Part 35

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.

I NOW turn to the consideration of the process of fertilization in plants and unicellular organisms.

With regard to plants, it can now be definitely asserted that in them, too, fertilization is essentially a conjugation of nuclei; it depends on the union of the nuclei of the two 'sex-cells.' These sex-cells are usually very small among lower plants, indeed up to the phanerogams; this is especially true of the zoosperm-like male germ-cells, but it usually holds also true of the ovum, which is but seldom burdened with an abundant supply of yolk. In spite of the many difficulties which this smallness of size puts in the way of observation, the untiring exertions of a host of excellent investigators have succeeded in following the process of fertilization in all the larger groups of plants--in algæ, fungi, mosses, ferns, and horse-tails among cryptogams, and in phanerogams.

I shall first give an example from among the lower plants (Fig. 81). In one of the lichens, _Basidiobolus ranarum_, each of two adjacent cells in the fungus-thread gives off a bill-like process, and the two processes become closely apposed (Fig. 81, _a_). The nucleus of each cell moves into the bill-shaped process, is there transformed into a nuclear spindle (_B_, _ksp_) and divides, so that one daughter-nucleus comes to lie in the apex point of the bill, the other at the base. The cell-body also divides, though very unequally, and the final outcome of the process is two cells in each, of which one is small and occupies the apex of the bill, while the other is large and fills all the rest of the cell-space. The former do not play any further part of importance, but break up, the latter are the sex-cells, the cytoplasm of which now coalesces through a gap in the cell-walls, while their nuclei become closely apposed and ultimately unite (_C_, ♂ and ♀ _k_). From this union arises the fertilized spore, the so-called 'zygote' (_D_). The two small abortive cells so greatly resemble in their origin the polar cells of the animal ovum that it is difficult to resist the supposition that they bring about a reduction in the number of chromosomes. But the number of the chromosomes has not yet been determined either in them or in the sex-nuclei.

We have come to know the processes of fertilization among phanerogams chiefly through Strasburger, Guignard, and more recently through the Japanese botanist Hirase. The agreement with the animal process is surprisingly great, notwithstanding the notable differences in the external conditions of fertilization.

As is well known, the male cells in the highest flowering plants are not zoosperms but roundish cells, each of which, enclosed, together with a sister-cell--the so-called 'vegetative' cell--in a thick cellulose capsule constitutes a pollen-grain. The pollen-grains reach the stigma, under which, buried deep within the 'ovule,' the female sex-cell rests, enclosed in a long, sac-like structure called the 'embryo-sac' (Fig. 82, _A_). Beside it (_eiz_) there lie several other cells, usually seven in number, two of which, the so-called 'synergidæ' (_sy_), have their place at one end of the embryo-sac, just in front of the ovum (_eiz_). Probably these give off a secretion which exercises an attractive (chemotactic) influence on the male fertilizing body ('the pollen-tube'), and thus, so to speak, show it the way to the ovum.

When a pollen-grain has reached the stigma it sends out a tube, usually after a few hours, which penetrates into the soft tissue of the style, and grows deep down into the interior of the ovule, ultimately penetrating as far as the embryo-sac through a special little opening in the covering of the ovule, the so-called 'micropyle' (Fig. 82_B_, _pschl_). Its blunt end is now closely apposed to this, so that the true sperm-nucleus (_B_, ♂_k_), surrounded by some protoplasm, can leave the pollen-tube and wander in among the cells of the embryo-sac. Later on we shall see that two generative nuclei migrate from the pollen-tube, but in the meantime we shall devote our attention only to one of them, the fertilizing nucleus, which immediately moves towards the ovum-nucleus and apposes itself closely to it. Then follows the fusion or conjugation of the two nuclei, which are alike in size and appearance, just as in the fertilization of the animal ovum (_C_, ♂ _k_ and ♀ _k_). Whether in this case, too, the sperm-nucleus brings with it a central corpuscle, or whether, as Guignard believed he observed, the ovum retains its central corpuscle (_C_, _csph_), or finally, whether both modes occur, is not yet known with certainty. The fact that, as a rule, seeds capable of reproduction only form in an ovule when the stigma has been previously dusted with pollen, leads us to suppose that, in this case, as among animals, the ovum lacks something that is necessary to induce embryonic development, only retaining this power in very exceptional cases, namely, when adapted for parthenogenesis. And this something may very well be the dividing apparatus of the cell, the centrosome with the centrosphere. But whether this supposition prove correct or not, a nuclear spindle always forms simultaneously with the fusion of the two sex-nuclei into a segmentation nucleus, and this spindle is the starting-point of the young plant, thus exactly corresponding to the first segmentation of the animal ovum. It agrees with it also in the important respect that it again contains the full number of chromosomes--twenty-four in the lily--while the two nuclei, male and female, only exhibit half the number each, that is, twelve.

Thus a reduction in the number of chromosomes to half takes place in plants also, but it is not yet known with certainty whether this is brought about in the same way as among animals, namely, by reducing divisions. Without entering more fully into this still unsolved and very complex problem, I should like to state that I consider this very probable; indeed, I agree with the view of V. Häcker[13], that the reducing divisions of plants are only more difficult to recognize as such, and, furthermore, are often disguised by the fact that they often occur alongside of, or between divisions which are not reducing. If it were possible to reduce the number of chromosomes in a cell to half without the aid of cell-division, if, for instance, only half were to integrate again from the chromatin-network, this must have been quite as possible in the case of animal cells, and then, moreover, the single chromosome would not have had the significance of an individuality, and no special form of nuclear division would have been introduced to reduce their number. That it has been introduced seems to me to prove that it was necessary, and since it was so among animals, it could not have been dispensed with among plants either.

[13] See V. Häcker, _Praxis und Theorie der Zellen- und Befruchtungslehre_, Jena, 1899, pp. 144-5.