Chapter XIX: THE GERM-PLASM THEORY (continued) 392 (14)

The comparatively small number is explained by the certainty with which each of them reaches the ovum, and the large size may be accounted for in part by the small number which suffices, and which, therefore, admits of the male cell also carrying a considerable portion of the material for the building up of the embryo. Probably, however, the thickness and firmness of the covering of the ovum has something to do with it, for it has no opening for the entrance of the male cell, and it is fully hardened by the time fertilization takes place. Perhaps nowhere can we see more clearly how every little detail of the structure of the organism is dominated by the principle of adaptation than in the arrangements for fertilization, and notably in those which obtain in the Ostracods. I pass by the complicated apparatus for copulation, since we do not yet understand it fully in all particulars. According to my own investigations and those of my former students, Dr. Stuhlmann and Dr. Schwarz, the essential point seems to be that the colossally large zoosperms, which show no activity within the body of the male, leave it one at a time, so to speak, in single file. In copulation they are pressed out singly, one after the other, through a very fine tube, and then they enter, still singly, through the reproductive aperture of the female into an equally fine passage with spiral windings, through which they ultimately reach a roomy pear-shaped receptacle, the 'receptaculum seminis' of the female. There they lie in a long band composed of several hundreds, and only now attain their full maturity by throwing off an outer cuticle--moulting, so to speak. It is only when they get into a fluid medium that they show the power of undulatory movement, feeble at first, but gradually more energetic and more violent. And these movements enable them to penetrate like gimlets into the calcareous egg-shell. In the normal course it happens that when a mature ovum is deposited from the opening of the oviduct, one of the giant zoosperms at the same time, or shortly afterwards, leaves the 'receptaculum seminis' of the female by way of the spiral passage, and reaches the exterior just behind the ovum. The actual process of penetration has not been observed as yet, but the zoosperm has been seen at a slightly later stage spirally coiled inside the ovum.

In these Ostracods the sperms are often visible with the naked eye, and in some species they are twice the length of the animal; they are thus emphatically giant cells, which can develop a very considerable boring power.

In respect to the various adaptations of the sperm-cells to the conditions of fertilization there is hardly any group more interesting than the water-fleas or Daphnids.

It is amazing how greatly the size of the sperms varies among the Daphnids, and how it stands in inverse proportion to their number, and how both are obviously regulated in relation to the difficulties which stand in the way of each sperm-cell before it can reach the ovum. In some species the sperm-cells are very large, in others extremely small. In the genera _Daphnia_, _Lynceus_, and others, copulation occurs as shown in Fig. 65; the sperm-cells (_sp_) are liberated by the male into the capacious brood-cavity of the female, which at the moment is closed to some extent by the abdomen of the male, in reality closed only partially at the posterior end and at the sides. It seems inevitable that a large proportion of the male elements should stream out again and be lost because of the violent movements of both animals. Accordingly, we find that the sperm-cells are only about the hundredth part of a millimetre in length and of round or rod-like form, and are voided in multitudes into the brood-cavity. Fig. 66, _f_, _g_, and _h_, show such cells in different species, as they occur in the testes to the number of many thousands. But in all the species in which the brood-cavity is _closed_, and in which therefore there is not such a serious loss of sperm-cells, the elements are much larger, and at the same time less numerous. They are largest and least numerous in species of genera like _Daphnella_, _Polyphemus_, and _Bythotrephes_, in which the males have a copulatory organ, so that the possibility of loss of the male cells is excluded. Thus the round, delicate, and viscid sperm-cells of _Bythotrephes_ (Fig. 66, _b_) are more than a tenth of a millimetre in length, but they are developed in proportionately smaller numbers, so that more than twenty are never found in the testis, and often only six or eight, while in copulation only from three to five are ejected. But as there are only two eggs to be fertilized at a time, and as the male cells are expressed into the brood-cavity directly upon the eggs, so that they immediately adhere to them, this small number is amply sufficient.

It is remarkable how different the sperm-cells sometimes are in quite nearly related species of Daphnids, as a glance at Fig. 66 will show; and, on the other hand, how similar they may be in two species which belong to different families, like _Bythotrephes longimanus_ (_b_), and _Daphnella hyalina_ (_c_). The last fact may be explained as an adaptation to similar conditions of fertilization. Both species have effective copulatory organs, and their large delicate sperm-cells must immediately adhere when they come into contact with the shell-less ovum, and penetrate into it by means of amœboid processes. Conversely, the difference between sperm-cells of allied species like _Sida crystallina_ (_a_), _Moina rectirostris_ (_e_) and _M. paradoxa_ (_d_) is related to different adaptations to nearly the same conditions of fertilization. In _Sida_ (Fig. 66 _a_) the large flat sperm-cells, with their fringed ends and their large soft surface, adhere easily to the ova, and the same end is attained in _Moina rectirostris_ by means of stiff radiating processes, while in the nearly related species, _Moina paradoxa_, the male cell (_d_) resembles an Australian boomerang and presses in like a wedge between the ova and the wall of the brood-sac.

In Fig. 67 a small selection of animal male cells is figured, all of which take the form of sperm-threads or spermatozoa, and yet they differ very much from one another in detail. It would undoubtedly be of great interest to follow out these minute adaptations of the sperm-cells to the conditions of fertilization, and to demonstrate that their size, and especially their form, in the different species of animals are adjusted to the special constitution of the ovum, its envelope, and its micropyles, and to the ease or difficulty with which it can be reached; but much information must be forthcoming before we can even suggest, for instance, why the sperm-cell of the salamander is so enormously long, large, and pointed at the head, while that of Man (Fig. 67, 1) is comparatively short, with broad, flat head and a recently discovered minute apex; or why those of Man and many fishes (such as _Cobitis_) should be so much alike, and so on. From many sides, however, we are led to conclude that even down to the minutest details nothing is in vain, and that everything depends on adaptation.

In general, even the peculiarities of form already indicate this; thus the spirally coiled structure of the head, which is especially well developed in the spermatozoa of birds (Fig. 67, 5), in those of the skate (7), and of the freshwater snail (_Paludina_) (10), works like a corkscrew, and makes it possible for the spermatozoon to pierce through the resistant envelope of the ovum. Similarly, the sharply pointed head of the insect spermatozoon (Fig. 67, 8 & 9) seems adapted for slipping through the minute pre-formed micropyle in the hard egg-shell.

Of the detailed and complicated structure of spermatozoa we have only recently been made aware through the increasing perfection of the microscope and of technical methods of investigation. Fig. 68 shows one after a diagrammatic figure by Wilson. We see the apical point (_sp_) for boring into the ovum, the nucleus (_n_) surrounded by a thin layer of protoplasm, which together form the head, then the middle portion (_m_) which contains the 'centrosome' (_c_), and the 'tail' or 'flagellum' which effects the movement of the whole and which itself possesses a complex structure with an 'axial filament' (_ax_) and an enveloping layer, the latter often drawn out into a spirally twisted, undulating membrane of the most extreme delicacy, as is most clearly seen in the newt (Fig. 67, 6).

Not in the Daphnids alone, but in other groups of Crustaceans as well, sperm-cells of quite peculiar form occur, as, for instance, in the crayfish and its marine relatives, the crabs and the long-tailed Decapods. In these cases the spermatozoa bear long and stiff thorn-like processes, which, as in the sperm-cells of _Moina_, make them adhesive, and, according to Brandes, render it possible for them to cling among the bristles on the abdomen of the female until one of the many eggs leaving the oviduct comes within reach. For among these Crustacea there is no true copulation, but the masses of sperm-cells are packed together into sperm-packets or 'spermatophores,' and are affixed by the male near the opening of the oviduct, where they burst and pour forth their contents between the appendages of the female.

All these remarkable and widely divergent structures and arrangements depend not upon chance or on the fantastic expression of a 'formative power,' as an earlier generation was wont to phrase it; they are undoubtedly without exception adaptations to the most intimate conditions of fertilization in each individual case. I lay particular stress upon a recognition of this, because it permits us to infer with certainty that even the variations of the single cell, if they are sufficiently important for the species, may be controlled by natural selection. It is obvious that the adaptations of the sex-cells must depend not on histonal selection, but only upon personal selection, since it is indifferent for the individual sperm-cells whether fertilization is accomplished successfully or not, while it is by no means indifferent for the species. The organism dies without descendants if its sperm-cells do not fertilize, and the carrying on of the species must be left to those of its fellows which produced sperm-cells which fertilize with more certainty; thus it is not the sperm-cells themselves, but the individual organisms which are selected, and that in relation to the quality of the sex-cells they produce.

In contrast with the great diversity of form exhibited by the spermatozoa, the differentiation of the ovum appears very uniform, at least in regard to form and activity. The main form is spherical, but it is subject to many variations in the way of elongation or flattening. In the lower forms of life, as, for instance, among the sponges, and also in the polyps and Medusæ the egg-cells possess, until they are mature, the locomotor capacity of unicellular organisms; they creep about after the manner of amœbæ, and indeed, as I showed years ago, this movement from place to place in many polyps is exactly regulated; thus at a definite time they may leave the place where they originated and may, for instance, creep from the outer layer of cells (ectoderm) of the animal into the inner layer (endoderm) by boring through the so-called 'supporting lamella,' then they may creep further in the endoderm, and finally return to quite definite and often remote spots in the ectoderm (_Eudendrium_, Fig. 95). In another hydroid polyp (_Corydendrium parasiticum_) the mature egg-cells leave their former position within the endoderm and creep entirely outside of the animal which produced them, establishing themselves in a definite spot on its external surface, where they await the fertilizing zoosperms. Many ova can accomplish slight amœboid movements, but in most animals these do not suffice for movement from place to place, and the ova remain quietly in the spot where they were developed, or are passively pushed to another. Cases such as that of the polyp I have cited, in which the ovum actually comes to meet the male element, are quite exceptional, for in general the ovum is the passive and the spermatozoon the active or exploring element in fertilization. The female cell is entrusted with procuring and storing the material necessary to the building up of the embryo; and its peculiarities depend chiefly on this.

It is true that in plants this stored material is seldom considerable, and that is because the ovum so frequently remains even after fertilization within the living tissues of the plant, and is thence supplied, often very abundantly, with food-stuffs; and, moreover, because the young plant that springs from the fertilized ovum maybe very small and simple, and yet capable of immediately procuring its own nourishment. But there are exceptions to this; thus the ova of the brown sea-wracks, or Fucaceæ, for instance, are quite twenty times as large as the ordinary cells of the algæ (Fig. 64), and contain a quantity of food-stuff within themselves. In this case the ova are liberated into the water even before fertilization, and the nutrition of the embryo from the mother-plant is excluded.

In these Algæ we meet, for the first time, with a special organ in which the ova arise. In animals this is much more generally the case, and from sponges upwards there are always quite definite parts and tissues of the body which are alone able to develop eggs, and these are usually well-defined organs of special structure, the ovaries. Similarly, in male animals the spermatozoa arise in special places, and usually in special organs, the spermaries or testes.

Animal ova often consist of more than the simple cell-body, the protoplasm and its nucleus; they almost always contain in the cell-body a so-called 'Deutoplasm,' as Van Beneden has fittingly named the yolk-substance. This consists of fats, carbohydrates, or albuminoids, which often lie in the cell-body in the form of spherules, flakes, or grains--a nutritive material that is often surrounded and enclosed by a small quantity of living matter or formative protoplasm. Apart from these stores of yolk it would be impossible for a young animal to develop from the ovum of a snake or a bird, for such highly differentiated animals could not be formed from an egg of microscopic dimensions if this remained without some supply of food from outside of itself during the period of development. There is obviously need for a considerable amount of building material, so that all the organs and parts, which are composed of thousands and millions of cells, may be developed.

Thus the size of the animal-ovum depends essentially on the quantity of yolk that has to be supplied to the egg, and this depends in the main on whether the egg is still drawing nourishment from the mother during the development of the young animal. Therefore, as a general rule, eggs which are laid, and are surrounded and protected by a shell, are usually much larger than the eggs of animals which go through their development within the body of the mother. The best known illustration of this proposition is offered by mammals and birds, animals of equally high organization and comparable in bodily size. While the eggs of birds may be as much as 15 centimetres in length, and may weigh 1½ kilogrammes, those of most mammals remain microscopically minute, and scarcely exceed a length of 0.3 millimetres. The same principle is often illustrated within one and the same small group of animals, and even in the same species. Here, again, the Daphnids or water-fleas may serve as an example.

Among these there are two kinds of eggs, summer and winter eggs, of which the former go through their development into a young animal within a brood-cavity on the back of the female, while the others are liberated into the water, and are surrounded by a hard shell. The summer eggs receive more or less nourishment from the mother by the extravasation of the nutritive constituents of the blood into the brood-cavity, and they thus require a smaller provision of yolk than the winter eggs, which are thrown entirely upon their own resources. Accordingly we find that in all Daphnids the summer eggs are at least a little smaller and have less yolk than the winter eggs, as in the genus _Daphnella_ (Fig. 70, _A_ and _B_), while in some species, e.g. of _Bythotrephes_, this difference increases so much that the summer eggs are almost without yolk, and therefore very minute (Fig. 71, _B_). The reason of this lies in the fact that in this case the brood-sac is filled with a nutritive fluid rich in albuminoid substances, so that the embryo during its development is continually supplied with concentrated nourishment. This is not the case with the winter eggs, because these are liberated into the water, and we therefore find that they are of enormous size and quite filled with yolk (Fig. 71, _A_).

In this instance, as in all the simpler eggs, the yolk constituents are secretions of the cell-body of the ovum; but nature employs many devices, if I may so speak, to bring up the mass of the egg, and especially of the yolk, to the highest attainable point. Thus in many orders of Crustaceans, for instance in the water-fleas just mentioned, there are special egg-nourishing cells, that is, young ovum-cells which do not differ from the rest either in origin or in appearance, only they do not become mature eggs, but at a definite time cease to make progress, and then slowly break up, so that their substance may be absorbed as food by the true ova. Thus there is a much greater and at the same time more rapid growth than could be attained through nourishment from the blood alone. In the Daphnids the ovaries consist of groups of four cells each, only one of which becomes an ovum (Fig. 72, _Ei_), while the other three (1, 2, and 4) form nutritive cells which break up. This is so in all summer eggs; but in the winter eggs a much larger number of nutritive cells may take part in equipping a single ovum, and in the genus _Moina_ over forty do so. But here the difference in size between the two kinds of eggs is very marked, the winter eggs being twice the diameter of the summer eggs.

In many insects also, e.g. in beetles and bees, similar nutritive cells occur, but there is in these forms a different arrangement which serves at the same time for the formation of the shell, and the supplying to the ovum of the necessary yolk-stuffs--the ovum is surrounded with a dense layer of epithelial cells, a so-called 'follicle.' In mammals and birds also these 'follicle cells' certainly play an important part in the nutrition of the ovum, though it is not yet quite clear how they act--whether they produce within themselves grains of yolk and other nutritive substances and convey these to the ovum by means of fine radiating processes, or whether they themselves ultimately migrate into the ovum and there break up. In any case it is worthy of note that all these follicular cells in insects and vertebrates have the same origin as the egg-cells, that is, they are modified germ-cells. The case is therefore essentially the same as in the nutritive cells of the Daphnids; nature sacrifices the greater number of the germ-cells in order to be able to provide more abundantly for the minority. She thus succeeds in raising the egg beyond itself, so to speak, and provides the means for a growth which could obviously not be attained by means of the ordinary nourishment supplied by the blood.

We now understand why the eggs of many animals should be of such enormous size and often of such complex structure. The eggs of birds are especially remarkable in this respect, and it has till recently been disputed whether they are really morphologically equivalent to a single cell. But this is undoubtedly the case, and though only the small thin germinal disk (Fig. 73, _Bl_) with its nucleus is the active part of this cell--the cell-body proper--yet all the rest--the enormous sphere of yolk with its regular layers of yellow (_GD_) and white (_WD_) yolk, the concentric layers of fluid albumen (_EW_) round about this, the chalazæ (_Ch_), and finally, the delicate shell membrane (_S_) and the limy shell (_KS_)--belong to this cell, and have arisen in connexion with it (Fig. 73).

LECTURE XV

THE PROCESS OF FERTILIZATION

Cell-division and nuclear division--The chromatin as the material basis of inheritance--The rôle of the centrosphere in the mechanism of division--The Chromosomes--Fertilization of the egg of the sea-urchin according to Hertwig--Of the egg of Ascaris according to Van Beneden--The directive divisions, or the extrusion of the polar bodies--Halving of the number of chromosomes--The same in the sperm-cell--Reducing division in parthenogenetic eggs--In the bee--Exceptional and artificial parthenogenesis--Rôle of the centrosphere in fertilization and in parthenogenesis.

NOW that we have made ourselves acquainted with the two kinds of germ-cells on the union of which 'sexual reproduction' depends, we may proceed to a more detailed discussion of the process of fertilization itself. But it is indispensable that we should take account of the processes of nuclear and cell-division, as these have been gradually recognized and understood in the course of the last decade. It may appear strange that the processes of division should throw light on the apparently opposite processes of cell-union, but it is the case, and no understanding of the latter is possible without a knowledge of the former.

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.

These processes of 'maturation of the ovum' are common to all animal ova which require fertilization, and they follow almost the same course, only that in many cases the second division of the first polar body does not take place, so that only two polar bodies in all are formed. All these processes have nothing directly to do with fertilization, but it is only through them that the ovum becomes capable of fertilization. This does not prevent the spermatozoon from previously making its way into the ovum, for this is usually the case (Fig. 75, _A_, _sp_); there it waits until the second 'directive division' of the ovum has been accomplished, utilizing the time to become transformed in the manner necessary for the conjugation of the two nuclei. Only in a few species, for example in the sea-urchin, does the egg complete its polar divisions within the ovary, therefore before it has come into contact with the sperm at all.

That we may be able to penetrate still more deeply into the processes of fertilization, the best illustration to take seems to me to be, as yet, the ovum of the thread-worm of the horse (_Ascaris_ _megalocephala_), which has become famous through the classical observations of Ed. van Beneden. Many favourable circumstances unite in this case to make the essentials of the process clearly recognizable. Fertilization takes place within the body of the female, in an enlarged portion of the oviduct, within which a number of the remarkable sperm-cells are always found in a mature female. They are remarkable in being not thread-like, but rather spheroidal cells, bearing, however, a small protuberance something like a pointed horn (Fig. 75, _A_, _sp_). When such a sperm-cell comes in contact with the upper surface of an ovum a swelling forms at the place touched, and the sperm-cell attaches itself firmly to this, and is drawn by it into the ovum. Without doubt, amœboid movements on the part of the sperm-cell itself play some part in this, as can be most plainly seen in the large sperm-cells of many Daphnids which we have already discussed. In the egg of the thread-worm the whole sperm-cell with its nucleus can soon be detected within the substance of the ovum, and it then changes rapidly. Its main body fades more and more completely, until at last it disappears altogether, while the nucleus becomes vesicle-like and soon attains a considerable size (Fig. 75, _B_, _spk_). Meanwhile the residue of the germinal vesicle which remained behind in the ovum after the second directive division (_B_, _Eik_) has changed into a large vesicle-like nucleus (_C_, ♀ _k_), which in the ovum of _Ascaris_, as well as in the spermatozoon, at first contains a nuclear reticulum with irregular fragments of chromatin. Later on, these form a spiral coil in the manner we have already described, and finally this breaks up into two large and relatively thick angular loops or chromosomes (Fig. 75, _C_ and _D_, _chr_).

At the same time a nuclear division apparatus has formed in the space between the two nuclei--the so-called male and female 'pronuclei' (♂ _k_, ♀ _k_)--two centrospheres (_csph_) become visible, at first lying close together, but afterwards moving apart (_D_) to form the poles of a nuclear spindle, in the equatorial plane of which the four chromosomes of the male and female pronuclei are now arranged. The nuclear membranes disappear, and the two nuclei now unite to form one, the segmentation nucleus (_D_). A dividing spindle then develops and brings about the first embryonic cell-division (_E_), and thus the beginning of the 'segmentation' of the ovum; each of the four chromatin loops splits longitudinally, and each of the split halves migrates, one to one, the other to the other daughter-nucleus (_F_). As this same method of distribution of the chromatin substance is repeated at every successive cell-division throughout embryogenesis, and indeed through the whole of development, it follows that the result of fertilization is, that all the cells of the body of the new animal which develops from the ovum contain an equal quantity of paternal and of maternal chromatin. If we are right in regarding the chromatin substance as the hereditary substance, it becomes immediately apparent that this equal division is of the most far-reaching importance, for it shows us that the so-called process of fertilization is the union of equal quantities of hereditary substance of paternal and maternal origin.

The process of fertilization is now known in all its details in a great number of animals in the most diverse groups; it is everywhere the same in its essential features; there is always only one sperm-cell which normally enters into conjugation with the ovum-nucleus, and in every case the sperm-cell, however minute it may be to begin with, forms a nucleus nearly or exactly as large as the nucleus of the ovum, and in all cases it contains the same number of chromosomes as the nucleus of the ovum. Of special interest, however, is the fact that this number is always half the number of the chromosomes exhibited by the somatic cells of the particular animal in question, and that the reduction of the number of chromosomes to half the normal is effected in both male and female germ-cells by the last divisions of these cells, which take place before they have attained to a state of maturity. In the ovum the reduction occurs in the directive divisions, to which we must therefore turn our attention once more, with special reference to the number of chromosomes.

We saw that, in the full-grown ovarian egg, the germinal vesicle rises to the surface and there becomes transformed into the first polar spindle. Now this shows, in its equatorial plane, double the number of chromosomes normal to the species. This duplication comes about, not directly before the nuclear division, but much earlier in the young mother-egg-cell; it is only the change in the time of the splitting of the chromosomes that is unusual. The first maturation division takes place nevertheless in accordance with the usual plan of nuclear division; it is, as I have called it, an 'equation division,' that is, both daughter-nuclei again receive the same number of chromosomes as the young mother-egg-cell had to start with, namely, the normal number of the species. Thus, if the young mother-egg-cell had four chromosomes (Fig. 76, _A_), this number would double to eight at an early stage (_B_), but the first maturing division would give each daughter-nucleus four (_C_ and _D_). In the second maturation division the case is different, for here no splitting and duplicating of the number of chromosomes takes place, but the existing number, by being distributed between the two daughter-nuclei, is reduced to half in each (_E_ and _F_). For this reason I have called it a 'reducing division.' In our example, therefore, the ovum, as well as the second polar body, would contain only two chromosomes (Fig. 76, _F_).

I cannot enter into the details of the process here, for we are dealing with essentials and not with isolated and, so to speak, chance details, but I must emphasize the fact that the same process of reduction of the number of chromosomes takes place in this or an analogous manner in all animal ova, and can be demonstrated also in most of the chief groups of plants. Whether it be, as many have maintained, that the reduction is not always first effected by the 'maturation divisions,' but in some cases takes place earlier in the primitive egg-cell[12], so much is certain, that the nuclei which come together for 'fertilization' only contain half the normal number of chromosomes, and this is true not only of the ovum but also of the sperm-nucleus.

[12] See the discussion of this point in chapter xxii.

Arguing from general considerations, but especially from the theory which regards the chromosomes as the bearers of the hereditary substance, I had come to the conclusion, before there was any full knowledge of the phenomena of the maturation of the ovum, that a reduction of the chromosomes by half _must_ take place, and had postulated a similar 'reducing division' for the sperm-cell, and further, for plants as well as animals--indeed, for all sexually reproducing forms of life. The two divisions in the sperm-cell corresponding to the polar divisions of the ovum with their reduction of chromosomes were demonstrated by Oscar Hertwig in the case of the thread-worm of the horse (_Ascaris megalocephala_)--a form which has proved so very important in relation to the whole theory of fertilization. It is true that in this case the course of the phenomena of reduction is less convincing than in some other forms which have been investigated more recently, as, for instance, the mole-cricket and the bugs. In these instances, at any rate, a 'reducing division' in spermatogenesis, quite corresponding to that of the egg-cell, has been demonstrated, and this demonstration is of particular value owing to the fact that the development of the sperm-cell, as we shall presently see, throws an entirely new light on that of the ovum, and especially on the phyletic significance of the polar bodies.