CHAPTER VIII
KARYOKINESIS
In 1873, Hermann Fol, writing of the eggs of Geryonia, thus describes the phenomenon of karyokinesis: "On either side of the residue of the nucleus there appears a concentration of plasma, thus forming two perfectly regular star-like figures, whose rays are straight lines of granulations. There are other curved rays which pass from one star or centre of attraction to the other. The whole figure is extraordinarily distinct, recalling in a striking manner the arrangement of iron filings surrounding the poles of a magnet. Sachs' theory is that the division of the nucleus is caused by centres of attraction, and I agree with him, not on theoretical grounds, but because I have actually seen these centres of attraction."
Since the discovery of Hermann Fol, a great number of explanations have been given, all of them theoretical, to account for the figures and phenomena of karyokinesis. Many of these so-called explanations are mechanical, while others invoke the aid of magnetism or electricity to account for the resemblance of the figures of karyokinesis to the magnetic or electric phantom or spectre. Among the authors who have dealt with this question we may mention Hartog of Cork, Gallardo of Buenos Ayres, and Rhumbler of Göttingen.
In 1904 I presented to the Grenoble Congress, and in 1906 to the Lyons Congress, a series of photographs and preparations of experimental karyokinesis. I showed how, in a solution analogous to that found in the natural cell, the simple processes of liquid diffusion, without the intervention of magnetism or electricity, may reproduce with perfect accuracy and in their normal sequence the whole of the movements and
figures which characterize the phenomenon of karyokinesis. This experiment consists not merely in the production of a certain figure, such as is obtained in the magnetic spectre, but in the reproduction of the movement itself, and of all the successive forms which are seen in the natural phenomenon. These are evolved before the eyes of the spectator in their regular order and sequence.
I may here reproduce the text of my communication at Grenoble: "Until I introduced the conception of a field of diffusion, there was no proper means of studying the phenomena of diffusion, which obey the laws of a field of force as expounded by Faraday. Moreover, no one suspected the possibility of reproducing by liquid diffusion a spectre analogous to the electro-magnetic phantom. Guided by this theory of a diffusion field of force, I have been able to reproduce experimentally the figures of karyokinesis by simple diffusion. With regard to the achromatin spindle, Professor Hartog has shown that the two poles of the spindle are of the same sign, and not of opposite signs as was at first supposed. In the process of karyokinesis the two centrosomes, i.e. the two poles of the achromatin spindle, repel one another. They must therefore be poles of the same sign. An electric or magnetic spectre showing a spindle between two poles of the same sign is unknown; such a thing would appear to be an absolute impossibility. What is impossible in electricity and magnetism, however, is quite possible in the artificial diffusion field; we can here have a spindle between two poles which repel one another—that is, between poles of the same sign. Fig. 31 is a photograph of such a spindle produced by diffusion. On either side are two poles of concentration, which represent the centrosomes, each pole being surrounded by a star-like radiation. These poles being alike, repel one another. In the preparation one may see the distance between the two poles slowly increase, the poles gradually separating from one another just as do the centrosomes of an ovum during karyokinesis. This preparation, then, which is produced entirely by diffusion, presents a perfect resemblance to the achromatin spindle in karyokinesis.
"The spindle of which we give a photograph in Fig. 31 was made by placing in salt water a drop of the same solution pigmented with blood or Indian ink, and placing on either side of this central drop a hypertonic drop of salt solution more lightly coloured. After diffusion had gone on for some minutes, we obtained the figure which we have photographed. I would draw your attention to the equatorial plane, which shows that the spindle is not formed by lines of force passing from one pole to the other, as would be the case between two poles of contrary sign, but by two forces acting in opposite directions. On either side the pigment of the central drop has been drawn towards the hypertonic centre nearest to it. In the median line, however, the pigment is attracted in opposite directions by equal forces, and therefore remains undisturbed, marking the position of the equatorial plane. This observation applies equally to the equatorial plane in natural karyokinesis, whose existence is thus readily explained.
"It is hardly necessary to insist on the fact that liquid preparations like these are of extreme delicacy and sensitiveness, and require for their production, and still more for their photography, the greatest care and skill, which can only be acquired by long practice.
"We are able to produce by diffusion not only the achromatin spindle, but also the segmentation of the chromatin, and the division of the nucleus. If in the saline solution we place a coloured isotonic drop between two coloured hypertonic drops, all the figures and movements of karyokinesis appear successively in their due order. The central drop, representing the nucleus between the two lateral drops or centrosomes, first becomes granular. Next we see what appears to be a rolled-up ribbon analogous to the chromatin band, which soon breaks into fragments analogous to the chromosomes. These arrange themselves around, and are gradually attracted towards the centrosomes, where they accumulate to form two pigmented nuclear masses. A partition then makes its appearance in the median line, and this partition becomes continuous with the boundary of the spheres around the centrosomes. Finally we have two cells in juxtaposition, each with its nucleus, its protoplasm, and its enveloping membrane. I have been able to photograph these successive stages of the segmentation of the chromatin just as I have those of the achromatin spindle" (Fig. 32).
This memoir, written in 1904, clearly asserts the homopolarity of the centrosomes, and shows that the nuclear division is the result of a bipolar action, two poles of the same sign exerting their influence on opposite sides of the nucleus. It also emphasizes the important fact that diffusion,
and as far as we know diffusion alone, is able to produce a spindle between homologous poles.
A glance at the photograph is enough to show that the spindle is formed between poles of the same sign. The lines of diffusion radiate from one centre and converge towards the other centre in curves, giving the double convergence characteristic of a spindle. The central drop merely supplies the necessary material, and should have a concentration but slightly less than that of the plasma, so as not to set up its own lines of diffusion. The photograph shows clearly that the rays of the spindle traverse the equator without any break. It has been objected that these lines form not so much a spindle as two hemi-spindles, but it is clear that these two hemi-spindles are continuous and form a single sheaf of rays uniting the two poles of concentration. This is a phenomenon entirely unknown in the magnetic or electric fields, where two poles of the same sign, one on either side of a pole of the contrary sign, give two separate spindles. In a magnetic field it is impossible to make the lines emanating from one pole converge, except to a pole of opposite sign. Hence if we admit the homopolarity of the centrosomes, we must also admit that diffusion is the vera causa of karyokinesis, since, as I showed at the Grenoble Congress in 1904, diffusion and diffusion alone is capable of producing a spindle between two poles of the same sign.
Nuclear Division.—In order to reproduce artificially the phenomena attending the division of the nucleus, we may proceed as follows. We cover a perfectly horizontal glass plate with a semi-saturated solution of potassium nitrate to represent the cytoplasm of the cell. The nucleus in the centre is reproduced by a drop of the same solution coloured by a trace of Indian ink, the solid particles of which will represent the chromatin granules of the nucleus. The addition of the Indian ink will have slightly lowered the concentration of the central drop, and this is in accordance with nature, since the osmotic pressure of the nucleus is somewhat less than that of the plasma. We next place on either side of the drop which represents the
nucleus a coloured drop of solution more concentrated than the cytoplasm solution. The particles of Indian ink in the central drop arrange themselves in a long coloured ribbon, apparently rolled up in a coil, the edges of the ribbon having a beaded appearance. After a short time the ribbon loses its beaded appearance and becomes smooth, with a double outline, as is shown in A, Fig. 32. This coil or skein of ribbon subsequently divides, forming a nuclear spindle, while the chromatin substance collects together in the equatorial plane as in B, Fig. 32.
A more advanced stage of the nuclear division is shown at C, Fig. 32, where the chromatin bands of artificial chromosomes are grouped in two conical sheafs converging towards the two centrosomes. For some considerable time these conical bundles remain united by fine filaments, the last vestiges of the nuclear spindle. The final stage is that of two artificial cells in juxtaposition, whose nuclei are formed by the original centrosomes augmented by the chromatin bands or chromosomes (Fig. 32, D).
The resemblance of these successive phenomena to those of natural karyokinesis is of the closest. The experiment shows that diffusion is quite sufficient to produce organic karyokinesis, and that the only physical force required is that of osmotic pressure. If in the cytoplasm of a cell there are two points of molecular concentration greater than that of the general mass, the nucleus must necessarily divide with all the phenomena which accompany karyokinesis. In nature these two centres of positive concentration are introduced into the protoplasm of the cell by fecundation—that is, by the entrance of the centrosomes of the sperm cell. In certain abnormal cases the concentration may be produced in the cell itself by the formation of two centres of catabolism or molecular disintegration, since, as we have seen, molecular disintegration raises the osmotic pressure. This phenomenon, namely the production of karyokinesis from centres of catabolism, may account for the abnormal karyokinesis of cancer cells and the like. The subject is one which would well repay further investigation.
It has been found in our experiments that in order to obtain the regular division of the artificial nucleus represented by the intermediary drop, the latter must have an osmotic pressure slightly below that of the plasma. This leads to the supposition that a similar condition must obtain in the natural cell. It may be noticed, moreover, that the grains of pigment follow the direction of the flow of water, being carried along by the stream. This would appear to show that the nucleus of a natural cell has also a molecular concentration less than that of the plasma—a result either of dehydration of the plasma, or of some diminution in the molecular concentration of the nucleus.
Other phenomena of karyokinesis may also be closely imitated by diffusion. For instance, in the diffusion preparation we notice at each extremity of the equator a V-shaped figure with its apex towards the centre, corresponding exactly to what in natural karyokinesis is called the equatorial crown.
We may also produce diffusion figures of abnormal karyokinesis. Fig. 34 represents such a form, a triaster produced by diffusion.
Artificial karyokinesis may also be produced by hypotonic poles of concentration—that is to say, when the central drop representing the ovum is positive and the lateral drops representing the centrosomes are negative with respect to the plasma. In this case, however, the resemblance to natural karyokinesis is less perfect.
Without attaching to it an importance which is not warranted by experimental results, it is interesting to note that we have here two methods of fertilization, hypertonic and hypotonic, i.e. by centrosomes of greater concentration and by centrosomes of less concentration than that of the plasma of the ovum, and that we have in nature two corresponding results, viz. two different sexes. It is possible that we have in these two methods of producing nuclear division the secret of the difference of sex.