Book - Text-Book of the Embryology of Man and Mammals 3
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Hertwig O. Text-book of the embryology of man and mammals. (1892) Translated 1901 by Mark EL. from 3rd German Edition. S. Sonnenschein, London.
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The Process of Cleavage
FERTILISATION is in most instances immediately followed by further development, which begins with the division of the egg-cell the simple elementary organism into an ever-increasing number of small cells the process of cleavage. We shall begin the study of cleavage with a very simple case, and here also choose as a foundation for the presentation of the subject the egg of an Echinoderm and the egg of the common Ascaris of the Horse.
In the living egg of the Echinoderm the cleavage-nucleus (fig. 26 fk}j which arose from the fusion of egg-nucleus and spermatic nucleus, is at first spheroidal, and lies exactly in the middle of the egg, where it forms the centre of a radiation which affects the whole yolk-mass ; but it soon begins to be slightly elongated, and at the same time to become less and less distinct, so that with the living object one might be misled into assuming that it had been completely dissolved. Before this, very regular changes in the distribution and arrangement of the protoplasm around the nucleus have taken place. The monocentric radiation resulting from fertilisation is divided. The two newly formed radiations thereupon move to the poles of the elongated nucleus. At first small and insignificant, they rapidly extend, and finally each occupies a half of the egg (fig. 27), and the rays of the two systems meet at a sharp angle in the median plane of the egg.
Just in proportion as the two radiations become more distinct, there arises, within the granular yolk, as the starting-point and centre of the radiations, a figure, which may be appropriately compared (fig. 27) with a dumb-bell. It arises by the accumulation of a largo amount of homogeneous protoplasm around the poles of the elongating nucleus, forming the two ends of the dumb-bell ; the poles may be regarded as if they were two centres of attraction. The non-granular streak, representing the handle of the dumb-bell. is the nucleus, which has meanwhile undergone a peculiar metamorphosis and has become indistinct.
A more accurate knowledge of the nuclear metamorphosis may be got by employing suitable reagents and dyes. By means of intermediate stages, which may be disregarded here, there arises out of
+++++++++++++++++++++++++++++++++++++++++ Fig. 26.
+++++++++++++++++++++++++++++++++++++++++ Fig. 27.
+++++++++++++++++++++++++++++++++++++++++ Fig. 26. Egg of a Sea-urchin immediately after the conclusion of fertilisation, fk, Cleavagenucleus.
+++++++++++++++++++++++++++++++++++++++++ Fig. 27. Eger of a Sea-urchin in preparation for division. The nucleus is no longer to be seen ; there has arisen in its place a dumb-bell figure. Both figures are drawn from the living object. +++++++++++++++++++++++++++++++++++++++++ the vesicular nucleus the nuclear spindle (fig. 31 .Z?), which is a typical structure for cell-division throughout the organic world. This (sp) consists of two substances, both of which, in my opinion, are derived from the quiescent condition of the nucleus namely, (1) of a non-chromatic substance, which does not show affinity for any dyes, and (2) of the stainable nuclein or cliromatin. The non-chromatic substance forms extraordinarily fine, and therefore at times scarcely discernible, " spindle-fibres" which are united into a bundle, and give rise to a spindle by the convergence of their ends to points. The chromatin, on the contrary, has assumed the form of small individual granules or chromosomes, which correspond in number with the spindle-fibres, and are so arranged that each granule adjoins a spindle-fibre at its middle point. In its totality, therefore, it constitutes at the middle of the spindle a plate composed of individual granules the nuclear plate of STRASBURGER. That which in the case of the Sea-urchin ordinarily appears as a chromatic granule is found, upon the employment of the highest magnifying powers, but especially in the study of objects (fig. 28 A] more suitable for this purpose, to be a small Y-shaped loop. The number of the loops or chromosomes appears to be very definite, and subject to law for each species of animal.
At the tips of the spindle there may be demonstrated, in addition, two special and exceedingly minute bodies, one of which occupies the exact centre of each of the two previously mentioned systems of rays ; they are, in fact, to be regarded as the cause of the
+++++++++++++++++++++++++++++++++++++++++ Fig. 28. Diagram of nuclear division, after RABL.
In figure A one sees the spindle, composed of delicate non-chromatic fibres, with the protoplasmic radiations at its tips and the chromatic loops at its middle. The splitting of the filaments of the latter has already taken place. In figure B the daughter-loops resulting from the fission have moved apart in opposite directions. In figure C'they begin to arrange themselves in a regular manner into two groups of loops. In figure D the. groups of daughter-loops lie near the two poles of the spindle. +++++++++++++++++++++++++++++++++++++++++
latter. Inasmuch as during the elongation of the nucleus they are to be found at each of its two poles, they may be especially designated as polar corpuscles [or centrosomes]. During the whole process of the division of nucleus and cell-body, it appears as though a directing influence belongs to the two polar corpuscles.
Important changes in the nuclear loops of the spindle take place during later stages of the process of division. Each loop is split lengthwise into two daughter-loops (fig. 28 A), as discovered by FLEMMING and as confirmed since then by numerous other investigators (STRASBURGER, HEUSER, VAN BENEDEN, RABL, and others). These daughter-loops soon move apart toward the opposite ends of the spindle (figs. 28 B, C ; see also the explanation of the figures), and approach very closely to the polar corpuscles at their tips (fig. 28 D}, Thus by a complicated process a division of the stainable nuclear substance into similar halves is brought about. As the immediate consequence of this the protoplasmic parts of the cell also begin at this time to be divided into halves by means of the process of cleavage, which is already recognisable externally. There is formed at the surface of the egg (fig. 29 A), in a plane passing between the two groups of loops through the middle of the spindle perpendicular to its long axis, a circular furrow, which rapidly cuts deeper and deeper into the substance of the egg, and in a short time divides it into two equal parts. Each of these contains half of the spindle
+++++++++++++++++++++++++++++++++++++++++ Fig. 29 A. Egg of a Sea-urchin at the moment of division.
A circular furrow cuts into the yolk and halves it in a plane which is perpendicular to the middle of the nuclear axis and to the long axis of the dumb-bell. +++++++++++++++++++++++++++++++++++++++++ B. Egg of a Sea-urchin after its division into two cells.
In each resultant of the division a vesicular daughter-nucleus has arisen. The radial arrange ment of the protoplasm begins to become indistinct. Both figures are drawn from the living object.
with half of the loops, half of the dumb-bell, and a protoplasmic radiation.
The resulting halves of the egg, still surrounded in common by the vitelline membrane, then closely apply to each other the surfaces resulting from the division, and become so flattened that each one of them forms approximately a hemisphere (fig. 29 B}. Internally, however, nucleus and protoplasm enter upon a brief transitory resting stage. There is developed out of the half of the nuclear spindle with its daughter-loops a vesicular homogeneous daughter-nucleus like the first, but in the protoplasm the radial arrangement becomes less and less distinct and at last entirely disappears.
The egg of the common Maw- worm of the Horse is also a very instructive object for the study of the process of cleavage, as it was for the study of fertilisation, for it allows a still deeper insight into this process. As has already been stated, the egg-nucleus and the spermatic nucleus remain for a time separate, even after they have approached each other. After a brief period of rest both of them begin to exhibit simultaneously the changes which precede the formation of the nuclear spindle. In each the chromatic substance is metamorphosed into a fine thread, which is arranged within the nuclear membrane in numerous windings. Each filament is thereupon divided into two equally large coiled loops, the chromosomes (fig. 25 ch). Now the two vesicular nuclei lose their delimitation from the surrounding yolk, in which there arise at a little distance from each other two polar corpuscles [centrosomes], surrounded by a system of rays, which is at first faint, but subsequently becomes more distinct. Between the two centrosomes, the method of whose development no one has as yet succeeded in observing, there are formed spindle-fibres, and the four loops (chromosomes), set free by the dissolution of the two nuclear membranes, so arrange themselves that they lie upon the outside of the spindle at its equator.
In the case of the egg of the Maw- worm, therefore, the union of the two sexual nuclei, which terminates the act of fertilisation, takes place only at the time of the metamorphosis to form the cleavagespindle, in which metamorphosis they take an equal share. In consequence of this remarkable deviation from the ordinary course of the process of fertilisation, VAN BENEDEN has been able to establish the interesting and important fact that half of the chromosomes of the first cleavage-spindle are derived from the egg-nucleus, and half from the spermatic nucleus, and that consequently they may be distinguished as female and male chromosomes. Since in this instance, just as in nuclear division ordinarily, the four loops are split lengthwise and then move apart toward the two polar corpuscles (centrosomes), there are formed two groups of four daughter-loops each, of which two are of male origin and two of female. Each group is then metamorphosed into the quiescent nucleus of the daughter-cell. This furnishes incontestable proof, that to each daughter-nucleus in each half of the egg, lohich arises as the result of the first cleavage, there is transmitted exactly the same amount of chromatic substance from the egg-nucleus as from the spermatic nucleus.
The first division is followed after a brief period of rest by the second, this by the third, the fourth, etc., during which are repeated the same series of changes in nucleus and protoplasm that have just been described. Thus in quick succession the 2 first daughter-cells are divided into 4, these into 8, 16, 32, 64, etc. (fig. 30), until there has resulted a large spheroidal mass, which has received the name morula or vmlberry-spkere, because the cells protrude as small elevations at its surface.
During the second and third stages of cleavage there is easily recognisable a rigidly observed order in the direction which the planes of cleavage sustain to each other. The second plane of cleavage always halves the first and cuts it perpendicularly ; the third plane, again, is perpendicular to the first two, and passes through the middle of the axis formed by their intersection. If one regards the ends of this axis as the poles of the egg, the first two planes of division may be designated as meridional, the third as equatorial.
This uniformity is caused by the mutual relation which subsists between nucleus and protoplasm, in which connection the two following laws are to be noted : (1) The plane of division always cuts the axis of the spindle perpendicularly at its centre. (2) The position of
+++++++++++++++++++++++++++++++++++++++++ Fig. 30. Various stages of the process of cleavage, after GEGENBAUR. +++++++++++++++++++++++++++++++++++++++++ the axis of the nuclear spindle in turn depends on the form and differentiation of the protoplasmic body which envelops it, and in such a manner that the two poles of the nucleus take the direction of the greatest protoplasmic masses. Thus, for example, in a sphere in which the protoplasm is uniformly distributed, the centrally situated spindle may come to lie in any radius ; but in an ovoid protoplasmic body, only in the longest diameter. In a circular protoplasmic disc the nuclear axis lies parallel to its surface in any diameter whatever of the circle, but in an oval disc, as before, in the longest diameter only.
Let us return now, after these general remarks, to the case under consideration. Each daughter-cell forms at the close of the first segmentation a hemisphere. According to the rule, the daughter-spindle cannot assume a position perpendicular to the flat surface of the hemisphere, but must lie parallel to it, so that a division into two quadrants must result. At the next segmentation the axis of the spindle must coincide with the long axis of the quadrant, whereby this becomes divided into two octants.
There are some important deviations from the process of division just described, which, affect the form of the cleavage products, although leaving unaltered the finer processes relating to the nucleus. The deviations are induced, as we shall show more in detail in the individual cases, by the variation in the amount of deutoplasni contained in the eggs, and by the previously described variability in its distribution. One may appropriately separate the various forms of the process of cleavage into two classes, and each class into two subclasses, although the forms merge into one another by means of transitional conditions.
To the first class we assign such eggs as are completely divided into segments by the process of cleavage. The cleavage itself we designate as total ; and according as the segments are of equal or unequal size, we distinguish as subdivisions equal cleavage and unequal cleavage.
With total is contrasted partial cleavage. This occurs in the case of eggs which are provided with very abundant deutoplasm, and are consequently of considerable size, and in which, at the same time, the previously described separation into formative yolk and nutritive yolk has been distinctly established. In this case the formative yolk alone undergoes a process of cleavage, whereas the chief mass of the egg, the nutritive yolk, remains undivided, and in general unaffected, by the processes of embryonic development ; hence the name partial cleavage. This, in turn, is resolvable into the two subtypes of discoidal and superficial cleavage, according as the formative yolk rests as a disc upon the nutritive yolk, or envelops the latter as a thick cortical layer. REMAK has designated eggs with total segmentation as holoblastic, those with partial segmentation us meroblastic.
We may therefore present the following scheme of cleavage : I. TYPE Total cleavage : ^ () Equal cleavage Holoblastic eggs.
(Z>) Unequal cleavage J II. TYPE Partial cleavage : () Discoidal cleavage Meroblastic eggs.
I a - Equal Cleavage.
In the general consideration of the process of cleavage we have already become acquainted with the phenomena of equal segmenta
tion. It remains to be added to what has been previously said, that this type is most frequent in the case of Invertebrates, and is to be encountered among Vertebrates only in the cases of Arnphioxus and Mammals. With the latter, however, there early appears a slight difference in the size of the segments ; this has induced many investigators to designate the cleavage of Amphioxus and Mammals as unequal also. If I have not followed this suggestion, it is because the differences are of a trivial nature, because the nucleus in the egg-cell and also in its segments still occupies a central position, and because the different methods of cleavage are in general not sharply definable, but connected by transitional conditions.
Concerning Amphioxus, HATSCHEK states that at the eight-cell stage four smaller and four larger cells are to be distinguished, and that from that time forward in all the subsequent stages there is to be observed a difference in size; and that the process of cleavage takes place in a manner similar to that which will be subsequently described for the Frog's egg. The egg of the Rabbit, concerning which we have the painstaking investigations of VAN BENEDEN, divides at the very outset into two segments of slightly different size ; moreover, from the third stage of division onward there occurs a difference in the rapidity with which the divisions follow each other in the different segments. After the four cleavage-spheres have been divided into eight, there is a stage with twelve spheres ; this is followed by another with sixteen, and afterwards another with twenty-four.
I b - Unequal Cleavage.
As a basis for the description of unequal cleavage we may employ the Amphibian egg, the structure of which has already been considered. As soon as the egg of the Frog or Triton is deposited in the water and is fertilised, and while the gelatinous envelope is swelling up, its black pigmented hemisphere or animal half becomes directed upward, because it contains more protoplasm and small yolk-spherules, and is specifically lighter. The want of uniformity in the distribution of the various components of the yolk also induces an altered position of the segmentation-nucleus. Whereas the latter assumes a central position in all cases in which the deutoplasm is uniformly distributed, it invariably alters its location whenever one half of the egg is richer in deutoplasm and the other richer in protoplasm ; it then migrates into the more protoplasmic territory.
In the case of the Frog's egg, consequently, we find it in the black pigmented hemisphere, which is turned upward.
When in this case the nucleus prepares to divide, its axis can no longer assume the position of any and every radius of the egg. In consequence of the want of uniformity in the distribution of the protoplasm, the nucleus comes under the influence of the more protoplasmic pigmented part, which rests on the more deutoplasmic portion like an inverted cup, and, on account of its less specific gravity, floats at the surface, and is spread out horizontally. But in a horizontal protoplasmic disc the nuclear spindle comes to occupy a horizontal position (fig. 31 A sp). Consequently the plane of division must be formed in a vertical direction. A small furrow now
+++++++++++++++++++++++++++++++++++++++++ Fig. 31. Diagram of the division of the Frog's egg.
A, Stage of the first division. B, Stage of the third division. The four segments of the second stage of division are beginning to be divided by an equatorial furrow into eight segments. P, pigmented surface of the egg at the animal pole ; pr, the part of the egg which is richer in protoplasm ; d, the part which is richer in deutoplasm ; sp, nuclear spindle.
begins to show itself at the animal pole first, because the latter is more under the influence of the nuclear spindle, which lies nearer to it, and because it contains more protoplasm, from which proceed the phenomena of motion during division. The furrow gradually deepens downward, and cuts through to the vegetative pole.
By the first act of division we get two hemispheres (fig. 32 2 ), each of which is composed of a quadrant richer in protoplasm and directed upward, and another poorer in protoplasm and directed downward. By this means both the position of the nucleus and the direction of its axis are again determined, when it prepares for the second division. According to the rule previously laid down, the nucleus is to be sought in the quadrant which contains the more protoplasm ; the axis of the spindle must take a position parallel to the long axis of the quadrant, and must therefore come to lie horizontally
The second plane of division is consequently, like the first, vertical, and cuts the latter at right angles.
After the conclusion of the second segmentation the Amphibian egg consists of four quadrants (fig. 32 4 ), which are separated from one another by vertical planes of division and possess two dissimilar poles, one richer in protoplasm, lighter, and directed upwards; the other richer in yolk, heavier, and directed downwards. In the case of equal segmentation we saw that at the stage of the third segmentation the axis of the nuclear spindle becomes parallel to the long axis of the quadrant. The same thing occurs here also, although in a somewhat modified manner. On account of the greater accumulation of protoplasm in the upper half of the quadrant, the spindle cannot, as
Fig. 32. Cleavage of Rana temporaria, after ECKER.
The numbers placed above the figures indicate the number of segments present in the corresponding stage. +++++++++++++++++++++++++++++++++++++++++ in the case of equal segmentation, lie in the middle of it, but must lie nearer to the animal pole of the egg (fig. 31 B sp). Moreover, it is exactly vertical, because the four quadrants of the Amphibian eggare definitely oriented in space on account of the difference in specific gravity of their halves. In consequence of this the third plane of division must be horizontal, and must also lie above the equator of the egy -sphere more or less toward its animal pole (fig. 32 8 ). The segments are very unlike both in size and composition ; and this is the reason why this form of segmentation has been called unequal. The four upper segments are smaller and contain less yolk, the four lower ones are much larger and richer in yolk. They are also distinguished from each other as animal cells and vegetative cells, according to the poles near which they lie.
In the course of further development, the distinction between animal and vegetative cells constantly increases, for the richer the cells are in protoplasm the more quickly and the more frequently do they divide. At the fourth stage the 4 upper segments are first divided by vertical furrows into 8, and then after an interval the 4 lower ones are divided in the same manner, so that the egg is now composed of eight smaller and eight larger cells (fig. 32 16 ). After a short resting stage the eight upper segments are again divided, this time by a horizontal furrow, and somewhat later a similar furrow divides the eight lower segments also (fig. 32 32 ). In the same manner the 32 segments are divided into 64 (fig. 32 64 ). In the stages which follow this, the divisions in the animal half of the egg are still more accelerated relatively to those of the vegetative half. While the 32 animal cells are divided into 128 segments by two divisions which follow each other in quick succession, there are still found in the lower half only 32 cells which are preparing for cleavage. It thus comes to pass that, as the final result of the process of cleavage, there exists a spheroidal mass of cells with entirely dissimilar halves, an upper, animal half with small, pigmented cells, and a vegetative half with larger, clear cells, containing more abundant yolk.
Erom the nature of the progress of unequal cleavage, as well as from a series of other phenomena, one may lay down a general law, first formulated by BALFOUR, that the rapidity of cleavage is proportional to the concentration of protoplasm in the segment. Cells which are rich in protoplasm divide more rapidly than those in which protoplasm is more scanty and deutoplasm more abundant.
As we have seen, the Frog's egg, by reason of the difference in specific gravity between its animal and vegetative halves, by reason of the heterogeneous pigmentation of its surface, by reason of the unequal distribution of protoplasm and deutoplasm, and by reason of the eccentric position of its nucleus, allows us to pass fixed and easily determinable axes through its spherical body. On this account it is an especially favourable object upon which to determine the question whether the egg allows one to recognise in the position of its parts, even before fertilisation, immediately after the same, and during the process of cleavage, fixed relations to the organs of the fully developed organism. This question has been tested by means of ingenious experiments, especially by PFLUEGER and Roux, by the latter in his " Beitrage zur Entwicklungsmechanik des Embryo." These have resulted in determining that the first cleavage plane of the egg corresponds to the median plane of the embryo, so that it separates the material of the right half of the body from that of the left. Secondly, according to Roux, the position of the head- and tail ends of the embryo may be determined in the fertilised egg. That half of the egg, namely, through which the spermatic nucleus migrates to reach the egg-nucleus, becomes the tail-end of the embryo ; the opposite half becomes the head-end. Every egg, however, can be fertilised in any meridian whatever, as was demonstrable experimentally, and thereby the tail-end of the embryo may be located at any chosen position in the egg. Thirdly, the plane in which the two sexual nuclei meet each other (copulation-plane) corresponds with the first plane of segmentation.
+++++++++++++++++++++++++++++++++++++++++ Fig. 33. Surface view of the first stages of cleavage in the Hen's egg, after COSTE. a, Border of the germ-disc ; b, vertical furrow ; c, small central segment ; d, large peripheral segment. +++++++++++++++++++++++++++++++++++++++++
II a - Partial Discoidal Cleavage.
The Hen's egg serves us as the classical example for the description of discoidal segmentation. In this instance the whole process of cleavage takes place while the egg is still in the oviduct, during the period in which the yolk is being surrounded by the albuminous envelope and the calcareous shell. It results simply in a cleavage of the germ-disc of formative yolk, whereas the greater part of the egg, which contains the nutritive yolk, remains unsegmented, and becomes subsequently enclosed in an appendage to the embryo, the so-called yolk-sac, and is gradually consumed as nutritive material. Just as in the case of the pigmented, animal half of the Frog's egg, so also in the case of the Hen's egg, turn it in whatever direction one will, the germ-disc floats on top, because it is the lighter part. As in the Frog's egg the first plane of cleavage is vertical and begins at the animal pole, so in the case of the Hen's egg (fig. 33 A) a small furrow (6) makes its appearance in the middle of the disc, and advances from above downward in a vertical direction. But whereas in the case of the Frog's egg the first plane of cleavage cuts through to the opposite pole, in the case of the Hen's egg it divides only the germ-disc into two similar segments, which like two buds rest upon the undivided yolk-mass with a broad base, by means of which they still have a physical connection with each other. Soon after this, there is formed a second vertical furrow, which crosses the first at right angles, and likewise remains limited to the germ-disc, which is now divided into four segments (fig. 33 }.
Each of the four segments is again divided into halves by a radial furrow. The segments thus formed correspond to sectors, which meet in the centre of the germ-disc with pointed ends, and have their broad ends turned toward the periphery. The apex of each of the segments is then cut oft* by a cross furrow, i.e., by one which is parallel to the equator of the egg (fig. 33 C), in consequence of which there are formed smaller central (c) and larger peripheral (d) segments. Since from this time forward radial furrows and those that are parallel to the equator make their appearance alternately, the germdisc is subdivided into more and more numerous segments, which are so arranged that the smaller lie at the centre of the disc, therefore immediately around the animal pole, the larger toward its periphery. With the advancing cleavage the smaller segments are entirely constricted off from the underlying yolk, whereas the larger peripheral ones still remain at first in continuit} 7 " with it (fig. 34). In this way we finally get a disc of small embryonic cells, which, toward the middle, are arranged in several superposed layers. +++++++++++++++++++++++++++++++++++++++++
Fig. 34. Section through the germ-disc of the Hen's egg during the later stages of segmentation after BALFOUR. The section, which represents rather more than half the breadth of the blastoderm (the middle line is at c), shows that the segments of the surface and of the centre of the disc are smaller than those below and toward the periphery. At the border they are still very large. One of the latter is indicated at a. tt, Large peripheral cell ; b, larger cells of the lower layers ; c, middle line of the blastoderm ; e, boundary between the blastoderm and the white yolk, w. +++++++++++++++++++++++++++++++++++++++++
The layer of yolk which immediately adjoins the periphery of the cellular disc, and which is very finely granular and especially rich in protoplasm, still merits particular consideration, for in it lie isolated nuclei (fig. 35 nx\ the muck-discussed yolk-nuclei or parablast-nuclei (the " merocytes" of RUCKERT). In the case of the Chick they are less striking than in Teleosts and Selachians, in which they have been accurately investigated by BALFOUR, HOFFMANN, RUCKERT, and KASTSCHENKO. Formerly these were held to arise spontaneously (free formation of nuclei) in the yolk, an assumption which in itself is very improbable, since, according to our present knowledge, the free formation of nuclei does not appear to occur anywhere in
+++++++++++++++++++++++++++++++++++++++++ Fig. 35. Section through the germ-disc of a Pristiurus embryo during segmentation, after BALFOUR. n, Nucleus; nx, modified nucleus prior to division; nx', modified nucleus in the yolk; f, furrows which appear in the yolk adjacent to the germ-disc. +++++++++++++++++++++++++++++++++++++++++ either animal or vegetable kingdom. Consequently the yolk-nuclei are now rightly held to be derived from the cleavage-nuclei. They are probably produced even at an early period, when the 'first-formed segments, which remain, as we have seen, for a long time in connection with the yolk, begin to be constricted off from the latter. This probably takes place in the following manner : there arise in" 1 the segments nuclear spindles, the halves of which go into the completely isolated embryonic cells at the time of their separation from the yolk, while the remaining halves go into the underlying yolk-layer, and are there converted into vesicular yolk-nuclei.
Their number subsequently increases by means of indirect division, as is established by the fact that in sections nuclear spindles have been observed in the yolk-layer (fig. 35 nx'").
While, on the one hand, there is an increase 4n the number of the yolk-nuclei, so, on the other hand, there is also a diminution in their
number, as is asserted by several authors (WALDEYER, RUCKERT, BALFOUR, etc.). This takes place by the constricting off of nuclei and surrounding protoplasm, which go to enlarge the cellular discWe may, with WALDEYER, designate these as secondary cleavage-cells, and regard the whole process as a kind of supplementary segmentation . By means of this a part of the voluminous yolk-material continues to be gradually individualised into cells. These annex themselves to the border of the germ-disc, which with their aid increases in extent and grows over a continually increasing territory of the unsegmented yolk-sphere. In still later stages of development, long after the cellular germ-disc has been differentiated into the germ -layers, the supplementary segmentation continues to go on at the margin of the disc in the neighbouring yolk-mass, and to furnish new cell-material. Therefore the layer which encloses the yolk-nuclei forms an important connecting link between the segmented germ and the unsegmented nutritive yolk; I shall come back to this subject later.
The appearance of merocytes and the supplementary cleavage which proceeds from them are phenomena which are induced by the vast accumulation of yolk-material, and which allow the latter to be divided up into cells, even though the process is a slow one.
The eggs of Selachians (KASTSCHENKO, RUCKERT) deviate a little from the usual method of partial cleavage in meroblastic eggs, and in a manner which recalls to a certain extent the processes of superficial cleavage, which are to be treated of later. The cleavage-nucleus, namely, is divided into two nuclei, these again into four and even a greater number, without an accompanying division of the germ -disc into a corresponding number of segments. In this case, therefore, there arises at first a multinuclear protoplasmic mass, a plasmodium, in which the nuclei are distributed at regular intervals. Subsequently furrows appear, generally in great numbers and all at once, by means of which the germ-disc becomes divided into cells from the centre to the periphery. Some of the nuclei always remain in the periphery outside the territory of cleavage, here undergo further division, migrate out of the germdisc into the surrounding nutritive yolk, and constitute the yolknuclei or merocytes. These cause and maintain in the yolk for a long time the process of supplementary cleavage.
When we institute a comparison between partial and unequal cleavage, for the, descriptions of which we have made use of the eggs of the Hen and the Frog, it is not difficult to derive the former from the latter, and to find a cause for the origin of the former,
It is the same as that which produced unequal cleavage from equal cleavage ; it is the great accumulation of nutritive yolk, the inequality in the distribution of the egg-substarices which goes hand in hand with it, and the alteration in the position of the cleavage-nucleus. The process of differentiation, which is still in a stage of transition in the case of the Frog's egg, is carried to an extreme in the case of the Hen's egg. Protoplasmic substance was already abundantly accumulated at the animal pole in the former case, but in the latter it is still more concentrated, and at the same time has become differentiated from the nutritive yolk as a disc enclosing the segmentation-nucleus. The yolk, accumulated to an enormous extent at the opposite pole, is, in consequence of this separation, relatively poor in protoplasmic substance, which only scantily fills the interstices between the large yolk- spheres.
Inasmuch as the phenomena of motion during the process of division emanate from the protoplasm and nucleus, whereas the deutoplasm remains passive, the active substance in the case of meroblastic eggs can no longer master the passive substance and cause it to participate in the cleavage. Even in the case of the Frog's egg a preponderance of the animal pole during cleavage is observable ; within its territory the nucleus lies, the radial figures of the protoplasm appear, and the first and second planes of division begin to arise, whereas they cut through at the vegetative pole last of all ; moreover the process of division during the later stages takes place there with greater rapidity, so that a distinction arises between the smaller animal cells and the larger vegetative ones. In the case of the Hen's egg, the preponderance of the animal pole is still further increased, and the contrast with the vegetative pole is most sharply expressed. The cleavage-furrows not only begin there, but they remain restricted to the territory immediately surrounding it. Thus we get on the one hand a disc composed of small animal cells, on the other an immense undivided yolk-mass, which corresponds to the larger vegetative cells of the Frog's egg. TJie yolk-nuclei enclosed in the periphery of the germ-disc are equivalent to the nuclei of the vegetative cells of the Frog's egg.
II b Partial Superficial Cleavage.
The second sub-type of partial cleavage is prevalent in the phylum of Arthropods, and occurs in centrolecithal eggs, where a central yolk-mass is enclosed in a cortical layer of formative yolk. Manifold variations are possible here, as well as transitions to equal and unequal cleavage. When the course pursued is quite typical, the segmentation-nucleus, surrounded by a mantle of protoplasm, lies in the middle of the egg in the nutritive yolk ; here it is divided into two daughter-nuclei, without the occurrence of a corresponding division of the egg-cell. The daughter-nuclei, in turn, undergo division into 4, these into 8, 16, 32 nuclei, etc., while the egg as a whole still remains unsegmented. Subsequently the nuclei move apart, the greater number gradually migrate to the surface, and penetrate into the protoplasmic cortical layer, where they arrange themselves at uniform distances from each other. It is only at this stage that the process of egg-segmentation takes place, for now the cortical layer is divided into as many cells as there are nuclei in it, ivhile the central yolk remains undivided. The latter is therefore suddenly enclosed in a sac formed of small cells a blastoderm (Keimhaut). Instead of a polar (telolecithal) yolk, we have a central (centrolecithal) yolk. Ordinarily yolk-nuclei or merocytes remain behind in the yolk, as in the meroblastic eggs of Vertebrates.
Now that we have become acquainted with the various forms of the process of segmentation, it will be expedient to dwell for a moment on its results. According as the process of cleavage takes place by one or the other of the four methods described, there arises a mass of cells with corresponding characteristics. From equal segmentation there arises a spherical germ with cells approximately uniform in size (Amphioxus, Mammals) (fig. 30, p. 56) ; from unequal segmentation, as well as from discoidal, there is produced a form of the germ with polar differentiation. This manifests itself in the first case (Cyclostomes, Amphibia) in the production of small cells at the animal pole and large yolk-laden elements at the opposite, vegetative pole (fig. 32 64 , p. 60). In the other case (fig, 35, p. 64) the vegetative pole is occupied by an unsegmented yolk-mass, in which at definite regions nuclei are found (Fishes, Reptiles, and Birds). Finally there is developed from superficial cleavage a germ composed of a mantle of cells, which envelops an unsegmented yolkmass in which also there are nuclei (Arthropods).
The multicellular germ undergoes further changes, sometimes in the earlier stages of the cleavage-process, sometimes only in the later stages, in that a small, fluid-filled cleavage-cavity is developed in its centre, by the separation of the embryonic cells. At first small, this cavity increases more and more in size, so that the surface of the whole germ is augmented, and the cells which were at first central come to the surface.
Fig. 36. Blastula of Amphioxus, after HATSCHEK. h, Segmentation-cavity ; az, animal cells ; dz, cells with abundant yolk.
Different names have been given to the solid and to the hollow mass of cells. A morula or mulberry -sphere is spoken of as long as the segmentation-cavity is either wanting or only slightly developed. But when a larger cavity has been formed, as is almost always the case toward the end of the cleavage-process, the germ is called a blastula or blastosphere (Keimblase). The latter in turn exhibits a four-fold variation of form, according to the abundance of yolk in the original egg and the method of the antecedent segmentation.
In the simplest case (fig. 36) the wall of the blastula is only one layer thick ; the cells are of uniform size and cylindrical, and are closely united to one another to form an epithelium (many of the lower animals, Amphioxus). In the case of lower, aquatic animals the blastulse at this stage abandon the egsr-envelopes, and, since their cylindrical cells develop cilia at the surface, swim, about with rotating motion in the water as ciliate spheres or blastospheres.
In eggs with Unequal seg- Fig. 37. Blastula of Triton tseniatus.
, . ih, Segmentation-cavity; /:., marginal zone ; dz, cells mentation the blastula is with abuildant yolk .
ordinarily formed of several layers of cells, as in the case of the Frog and Triton, and at the same time it exhibits in different regions different thicknesses (fig. 37). At the animal pole the wall is thin ; at the vegetative pole, on the contrary, it is so much thickened that an elevation, composed of large yolk-cells, protrudes from this side far into the cleavage-cavity, thus considerably diminishing it.
The eggs \vith partial discoidal segmentation (fig. 38) are modified most of all, and are therefore scarcely to be recognised as blastulre. In consequence of the immense accumulation of yolk on the ventral (vegetative) side, the cleavage-cavity (B] is extraordinarily constricted, and is still preserved only as a narrow fissure filled with albuminous fluid. Dorsally its wall consists of the small embryonic cells (kz) resulting from the process of cleavage, which are accumulated in several superposed layers ; at the surface they join each other closely, deeper they lie more loosely associated. The floor of the cleavagecavity is formed of a yolk-mass, scattered through which are to be found the yolk-nuclei or merocytes (dk), which likewise result from the cleavage-process. It is to be seen that they are especially nurrterous at the place of transition frOUl the ^ig- 38. Median section through a germ-disc of Pristiurus in the o-erm-disC to the Wastula stage, after RUCKERT.
B, Cavity of the blastula ; /,:, s^'inenU-d ^enu ; ill-, finely granular yolk-maSS. y..lk with yolk-nuclei.
This nucleated yolk-mass very evidently corresponds to the large vegetative cells which constitute the floor of the cleavage-cavity in the case of the Amphibian egg (fig. 37).
In the case of superficial cleavage there is formed, strictly speaking, no blastula, since the place where the segmentation-cavity should be developed is filled with nutritive yolk. The latter either remains unsegmented or is subsequently divided, as in the Insects, into individual yolk-cells.
History of the Process of Cleavage
The investigation and right comprehension of the process of cleavage have been attended with manifold difficulties. A voluminous literature has arisen on this subject. We limit ourselves to pointing out the most important discoveries and the chief questions which have been discussed.
The first observations on the process of segmentation were made on the Frog's egg. Aside from short statements by SWAMMERDAM and RO'SEL vox EOSENTTOF, it was PREVOST ET DUMAS who were the first to describe, in 1824, the manner in which regular furrows arise on the Frog's egg, and how by means of these the whole surface is divided into smaller and smaller areas. According to the French investigators, the furrows were restricted to the surface of the egg. However, only a few years later, RUSCONI (182(5) and C. E. V. BAER recognised that the furrows visible at the surface correspond to fissures which extend through the whole mass of the yolk, and divide it into separate parts. Even in his time VON BAER rightly characterised the whole process of segmentation, in which he discerned the first impulse of life, as an automatic division of the egg-cell, but subsequently he abandoned this, the right path, since he sought for the meaning of division in the dictum : that "all yolk-masses are subject to the influence of the fluid and volatile components of the fertilising material." In the next decennary there followed numerous discoveries of the process of segmentation in other animals. During this period acquaintance was also gained with partial segmentation. After RUSCONI and VOGT had seen it in the case of fish eggs, KOLLIKER gave, in the year 1844, the first detailed description of it as seen in the eggs of Cephalopods, and four years later COSTE described it in the Hen's egg.
The question of the significance of the cleavage-process has engaged the earnest attention of investigators, and has given rise to many controversies. The discussion first took a definite turn upon the establishment of the celltheory. The question was, to determine whether and in what manner cleavage was a process of cell-formation. Although there were already many observations on the division of eggs, SCHWANN himself took no definite position on this question. The views of other investigators were at variance for years. There was a difference of opinion as to whether the egg or the germinative vesicle was a cell, whether the segments resulting from cleavage possessed a membrane or not, and whether these segments were to be regarded as cells or not. In the earlier literature the germinative vesicle and the nuclei of the cleavage-spheres were often designated as embryonic cells, and the surrounding yolk-mass as an enveloping sphere. The difficulty of comprehending the process of segmentation was also aggravated by the false doctrine of free cell-formation from an organic matrix the cytoblastema founded by SCHWANN. It remained for a long time a controverted point whether the tissue-cells of the adult organism were the direct descendants of the segmentation-spheres, or whether they arose at a later period by means of free cell-formation from cytoblastema. After NAGELI on the botanical side had adopted the right course, it was the service of KOLLIKER, EEICHERT, REMAK, and LEYDIG to have paved the way to a comprehension of cleavage, and to have shown that free cell-formation does not take place, but that all cellular elements arise in uninterrupted sequence from the egg-cell.
As far as regards the different kinds of cleavage, KOLLIKER designated them as total and partial. VAN BENEDEN has given in his " Recherches sur la composition et la signification de 1'oeuf " a more exhaustive review of the subject, and has also expounded in a clear way the signification of the deutoplasm for the different kinds of cleavage. Subsequently HAECKEL materially simplified the categories of segmentation recognised by VAN BENEDEN, and proposed in his " Anthropogenic " and in his paper " Die Gastrula und die Eif urchung " the classification of the methods of cleavage on which is based the scheme previously given, and according to which total cleavage is divided into equal and unequal, and partial into discoidal and superficial. At the same time HAECKEL endeavoured to derive the different methods of cleavage from one another, and apropos of this directed attention to the important role of the nutritive yolk.
The processes which take place within the yolk have eluded observation and a correct interpretation even more than the external phenomena of cleavage, so that it is only in the most recent times that we have acquired a satisfactory insight into them. It is true that the problem, as to what part the nucleus plays in segmentation, has had the uninterrupted attention of investigators, but without any solution having been found. For years there were in the literature two opposing views : sometimes one of them, sometimes the other, attained temporarily greater currency. According to one view which was almost universally adopted by the botanists, and was defended on the zoological side principally by REICHERT, and even recently by AUERBACH the nucleus disappears before every division, and is dissolved, to be afterwards formed anew in each daughter-segment ; according to the other view the nucleus, on the contrary, is not dissolved, but is constricted, becomes dumb-bell-shaped, and is divided into halves, and thereby induces cell-division. This view was taught especially by such zoologists and anatomists as C. E. v. BAEE, JOH. MULLER, KOLLIKER, LEYDIG, GEGENBAUR, HAECKEL, VAN BENEDEN, and others, who were supported by the observations which they had made on transparent eggs of the lower animals.
Light was first thrown on the disputed question at the moment when suit able objects were studied with the aid of higher magnifications, and especiall with the employment of modern methods of preparation (fixing and staining reagents).
The works of FOL, FLEMMING, SCHNEIDER, and AUERBACH on the cleavage of the eggs of various animals mark a noteworthy advance. They still maintained, it is true, that the nucleus is dissolved at the time of cleavage, but they gave a detailed and accurate description of the striking radiation which arises in the yolk upon the disappearance of the nucleus, and which during the constriction of the egg soon becomes visible in the region of the daughternuclei.* SCHNEIDER observed parts of the spindle-stage.
Soon after this a more exact insight into the complicated and peculiar nuclear changes was obtained by means of three investigations, which were carried out independently and simultaneously on different objects, and were published in rapid succession by BUTSCHLI, STRASBURGER, and the author. It was definitely established by these observations that there is no dissolution of the nucleus at the time of division, but a metamorphosis, such as has been described in the preceding pages. At the same time I likewise proved that the egg-nucleus is not a new formation, but is derived from parts of the germinative vesicle. From this resulted the important doctrine that, just as all cells, so also all nuclei of the animal organism are derivatives in an uninterrupted sequence, the one from the egy-cell and the other from its nucleus. (Omnis cellula e cellula, omnis nucleus e nucleo.) Through these researches there was furnished for the first time a scheme of nuclear division and cell-division, which has since proved to be correct in all essentials, even though it has undergone important improvements and additions at the hands of FOL, FLEMMING, VAN BEXEDEN, and IxABL.
- Radiating structures had already been observed in the yolk before this, but in an incomplete manner, by different authors by GRUBE in the Hirudinea, by DERBES and MEISSNER in the Sea-urchin, by GEGENBAUR in Sagitta, by KROHN, KOWALEVSKY, and KUPFFER in Ascidians, by LEUCKAET in Nematodes, by BALBIANI in Spiders, and by OELLACHER in the Trout.
FOL published an extended monographic investigation of the process of cleavage, which he had observed in many invertebrated animals. FLEMMING, starting with nuclear division in tissue-cells, distinguished with great acumen the non-chromatic and the chromatic parts of the nuclear figure, the nonstainable nuclear spindle-fibres, and the stainable nuclear filaments and loops, which are located upon the surface of the former. He made the interesting discovery concerning the latter, that they become split lengthwise. Light was soon thrown upon this peculiar phenomenon, when HEUSER, VAN BENEDEN, and RABL, independently of each other, discovered that the halves of the split filaments moved apart toward the poles of the nucleus, and furnished the fundament for the daughter-nuclei. VAN BENEDEN at the same time made the additional and important observation on the egg of Ascaris megalocephala, that of the four chromatic loops, which are constantly to be observed in the case of the cleavage-nucleus, two are derived from the chromatic substance of the spermatic nucleus, the other two from the chromatic substance of the egg-nucleus ; and that, in consequence of the longitudinal splitting, each daughter-nucleus receives at the time of division two male and two female nuclear loops. In addition there have appeared many other recent works of value on the process of cleavage by NUSSBAUM, RABL, CARNOY, BOVERT, TLATNER, and others.
Within the last few years PFLUGER has endeavored to prove by interesting experiments that gravitation exercises a determining influence on the position of the planes of cleavage. BORN, Roux, and the author, on the contrary, thought they were able to explain division from the organisation of the eggcell itself. In the author's article, " Welchen Einfluss iibt die Schwerkraft auf die Theilung der Zellen ? " he recognised the causes which determine the various directions of the planes of division, (1) in the distribution of the lighter egg-plasm and the heavier deutoplasm, and (2) in the influence which the spatial arrangement of the egg-plasm exercises on the position of the nuclear spindle, and that which the position of the latter exercises upon the direction of the plane of cleavage.
1. In the process of cleavage the internal and the external phenomena of segmentation are to be distinguished from each other.
2. The internal phenomena of cleavage find expression in changes (a) of the nucleus, (6) of the protoplasm.
3. The nucleus while in the process of division consists of a nonchromatic and a chromatic nuclear figure. The non-chromatic figure is a spindle composed of numerous fibres. The chromatic figure is formed of bent, Y-shaped nuclear filaments (chromosomes), which lie upon the surface of the middle of the spindle. At the two ends of the spindle there is found a special polar corpuscle [centrosome].
4. The division of the nucleus takes place in the f ollowing manner : the nuclear filaments split lengthwise, and their halves move apart in opposite directions toward the ends of the spindle, and are there converted into vesicular daughter-nuclei.
5. The protoplasm arranges itself around the ends of the spindle in filaments having the form of a stellate figure (an aster), so that a double radiation or an amphiaster arises in the egg.
6. The external phenomena of cleavage consist in the division of the egg-contents into individual parts, the number of which corresponds to that of the daughter-nuclei. They exhibit various modifications, which are dependent on the arrangement and distribution of the egg-plasm and the deutoplasm, as is to be seen from the following scheme of segmentation.
Scheme of the Various Modifications of the Process of Cleavage.
I. Total Cleavage. (Holoblastic eggs.) The eggs, which for the most part are small^ contain a small or moderate amount of deutoplasm, and are completely divided into daughter-cells.
1. Equal Cleavage.
This takes place in eggs with meagre and uniformly distributed deutoplasm (alecithal). By the process of cleavage there are formed segments which, in general, are of uniform size. (Amphioxus, Mammalia.) 2. Unequal Cleavage.
This occurs in eggs in which a more abundant deutoplasm is unequally distributed, being concentrated toward the vegetative pole, and in which the cleavage-nucleus is located nearer the animal and more protoplasmic pole. Usually the segments become unequal in size only with and after the third act of division. (Cyclostomes, Amphibia.) II. Partial Cleavage. (Meroblastic eggs.) The eggs, which are often very large, ordinarily contain considerable quantities of deutoplasm. In consequence of the unequal distribution of this, the egg-contents are separated into a formative yolk, in which alone the process of cleavage is manifested, and a nutritive yolk, which remains undivided, and is used up during embryonic development for the growth of the organs.
1. Discoidal Cleavage.
This takes place in eggs with nutritive yolk in a polar position The process of cleavage remains confined to the formative yolk accumulated at the animal pole, which has the form of a disc and contains only a small amount of deutoplasm. There is formed, consequently, a cellular disc. (Fishes, Reptiles, Birds.)
2. Superficial Cleavage.
This occurs in the case of eggs with central yolk. In typical cases the nucleus alone, which occupies the middle of the egg, undergoes repeated division. The numerous daughter-nuclei which arise in this manner migrate into the layer of protoplasm which invests the central nutritive yolk, and the protoplasm is thereupon divided into as many segments as there are nuclei lying in it. There is formed a germ-membrane (Keimhaut). (Arthropods.) 7. Eggs with total cleavage are designated as holoblastic, eggs with partial cleavage as meroblastic.
8. The direction and position of the first cleavage-plane are strictly conformable to laws which are founded in the organisation of the cell ; they are determined by the following three factors : First factor. The cleavage-plane always divides the axis of the nucleus which is preparing for division perpendicularly at its middle.
Second factor. The 'position of the axis of the nucleus during division is dependent upon the form and differentiation of the enveloping protoplasm.
In a protoplasmic sphere the axis of the nuclear spindle, occupying the centre of the sphere, can lie in the direction -of any radius whatever ; but in an oval protoplasmic body, only in the longest diameter. In a circular disc the nuclear axis lies parallel to its surface in any diameter of the circle, but in an oval disc only in the longest diameter.
Third factor. In the case of eggs of unequal segmentation, which, in consequence of their unequally distributed, polar deutoplasm, are geocentric, and therefore assume when in equilibrium a particular position, the first two planes of cleavage must be vertical, and the third must be horizontal and placed above the equator of the sphere.
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