Part 33

From the time of the discovery of the cell until well on in the sixties the process of cell-division was looked on as a perfectly simple process, as a mere constriction in the middle of the cell. It was observed that a cell in the act of dividing (Fig. 59, _A_) stretched itself out, that its nucleus also became longer, became thinner in the middle, assumed a dumb-bell form, and was then gradually constricted, giving rise to two nuclei (_B_), whereupon the body of the cell also constricted and the two daughter-cells were formed (_C_). In certain worn-out or highly differentiated cells a cell-division of this kind really seems to occur--the so-called 'direct' division--but in young cells, and indeed in all vigorous cells, the process, which looks simple, is, in reality, exceedingly complex. Not only is the structure of the nucleus incomparably more complex than was recognized a quarter of a century ago, but nature has placed within the cell a special and marvellously intricate apparatus, by means of which the component parts of the nucleus are divided between the two daughter-nuclei.

For a long time all that was distinguished in the cell-nucleus was the nuclear membrane and a fluid content in which one or more nuclear bodies or nucleoli float. But this does not by any means exhaust what can now be recognized in the structure of the nucleus, and the most important constituents are not even among these, for recent researches, especially those of Häcker, have shown that the nucleolus or the nucleoli, to which there was formerly an inclination to attach a very high importance, must be regarded as only transient formations and not living elements--in fact, as mere collections of organic substance--'bye-products of the metabolism,' which at a definite time, that is just before the division of the nucleus, disappear from the nuclear space and are used up. We now know that in the resting cell, that is, in the cell which is not in the act of dividing (Fig. 74, _A_), a very fine network of pale threads, often very difficult to make visible, fills the whole nuclear cavity, like a spider's web or the finest soap bubbles, and that in this so-called nuclear framework there are embedded granules of rounded or angular form (_A_, _chr_) which consist of a substance which stains deeply with such pigments as carmine, hæmatoxylin, all aniline dyes, &c., and which has therefore received the name of chromatin. Often, indeed generally, these granules are exceedingly small, but sometimes they are bigger, and in that case they are less numerous and more easily made visible; in all cases, however, they are in a certain sense the most important part of the nucleus, for we must assume that it is their influence which determines the nature of the cell, which, so to speak, impresses it with the specific stamp, and makes the young cell a muscle-cell or a nerve-cell, which even gives the germ-cell the power of producing, by continued multiplication through division, a whole multicellular organism of a particular structure and definite differentiation, in short, a new individual of the particular species to which the parents belong. We call the substance of which these chromatin granules consist by the name first introduced into science by Nägeli, though only to designate a postulated substance which had not at that time been observed, but which he imagined to be contained within the cell-body--by the name _Idioplasm_, that is to say, a living substance determining the individual nature (εἶδος = form). I am anticipating here, and I reserve a more detailed explanation until I can gradually bring together all the facts which justify the conception I have just indicated of the 'chromatin grains' as an 'idioplasm,' or, as we may also call it, a 'hereditary substance.'

That this chromatin must be something quite special we see from the processes of cell and nuclear division, which I shall now briefly describe.

When a cell is on the eve of dividing we observe first that the chromatin grains, which have till then been scattered throughout the network of the nucleus, approach each other and arrange themselves into a long thin thread which, irregularly intertwined, forms a loose skein, the so-called coil-stage (Fig. 74, _B_). The thread then begins to thicken, and somewhat later it can be seen to have broken up into a number of pieces of equal length, as if it had been cut into equal pieces with scissors (_C_).

These pieces or chromosomes become shorter by slowly contracting, and thus each takes the form of an angular loop, a straight rod, or a roundish, oval, or spherical body (Fig. 74, _C_, _chrs_). While this is happening, we can see at the side of the nucleus, and closely apposed to it, a pale longitudinally striped figure with a swelling, similar to a handle, at both ends--the so-called nuclear spindle or central spindle (_ksp_). This is the apparatus for the division of the nucleus, and it was previously represented by a small body susceptible to certain stains--the centrosome, which was surrounded by a halo-like zone, the centrosphere or 'sphere.' This body was long overlooked, but now the majority of investigators assume that, though it is often inconspicuous and very difficult to make visible, it is nevertheless present in every cell which is capable of division, and that it is therefore a permanent and indispensable constituent of the cell (Fig. 74, _A_ and _B_, _csph_).

When a cell is on the point of dividing, this remarkable cell-organ, which has hitherto seemed no more than an insignificant, pale, little sphere, now becomes active. First of all, often before the formation of the chromatin coil, it doubles by division (_A_ and _B_ _csph_), at first only as regards the centrosome, and then as regards the sphere also (_B_); and while division is going on fine protoplasmic filaments issue from the dividing sphere and radiate like rays from a sun into the cell-substance. As they only retain their connexion with each other at the surfaces of the dividing halves of the sphere which are turned towards each other, we might almost say that fine threads are drawn out between the two halves as they separate, and these become longer the further apart the halves diverge. In this manner the much-talked-of 'spindle figure' arises, which was first described in the seventies through the researches of A. Schneider, Auerbach, and Bütschli, but the significance and origin of which have claimed the labours of many later investigators down to our own day.

The processes now to be described do not always take place in exactly the same manner, but the gist of the business is everywhere the same, and it consists in this, that the two ends or 'poles' of the spindle diverge further and further apart, and between them lies the nucleus whose membrane now disappears (_C_, _km_) while the spindle threads traverse its interior. Sometimes the membrane is retained, but nevertheless the spindle threads penetrate into the interior of the nucleus. But the chromosomes always range themselves quite regularly in the 'equatorial plane' of the spindle (_D_, _aeq_)--a process the precise mechanism of which is by no means clearly understood, and indeed the play of the forces in the whole process of nuclear division is still very imperfectly revealed to our intelligence.

Thus we have now before us a pale, spindle-shaped figure, which takes only a faint stain, with the 'suns' (_cs_) at its 'poles,' and in its equatorial plane the loop- or rod-shaped, or spherical chromosomes (_chrs_). The whole is designated the 'karyokinetic,' the 'mitotic,' or the 'nuclear division figure.'

The meaning and importance of this, at first sight, puzzling figure will at once become clear from what follows. It may be observed at this stage, if not even long before, that each of the chromatin rods or loops has split along its whole length like a log of wood, and that the split halves are beginning slowly and hardly noticeably to move away from each other, one half towards one, the other towards the other pole of the spindle (Fig. _D_ and _F_). Directly in front of the centrosome they make a halt, and now the material for the two daughter-nuclei is in its proper place (_F_, _chrs_). These develop quickly, each chromosome group surrounding itself with a nuclear membrane (Fig. _G_) within which the chromosomes gradually become transformed again into a nuclear network. Within the chromatin substance proper this is scattered about in small roundish or angular granules, lying especially at the intersecting points of the network. It may be stated at once, though the full significance of the statement can only be appreciated later, that we may assume with probability that this breaking up of the chromosomes is only apparent, and that these rods or spheres really continue to exist in the nuclear network, only in a different form, greatly spread out, somewhat after the manner of a Rhizopod which stretches out fine processes in all directions. These processes branch and anastomose, so that the body, which previously seemed compact, now appears as a fine network. In point of fact, it can be directly observed that the chromosomes, after the nucleus is completely divided into two daughter-nuclei, send out pointed processes (_F_ and _G_) which gradually increase in length and branch, while the body of the chromosome itself becomes gradually smaller. It is thus probable that, when such a daughter-nucleus is on the point of dividing anew, it may, by a drawing together of the processes or pseudopodia of the chromosomes, produce the same rods or spheres as those which previously gave rise to the network. More definite reasons for this interpretation will be adduced later on. In any case, the chromosomes, even in their compact rod-like state, consist of two kinds of substance, the chromatin proper, which stains deeply, and the linin, which is difficult to stain; and it is the latter which, by breaking up, forms the pale part of the nuclear network.

Thus we can understand that the number of chromosomes remains the same in every cell-generation throughout development, as it is the same in all the individuals of a species. The numbers are known for many species: in some worms there are only two or four chromosomes, while in other related worms there are eight; in the grasshopper there are twelve, and in a marine worm, _Sagitta_, eighteen; in the mouse, the trout, and the lily there are twenty-four; in some snails thirty-two; in the sharks thirty-six, and in _Artemia_, a little salt-water crustacean, 168 chromosomes. In Man the chromosomes are so small that their normal number is not certain--sixteen have been counted. This counting can only be done during the process of nuclear division, for afterwards the chromosomes flow indistinguishably together, or rather apart, only to reappear, however, in the old form and number whenever the nucleus again begins to divide.

It remains to be told what becomes of the centrosphere in cell-division. As soon as the formation of the daughter-nuclei has been brought about by the divergence of the split halves of the loops, the spindle figure begins to retrograde, its threads become pale and gradually disappear, as does the whole radiate halo of the centrosphere (Fig. _F_ and _G_). The cell-body has by this time also divided in the equatorial plane of the nuclear spindle, and the centrosome remains usually as a very inconspicuous pale body lying in the cytoplasm close to the nucleus, reawakening to renewed activity when cell-division is about to recommence (_G_, _csph_).

These, briefly, are the remarkable processes of nuclear division. Their net result is obvious; the chromatin substance is divided between the daughter-nuclei with the greatest conceivable accuracy.

It is not so easy to understand the mechanism of this partition, and there are various divergent theories on this point. According to the older idea of Van Beneden, the spindle fibres work like muscles, and by contracting draw the halves of the chromosomes which adhere to them towards the pole, while the rest of the fibres radiating out from the polar corpuscles act as resisting and supporting elements. This view, with many modifications however, has still its champions, and M. Heidenhain in particular has made a notable attempt to establish it and to work it out in detail. Opposed to it stand the views of those who, like O. Hertwig, Bütschli, Häcker, and others regard the rays not as specific elements which were pre-formed in the cell, but as the expression of the orientation of certain protoplasmic particles--an orientation evoked by forces which have their seat within the central corpuscles, and act in the manner of magnetic or electric forces. That the central corpuscles are centres of attraction seems to me hardly open to doubt, and I cannot regard the regular arrangement of the chromosomes in the equatorial plane of the spindle as due to a mere adhesion to contractile threads. Some still unknown forces--chemotactic or otherwise--must be at work here. Later on we shall study the phenomenon of the migration of the sperm-nucleus into the ovum, when it is accompanied by its central body and its halo of rays. Häcker seems to me justified in inferring from this phenomenon alone that the sudden origin of the rays is due to forces resident in the central corpuscle. But undoubtedly even this 'dynamic' explanation of karyokinesis is still only at the stage of hypothesis and reasoning from analogy, and is far removed from a definite knowledge of the forces at work.

For the problems with which we are here chiefly concerned, the problems of heredity, it is enough to know that the cells of multicellular organisms possess an extremely complex apparatus for division, whose chief importance lies in the fact that through it the chromatin units of the nucleus are divided into precisely equal parts, and so separated from each other that one half forms one daughter-nucleus, the other half the other. It is not merely that there is an exact division of the whole chromatin in the mass, which could have been effected much more simply, but that there is _a regulated distribution of the different qualities of the chromatin_, as we shall see later.

It must here be emphasized that the splitting of the chromosomes does not depend on external forces, but on internal ones involved in their organization, and in the definite attractions and repulsions of their component particles which come about in the course of growth. The chromosomes do not split like a trunk that has been broken open with an axe, but rather like a tree burst apart by the frost, that is, by the freezing of the water within itself. I consider it very important that we should recognize this, even though we do not yet know what the forces are that have control in this case, because it leads us to conclude that the structure of the chromosomes is extremely complex, that they are, so to speak, a world in themselves, that they possess an infinitely complex and delicate though invisible organization, in which intrinsic chemico-physical forces produce the regulated succession of changes which we observe. We shall afterwards see that we are led to the same conclusion from another direction--that is, from the phenomena of inheritance. We shall then recognize that the rod- or loop-shaped chromosomes cannot be simple elements, but are composed of linear series of 10, 20, or more globular single-chromosomes, each of which represents a particular kind of chromatin or hereditary substance. If we consider this carefully, we shall see that it would hardly be possible to think out a mode of nuclear division which would so exactly and securely fulfil the purpose of conveying these many kinds of chromatin to the two daughter-nuclei in like proportions as does the mechanism of distribution actually brought about by nature. The longitudinal splitting of the rods halves the chromosomes, and the spindle apparatus secures the proper distribution of the halves between the two daughter-nuclei.

So much, at least, is certain, that no such complicated mechanism for 'mitotic' division would have arisen if the very precise division of a substance _of the highest importance_ had not been concerned, and in this conclusion lies the first hint of the interpretation of the chromatin substance as the bearer of the hereditary qualities.

We are now familiar with the cell-nucleus and the apparatus for its division, and we are thus fully prepared to begin the study of the phenomena of 'fertilization.' Here also the processes depend essentially on the behaviour of the cell-nuclei, for even the first observations made by O. Hertwig on the behaviour of the spermatozoon after it has penetrated into the ovum led to the suggestion that the essential fact is the union of two nuclei; and numerous later, more and more deeply penetrating researches have furnished abundant evidence that the so-called 'fertilization' _is essentially a nuclear fusion_.

Let us begin with O. Hertwig's observations on the ovum of the sea-urchin. Eggs of this animal, which have been taken out of the ovary of the female, may easily be fertilized artificially by pouring over them spermatic fluid taken from a male, and diluted with sea-water. Before this is done only one nucleus can be observed in the ovum, but shortly afterwards two nucleus-like structures of unequal size can be seen within the ovum, and the smaller is surrounded by a circle of rays. Hertwig rightly interpreted this smaller nucleus as the modified remains of the penetrating spermatozoon, which then slowly approaches the nucleus of the egg, and ultimately fuses with it to form a 'segmentation nucleus.' From this starts the so-called 'segmentation' of the ovum, that is, the series of repeated divisions resulting in the formation of an ordered mass of cells, which by continued division of cells builds up the embryo.

Simple as this process of nuclear conjugation may seem, it was by no means so easy to recognize, and several investigators, especially Auerbach, Schneider, and Bütschli, had seen stages of the process at an earlier date without arriving at the true interpretation of the phenomena. This was chiefly due to the fact that, in addition to the phenomena of fertilization proper, which we have briefly sketched, other nuclear changes take place in the maturing ovum, and these are not very easy to distinguish from the former; we refer to the phenomena of the so-called 'maturation of the ovum.' When the ovum-cell has attained its full size within the ovary it is not yet capable of being fertilized, but must first undergo two processes of division, to the right understanding of which Hertwig's investigations, and afterwards those of Fol, have contributed much.

For a long time it had been a familiar observation that small refractive corpuscles were extruded from one pole of the ovum shortly before the beginning of embryonic development. These were called 'polar bodies,' because it was believed that they marked the place which would afterwards be intersected by the first plane of division; it was only known at that time that they had to be extruded from the egg, but no one had the remotest idea of their real nature.

We now know that they are cells, and that their origin depends on a twice repeated division of the egg-cell; but it is a very unequal division, for these 'directive cells' or 'polar bodies' are always much smaller than the ovum, and indeed are usually so small that it is easy to understand why their cellular nature was for so long overlooked. Yet they have always a cell-body, and in many ova, for instance those of certain marine Nudibranchs, this is quite considerable; and they have likewise always a nucleus, which, notwithstanding the smallness of the cell-body, is in all cases exactly of the same size as the sister nucleus which remains behind in the ovum after division--a fact which is in itself enough to indicate that we have here to do essentially with readjustments and changes in the nucleus of the ovum.

Long before the polar or directive divisions were recognized as divisions of the egg-cell it was known that the nucleus of the ovum disappeared as soon as the latter attained to its full size within the ovary. It was also known that this nucleus--the large so-called 'germinal vesicle' lying in the middle of the ovum--left its central position and moved to the upper surface of the ovum, there to become paler and paler, and ultimately to disappear altogether from the sight of the observer. By many it was believed that it broke up, and that the 'segmentation nucleus,' which is afterwards obvious, is a new formation. The truth is that the germinal vesicle, at the time of its disappearance, is transformed into a division figure which is invisible without the aid of artificial staining. The nuclear membrane breaks up; the centrosome of the ovum, which, although hardly visible, had previously lain beside the germinal vesicle, divides into two centrosomes and their centrospheres, and these now form the 'mitotic figure' by moving away from each other and sending out their protoplasmic rays. This nuclear spindle soon ranges itself at right angles to the surface of the egg, which at the same time arches itself into a protuberance, and soon two daughter-nuclei are formed, one of them lying within the protuberance (Fig. 75, _A_, _Rk1_). This soon separates itself off from the ovum, surrounded by a small quantity of cell-substance. The other daughter-nucleus remains within the ovum, but neither of them remains in a state of rest; both are again transformed into a spindle and divide once more; the minute first 'polar body' dividing into two 'secondary polar bodies' of half the size (_B_, _Rk1_), while the nuclear spindle within the egg brings about a second division of the ovum (_B_, _Rk2_) whose unequal products are the second polar cell and the mature ovum--that is, the ovum ready for fertilization. The process is now complete; the egg-cell, which has lost very little plasmic material through the 'polar bodies' and has not become visibly smaller, has now a nucleus (_B_, _Eik_) which has become considerably smaller through the two rapidly successive divisions, and, as we shall see later, has also undergone internal changes. In this state it is 'ripe,' that is, it is ready to enter into conjugation with the nucleus of a male cell, and this we have already recognized as the essential element in the process of fertilization.