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.
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,ny, 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.]
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 (videPl.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 inPl.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 (videfig. 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 wasproduced 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.”
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.
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 anextrusion of the vesicle in the way described by Oellacher would have escaped his notice.
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-calledRichtungskörperhas 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].
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 theRichtungskörper.
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 0.06mm.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.”
Kleinenberg clearly finds that the germinal vesicle disappears completely before the appearance of theRichtungskörper, in which he states a pseudocell or yolk-sphere is usually found.
The connection between theRichtungskörperand 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 asRichtungskörperthis 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 theRichtungskörper.
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.
If we suppose, and this is not contradicted by observation, that theRichtungskörperis 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 observationsonly 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 itscontentsbecome 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 Iknow, 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.
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.
[70]Archiv für Micro. Anat.Vol.XI.1875.
[71]Quart. Journ. Micro.Science,Oct.1874. [This edition,No.V.]
[72]Archiv für Micr. Anat.Vol.VIII.p. 1.
[73]Entwicklungsgeschichte der Unke.
[74]Recherches sur la Composition et la Signification de l'Œuf.
[75]“Studien in der Entwicklungsgeschichte der Najaden,”Sitz. d. k. Akad. Wien, Bd.LXXI.1875.
[76]Hydra.Leipzig, 1872.
[77]“Die Eier von Raja quadrimaculata,”Sitz. der k. Akad. Wien, Bd.LXVIII.
[78]Agassiz,Embryology of the Star-Fish.
I have not been fortunate enough to obtain an absolutely complete series of eggs during segmentation.
In the cases of Pristiurus and Scyllium only have I had any considerable number of eggs in this condition, though one or two eggs of Raja in which the process was not completed have come into my hands.
In the youngest impregnated Pristiurus eggs, which I have obtained, the germinal disc was already divided into four segments.
The external appearance of the blastoderm, which remains nearly constant during segmentation, has been already well described by Leydig[79].
The yolk has a pale greenish tinge which, on exposure to the air, acquires a yellower hue. The true germinal disc appears as a circular spot of a bright orange colour, and is, according to Leydig's measurements, 1½m. in diameter. Its colour renders it very conspicuous, a feature which is further increased by its being surrounded by a narrow dark line (Pl.6, fig. 2), the indication of a shallow groove. Surrounding this line is a concentric space which is lighter in colour than the remainder of the yolk, but whose outer border passes by insensible gradations into the yolk. As was mentioned in my preliminary paper (loc. cit.), and as Leydig (loc. cit.) had before noticed, the germinal disc is always situated at the pole of the yolk which is near the rounded end of the Pristiurus egg. It occupies a corresponding position in the eggs of both species of Scyllium (stellare and canicula) near the narrower end of the egg to which the shorter pair of strings is attached. The germinal disc in the youngest eggexamined, exhibited two furrows which crossed each other at right angles in the centre of the disc, but neither of which reached its edge. These furrows accordingly divided the disc into four segments, completely separated from each other at the centre of the disc, but united near its circumference.
I made sections, though not very satisfactorily, of this germinal disc. The sections shewed that the disc was composed of a protoplasmic basis, in which were imbedded innumerable minute spherical yolk-globules so closely packed as to constitute nearly the whole mass of the germinal disc.
In passing from the coarsest yolk-spheres to the fine spherules of the germinal disc, three bands of different-sized yolk-particles have to be traversed. These bands graduate into one another and are without sharp lines of demarcation. The outer of the three is composed of the largest-sized yolk-spherules which constitute the greater part of the ovum. The middle band forms a concentric layer around the germinal disc, and is composed of yolk-spheres considerably smaller than those outside it. Where it cuts the surface it forms the zone of lighter colour immediately surrounding the germinal disc. The innermost band is formed by the germinal disc itself and is composed of spherules of the smallest size. These features are shewn inPl.6, fig. 6, which is the section of a germinal disc with twenty-one segments; in it however the outermost band of spherules is not present.
From this description it is clear, as has already been mentioned in the description of the ripe unimpregnated ovum, that the germinal disc is not to be looked upon as a body entirely distinct from the remainder of the ovum, but merely as a part of the ovum in which the protoplasm is more concentrated and the yolk-spherules smaller than elsewhere. Sections shew that the furrows visible on the surface end below, as indeed they do on the surface, before they reach the external limit of the finely granular matter of the germinal disc. There are therefore at this stage no distinct segments: the otherwise intact germinal disc is merely grooved by two furrows.
I failed to observe any nuclei in the germinal disc just described, but it by no means follows that they were not present.
In the next youngest of the eggs[80]examined the germinal disc was already divided into twenty-one segments. When viewed from the surface (Pl.6, fig. 3), the segments appeared divided into two distinct groups—an inner group of eleven smaller segments, and an outer group of segments surrounding the former. The segments of both the inner and the outer group were very irregular in shape and varied considerably in size. The amount of irregularity is far from constant and many germinal discs are more regular than the one figured.
In this case the situation of the germinal disc and its relations to the yolk were precisely the same as in the earlier stage.
In sections of this germinal disc (Pl.6, fig. 6), the groove which separates it from the yolk is well marked on one side, but hardly visible at the other extremity of the section.
Passing from the external features of this stage to those which are displayed by sections, the striking point to be noticed is the persisting continuity of the segments, marked out on the surface, with the floor of the germinal disc.
The furrows which are visible on the surface merely form a pattern, but do not isolate a series of distinct segments. They do not even extend to the limit of the finely granular matter of the germinal disc.
The section represented,Pl.6, fig. 6, bears out the statements about the segments as seen on the surface. There are three smaller segments in the middle of the section, and two larger at the two ends. These latter are continuous with the coarser yolk-spheres surrounding the germinal disc and are not separated from them by a segmentation furrow.
In a slightly older embryo than the one figured I met with a few completely isolated segments at the surface. These segments were formed by the apparent bifurcation of furrows as they neared the surface of the germinal disc. The segments thus produced are triangular in form. They probably owe their origin to the meeting of two oblique furrows. The last-formed of these furrows apparently ceases to be prolonged after meeting the first-formed furrow. I have not in any caseobserved an example of two furrows crossing one another at this stage.
The furrows themselves for the most part are by no means simple slits with parallel sides. They exhibit a beaded structure, shewn imperfectly inPl.6, fig. 6, but better inPl.6, fig. 6a, which is executed on a larger scale. They present intervals of dilatations where the protoplasms of the segments on the two sides of the furrow are widely separated, alternating with intervals where the protoplasms of the two segments are almost in contact and are only separated from one another by a very narrow space.
A closer study of the germinal disc at this period shews that the cavities which cause the beaded structure of the furrows are not only present along the lines of the furrows but are also found scattered generally through the germinal disc, though far more thickly in the neighbourhood of the furrows. Their appearance is that of vacuoles, and with these they are probably to be compared. There can be little question that in the living germinal disc they are filled with fluid. In some cases, they are collected in very large numbers in the region of a furrow. Such a case as this is shewn inPl.6, fig. 6b. In numerous other cases they occur, roughly speaking, alternately on each side of a furrow. Some furrows, though not many, are entirely destitute of these structures. The character of their distribution renders it impossible to overlook the fact that these vacuole-like bodies have important relations with the formation of the segmentation furrows.
Lining the two sides of the segmentation furrows there is present in sections a layer which stains deeply with colouring reagents; and the surface of the blastoderm is stained in the same manner. In neither case is it permissible to suppose that any membrane-like structure is present. In many cases a similar very delicate, but deeply-stained line, invests the vacuolar cavities, but the fluid filling these remains quite unstained. When distinct segments are formed, each of these is surrounded by a similarly stained line.
The yolk-spherules are so numerous, and render even the thinnest section so opaque, that I have failed to make satisfactory observations on the behaviour of the nucleus. I findnuclei in many of the segments, though it is very difficult even to see them, and only in very favourable specimens can their structure be studied. In some cases, two of them lie one on each side of a furrow; and in one case at the extreme end of a furrow I could see two peculiar aggregations of yolk-spherules united by a band through which the furrow, had it been continued, would have passed. The connection (if any exists) between this appearance and the formation of the fresh nuclei in the segments, I have been unable to elucidate.
The peculiar appearances attending the formation of fresh nuclei in connection with cell-division, which have recently been described by so many observers, have hitherto escaped my observation at this stage of the segmentation, though I shall describe them in a later stage. A nucleus of this stage is shewn onPl.6, fig. 6c. It is lobate in form and is divided by lines into areas in each of which a deeply-stained granule is situated.
The succeeding stages of segmentation present from the surface no fresh features of great interest. The somewhat irregular (Pl.6, figs. 4 and 5) circular line, which divides the peripheral larger from the central smaller segments, remains for a long time conspicuous. It appears to be the representative of the horizontal furrow which, in the Batrachian ovum, separates the smaller pigmented spheres from the larger spheres of the lower pole of the egg.
As the segments become smaller and smaller, the distinction between the peripheral and the central segments becomes less and less marked; but it has not disappeared by the time that the segments become too small to be seen with the simple lens. When the spheres become smaller than in the germinal disc represented onPl.6, fig. 5, the features of segmentation can be more easily and more satisfactorily studied by means of sections.
To the features presented in sections, both of the latter and of the earlier blastoderms, I now return. A section of one of the earlier germinal discs, of about the age of the one represented onPl.6, fig. 4, is shewn inPl.6, fig. 7.
It is clear at a glance that we are now dealing with true segments completely circumscribed on all sides. The peripheralsegments are, as a rule, larger than the more central ones, though in this respect there is considerable irregularity. The segments are becoming smaller by repeated division; but, in addition to this mode of increase, there is now going on outside the germinal disc a segmentation of the yolk, by which fresh segments are being formed from the yolk and added to those which already exist in the germinal disc. One or two such segments are seen in the act of being formed (Pl.6, fig. 7,f); and it is to be noticed that the furrows which will eventually mark out the segments, do so at first in a partial manner only, and do not circumscribe the whole circumference of the segment in the act of being formed. These fresh furrows are thus repetitions on a small scale of the earliest segmentation furrows.
It deserves to be noticed that the portion of the germinal disc which has already undergone segmentation, is still surrounded by a broad band of small-sized yolk-spherules. It appears to me probable that owing to changes taking place in the spherules of the yolk, which result in the formation of fresh spherules of a small size, this band undergoes a continuous renovation.
The uppermost row of segmentation spheres is now commencing to be distinguished from the remainder as a separate layer which becomes progressively more distinct as segmentation proceeds.
The largest segments in this section measure about the 1/100th of an inch in diameter, and the smallest about 1/300th of an inch.
The nuclei at this stage present points of rather a special interest. In the first place, though visible in many, and certainly present in all the segments[81], they are not confined to these: they are also to be seen, in small numbers, in the band of fine spherules which surrounds the already segmented part of the germinal disc. Those found outside the germinal disc are not confined to the spots where fresh segments are appearing,but are also to be seen in places where there are no traces of fresh segments.
This fact, especially when taken in connection with the formation of fresh segments outside the germinal disc and with other facts which I shall mention hereafter, is of great morphological interest as bearing upon the nature and homologies of the food-yolk. It also throws light upon the behaviour and mode of increase of the nuclei. All the nuclei, both those of the segments and those of the yolk, have the peculiar structure I described in the last stage.
In specimens of this stage I have been able to observe certain points which have an important bearing upon the behaviour of the nucleus during cell-division.
Three figures, illustrating the behaviour of the nucleus, as I have seen it in sections of blastoderms hardened in chromic acid, are shewn inPl.6, figs. 7a, 7band 7c.
In the place of the nucleus is to be seen a sharply defined figure (Fig. 7a) stained in the same way as the nucleus or more deeply. It has the shape of two cones placed base to base. From the apex of each cone there diverge towards the base a series of excessively fine striæ. At the junction between the two cones is an irregular linear series of small deeply stained granules which form an apparent break between the two. The line of this break is continued very indistinctly beyond the edge of the figure on each side.
From the apex of each cone there diverge outwards into the protoplasm of the cell a series of indistinct markings. They are rendered obscure by the presence of yolk-spherules, which completely surround the body just described, but which are not arranged with any reference to these markings. These latter striæ, diverging from the apex of the cone, are more distinctly seen when the apex points to the observer (Fig. 7b), than when a side of the cone is in view.
The striæ diverging outwards from the apices of the cones must be carefully distinguished from the striæ of the cones themselves. The cones are bodies quite as distinctly differentiated from the protoplasm of the cell as nuclei, while the striæ which diverge from their apices are merely structures in the general protoplasm of the cell.
In some cells, which contain these bodies, no trace of a commencing line of division is visible. In other cases (Fig. 7c), such a line of division does appear and passes through the junction of the two cones. In one case of this kind I fancied I could see (and have represented) a coloured circular body in each cone. I do not feel any confidence that these two bodies are constantly present; and even where visible they are very indistinct.
Instead of an ordinary nucleus a very indistinctly marked vesicular body sometimes appears in a segment; but whether it is to be looked on as a nucleus not satisfactorily stained, or as a nucleus in the act of being formed, I cannot decide.
With reference to the situation of the cone-like bodies I have described I have made an observation which appears to me to be of some interest. I find that bodies of this kind are found in the yolkcompletely outsidethe germinal disc. I have made this observation, in at least two cases which admitted of no doubt (videFig. 7,nx´).
We have therefore the remarkable fact, that whatever connection these bodies may have with cell-division, they can occur in cases where this is altogether out of the question and where an increase in the number of nuclei can be their only product.
These are the main facts which I have been able to determine with reference to the nuclei of this stage; but it will conduce to clearness if I now finish what I have to say upon this subject.
At a still later stage of segmentation the same peculiar bodies are to be seen as during the stage just described, but they are rarer; and, in addition to them, other bodies are to be seen of a character intermediate between ordinary nuclei and the former bodies.
Three such are represented inPl.6, figs. 8a, 8b, 8c. In all of these there can be traced out the two cones, which are however very irregular. The striation of the cones is still present, but is not nearly so clear as it was in the earlier stage.
In addition to this, there are numerous deeply stained granules scattered about the two figures which resemble exactly the granules of typical nuclei.
All these bodies occupy the place of an ordinary nucleus, they stain like an ordinary nucleus and are as sharply defined as an ordinary nucleus.
There is present around some of these, especially those situated in the yolk, the network of lines of the yolk described by me in a preliminary paper[82], and I feel satisfied that there is in some cases an actual connection between the network and the nuclei. This network I shall describe more fully hereafter.
Further points about these figures and the nuclei of this stage I should like to have been able to observe more completely than I have done, but they are so small that with the highest powers I possess (Zeiss, ImmersionNo.2 = 1/15 in.) their complete and satisfactory investigation is not possible.
Most of the true nuclei of the cells of the germinal disc are regularly rounded; those however of the yolk are frequently irregular in shape and often provided with knob-like processes. The gradations are so complete between typical nuclei and bodies like that shewn (Pl.6, fig. 8c) that it is impossible to refuse the name of nucleus to the latter.
In many casestwo nucleiare present in one cell.
In later stages knob-like nuclei of various sizes are scattered in very great numbers in the yolk around the blastoderm (videPl.7). In some cases it appears to me that several of these are in close juxtaposition, as if they had been produced by the division of one primitive nucleus. I do not feel absolutely confident that this is the case, owing to the fact that in the investigation of a knobbed body there is great difficulty in ascertaining that the knobs, which appear separate in one plane, are not in reality united in another.
I have, in spite of careful search, hitherto failed to find amongst these later nuclei cone-like figures, similar to those I found in the yolk during segmentation. This is the more remarkable since in the early stages of segmentation, when very few nuclei are present in the yolk, the cone-like figures are not uncommon; whereas, in the latter stages of development when the nuclei of the yolk are very common and obviously increasing rapidly, such figures are not to be met with.
In no case have I been able to see a distinct membrane round any of the nuclei.
I have hitherto attempted to describe the appearances bearing on the behaviour of the nuclei in as objective a manner as possible.
My observations are not as complete as could be desired; but, taken in conjunction with those of other investigators, they appear to me to point towards certain definite conclusions with reference to the behaviour of the nucleus in cell-division.
The most important of these conclusions may be stated as follows. In the act of cell-division the nuclei of the resulting cells are formed from the nucleus of the primitive cell.
This may occur:—
(1) By the complete solution of the old nucleus within the protoplasm of the mother cell and the subsequent reaggregation of its matter to form the nuclei of the freshly formed daughter cells,
(2) By the simple division of the nucleus,
(3) Or by a process intermediate between these two where part of the old nucleus passes into the general protoplasm and part remains always distinguishable and divides; the fresh nucleus being in this case formed from the divided parts as well as from the dissolved parts of the old nucleus.
Included in this third process it is permissible to suppose that we may have a series of all possible gradations between the extreme processes 1 and 2. If it be admitted, and the evidence we have is certainly in favour of it, that in some cases, both in animal and vegetable cells, the nucleus itself divides during cell division, and in others the nucleus completely vanishes during the cell-division, it is more reasonable to suspect the existence of some connection between the two processes, than to suppose that they are entirely different in kind. Such a connection is given by the hypothesis I have just proposed.
The evidence for this view, derived both from my own observations and those of other investigators, may be put as follows.
The absolute division of the nucleus has been stated to occur in animal cells, but the number of instances where theevidence is quite conclusive are not very numerous. Recently F. E. Schultze[83]appears to have observed it in the case of an Amœba in an altogether satisfactory manner. The instance is quoted by Flemming[84]. Schultze saw the nucleus assume a dumb-bell shape, divide, and the two halves collect themselves together. The whole process occupied a minute and a half and was shortly followed by the division of the Amœba, which occupied eight minutes. Amongst vegetable cells the division of the nucleus seems to be still rarer than with animal cells. Sachs[85]admits the division of the nucleus in the case of the parenchyma cells of certain Dicotyledons (Sambucus, Helianthus, Lysimachia, Polygonum, Silene) on the authority of Hanstein.
The division of the nucleus during cell-division, though seemingly not very common, must therefore be considered as a thoroughly well authenticated occurrence.
The frequent disappearance of the nucleus during cell-division is now so thoroughly recognised, both for animal and vegetable cells, as to require no further mention.
In many cases the partial or complete disappearance of the nucleus is accompanied by the formation of two peculiar star-like figures. Appearances of the kind have been described by Fol[86], Flemming[87], Auerbach[88]and possibly also Oellacher[89]as well as other observers.
These figures[90]are possibly due to the streaming out of theprotoplasm of the nucleus into that of the cell[91]. The appearance of striation may on this hypothesis be explained as due to the presence of granules in the protoplasm. When the streaming out of the protoplasm of a nucleus into that of a cell takes place, any large granule which cannot be moved by the stream will leave behind it a slack area where there is no movement of the fluid. Any granules which are carried into this area will remain there, and by the continuation of a process of this kind a row of granules may be formed, and a series of such rows would produce an appearance of striation. In many cases,e.g.Anodon,videFlemming[92], even the larger yolk-spherules are arranged in this fashion.
On the supposition that the striation of these figures is due to the outflow from the nucleus, the appearances presented in Elasmobranchii admit of the following explanation.
The central body consisting of two cones (figs. 7a, 7c) is almost without question the remnant of the primitive nucleus. This is shewn by its occupying the same position as the primitive nucleus, staining in the same way, and by there being a series of insensible gradations between it and a typical nucleus. The contents must be supposed to be streaming out from the two apices of the cones, as appears from the striæ in the body converging on each side towards the apex, and then diverging again from it. In my specimens the yolk-spherules are not arranged with any reference to the radiating striation.
It is very likely that in the cases of the disappearance of the nucleus, its protoplasm streams out in two directions, towards the two parts of the cell which will eventually become separated from each other; and probably, after the division, the matter of the old nucleus is again collected to form two fresh nuclei.
In some cases of cell-division a remnant of the old nucleus is stated to be visible after the fresh nuclei have appeared. These cases, of which I have not seen full accounts, are perhaps analogous to what occasionally happens with the germinalvesicle of an ovum. The whole of the contents of the germinal vesicle become at its disappearance mingled with the protoplasm of the ovum, but the resistant membrane remains and is eventually ejected from the egg,videp.215et seq. If the remnant of the old nucleus in the cases described is nothing more than its membrane, no difficulty is offered to the view that the constituents of the old nucleus may help to form the new ones.
In many cases the total bulk of the new nuclei is greater than that of the old one; in such instances part of the protoplasm of the cell necessarily has a share in forming the new nuclei.
Although, in instances where the nucleus vanishes, an absolute demonstration of the formation of the fresh nuclei from the matter of the old one is not possible; yet, if cases of the division of the old nucleus to form the new ones be admitted to exist, the derivation in the first process of the fresh nuclei from the old ones must be postulated in order to maintain a continuity between the two processes of formation; and, as I have attempted to shew, all the circumstantial evidence is in favour of it.
Admitting the existence of the two extreme processes of nuclear formation, I wish to shew that my results in Elasmobranchii tend to demonstrate the existence of intermediate steps between them. The first figures I described of two opposed cones, appear to me almost certainly to represent nuclei in the act of dissolution; but though a portion of the nucleus may stream out into the yolk, I think it impossible that the whole of it does[93].
I described these bodies in two states. An earlier one, in which the two cones were separated by an irregular row of deeply stained granules; and a later one in which a furrow had already appeared dividing the cones as well as the cell. In neither of these conditions could I see any signs of the body vanishing completely. It was as clearly defined and as deeply stained as an ordinary nucleus, and in its later condition the signs of the streaming out of material from its pointed extremities were less marked than in the earlier stage.
All these facts, to my mind, point to the view that these cone-like bodies do not disappear, but form the basis for the new nuclei. Possibly the body visible in each cone in the later stage, was the commencement of this new nucleus. Götte[94]has figured structures somewhat similar to these bodies, but I hardly understand either his figure or his account sufficiently clearly to be able to pronounce upon the identity of the two. In case they are identical, Götte gives a very different explanation of them from my own[95].
A second of my results, which points to a series of intermediate steps between division and solution of the nucleus, is the distribution in time of the peculiar cone-like bodies. These are present in fair abundance at an early period of segmentation, when there are but few nuclei either in the blastoderm or the yolk. But at later periods, when there are both more nuclei, especially in the yolk, and they are also increasing in numbers more rapidly than before, no bodies of this kind are to be seen. This fact becomes the more striking from the lobate appearance of the later nuclei of the yolk, an appearance which exactly suits the hypothesis of the rapid budding off of fresh nuclei.
The observations of R. Hertwig[96]on the gemmation ofPodophrya gemmipara, support my interpretation of the knobbed condition of the nuclei. Hertwig finds (p. 47) that
The horse-shoe shaped nucleus grows out into numerous anastomosing projections. Over the free ends of the projections little knobs appear on the surface of the body, into which the lengthening ends of the processes of the nucleus grow up. Here they bend themselves into a horse-shoe form. The newly-formed nucleus then separates from the original nucleus, and afterwards the bud containing it from the body.
From the peculiar arrangement of the net-work of lines of the yolk around these knobbed nuclei, it is reasonable to conclude that interchange of material between the protoplasm ofthe yolk and the nuclei is still taking place, even during the later periods.
These facts about the distribution in time of the cone-like bodies afford a strong presumptive evidence of a change in the manner of nuclear increase.
The last argument I propose urging on this head is derived from the bodies (Pl.6, fig. 8a,b,c) which I have described as intermediate between the true cone-like bodies and typical nuclei. They appear to afford evidence of less and less of the matter of the nucleus streaming out into the yolk and of a large proportion of it becoming divided.
The conclusion to be derived from all these facts is that for Elasmobranchii in the earlier stages of segmentation, and during the formation of fresh segments, a partial solution of the old nucleus takes place, but all its constituents serve for the reconstruction of the fresh nuclei.
In later periods of development a still smaller part of the nucleus becomes dissolved, and the rest divides; but the two fresh nuclei are still derived from the two sources. After the close of segmentation the fresh nuclei are formed by a simple division of the older ones.
The appearance of the cone-like bodies in the yolk outside the germinal disc is a point of some interest. It demonstrates in a conclusive manner that whatever influence (if any) the nucleus may have in ordinary cases of cell division, yet it may undergo changes of a precisely similar character to those which it experiences during cell division, without exerting any influence on the surrounding protoplasm[97]. If the lobate nuclei are also nuclei undergoing division, we have in the egg of an Elasmobranch examples of all the known forms of nuclear increase unaccompanied by cell division.
The next stage in the segmentation does not present so many features of interest as the last one. The segments arenow so small, as to be barely visible from the surface with a simple lens. A section of an embryo of this stage is represented inPl.6, fig. 8. The section, which is drawn on the same scale as the section belonging to the last stage, serves to shew the relative size of the segments in the two cases.
The epiblast is now more distinct than it was. The segments composing it are markedly smaller than the remainder of the cells of the germinal disc, but possess nuclei of an absolutely larger size than do the other cells. They are irregular in shape, with a slight tendency to be columnar. An average segment of this layer measures about 1/700 inch.
The cells of the lower layer are more polygonal than those of the epiblast, and are decidedly larger. An average specimen of the larger cells of the lower layer measures about 1/400in.in diameter, and is therefore considerably smaller than one of the smallest cells of the last stage. The formation of fresh segments from the yolk still continues with fair rapidity, but nearly comes to an end shortly after this.
Of the nuclei of the lower layer cells, there is not much to add to what has already been said. Not infrequently two nuclei may be observed in a single cell.
The nuclei in the yolk which surrounds the germinal disc are more numerous than in the earlier periods, and are now to be met with in fair numbers in every section (fig. 8,n´).
These are the main features which characterise the present stage, they are in all essential points similar to those of the last stage, and the two germinal discs hardly differ except in the size of the segments of which they are composed.
In the last stage which I consider as belonging to the segmentation, the cells of the whole blastoderm have become smaller (Pl.6, fig. 9).
The epiblast (ep) now consists of a very marked layer of columnar cells. It is, as far as I have been able to observe, never more than one cell deep. The cells of the lower layer are of an approximately uniform size, though a few of those at the circumference of the blastoderm considerably exceed the remainder in the bulk.
There are two fresh features of importance in germinal discs of this age.
Instead of being but indistinctly separated from the surrounding yolk, the blastoderm has now very clearly defined limits.
This is an especially marked feature of preparations made with osmic acid. In these there may frequently be seen a deeply stained doubly contoured line, which forms the limit of the yolk, where it surrounds the germinal disc. Lines of this kind are often to be seen on the surface of the yolk, or even of the blastoderm, but are probably to be regarded as products of reagents, rather than as organised structures. The outline of the germinal disc is well rounded, though it is occasionally broken, from the presence of a larger cell in the act of being formed from the yolk.
It is not probable that any great importance is to be attached to the comparative distinctness of the outline of the germinal disc at this stage, which is in a great measure due to a cessation in the formation of fresh cells in the surrounding yolk, and in part to the small and comparatively uniform size of the cells of the germinal disc.
The formation of fresh cells from the yolk nearly comes to an end during this period, but it still continues on a small scale.
The number of the nuclei around the germinal disc has increased.
Another feature of interest which first becomes apparent during this stage is the asymmetry of the germinal disc. If a section were made through the germinal disc, as it layin situin the egg capsule, parallel or nearly so to the long axis of the capsule, one end of the section would be found to be much thicker than the other. There would in fact be a far larger collection of cells at one extremity of the germinal disc than at the other. The end at which this collection of cells is formed points towards the end of the egg capsule opposite to that near which the yolk is situated. This collection of cells is the first trace of the embryo; and with its appearance the segmentation may be supposed to terminate.
The section I have represented, though not quite parallel to the long axis of the egg, is sufficiently nearly so to shew the greater mass of cells at the embryonic end of the germinal disc.
This very early appearance of a distinction in the germinal disc between the extremity at which the embryo appears and the non-embryonic part of the disc, besides its inherent interest, has a further importance from the fact that in Osseous Fishes a similar occurrence takes place. Oellacher[98]and Götte[99]both agree as to the very early period at which a thickening of one extremity of the blastoderm in Osseous Fishes is formed, which serves to indicate the position at which the embryo will appear. There are many details of development in which Osseous Fish and Elasmobranchii agree, which, although if taken individually are without any great importance, yet serve to shew how long even insignificant features in development may be retained.
* * * * *
The segmentation of the Elasmobranch egg presents in most of its features great regularity, and exhibits in its mode of occurrence the closest resemblance to that in other meroblastic vertebrate ova.
There is, nevertheless, one point with reference to which a slight irregularity may be observed. In almost all eggs segmentation commences by, what for convenience may be called, a vertical furrow which is followed by a second vertical furrow at right angles to the first. The third furrow however is a horizontal one, and cuts the other two at right angles. This method of segmentation must be looked on as the normal one, in almost all the important groups of the animal kingdom, both for the so-called holoblastic and meroblastic eggs, and the gradations intermediate between the two. The Frog amongst vertebrates exhibits a most typical instance of this form of segmentation.
In Elasmobranchii the first two furrows are formed in a perfectly normal manner, but though I have not observed the actual formation of the next furrow, yet from the later stages, which I have observed, I conclude that it is parallel to one of the first formed furrows; and it is fairly certain that, not till a considerably later period, is a furrow homologous with the horizontal furrow of the Batrachian egg formed. This furrow appears to be represented in the Elasmobranch segmentationby the irregular circumscription of a body of central smaller spheres from a ring of peripheral larger ones (videPl.6, figs. 3, 4 and 5).
In the Bird the representative of the horizontal furrow appears relatively much earlier. It is formed when there are eight segments marked out on the surface of the germinal disc[100]. From Oellacher's[101]account of the segmentation in the fowl[102]it seems certain, as might be anticipated, that this furrow is nearly parallel to the surface of the disc, so that it cuts the earlier formed vertical furrows and causes the segments of the germinal disc to be completely circumscribed below as well as at the surface. In the Elasmobranch egg this is not the case; so that, even after the smaller central segments have become separated from the outer ring of larger ones, none of the segments of the disc are completely circumscribed, and only appear to be so in surface views (videPl.6, fig. 6). Segmentation in the Elasmobranch egg differs in the following particulars from that in the Bird's egg:
(1) The equivalent of the horizontal furrow of the Batrachian egg appears much later than in the Bird.
(2) When it has appeared it travels inwards much more slowly.
As a result of these differences, the segments of the germinal disc of the Birds' eggs are much earlier circumscribed on all sides than those of the Elasmobranch egg.
As might be expected, the segmentation of the Elasmobranch egg resembles in many points that of Osseous Fishes (videOellacher[103]and Klein[104]). It may be noticed, that with Osseous as with Elasmobranch Fishes, the furrow corresponding with the horizontal furrow of the Amphibian's egg does not appear at as early a period as is normal. The third furrow of an Osseous Fish egg is parallel to one of the first formed pair.
In Oellacher's[105]figures,Pl.23, figs. 19-21, peculiar beadingsof the sides of the earlier formed furrows are distinctly shewn. No mention of these is made in the text, but they are unquestionably similar to those I have described in the Elasmobranch furrows. In the case of Elasmobranchii I pointed out that not only were the sides of the furrow beaded, but that there appeared in the protoplasm, close to the furrows, peculiar vacuole-like cavities, precisely similar to the cavities which were the cause of the beadings of the furrows.
The presence of these seems to shew that the molecular cohesion of the protoplasm becomes, as compared with other parts, much diminished in the region where a furrow is about to appear, so that before the protoplasm finally gives way along a particular line to form a furrow, its cohesion is broken at numerous points in this region, and thus a series of vacuole-like spaces is formed.
If this is the true explanation of the formation of these spaces, their presence gives considerable support to the views of Dr Kleinenberg upon the causes of segmentation, so clearly and precisely stated in his monograph upon Hydra; and is opposed to any view which regards the forces which come into play during segmentation as resident in the nucleus.
I have not observed the peculiar threads of protoplasm which Oellacher[106]describes as crossing the commencing segmentation furrows. I have also failed to discover any signs of a concentration of the yolk-spherules, round one or two centres, in the segmentation spheres, similar to that observed by Oellacher in the segmenting eggs of Osseous Fish. The appearances observed by him are probably connected with the behaviour of the nucleus during segmentation, and are related to the curious bodies I have already described.
With reference to the nuclei which Oellacher[107]has described as occurring in the eggs of Osseous Fish during segmentation, there can, I think, be little doubt that they are identical with the peculiar nuclei in the Elasmobranch eggs.
He[108]says: