The Mechanistic Conception of Life

2. In adopting the theory of Sachs and applying it to animal morphology, we must avoid a mistake very often made even in the case of good theories, namely, the endeavor to explain special cases which are complicated by unknown conditions. Huyghens explained by his theory of light the phenomena of refraction, but he could not and did not attempt to explain the sensations of color. For these phenomena the wave theory of 1 J. Sachs, Stoff und Form der Pflanzenorgane, Gesammelte Abhandlungen,

light remains true, but color sensations depend not only on the wave motion of the ether, but also on the chemical and physical structure of the retina. I think it perfectly safe to say that every animal has specific germ substances, and that the germ substances of different animals differ chemically. Its chemical qualities determine that from a chick's egg only a chick can arise. But it would be a mistake to attempt at present an explanation of how the unknown chemical nature of the germ determines all the different organs and characters that belong to the species. For instance, the yolk sac of the Fundulus embryo has a tiger-like coloration. We might say that these markings may be due to a certain arrangement of molecules or complexes of molecules (determinants), which later on give rise to the colored places of the yolk sac, but I found that this coloration originates in a manner much more simple. The pigment cells are formed irregularly on the surface of the yolk. The pigment is chemically closely related to hemoglobin, and so its formation may from the first be connected with the formation of the blood corpuscles. But the arrangement of the pigment cells during the first days of development is not such as to produce any definite markings. They lie upon the walls of the blood-vessels as well as in the spaces between the capillaries (Fig. 36). Later on, however, all of the pigment cells have crept upon the surface of the neighboring bloodvessels (Fig. 38). I succeeded experimentally in showing it to be probable that some of the substances contained in the blood determine this reaction.

These substances, if they diffuse from the blood-vessel and touch the chromatophore, make, according to the laws of surface tension, the protoplasm of the chromatophore flow toward and at last over the blood-vessel and form a sheath around it, while the gaps between the blood-vessels become empty of chromatophores. In this way the chromatophores are arranged in stripes, and possibly changes in the surface tension, and not a preformed arrangement of the germ.

Figs. 36, 37, and 38. — From nature. The origin of the pattern on the yolk sac of a fish embryo {Fundulus heteroclitus). Fig. 36 is a drawing of the bloodvessels at the surface of the yolk sac at an early stage of development. The black pigment cells show no definite orientation in regard to the blood-vessels. Fig, 37, the same egg a few days later. Here we notice that some pigment ceUs show a tendency to creep on the blood-vessels. Fig. 38, the same egg still a few days later. The black pigment cells have completely crept on the blood-vessels and formed a sheath around them. The red chromatophores are omitted in this drawing.

This was the first observation pro\'ing that tropisms play a role in the arrangement of the organs in the body. determine the marking. We do not know what processes determine the coloration of animals which owe their markings to interference colors, but the task of deriving such a coloration in the adult from a similar arrangement of molecules in the germ plasm would prove too much even for a genius like Huyghens, and without the possibility of such a derivation the theory is of no use.

3. The reasons why roots grow on the under side of the stem of Antennularia and stems on the upper side can only be given when the special physical and chemical conditions inside the stem of Antennularia have been worked out. At present we can only think of possibiHties. It is possible that the hj'pothetical root substances of Sachs may have a greater specific gravity than the substances which form stems, and therefore take the lowest position in the cell. Since outgro\\i:h can take place only at the free surface of a stem or branch, roots can on this assumption grow only at the under side and stems only at the upper side of an element. But there are still other possibilities which we must omit here. In the case of Margelis and other hydroids, it might happen that contact with solid bodies produced an increase of surface in the touched elements in case they contained specific root substances, while the opposite took place in the case of elements containing polyp substances. The consequence would be an increase in the surface of the roots if they came into contact with solid bodies, while polyps only would grow out in the opposite direction. I found, indeed, in some forms at Naples that roots of hydroids which grew free in the water began to grow much faster and to branch off more abundantly when brought into contact with solid bodies. But in these cases we must wait with our attempts at explanation until the physical and chemical conditions for the form are worked out. For the same reasons I will not go into a discussion of the question of what determines the polarization of animals like Cerianthus. It may suffice to suggest the possibility

that in polarized animals the tissues or cells may have such a peculiar structure as to allow the specific formative substances to migrate or arrange themselves only in one direction, while in cases of heteromorphosis migration or arrangement in every direction or in several directions is possible. 4. The egg of a sea-urchin under normal conditions gives rise to but one embryo. This circumstance is due simply to the geometrical shape of the protoplasm, which, under normal conditions, is that of a sphere. When we make the eggs burst, the protoplasm outside the egg membrane and that which remains within it assume spherical forms, by reason of the surface tension of the protoplasm. When this happens, as a rule, we get twins, if two separate segmentation cavities are formed, and only one embryo, if both cavities communicate with one another. Whether the first or the second case will happen depends upon the molecular condition of the part of the protoplasm connecting the two drops. Therefore, the number of embryos which come from one egg is not determined by the preformation of germ-regions in the protoplasm, or nucleus, but by the geometrical shape of the egg and the molecular condition of the protoplasm, in so far as these circumstances determine the number of blastulae. In my experiments, I got double or triple embryos when the egg formed two or three droplets or spheres, as every sphere gives rise to a blastula. In Driesch's experiments, one single cell of the four-cell stage necessarily formed a whole embryo after it had been isolated, as it assumed the shape of a single sphere or ellipsoid. Of course, there must be a limit to the number of embryos that can arise from one egg; but the limit is not due to any preformation, but to other circumstances, the chief one being that with too small an amount of protoplasm the formation of a blastula — from merely geometrical reasons, as there must be a minimum size for the cleavage-cells — becomes impossible.^ Without the

1 1 stated that the minimal size is about one-eighth of the mass of the seaurchin's egg and I do not think that this is very far off the limit. formation of the blastula, of course it is not possible to get the later stages which are determined by the blastula. I have chosen the name Physiological Morphology for these investigations, inasmuch as their object has been to derive the laws of organization from the common source of all life phenomena, i.e., the chemical activity of the cell. In what way this is to be done is indicated in the chapter on the mechanics of growth.

But the aim of Physiological Morphology is not solely analytical. It has another and higher aim, which is synthetical or constructive, that is, to form new combinations from the elements of living nature, just as the phj'sicist and chemist form new combinations from the elements of non-living nature. Experimental biology is a very recent science. Not until recently have biologists begun to become conscious of the uncertainty of conclusions which are not tested and verified by adequate experiments.

Leeuwenhook demonstrated in 1677 the existence of motile elements in the sperm, the so-called spermatozoa. He believed that the spermatozoa represented the future embryo. The majority of his contemporaries assumed that the spermatozoa were parasitic organisms which had nothing to do with fertilization. The idea that spermatozoa are not parasites did not subside until it was proved about 160 years later that the spermatozoa originate from the cells of the testes.

That sperm was needed to bring about fertilization of the egg was too obvious a fact to escape even those biologists who never made an experiment, but that the spermatozoa and not the liquid constituents were the essential element in the sperm was a fact which could not be established except experimentally. It was generally assumed that no direct contact between sperm and egg was necessary and that something volatile contained in the sperm, the imaginary ''aura seminalis" was sufficient for the act of fertilization. That contact between sperm and egg was really necessary for fertilization was at last proved experimentally by Jacobi (1764) who showed that fish eggs can only be fertilized if the sperm is brought into direct contact with the eggs; and by Spallanzani who put the males of frogs during the act of cohabitation into trousers and convinced

1 Reprinted, from Biological Lectures delivered at Woods Hole, 1899, by courtesy of Ginn & Co., Boston. himself that under such conditions the eggs remained unferti- Hzed although the "aura seminalis" was not prevented from acting upon the eggs. This ended the reign of the ''aura seminalis.'' It was reserved to two experimenters, Prevost and Dumas (the latter the famous chemist) to prove that the spermatozoa are the essential element in the sperm. They made the simple experiment of filtering the sperm and demonstrated that the sperm whose spermatozoa had been retained by the filter had lost its power of fertilizing the eggs. But even this did not convince many of the descriptive biologists and nine years later K. E. von Baer still expressed the opinion that spermatozoa had nothing to do with fertilization. In 1843 the entrance of the spermatozoon into the egg was directly observed by Barry and this fact has since been verified by an endless number of investigators for the egg of all kinds of animals. It is probably no exaggeration to say that with the general recognition of the experimental method in biology it would probably have taken about as many years as it took centuries to establish the simple fact that the spermatozoon is the essential element in sperm.

The mere observation of the fact that the spermatozoon must enter the egg in order to bring about fertilization did not lead to any understanding of the mechanism of the activation of the egg. Nevertheless four theories or rather suggestions were offered. The first theory of fertilization is a morphological one. According to this theory, it is the morphological structure of the spermatozoon which is responsible for the process of fertilization.

The second theory is a chemical one. According to this theory it is not a definite morphological or structural element of the spermatozoon, but a chemical constituent, that causes the development of the egg. Against this second view Miescher raised the objection that his investigations showed the same compounds in the egg and the spermatozoa. I do not beheve that this objection is valid. We know that simple variations in the configuration of a molecule have an enormous effect upon life phenomena. This is shown among others by the work of Emil Fischer on the relation between the molecular configuration of sugars and their fermentability. When Miescher made his experiments he was not familiar with such possibilities. Moreover, Miescher was not able to state whether the spermatozoa contain enzymes or not.

A third theory was a physical theory (Bischof). This theory assumes that a peculiar condition of motion exists in the spermatozoon which is transmitted to the egg and causes its development. It should be said, however, that this idea is not so very different from the chemical conception, because it assumes exactly the same for the spermatozoon that Liebig assumes for the enzymes. Liebig thought that the enzymes owed their power of producing fermentation to the motions of certain atoms or groups of atoms.

The fourth conception is the stimulus conception, which was originated by His. According to this conception the egg is considered as a definite machine which if once wound up will do its work in a certain direction. The spermatozoon is the stimulus which causes the egg to undergo its development. It is to be said in connection with this stimulus conception that the main point at issue is omitted as to whether the stimulus carried by the spermatozoon is of a physical or a chemical character, and in this way, of course, the stimulus conception is nothing but a disguised repetition of the chemical or physical theory of fertilization.

All these theories are so vague that we do not need to be surprised that none of them has led to any further discovery. If we want to make new discoveries in biology, we must start from definite facts and observations, and not from vague speculations. Among these observations the most important are those on parthenogenesis. It had been observed for a long time that the imfertihzed egg of the silkworm can develop parthenogenetically. It was, moreover, kno^\^l that plant lice can give rise to new generations without fertilization. The most impressive fact concerning the parthenogenesis of animals was contributed by Dzierzon, who discovered that the unfertilized eggs of bees develop and give rise to males, while the fertilized eggs give rise to females. Similar conditions seem to exist in wasps. It is, moreover, certain that a few crustaceans show parthenogenesis.

A beginning of parthenogenetic development had been observed in the case of a great many marine animals which develop outside of the female in sea-water. It was found that such eggs when left long enough in sea-water may divide into two or three cells, but no farther. On the other hand, in ovaries of mammals now and then eggs were found that were segmented into a small number of cells. ^ These facts and the occurrence of a certain class of tumors in the ovary, the so-called teratomata, suggest the possibility of at least partial parthenogenesis in the eggs of mammals. But all these phenomena were considered to be of a pathological character. It must be, however, admitted that we cannot utilize these facts with any degree of certainty for the theory of fertilization, as in this case certainty can only be obtained bj^ the experiment. It was not until very recently that such experiments were made.

Eight years ago I observed that if the fertilized eggs of the sea-urchin were put into sea-water whose concentration was raised by the addition of some neutral salt they were not able to segment, but that the same eggs, when put back after they had been in such sea-water for about two hours, broke up into a large number of cells at once instead of dividing successively into two, four, eight, sixteen cells, etc. Of course it is necessary for this experiment that the right increase in the concentration of the sea-water be selected. The explanation of this fact is as follows: The concentrated sea-water brings about a change in the condition of the nucleus which permits a division and a scattering of the chromosomes in the egg.^ As soon as the egg is put back into normal sea-water it at once breaks up into as many cleavage-cells as nuclei or distinct chromatin masses had been preformed in the egg. Morgan tried the same experiment on the unfertilized eggs of the sea-urchin, and found that the unfertilized egg, if treated for several hours with concentrated sea-water, was able to show the beginning of a segmentation when put back into normal sea-water. A small number of eggs divided into two or four cells, and, in a few cases, went as far as about sixty cells, but no larvae ever developed from these eggs. Morgan^ had used the same concentration of sea-water as Norman^ and I had used in our previous experiments. I had added about 2 grams of sodium chloride to 100 c.c. of sea-water. Norman used instead of this 3§ grams of MgCl2 to 100 c.c. of sea-water, and Morgan used the same concentration. Mead'' made an observation somewhat similar to Morgan's upon Chaetopterus. He found that by adding a very small amount of KCl to sea-water he could force the unfertilized eggs of Chaetopterus to throw out their polar bodies. The substitution of a little NaCl for KCl did not have the same effect. While continuing my studies on the effects of salts upon life phenomena, I was led to the fact that the peculiar actions of protoplasm are influenced to a great extent by the ions contained in the solutions which surround the cells. As is

3 Norman, W. W., " Segmentation of the Nucleus without Segmentation of the Protoplasm," Arch. f. E ntwickelungsmechanik. III, 1896. * Mead, A., " The Rate of Cell-Division and the Fimction of the Centrosome," Woods Hole Biol. Led., 1898. well kno^Mi, if we have a salt in solution, e.g., sodium chloride, we have not only NaCl molecules in solution, but a certain number of NaCl molecules are split up into Na ions (Na atoms charged with a certain quantity of positive electricity) and CI ions (CI atoms charged with the same amount of negative electricity). When an egg is in sea-water, the various ions enter it in proportions determined by their osmotic pressure and the permeability of the protoplasm. It is probable that some of these ions are able to combine with, the proteins of the protoplasm. At any rate, the physical qualities of the proteins of the protoplasm (their state of matter and power of binding water) are determined by the relative proportions of the various ions present in the protoplasm or in combination with the proteins.^ By changing the relative proportions of these ions we change the physiological properties of the protoplasm, and thus are able to impart properties to a tissue which it does not possess ordinarily. I have found, for instance, that by changing the amount of sodium and calcium ions contained in the muscles of the skeleton we can make them contract rhythmically like the heart. It is only necessary to increase the number of sodium ions in the muscle or to reduce the number of calcium ions or do both simultaneously.- On the basis of this and similar observations I thought that by changing the constitution of the sea-water it might be possible to cause the eggs not only to show a beginning of development but to develop into living larvae, which were in every way similar to those produced by the fertilized egg.

There seemed to be three ways in which this might be accomplished. The first way was a simple change in the constitution of the sea-water without increasing its osmotic pressure. The second way was to increase the osmotic pressure 1 Loeb, J., "On lon-Proteid Compounds and Their Role in the Mechanics of Life-Phenomena," Amer. Joum. of Phys., Ill, 1900. - It is due to the Ca ions of our blood that the muscles of our skeleton do not beat rhythmically, like our heart .

of the sea-water by adding a certain amount of a certain salt. The third way was by combining both of these methods. The first way did not lead to the result I desired.^ All the various artificial solutions I prepared had only the one effect of causing the unfertilized egg to divide into a few cells, but I was not able to produce a blastula. I next tried the effects of an increase in the sea-water by adding a certain amount of magnesium chloride. In this case I had no better results than Morgan. Very few eggs began to divide, but these did not develop beyond the first stages of segmentation. I then tried the combination of both methods. The osmotic pressure of ordinary sea-water is roughly estimated to be the same as that of a f n NaCl solution or a ^-n MgCl.^ solution. I found, after a number of experiments, that by putting the unfertiHzed eggs of the sea-urchin into a solution of 60 c.c. of -^^-n MgClg solution and 40 c.c. of sea-water for two hours the eggs began to develop when put back into normal sea-water. Such eggs reached the blastula stage. I do not think that anybody has ever seen before such blastulae as resulted from these unfertilized eggs. As these eggs had no membrane, the amoeboid motions of the cleavage-cells led very frequently to a disconnection of the various parts of one and the same egg, and the outlines of the egg became extremely irregular. The blastulae showed, as a rule, the same outline as the egg had in the morula stage. It was, moreover, a rare thing that the whole mass of the egg developed into one blastula. The disconnection of the various cleavage-cells led, as a rule, to the formation of more than one embryo from one egg. The results were in a certain way similar to those I had obtained when I caused the fertilized eggs of sea-urchins to burst.

In such cases a part of the protoplasm flowed out from the egg but was able to develop. These extraovates had no membrane, and of course showed some irregularity in their outlines, but the irregularity in this case 1 Later experiments gave, however, positive results. See next chapter. was far less than that observed in the unfertilized eggs of my recent experiments. But although I had thus far satisfied my desire to see the unfertilized eggs of the sea-urchin reach the blastula stage, I was not able to keep these eggs alive long enough to see them grow into the pluteus stage. They developed more slowly than the normal eggs, and died, as a rule, on the second day.

It was my next task to find a solution which would allow the eggs to reach the pluteus stage. I found that this can be done by reducing the amount of magnesium chloride and increasing the amount of sea-water. By putting the unfertilized eggs for about two hours into a mixture of equal parts of -2^-n MgCl^ and sea-water, the eggs, after they were put back into normal sea-water, not only reached the blastula stage, but went into the gastrula and pluteus stages. The blastulae that originated from these eggs looked much healthier and more normal than those of the former solution with more MgCla- Of course as these unfertilized eggs had no membrane it happened but rarely that the whole mass of an egg developed into one single embryo. Quadruplets, triplets, and twins were much more frequently produced than a single embryo. The outlines of each blastula were much more spherical than in the previous experiment. These eggs reached the pluteus stage on the second day (considerably later than the fertilized eggs do). Thus I had succeeded in raising the unfertilized eggs of seaurchins to the same stage to which the fertifized eggs can be raised in the aquarium. I have not yet succeeded in raising the fertilized eggs in my laboratory dishes beyond the pluteus stage.

Though I do not wish to go into the technicalities of these experiments, I must mention a few of the precautions that I took in order to guard against the possible presence of spermatozoa in the sea-water.^ The reader who is interested in this 1 Today it may seem strange that I had to meet such objections, but when my first papers on artificial parthenogenesis appeared, very few biologists were willing to accept the correctness of my statements. The most absurd sources of error were suggested.

technical side of the experiments will find all the necessary data in my publication in the American Journal of Physiology} Here I wish only to mention the following points: 1. These experiments were made after the spa^\Tiing season was practically over. 2. Bacteriological precautions were taken against the possibility of contamination of the hands, dishes, or instruments with spermatozoa. 3. The spermatozoa contained in the sea-water lose, according to the investigation of Gemmill,^ their fertilizing power within five hours if distributed in large quantities of seawater.

4. We have a criterion by which we can tell whether the egg is fertilized or not in the production of a membrane. The fertilized egg forms a membrane and the unfertilized egg has no distinct membrane. None of the unfertilized eggs that developed artificially had a membrane.^ 5. With each experiment a number of control experiments were made. Part of the unfertilized eggs were put into the same normal sea-water that was used for the eggs that did develop. None of these eggs that remained in normal sea-water formed a membrane or showed any development, except that a few of them were divided into two cells after about twentyfour hours.

6. I made another set of control experiments by putting a lot of eggs of the same female into a solution w^hich differed less from the normal sea-water than the one which caused the formation of blastulae or plutei from the unfertilized eggs. 1 Loeb, J., "On the Artificial Production of Normal Larvae from the Unfertilized Egg of the Sea-Urchin," Amer. Joum. of Phys., III. 2 Gemmill, "The Vitality of the Ova and Spermatozoa of Certain Animals," 3 The method used in these experiments was primitive inasmuch as no fertilization membrane was formed. A few years later I found a method for the artificial production of a fertilization membrane, which is described in the next paper. In the earlier experiments in which no fertilization membrane was developed, nevertheless a change in the cortical layer of the egg was brought about by the combined action of the hydroxyl-ions of the solution, and the increased osmotic pressure.

In this case it was shown, that although these eggs received the same sea-water as the ones which developed, and although they were injured less than the ones which developed, yet not one single egg formed a membrane or reached the blastula stage. If the sea-water had contained any spermatozoa these eggs should have reached the blastula stage.^ Hence, as in nine different series of experiments these results were confirmed, we may assume that by treating the eggs for two hours with a solution of equal parts of a ^ n MgCl, solution and sea-water we can cause them to develop parthenogenetically into plutei.

What conclusions may we draw from these results ? If we wish to avoid wild and sterile speculations, I think we should confine ourselves to the following question: What alterations can be produced in an egg by treating it for two hours with a solution of equal parts of ^^n MgClg and of sea-water ? Even in this regard we can only give a very indefinite answer which, however, will have to be in the following direction: The bulk of our protoplasm consists of colloidal substances. This material easily changes its state of matter and its power of binding water. It seems probable that changes of these two qualities are mainly responsible for muscular contraction and perhaps amoeboid motions. Among the agencies that cause changes of these physical qualities we know of three that are especially powerful. The one is specific enzymes (trypsine, plasmase, etc.). The second is ions in definite concentration. The concentration varies for various ions. The third agency is temperature. In our experiments it is obvious that only the second possibility can have been active. I do not consider it advisable to enter into theoretical discussions beyond these