Chapter XIX: THE GERM-PLASM THEORY (continued) 392 (15)

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-grown _mother-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 this _mother-sperm-cell_ there 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 is _four_ cells capable of fertilizing, in the latter _one_ cell 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.

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 on _pari passu_ with 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 the ovum 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 purely _phyletic reminiscences_ without 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, since _this 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 a permanent 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 as _unisexual_, 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 concluding _a posteriori_ that 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.

In _Artemia_, 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.

Among bees the state of affairs is again exceptional. Here the female, the so-called queen bee, possesses a capacious sperm-sac, in which the spermatozoa received in copulation remain living for years, and the fertilization of an ovum is effected in the usual way from this sac while the egg from the ovary is passing down the oviduct. The queen bee has the power of releasing some spermatozoa from the receptacle, or of not doing so, and thus of fertilizing the egg, or of not fertilizing it. Since the notable observations of Dzierzon and the investigations of von Siebold and Leuckart which followed them, it has been assumed that only those eggs were fertilized which were laid in the cells destined for rearing females (workers or queens), while those which were to give rise to 'drones' or males remained normally unfertilized. Only in the last decade of the past century did the bee-keepers begin to cast doubt on this so-called 'Dzierzon theory'; various violent and obstinate attacks were made upon it, and these were supported by new and apparently convincing experiments. Dickel, a teacher in Darmstadt, has been particularly strenuous in attempting to overthrow the old theory, by emphasizing the fact that von Siebold's old investigations on bee eggs afforded no convincing proof. Von Siebold made his investigations on eggs freshly taken from the hive, and was never able to find spermatozoa in 'drone eggs' (that is, eggs laid in drone cells and therefore destined to develop into drones), while he was often able to demonstrate the presence of from one to four spermatozoa in 'worker eggs.' But he only examined 'drone eggs' which were already twelve hours old, and in these, as we now know, he would not have found spermatozoa in any case, even if they had been fertilized, because in ova at that stage the development of the embryo has already fully begun, and nothing remains of the spermatozoa. In the bee, according to Buttel-Reepen, the fertilizing spermatozoon is transformed in twenty minutes after it has penetrated into the egg into a minute 'sperm-nucleus' which is almost invisible even in sections, and certainly nothing whatever could be seen of it by the old method of squeezing the fresh ovum.

It had therefore to be admitted that Dzierzon's theory rested on an insecure foundation, and I accordingly set two of my students at that time, Dr. Paulcke and Dr. Petrunkewitsch, to examine the eggs of the bee anew with regard to the point in question, using the greatly improved methods at their disposal. These investigations have been carried out in the Freiburg Zoological Institute during the last three years, and have resulted in establishing the absolute correctness of Dzierzon's theory: the 'drone eggs' do remain unfertilized, while the eggs from which females are to develop are fertilized without exception.

In this case, therefore, we have, in the same animal, eggs which can be fertilized and eggs which, without fertilization, develop parthenogenetically, and it is therefore of the greatest possible interest to know the state of matters in them in regard to the directive divisions and the reduction of the chromosomes.

Dr. Petrunkewitsch's investigations have shown that in both cases, that is, whether a spermatozoon penetrates into the ovum or does not, a twice-repeated division of the nuclear material in the ovum takes place. Moreover, the two daughter-nuclei which result from the second division do not, as Brauer showed was sometimes the case in _Artemia_, unite again afterwards; they remain separate, and the number of chromosomes--there are sixteen of them--is thereby reduced to half in the segmentation nucleus. But this is not all, for before embryonic development has begun the normal number can be again seen in the segmentation nucleus; the chromosomes must therefore have _doubled their number by division within the nucleus_.

It is probable that something similar takes place in the cases of exceptional parthenogenesis which have long been known, but this point has not yet been sufficiently investigated. Nevertheless I cannot pass them over, as they are instructive from another point of view.

In some silk-moths (Bombycidæ) and hawk-moths (Sphingidæ), especially in the silk-moth proper (_Bombyx mori_), in _Liparis dispar_, and in quite a number of other Lepidoptera, it sometimes happens that, out of a large number of unfertilized eggs, a few will develop and produce caterpillars. This is interesting enough, but it gains increased importance through the investigations of the Russian naturalist, Tichomiroff, who succeeded in considerably increasing the number of unfertilized eggs that developed by gently rubbing them with a paint-brush, or by dipping them for a little in dilute sulphuric acid. It is thus possible to make eggs, which would not ordinarily develop without being fertilized, capable of parthenogenetic development by means of mechanical or chemical stimulus. This sounds almost incredible, but it is beyond a doubt, and it is still further corroborated by the fact that Prof. Jacques Loeb has succeeded in inciting the eggs of a sea-urchin to parthenogenetic development by means of a chemical stimulus. When he added to the sea-water in which the eggs were laid a certain quantity of chloride of magnesium the ova developed, and not only went through the process of segmentation, but even reached the stage of the quaint easel-like Pluteus larva. Quite recently Hans Winkler has made the interesting observation that from sea-urchin sperms which have been killed by heat it is possible to extract in aqueous solution a substance capable of exciting unfertilized sea-urchin eggs to development, although they only go as far as to the sixteen-cell stage.

From all these results we can at least infer so much, that chemical changes and influences may determine whether the ripe ovum shall go on to embryonic development or not, and that these influences, may be very diverse in nature in different cases. I shall return later to these important facts.

When we sum up the facts we have cited with reference to the reduction of the number of chromosomes, it appears that nature is, as it were, striving to keep the number constant for each species; that in germ-cells which are destined for amphimixis they are reduced to half the normal number, but that this halving of the number is suppressed where fertilization is always absent, or that the reduction to half is compensated for again in various ways, whether by subsequent fusion of the two daughter-nuclei, which have arisen from the process of reduction, or by an independent duplicating of the chromosomes in the segmentation nucleus.

We might perhaps be inclined to conclude from all this that the occurrence of development depended on the presence of the normal number of chromosomes; and I used to regard this as possible. But facts which have been more recently brought to light have excluded this view. Above all, we now know that every nuclear division depends on the presence of a dividing apparatus, a centrosphere, but that this organ degenerates in the ova of most animals and is completely lost after the second polar division has been effected. The mature ovum is therefore in itself incapable of entering on its embryonic development, no matter how many chromosomes its nucleus contains; it is only capable of further division when the fertilizing sperm-cell brings with it its dividing apparatus or centrosphere. In thread-like sperms this lies in the median portion (Fig. 68_C_), and after the tail-piece has been dissolved, which happens soon after the sperm enters the egg, the central corpuscle, at first very small, can be recognized in front of the sperm-nucleus, where it is soon transformed into an 'aster' and divides into two. Then both spheres move apart (Fig. 75_D_, p. 296) and form the nuclear spindle between them by the confluence of their rays.

From this the division of the ovum into the two first embryonic cells proceeds. The two pronuclei in the ovum, the male and the female, are thus exactly alike as to number of the chromosomes, and frequently at least as to size and appearance (Fig. 75_C_). But they differ in the possession or absence of a dividing apparatus, and in the great majority of cases it is the male nucleus that brings with it the central corpuscle which seems to be indispensable to embryonic development (_B_, _cspt_). Hitherto, at least, only two exceptions to this are known. In the little segmented worm, _Myzostoma_, which is parasitic on sea-lilies or Crinoids, Wheeler observed that the ovum retained its central corpuscle even after the polar divisions, while the sperm-cell which penetrated into the egg had none. More recently Conklin made the interesting discovery that in the egg of a marine Gasteropod (_Crepidula_) both the egg-nucleus and the sperm-nucleus retain their centrosphere and together form the segmentation spindle, one lying at one pole and the other at the opposite.

All these observations confirm the view that the sperm and the egg-cell are alike in this respect also. Each of them can, in certain circumstances, bring with it the dividing apparatus indispensable to development, though it is usually the sperm-cell that does so.

I should indeed assume that the sperm-cell and the egg-cell were essentially alike, even although there were no exception to this rule, that is, although the centrosome of the ovum perished in all eggs which were fertilized. For this is obviously a secondary arrangement, an adaptation to fertilization, that the ovum should be incapable of development without fertilization, and it is made so by the disappearance of its centrosome. In all other cells, as far as is known, the central corpuscle persists after division, so that this remarkable cell-organ is transmitted from cell to cell just like the nucleus, and like it, never rises _de novo_. It is only in the egg-cell that it disappears, though even there often very late, for it may be present, as an aster, even after the sperm has penetrated into the ovum and disclosed its own central body, or even brought it the length of dividing into two (Fig. 80, _A_ and _B_). But the ovum-centrosome disappears as soon as the second polar division is accomplished.

That this disappearance is really a secondary arrangement, which may be again departed from, is proved by the case of those eggs which are able to develop parthenogenetically, for in them the central body does not disappear, but persists in the ovum after the first polar division, as Brauer showed in _Artemia_. It then behaves exactly like the sphere of the sperm-nucleus in the fertilized ovum, that is, it duplicates itself and forms the segmentation spindle.

Thus the beginning of embryonic development in the ovum depends not on a definite number of chromosomes, but on the presence of an apparatus for division. Upon what the awakening of this to activity just at that time depends cannot as yet be exactly stated; we can only indicate that all parts of the cell have interrelations with each other, and that, therefore, the division mechanism is dependent on the condition of the rest of the cell-parts at the moment, and on the substances which they contain or produce. From what we know experimentally in regard to artificial parthenogenesis it is not difficult to imagine that some sort of chemical substances are necessary to stimulate the central corpuscle to activity. In any case, the whole nutrition of the central corpuscle depends on the cell in which it lies, as is shown by the fact that the sperm-nucleus, whose centrosome before the entrance of the sperm into the ovum was inactive and scarcely recognizable, grows rapidly after entrance and forms a large aster round itself--is, in short, in the highest degree active (Fig. 80). As the chromosomes certainly play an important part in the life of the cell, and materially help to determine its various phases, it cannot be disputed that they also may share in awakening the activity of the central corpuscle. But this influence is only indirect; it is not the mere number of chromosomes that decides whether the central corpuscle is to become active or remain inactive. This cannot be assumed, because we have in the maturation divisions a proof that division may take place with a double number of chromosomes as well as with the undoubled number; while in the divisions of the mother-egg-cells and the mother-sperm-cells we have proof that a doubled number of chromosomes does not in itself compel to division.

The exceptional and artificially produced cases of parthenogenesis which we have discussed above are probably to be interpreted thus: through slight differences in the constitution of the ovum, or through certain mechanical or chemical stimuli, the metabolic processes in the ovum are so altered that the centrosome of the ovum, instead of breaking up, is stimulated to growth, and thus produces the active dividing apparatus which is otherwise only brought into it by the sperm. This is a more exact definition of the interpretation I gave earlier (1891) of the 'chance' parthenogenesis of the silk-moth, which was then the only case known, when I said 'the nucleoplasm of some ova must possess the power of growth in a greater degree than the majority.'

But we are not yet in a position to go further, or to define more exactly the nature of the processes of metabolism which are involved.

LECTURE XVI

FERTILIZATION IN PLANTS AND UNICELLULAR ORGANISMS, AND ITS IMMEDIATE SIGNIFICANCE

Fertilization in a lichen, Basidiobolus--In Phanerogams--Here too there is reduction of the number of chromosomes by a half--'Polar cells' in lower and higher plants--Conjugation among unicellular organisms--Noctiluca--The maternal and paternal chromosomes remain apart--Actinophrys--Infusoria--Sexual differentiation of the two conjugates in Vorticella--Importance of the process of Amphimixis--Not a 'life-awakening' process--May occur independently of multiplication--The Rejuvenescence hypothesis--Pure parthenogenesis--The cycle idea--Does Amphimixis prevent natural death?--Maupas' experiments with Infusorians--Bütschli's view--Potential immortality of unicellular organisms--The immortality of unicellular organisms and of the germ-cells depends on the fact that there is no time-limit to the multiplication of the smallest living particles--Parthenogenesis is not self-fertilization--Petrunkewitsch's observations on the ova of bees--Is the chromatin really the 'hereditary substance'?--Nägeli's conclusion from the difference in size between ovum and spermatozoon--Artificial division of Infusorians--Boveri's experiments with the fertilization of pieces of ova not containing a nucleus--Fertilization gives an impulse to development even to non-nucleated pieces of ova--Merogony--The female and male nuclear substances are essentially alike--Summary.