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.

Nowthat 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 wasthe 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 nameIdioplasm, 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.

Fig. 74.Diagram of nuclear division, adapted from E. B. Wilson.A, resting cell with cell-substance (zk), centrosphere (csph) which contains two centrosomes, nucleolus (kk); and chromosomes (chr), the last distributed in the nuclear reticulum.B, the chromatin united in a coiled thread; the centrosphere divided into two and giving off rays which unite the halves.C, the nuclear spindle (ksp) formed, the rays more strongly developed, the nuclear membrane (km) in process of dissolution, the chromatin thread divided into eight similar pieces (chrs), the rays are attaching themselves to the chromosomes.D, perfected nuclear spindle with the two centrospheres at the poles (csph) and the eight chromosomes (chrs) in the equator of the spindle, all now longitudinally split.E, daughter-chromosomes diverging from one another, but still united by filaments, the centrosomes (cs) are already doubled for the next division.F, daughter-chromosomes, quite separated from one another, are already beginning to give off processes; the cell-substance is beginning to be constricted.G, end of the process of division: two daughter-cells (tz) with similar nuclear reticulum (tk) and centrospheres (csph), as inA.

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 themselvesinto 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,AandB,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 (AandBcsph), 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.DandF). 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 (FandG) 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. Inany 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 inArtemia, 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.FandG). 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 thecell, 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 isa 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, fromthe 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 substanceof the highest importancehad 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, especiallyAuerbach, 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 invisiblewithout 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.

Fig. 75.Process of fertilization inAscaris megalocephala, the thread-worm of the horse, adapted from Boveri and Van Beneden.A, ovum in process of the first directive division;Rk1, first polar body;sp, spermatozoon with two chromosomes in its nucleus, attaching itself to the ovum, and about to penetrate into it; a protrusion of the egg-protoplasm is meeting it.B, the second directive division has been completed;Rk2, the second polar body;Eik, the reduced nucleus of the ovum. The first polar body (Rk1) has divided into two daughter-cells,spk; the nucleus of the spermatozoon remains visible with its two centrospheres (csph).C, the sperm nucleus (♂k) and the ovum nucleus (♀k) have grown, each has two loop-like chromosomes; only the male nucleus has a centrosphere, which has already divided into two (csph).D, the two nuclei lie apposed between the poles of the nuclear spindle.E, the four chromosomes have split longitudinally; the spindle for the first division of the ovum (the segmentation spindle,fsp) has been formed.F, divergence of the daughter-chromosomes towards the two poles; division of the ovum into the first two cleavage cells or embryonic cells.

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 (Ascarismegalocephala), 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 anenlarged 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 ofAscaris, 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,CandD,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 apparentthat 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 (CandD). 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 (EandF). 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).

Fig. 76.Diagram of the maturation divisions of the ovum.A, primitive germ-cell.B, mother-egg-cell, which has grown and has doubled the number of its chromosomes.C, first maturation division.D, immediately thereafter;Rk1, the first directive cell or polar body.E, the second maturation spindle has been formed; the first polar body has divided into two (2 and 3); the four chromosomes remaining in the ovum lie in the second directive spindle.F, immediately after the second maturation division; 1, the mature ovum; 2, 3, and 4, the three polar cells, each of these four cells containing two chromosomes.

I cannot enter into the details of the process here, for we aredealing 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 fullknowledge of the phenomena of the maturation of the ovum, that a reduction of the chromosomes by halfmusttake 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.

We began our consideration of the processes of reduction with the full-grown egg-cell, but now let us go back to the earliest rudiments of the ovary of the embryo, and we find that it consists of a single primitive egg-cell, from which, by division, all the other egg-cells arise. In the same way the first rudiment in the testis or spermary is formed by a primitive sperm-cell, which does not differ visibly from the primitive egg-cell. Both now multiply by division for a considerable time, and in the ovary this is followed by a period of growth, during which multiplication ceases, and each cell increases considerably in size and lays in a store of yolk. Each cell thus ultimately reaches the condition with which we started previously, that of the full-grownmother-egg-cell.

Although the primitive sperm-cells do not exhibit such pronounced growth as the ova, they have likewise their period of growth, during which multiplication by division ceases, and the cells increase only in size (Fig. 77,A). When they have attained their maximum of growth the number of chromosomes is seen to have been doubled by longitudinal splitting (as in the diagram, Fig. 77,B, from four to eight). From thismother-sperm-cellthere now arise by two divisions in rapid succession (C-F) four sperm-cells, and the same reduction of the number of chromosomes to half is effected as in the polar divisions of the egg-cell. In the first division, four chromosomes go to each daughter-cell (D), in the second, two (F). The only essential difference between the corresponding processes in the egg-cell and the sperm-cell lies in the fact that the divisions of the so-called 'spermatocyte' or mother-sperm-cell are equal, so that four granddaughter-cells of equal size arise, while in the mother-egg-cell or 'ovocyte' the divisions are very unequal. In the former the result of the divisions isfourcells capable of fertilizing, in the latteronecell capable of being fertilized and three minute 'polar cells' which are incapable of conjugating with a sperm-cell and giving rise to a new individual.

Fig. 77.Diagram of the maturation-divisions of the sperm-cell, adapted from O. Hertwig.A, primitive sperm-cell.B, mother-sperm-cell.C, first maturation division.D1 and 2, the two daughter-cells.E, the second maturation division, by which the four cells ofFarise, each with half the number of chromosomes.

There can thus be no doubt that the polar cells, as Mark and Bütschli long ago suggested, are abortive ova, that is, that, at a remote period in the evolution of animal life, each of these four descendants of a mother-egg-cell became a germ-cell capable of development. It is not difficult to infer that the unequal division, which now leads to an insufficient size in three of these descendants, has gone onpari passuwith the continually increasing size of the mature ovum, and had its reason in the fact that it was important above all things to store in the ovum as much protoplasm and yolk as possible. We have already seen that even the dissolution of a number of the sister-cells of the ovum is sometimes demanded, so that the ovum may be surrounded by nutritive follicular cells. In short, the greatest possible quantity of nourishment is conveyed to theovum in every conceivable way, and it is thus stimulated to a growth which no single cell could attain to if it were dependent on the ordinary nutrition supplied by the blood. And we can understand that nature—to speak metaphorically—did not wish to destroy her own work by finally distributing among four ova all the nourishment she had succeeded in heaping up in all sorts of ways within the mother-egg-cell.

But it may be asked, Why have all these unnecessary divisions been maintained up till the present day? Why have they not long ago been given up, since they can and do only lead to the production of three abortive ova, which are foredoomed to perish? Are they mere vestiges, processes which are in themselves meaningless, but have, so to speak, been maintained by the principle of inertia? This principle is certainly operative in some sense and to some extent even in living nature; a process which has been regularly repeated through a long series of generations does not at once cease to be performed when it is no longer of use to the organism concerned. The eyes of animals which have migrated to lightless depths do not disappear all at once and leave no trace; they degenerate very gradually and only in the course of many generations; and it would thus be quite possible to defend the position that these polar or 'maturation divisions' of the ovum are purelyphyletic reminiscenceswithout actual significance.

But I cannot agree with this opinion. If it were actually so we should expect that the formation of the polar bodies would not still take place in all cases in almost the same manner, for all rudimentary parts and processes vary greatly; we should expect that in many animal groups the polar divisions would not occur, or perhaps that only half the number would occur. But this is not so; in all multicellular organisms, from the lowest to the highest, two reducing divisions take place, and always in almost the same manner, with the exception of a single category of ova, of which I shall presently have to speak. We shall see later that even in unicellular organisms analogous processes may be observed.

But it is also intelligible that this twice repeated division of the mother-egg-cell is necessary if the reduction in the number of chromosomes to half is only possible in this way, sincethis reduction is indispensable. If each of the two conjugating germ-cells contained the full normal number of chromosomes, the segmentation-nucleus would contain a double number, and if that went on, the number of chromosomes would increase in arithmetical proportion from generation to generation, and would soon become enormous. Even though we were not otherwise certain that these chromosomes are units of apermanent nature, which only apparently break up in the nuclear reticulum, but in reality persist, the fact of reduction would point in this direction. For if they were not permanent structures and distinct from one another, and if their number depended solely on the quantity of chromatin which the nucleus contains, the reduction in number might be secured if the chromosomes in the growing egg and sperm-cells increased in size more slowly than the cell-body and the other parts of the cell. But from the fact that the reduction takes place not in this simple way, but, in sperm-cells and in ova which require to be fertilized, only through cell-division and a specific mode of nuclear division, we may conclude that it cannot happen otherwise, that chromosomes are not mere aggregates of organic substance, but organs whose number can only be reduced by the extrusion of some of them from the cell.

It is true that there are ova in which the process of reduction does not follow the course we have described, but the exceptions only serve to confirm our view of the reducing significance of the polar divisions, and of their persistence because of the necessity for reduction.

As far back as the middle of the nineteenth century it was known that in various animals the eggs develop without fertilization. This reproduction by 'parthenogenesis' was first established with certainty by the German bee-keeper Dzierzon in 1845, and then scientifically corroborated by Rudolph Leuckart and C. Th. von Siebold. Although parthenogenesis was at first observed only in a few groups of the animal kingdom, in bees and some nocturnal Lepidoptera (Psychidæ and Tineidæ), it has become more and more apparent in the course of years that this 'virgin reproduction' is by no means a rare form of reproduction, and that it occurs regularly and normally in many cases, especially in the very diverse groups of the great series of Arthropoda. Thus among insects it is found in certain saw-flies, gall-flies, ichneumon-flies, in the honey bee, and in common wasps, and it is particularly widespread among plant-lice (Aphides) such as the vine-aphis (Phylloxera), whose prodigious multiplication in a short time depends partly on the fact that all the generations, with the exception of one, consist only of females with a parthenogenetic mode of reproduction.

Among the lower Crustaceans also parthenogenesis plays a large rôle, and in many species it even occurs as the sole mode of reproduction, but more often—as is also the case among insects—it occurs alternately with bi-sexual reproduction. For parthenogenesis must not be regarded as asexual reproduction, but rather asunisexual, that is,as arising from sexually differentiated individuals (females), and from germ-cells (true ova), but brought about by the agency of individuals of only one sex, the female. These parthenogenetic eggs emancipate themselves, so to speak, from the law that was previously regarded as without exception, that all ova require fertilization to enable them to develop. That this law admits of many exceptions is now universally admitted; thus in the small family of water-fleas (Daphnids) there are even two kinds of eggs, the summer and winter eggs we have already mentioned, which are produced by the same female, and yet the former kind develop without fertilization, while the latter require to be fertilized before they can develop.

It was obviously important to learn the state of affairs in regard to reducing divisions in parthenogenetic ova, to find out whether here also, three, or, in some circumstances, two polar bodies were formed, and whether the second polar division reduced the number of chromosomes to half. If the theory previously advanced as to the importance of the chromatin, and especially of the reducing effect of the second maturing division be correct, we should expect the second division to be wanting in parthenogenetic eggs, since otherwise the number of chromosomes would be reduced to half in each generation, and would thus gradually disappear or sink to one.

Having directed my attention to this problem, I succeeded in establishing for a Daphnid,Polyphemus, that the second polar division does not occur, and that only one polar body is formed. Blochmann found the same in the parthenogenetic eggs of plant-lice or Aphides, in which, moreover, the eggs requiring fertilization exhibit, like the winter eggs of Daphnids, two polar divisions. It was thus established that at least those eggs of Aphides and Daphnids which are wholly parthenogenetic retain the full number of chromosomes of their species, as is represented in the diagram,Fig. 78. When parthenogenesis set in the polar divisions were limited to one, and that this could happen justifies us in concludinga posteriorithat it could have happened also in the case of ova which required fertilization if that had been necessary or even merely indifferent. The polar divisions are thus not mere 'vestigial' processes; they have an immediate significance, and it lies in the reduction of the number of chromosomes.

But I must make a reservation here; it is not universally true of parthenogenetic eggs that maturation takes place without the second polar division. The first exception was observed in the salt-water crustacean,Artemia salina. In this case only one polar body is actually extruded and the number of chromosomes remains normal,as I was able to demonstrate with the small number of ova at my disposal; but according to the investigations of Brauer on more abundant material it appears that, while the second polar division is suppressed in the majority of the ova, and the external extrusion of a second polar body never occurs, the second polar division does nevertheless sometimes take place. The two daughter-nuclei arising from this division unite again immediately afterwards to form a single nucleus, and this now functions as a segmentation nucleus. Of course it again contains the full number of chromosomes, namely, twice 84=168.

InArtemia, therefore, the adaptation of the ova to parthenogenetic development is not yet fully established, and the complete abandonment of the second polar division seems to be phyletically striven for, since, although the division still takes place, its effect is neutralized immediately afterwards.

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