The Mechanism of Life

Artificial Parthenogenesis. The experimental partheno- genesis of Loeb and Uelagc consists in plunging the egg into a liquid other than sea water, and returning it again to its original medium. This operation will necessarily determine slow movements of diffusion in the egg, which will give rise to segmentation. It may be objected that segmentation is also produced by a solution which is isotonic with sea water. Such a solution would not indeed produce an exchange of water with the egg, but it would set up an exchange of electrolytes, since there would be a difference of their osmotic pressure in the egg and in the new isotonic medium. The extremely slow movements of diffusion thus produced would be very favourable to the action of the cohesive force on the particles in suspension, and hence to the segmentation of the egg.

Few physical phenomena give us a deeper insight into the phenomena of life than those which we here contemplate. There is still another experiment which is even more convinc- ing. On the surface of our horizontal salt solution we sow a number of drops of a more concentrated salt solution at equal distances around the circumference of a circle. Movements of diffusion arc thus set up in the interior of the circle, and after a time, when this diffusion has become so slow as to be almost imperceptible, a furrow begins to appear in the coloured mass. Then a second and third appear, and others crossing the former break up the mass

into segments. Finally the segmentation becomes complete, and the preparation presents a muriform appearance, looking in fact something like a mulberry (Fig. 24). If the prepara- tion is preserved for several hours longer, we may see the cells formed by segmentation unite around the circumference so as to form a hollow bag corresponding to a gastrula, as shown in Fig. 25. FIG. 24. A circle of eight drops 01 Indian ink 30 minutes after they have been sown in a salt solution. The drops have undergone diffusion and sub- sequent cohesion, resulting in a reticulate structure.

influences, which renders the demonstration of cohesion phenomena difficult. I have nevertheless on several occasions been able to project the experiment on the screen during a lecture. The segmentation is influenced by very slight currents of diffusion, and I have many preparations showing the segmentation regularly distributed in various ways along radial diffusion lines. We may in this way produce many varieties of structure lamellar, vacuolate, or cellular, in fact

all the tissue structures which are met with in living organisms. All these structures are retractile, the retraction going on very slowly for a long time, as if the force of cohesion continued to act in the web of the structure even after its formation was complete. The phenomenon is a purely physical synthetic reproduction of the phenomenon of coagula- tion, the cohesion figure being in fact a retractile clot. Crystallization. When we evaporate a solution of a crystalloid it becomes more concentrated, slow movements of

FlG. 25. The same preparation several hours later, showing a cellular gastrula-like structure. diffusion are set up, and at a given moment agglomeration occurs, the agglomerates taking the form of crystals. Thus crystallization may be regarded as a particular case of con- glomeration by cohesion, differing only in the regularity of the arrangement of the molecules, which gives the geometrical form of the crystal. Hence we can easily understand how the presence of a crystalline fragment may facilitate the process of crystalli/ation. Consider a liquid in which extremely slow movements of diffusion are taking place. If the liquid is perfectly homogeneous there will be no centre of attraction to which the molecules may become attached.

If, however, a crystal or other heterogeneous structure is present, the arrangement of the molecules of a liquid around a crys- tal in the act of formation (Fig. 26). For this purpose we add to the solution traces of some col- loidal substance, such as gelatine or gum, so as to delay the crystallization. FIG. 26. Field of crystallization of sodium chloride (magnified 60 diameters). It may thus be shown that the molecules of the surrounding liquid are already arranged in crystalline order for some distance from the crystal, forming a sort of field of crystallization. The arrangement of this regular field varies in ( 1 i fferen t cases, and is more or less com- plicated according to circumstances. One of the most frequent forms is that shown in Fig. 27, which is the field around a crystal of sodium chloride. In the centre

FIG. 27. Field of crystallization around a crystal of sodium chloride in process of formation. of the crystal is a square with well-marked outline. At each corner of this square there is a straight line at right angles to the diagonal, which will form the sides of the crystal in process of formation. From the middle of each side arise yet other perpendiculars, which in their turn bear other cross lines, each new line being set at right angles to its predecessor. A later stage of crystallization is shown in Fig. 27, where the two squares one inside the other at an angle of 45 are clearly indicated.

FIG. 28. Three crystals of sodium chloride in process of formation, each in the centre of a field of crystallization. Every crystallizable substance gives a different characteristic field of crystallization. In 1903, at the Congress of Angers, I terminated my address by these words : " The field of crystallization may serve to determine the character of a substance in solution." I have subsequently received from Carbonell y Soles of Barcelona an interesting work on this subject, which he contributed to the International Congress of Medicine at Madrid in 1903, entitled Application de la crystalogenia experimental a la investigation toxicologica de cas alcalo'ides.

Six years ago I received from Australia an exceedingly beautiful photograph of a thin pellicle found in a rain gauge. My correspondent supposed that this strange figure might have been produced under the influence of an electric or magnetic field. I was able to assure him by return of post that the figure was the result of the crystallization of copper sulphate in a colloidal medium. In return I received a letter verifying this fact, and saying that there were copper works in the neigh- bourhood, and the air was filled with the dust of copper sulphate.

Living beings are but solutions of colloids and crystal- loids, and their tis- sues are built up by the aggregation of these solutes. We have already seen how the forces of crystallization are modified in colloid FIG. 29. Crystallization of sodium chloride in a col- solutions. This loidal solution, giving a plant- like form. force of crystalliza- tion must play an important role in the metamorphoses of the living organism, and influence their morphology. It may therefore be of interest to investigate some of the numberless forms of crystallization in colloidal solutions.

Figs. 9 and 30 represent the forms produced by chloride of sodium and chloride of ammonium respectively, in solutions of gelatine of different degrees of concentration. Their resemblance to vegetable growth is so remarkable that several observers on first seeing them have called them " Fern- crystals. 1 ' I should like here to recall to your notice the work of an English observer. Dr. E. Montgomery of St. Thomas's Hospital, which was published as long ago as 1865. This work was recently brought to my notice by the kindness of Professor Baumlcr of Freiburg. He says : " Crystals are not strangers in the organic world. Many organic compounds are able to assume crystalline forms under certain conditions.

Rainey has shown that many shells consist of globular crystals FIG. 30. Form produced by the crystallization of chloride of ammonium in a colloidal solution, i.e. of mineral substances made to crystallize by the influence of viscid material.''' In this connection I may also mention the interesting work of Otto Lehman n of Karlsruhe on liquid crystals. In conclusion, we may recall the words of Schwann himself, the originator of the cell theory : " The formation of the elementary shapes of an organism is but a crystallization of substances capable of imbibition. The organism is but an aggregate of such imbibing crystals/ 1

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 arc mechanical, while others invoke the aid of magnetism or electricity to account for the resemblance of the figures of karyokinesis to the mag- netic 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 Gottingen.

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, hut 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 thefr 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 pro- duced entirely by diffusion, presents a perfect resemblance to the achromatin spindle in k^irineyoksis. . ,

u 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 liypertonic 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

FlG. 31. Diffusion figure representing karyokinesis. Achromatin spindle between two similar poles of concentration. 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 ex- plained.

"It is hardly necessary to insist on the fact that liquid preparations like these are of extreme delicacy and sensitive- ness, 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 chroniatin, 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 be- comes granular. Next we see what appears to be a rolled- up ribbon analogous to the chroniatin band, which soon breaks into fragments analo- gous to the chromosomes. These arrange themselves around, and are gradually attracted towards the cen- trosomes, where they accumu- late to form two pigmelited 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 centro- somes. Finally we have two ^Us in juxtaposition, each with its nucleus, its proto- plasnij and its enveloping

membrane. I have been able to photograph these successive stages of the segmentation of the chroniatin just as I have those of the achromatin spindle" (Fig. 32). This memoir, written in 1904, clearly asserts the homo- polarity 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,

FIG. 32. Four successive stages in the production of artificial karyo- kinesis by 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-spindlcs, but it is clear that these two hemi-spindles arc 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 chromo- somes 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 chromo- somes (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 cata- bolism, 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 supposi- tion 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.

imitated by diffusion. For instance, in the diffusion prepara- tion 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 karyo- kinesis. 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 hypotonie, 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, vix. two different sexes. It is possible that we have in these two methods of producing nuclear division the secret of the difference of sex.