view that the contents will be left behind and absorbed when the membrane is pushed out, are the following features of my sections:

(1) The rupture of the membrane of the germinal vesicle on its lower surface. (2) The position of the contents almost completely below the membrane of the vesicle and surrounded by yolk.

In connection with this subject, Oellacher's valuable observations upon the behaviour of the germinal vesicle in Osseous Fishes and in Birds at once suggest themselves[72]. Oellacher sums up his results upon the behaviour of the germinal vesicle in Osseous Fishes in the following way (p. 12):

"The germinal vesicle of the Trout's egg, at a period when the egg is very nearly ripe, lies near the surface of the germinal disc which is aggregated together in a hollow of the yolk.... After this a hole appears in the membrane of the germinal vesicle, which opens into the space between the egg-membrane and the germinal disc. The hole widens more and more, and the membrane frees itself little by little from the contents of the germinal vesicle, which remain behind in the form of a ball on the floor of the cavity formed in this way. The cavity becomes flatter and flatter and the contents are pushed up further and further from the germinal disc. When the hollow, in which lie the contents of the original germinal vesicle, completely vanishes, the covering membrane becomes inverted ... and the membrane is spread out on the convex surface of the germinal disc as a circular, investing structure. It is clear that by the removal of the membrane the contents of the germinal vesicle become lost."

Footnote 72: _Archiv für Micr. Anat._ Vol. VIII. p. 1.

These very definite statements of Oellacher tell strongly against my interpretation of the appearance presented by the germinal vesicle of the ripe Skate's egg. Oellacher's account is so precise, and his drawings so fully bear out his interpretations, that it is very difficult to see where any error can have crept in.

On the other hand, with the exception of those which Oellacher has made, there cannot be said to be any satisfactory observations demonstrating the extrusion of the germinal vesicle from the ovum. Oellacher has observed this definitely for the Trout, but his observations upon the same point in the Bird would quite as well bear the interpretation that the membrane alone became pushed out, as that this occurred to the germinal vesicle, contents and all.

While, then, there are on the one hand Oellacher's observations on a single animal, hitherto unconfirmed, there are on the other very definite observations tending to shew that the germinal vesicle has in many cases an altogether different fate. Götte[73], not to mention other observers before him, has in the case of Batrachian's eggs traced out with great precision the gradual atrophy of the germinal vesicle, and its final absorption into the matter of the ovum.

Footnote 73: _Entwicklungsgeschichte der Unke._

Götte distinguishes three stages in the degeneration of the germinal vesicle of Bombinator's egg. In the first stage the germinal vesicle has begun to travel up towards the surface of the egg. It retains nearly its primitive condition, but its contents have become more opaque and have partly withdrawn themselves from the thin membrane. The germinal spots are still circular, but in some cases have increased in size. The most important feature of this stage is the smaller size of the germinal vesicle than that of the cavity of the yolk in which it lies, a condition which appears to demonstrate the commencing atrophy of the vesicle.

In the next stage the cavity containing the germinal vesicle has vanished without leaving a trace. The germinal vesicle itself has assumed a lens-like form, and its borders are irregular and pressed in here and there by yolk. Of the membrane of the germinal vesicle, and of the germinal spots, only scanty remnants are to be seen, many of which lie in the immediately adjoining yolk.

In the last stage no further trace of a distinct germinal vesicle is present. In its place is a mass of very finely granular matter, which is without a distinct border and graduates into the surrounding yolk and is to be looked on as a remnant of the germinal vesicle.

This careful investigation of Götte proves beyond a doubt that in Batrachians neither the membrane, nor the contents of the germinal vesicle, are extruded from the egg.

In Mammalia, Van Beneden[74] finds that the germinal vesicle becomes invisible, though he does not consider that it absolutely ceases to exist. He has not traced the steps of the process with the same care as Götte, but it is difficult to believe that an extrusion of the vesicle in the way described by Oellacher would have escaped his notice.

Footnote 74: _Recherches sur la Composition et la Signification de l'OEuf._

Passing from Vertebrates to Invertebrates, we find that almost every careful investigator has observed the disappearance, apparent or otherwise, of the germinal vesicle, but that very few have watched with care the steps of the process.

The so-called Richtungskörper has been supposed to be the extruded remnant of the germinal vesicle. This view has been especially adopted and supported by Oellacher (_loc. cit._), and Flemming[75].

Footnote 75: "Studien in der Entwicklungsgeschichte der Najaden," _Sitz. d. k. Akad. Wien_, Bd. LXXI. 1875.

The latter author regards the constant presence of this body, and the facility with which it can be stained, as proofs of its connection with the germinal vesicle, which has, however, according to his observations, disappeared before the appearance of the Richtungskörper.

Kleinenberg[76], to whom we are indebted for the most precise observations we possess on the disappearance of the germinal vesicle, gives the following account of it, pp. 41 and 42.

"We left the germinal vesicle as a vesicle with a distinct doubly contoured membrane, and equally distributed granular contents, in which the germinal spot had appeared.... The germinal vesicle reaches 0.06 mm. in diameter, and at the same time its contents undergo a separation. The greater part withdraws itself from the membrane and collects as a dense mass around the germinal spot, while closely adjoining the membrane there remains only a very thin but unbroken lining of the plasmoid material. The intermediate space is filled with a clear fluid, but the layer which lines the membrane retains its connection with the mass around the germinal vesicle by means of numerous fine threads which traverse the space filled with fluid.... At about the time when the formation of the pseudocells in the egg is completed the germinal spot undergoes a retrogressive metamorphosis, it loses its circular outline and it now appears as if coagulated; then it breaks up into small fragments, and I am fairly confident that these become dissolved. The germinal vesicle ... becomes, on the egg assuming a spherical form, drawn into an eccentric position towards the pole of the egg directed outwards, where it lies close to the surface and only covered by a very thin layer of plasma. In this situation its degeneration now begins, and ends in its complete disappearance. The granular contents become more and more fluid; at the same time part of them pass out through the membrane. This, which so far was firmly stretched, next collapses to a somewhat egg-like sac, whose wall is thickened and in places folded.

"The inner mass which up to this time has remained compact now breaks up into separate highly refractive bodies, of spherical or angular form and of very different sizes; between them, here and there, are scattered drops of a fluid fat.... I am very much inclined to regard the solid bodies in question as fat or as that peculiar modification of albuminoid bodies which we recognise as the certain forerunner of the formation of fat in so many pathologically altered tissues; and therefore to refer the disappearance of the germinal vesicle to a fatty degeneration. On one occasion I believe that I observed an opening in the membrane at this stage; if this is a normal condition it would be possible to believe that its solid contents passed out and were taken up in the surrounding plasma. What becomes of the membrane I am unable to say; in any case the germinal vesicle has vanished to the very last trace before impregnation occurs."

Footnote 76: _Hydra._ Leipzig, 1872.

Kleinenberg clearly finds that the germinal vesicle disappears completely before the appearance of the Richtungskörper, in which he states a pseudocell or yolk-sphere is usually found.

The connection between the Richtungskörper and the germinal vesicle is not a result of strict observation, and there can be no question that the evidence in the case of invertebrates tends to prove that the germinal vesicle in no case disappears owing to its extrusion from the egg, but that if part of it is extruded from the egg as Richtungskörper this occurs when its constituents can no longer be distinguished from the remainder of the yolk. This is clearly the case in Hydra, where, as stated above, one of the pseudocells or yolk-spheres is usually found imbedded in the Richtungskörper.

My observations on the Skate tend to shew that, in its case, the membrane of the germinal vesicle is extruded from the egg, though they do not certainly prove this. That conclusion is however supported by the observations of Schenk[77]. He found in the impregnated, but not yet segmented, germinal disc a cavity which, as he suggests, might well have been occupied by the germinal vesicle. It is not unreasonable to suppose that the membrane, being composed of formed matter and able only to take a passive share in vital functions, could, without thereby influencing the constitution of the ovum, be ejected.

Footnote 77: "Die Eier von Raja quadrimaculata," _Sitz. der k. Akad. Wien_, Bd. LXVIII.

If we suppose, and this is not contradicted by observation, that the Richtungskörper is either only the metamorphosed membrane of the germinal vesicle with parts of the yolk, or part of the yolk alone, and assume that in Oellacher's observations only the membrane and not the contents were extruded from the egg, it would be possible to frame a consistent account of the behaviour of the germinal vesicle throughout the animal kingdom, which may be stated in the following way.

The germinal vesicle usually before, but sometimes immediately after impregnation undergoes atrophy and its _contents_ become indistinguishable from the remainder of the egg. In those cases in which its membrane is very thick and resistent, _e.g._ Osseous and Elasmobranch Fishes, Birds, etc., this may be incapable of complete resorption, and be extruded bodily from the egg. In the case of most ova, it is completely absorbed, though at a subsequent period it may be extruded from the egg as the Richtungskörper. In all cases the contents of the germinal vesicle remain in the ovum.

In some cases the germinal vesicle is stated to persist and to undergo division during the process of segmentation; but the observations on this point stand in need of confirmation.

My investigations shew that the germinal vesicle atrophies in the Skate before impregnation, and in this respect accord with very many recent observations. Of these the following may be mentioned.

(1) Oellacher (Bird, Osseous Fish). (2) Götte (Bombinator igneus). (3) Kupffer (Ascidia canina). (4) Strasburger (Phallusia mamillata). (5) Kleinenberg (Hydra). (6) Metschnikoff (Geryonia, Polyzenia leucostyla, Epibulia aurantiaca, and other Hydrozoa).

This list is sufficient to shew that the disappearance of the germinal vesicle before impregnation is very common, and I am unacquainted with any observations tending to shew that its disappearance is due to impregnation.

In some cases, _e.g._ Asterocanthion[78], the germinal vesicle vanishes after the spermatozoa have begun to surround the egg; but I do not know that its disappearance in these cases has been shewn to be due to impregnation. To do so it would be necessary to prove that in ripe eggs let loose from the ovary, but not fertilized, the germinal vesicle did not undergo the same changes as in the case of fertilized eggs; and this, as far as I know, has not been done. After the disappearance of the germinal vesicle, and before the first act of division, a fresh nucleus frequently appears [--vide--Auerbach (Ascaris nigrovenosa), Fol (Geryonia), Kupffer (Ascidia canina), Strasburger (Phallusia mamillata), Flemming (Anodon), Götte (Bombinator igneus)], which is generally stated to vanish before the appearance of the first furrow; but in some cases (Kupffer and Götte, and as studied with especial care, Strasburger) it is stated to divide. Upon the second nucleus, or upon its relation to the germinal vesicle, I have no observations; but it appears to me of great importance to determine whether this fresh nucleus arises absolutely de novo, or is formed out of the matter of the germinal vesicle.

Footnote 78: Agassiz, _Embryology of the Star-Fish_.

The germinal vesicle is situated in a bed of finely divided yolk-particles. These graduate insensibly into the coarser yolk-spherules around them, though the band of passage between the coarse and the finer yolk-particles is rather narrow. The mass of fine yolk-granules may be called the germinal disc. It is not to be looked upon as diverging in any essential particular from the remainder of the yolk, for the difference between the two is one of degree only. It contains in fact a larger bulk of active protoplasm, as compared with yolk-granules, than does the remainder of the ovum. The existence of this agreement in kind has been already strongly insisted on in my preliminary paper; and Schultz (_loc. cit._) has arrived at an entirely similar conclusion, from his own independent observations.

One interesting feature about the germinal disc at this period is its size.

My observations upon it have been made with the eggs of the Skate (Raja) alone; but I think that it is not probable that its size in the Skate is greater than in Scyllium or Pristiurus. If its size is the same in all these genera, then the germinal disc of the unimpregnated ovum is very much greater than that portion of the ovum which undergoes segmentation, and which is usually spoken of as the germinal disc in impregnated ova.

I have no further observation on the ripe ovarian ovum; and my next observations concern an ovum in which two furrows have already appeared.