The Mechanistic Conception of Life

1 Through other control experiments I convinced myself that a treatment of eggs or spermatozoa with equal parts of a -g'^n MgClg solution and sea-water diminishes the impregnability of the eggs and annihilates the fertilizing power of spermatozoa in a very short time. statements. The next question that should be raised would be whether the spermatozoa act in the same way. It is true that the spermatozoon contains a considerable proportion of salts, especially KgPO^, but it may contain enzj-mes or it may contain substances which have similar effects upon the physical qualities of the colloids, like the three agencies mentioned above. In the last volume of these lectures I pointed out that it is impossible to derive all the various elements that constitute heredity from one and the same condition of the egg.^ Our recent experiments suggest the possibility that different constituents of the egg are responsible for the process of fertilization and for the transmission of the hereditary qualities of the male. While we are able to produce the process of fertilization by a treatment of the unfertilized egg with certain salts in certain concentrations, we cannot hope to bring about the transmission of the hereditary qualities of the male by any such treatment. Hence, the inference must be that the transmission of the hereditary qualities of the male and the agency that causes the process of fertilization are not necessarily one and the same thing. I consider the chief value of the experiments on artificial parthenogenesis to be the fact that they transfer the problem of fertilization from the realm of morphology into the realm of physical chemistry .^

1 Loeb, J., " The Heredity of the Markmg in Fish Embryos," Woods Hole Biol. Led., Boston, 1899. 2 This paper was written immediately after I had succeeded in producing larvae from the unfertilized egg. In the following years the methods of artificial parthenogenesis were improved and this led to the unraveling of the mechanism by which the spermatozoon causes the egg to develop. An account of this work is given in the two following papers. The title of this paper was chosen in reference to Virchow's paper on ''Stimulation and Irritability" {Virchow's Archiv, XIV, 1, 1858) in which he discriminates between three forms of stimulation : functional, nutritive, and formative. By formative stimuli he means those which give rise to nuclear and cellular division. He considers as the classic example for formative stimulation the fertilization of the egg and the parallel drawn by him between this process and the causation of a pathological process of growth is so characteristic that we may quote it in full :

If we admit the identity between the pathological and the embryonic neoformation, the egg will have to be considered as the analogue of the pathological mother cell and the act of impregnation as the analogue of pathological stimulation. This view is not essentially altered through the discovery of the entrance of the spermatozoon into the egg, since there is no reason to consider the spermatozoon as the direct morphological starting-point for the development of definite parts of the egg. If, as seems to be the case, the spermatozoa are dissolved in the egg, they carry into it only certain chemical substances, which serve as specific stimuli, by calling forth new chemical and morphological arrangements of the atoms. Each specific contagium offers the same possibilities.

The supposition prevalent at Virchow's time, that the spermatozoon is entirely dissolved in the egg was not correct; but his view, that the spermatozoon carries chemical substances into the egg, which form the stimulus for its development, is perfectly correct; and like\\ise the analogy between the causation of the development of the egg by a spermatozoon and the causation of a pathological growth seems correct. I therefore 1 Address delivered at the International jSIedical Congress at Budapest, 1909.

believe that it may be of interest to the medical profession to follow me in a brief survey of my experiments on artificial parthenogenesis and the causation of the development of the egg by a spermatozoon. Cellular physiology has shown that tissues and organs develop only from cells through nuclear and cellular division. The conditions which cause cells to divide and to develop into new normal or pathological tissues have, since Virchow, been called formative stimuli. It is the task of modern biology to ascertain first what is the nature of these stimuli, and second, which change occurs in the cell in the process of formative stimulation. Virchow already emphasized the fact that the fertilization of the egg is the model of all phenomena of formative stimulation and that the spermatozoon may be considered as the formative stimulus in this case.

Pathologists have not yet succeeded in determining what the physico-chemical nature of the formative stimulus in the case of a tumor is, or what changes a cell undergoes in such a process. This task has, however, been accomplished to a high degree in the animal egg, and it may therefore interest the pathologist and the physician in general to become familiar with the essential features of the data thus obtained. It is known that aside from a few exceptions the animal egg can only develop if a spermatozoon enters into it. If no spermatozoon enters, as a rule no segmentation of the egg takes place and it perishes after a comparatively short period of time. The questions which I tried to solve were the following: B}^ which physico-chemical agencies does the spermatozoon cause the egg to divide and to develop into an embryo; and second, which changes does the egg undergo in this formative stimulation by a spermatozoon ? Or in other words, what is the mechanism by which the unfertilized egg is caused to segment and to develop ?

Two ways were open to find an answer to this question: first to try to cause the development of the unfertiUzed egg with extracts from sperm. I have spent a good deal of time in trying to succeed in this task, but met at first with only negative results for the reason that I used at first only extracts from the sperm of the same species of animals from which the eggs were taken. Only recently have I found that the extract of sperm is effective only if it is taken from a foreign species. We shall return to this curious fact later on and show that it has a bearing upon the problem of the immunity of our cells to the lysins of our body.

The second way which could lead to a decision of the question concerning the nature of formative stimulation lay in the direction of artificial parthenogenesis, i.e., of the causation of the development of the animal egg, not by extracts of sperm but directly by physico-chemical agencies. This method of procedure has a special advantage. Since in this case we know the nature of the agencies we employ, it is easier to get an insight into the mechanism by which they cause the development of the egg; while if we work with extracts of sperm we are in the dark as to the chemical character of the active substances.

We will begin with a description of the method of artificial parthenogenesis in the egg of the Cahfornian sea-urchin, since here this method has been worked out most completely. It may be mentioned that in the eggs of many animals the effect of the entrance of the spermatozoon manifests itself almost instantly by a characteristic change, namely, the formation of the so-called membrane of fertilization. Briefly stated this process may possibly consist in the entrance of sea-water between the surface film and the protoplasm of the egg, whereby the former is lifted up from the protoplasm of the egg and separated from it by a more or less wide, clear space. Figs. 1

and 2 (page 7) show these changes m the sea-urchin egg. Fig. 1 represents the unfertilized egg, Fig. 2 shows the same egg after the entrance of the spermatozoon. In 1905 I succeeded in finding a method by which it is possible to call forth the formation of a membrane of fertilization without apparent injury to the egg. This method consists in putting the eggs for about two minutes (at a temperature of 15°) into a mixture of 50 c.c. of sea-water-f3 c.c. of an n/10 lower monobasic fatty acid, e.g., acetic, propionic, butyric, or valerianic acid. In this mixture no membrane formation takes place; if, however, the eggs are transferred into normal sea-water all the eggs form a perfect fertilization membrane. The experiments showed that this process of membrane formation is the essential condition which causes the egg to develop. In all these eggs in the course of the next hours after the membrane formation those changes begin which lead to a celldivision. If the temperature is very low not only cell-divisions begin but the egg may develop into a swimming larva; it reaches the so-called blastula stage. At room temperature, however, the artificial production of a membrane in the egg by fatty acid only calls forth a nuclear and possibly a celldivision; after this the egg slowly begins to disintegrate.

We therefore see that the artificial membrane formation by a fatty acid induces the developmental process, but that at ordinary temperature the latter does not go far. In order to cause a complete development, a second influence is needed, as we shall see later. Before we describe this second influence, another question has to be settled, namely, how we know that the membrane formation and not any other action of the acid, e.g., a catalytic, is the formative stimulus in this case. The answer is, that if we apply the acid but prevent the changes leading to a membrane formation, divisions of the nucleus and of the cell do not occur. On the other hand, we shall see later on that we can

call forth the membrane formation not alone by fatty acids but by a number of different agencies and that all these means act as formative stimuli. The causation of the membrane formation by a fatty acid starts, therefore, the development in the sea-urchin egg, but this development is abnormal and the egg is sickly and perishes the more rapidly the higher the temperature. The question arises, how can we inhibit this sickliness and grant a normal development to the egg?

I found that two different means are at our disposal for this purpose. The one which never fails consists in putting the eggs about twenty minutes after the artificial membrane formation into hypertonic sea-water (or any other hj-pertonic solution, e.g., sugar solution), i.e., into sea-water or any other solution the osmotic pressure of which has been rendered 50 per cent higher than that of the sea-water. In this solution the eggs remain from twenty to sixty minutes — according to the temperature and the concentration of hydroxj'1-ions in the solution. If after this time the eggs are transferred into normal sea-water they develop at room temperature in a wa}' similar to the eggs which are fertilized by sperm. ^

The second method of causing the eggs to develop normally at room temperature after the artificial causation of the membrane formation consists in putting these eggs for three hours in sea-water free from oxygen or into sea-water to which a trace of KCN has been added. After the eggs are transferred into normal seawater they develop often but not always. This method is, therefore, not quite as reliable as the other method mentioned previously.

We see, therefore, that the formative stimulus in the artificial activation of the egg of the sea-urchin consists of two phases, 1 The larvae originating from eggs fertilized by sperm live no longer than those originating from eggs which develop parthenogenetically, if the larvae are not fed. The feeding of these larvae is a tedious process and for this reason I have not undertaken the task. Delage has, however, raised two such larvae until they were sexually mature.

namely, first the artificial causation of the membrane formation and second the subsequent short treatment of the egg with a hypertonic solution; (or a longer treatment with an isotonic solution free from oxygen or containing KCN). We may add that these observations do not hold good for the sea-urchin egg only. Similar observations were made on the eggs of annelids (Polynoe) and of star-fish (Asterina). In Polynoe and star-fish the artificial membrane production is often sufficient to allow the eggs to develop into larvae. But the number of eggs which reach the larval stage and the type of segmentation is improved if the eggs are treated subsequently with one of the above-mentioned methods, as R. Lillie found for Asterias and I for Polynoe. The experiments on annelids and star-fish, therefore, confirm the fact, that the calling forth of the membrane is the essential feature in formative stimulation and that the subsequent treatment of the eggs with a hypertonic solution (or an isotonic solution free from oxygen) has merely a corrective effect; it probably counteracts a secondary detrimental effect connected with the membrane formation.

We will now try to gain some insight into the mechanism of these two agencies. How can the fatty acid cause the formation of a membrane? In order to get an answer to this question we must find out whether there are other agencies which act like fatty acids. It was noticed that all the agencies which cause cytolysis also cause membrane formation, namely, first the specifically cytolytic agencies like saponin, solanin, digitalin, bile salts, and soaps. Experiments with these agencies, especially with saponin, solanin, and digitalin, yielded a curious result. If the unfertilized eggs are put into a weak solution of saponin in sea-water we notice as the first effect on the eggs the formation of a fertilization membrane. Then ensues a pause of sometimes several minutes and after this pause a sudden

cytolysis of the whole egg follows. If we take the eggs during this pause (i.e., after the membrane is formed, but before the Figs. 39-43. — Membrane formation and subsequent cytolysis of the seaurchin egg in a weak solution of saponin in sea-water. Camera drawings from nature. Fig. 39, unfertilized egg at the beginning of the experiment. In this condition the egg was put into sea-water containing a small amount of saponin. The following figvires show the changes it underwent in this solution. Fig. 40, membrane formation under the influence of saponin, eight minutes later. If the egg is taken out of the saponin-sea-water in this stage, washed and put into a hypertonic solution for about one half-hour, it will develop into a larva, after it is put back into normal sea-water. If, however, it is left in the saponin solution it undergoes the rapid cytolysis represented in Figs. 41, 42, and 43. In the above drawing of the egg, cytolysis began at G, Fig. 41, five minutes after the membrane formation. The stages represented in Figs. 42 and 43 were reached a few minutes later.

cytolysis of the egg occurs) out of the sea-water containing saponin and free them from all traces of saponin by washing them repeatedly with sea-water, they behave as if the membrane formation had been called forth by a fatty acid. Such eggs begin to develop, but do not go beyond the first nuclear division at room temperature. If, however, the eggs are treated for half an hour Tvdth hypertonic sea-water they can develop to normal plutei, i.e., larvae with skeletons.

The second group of cytolytic agencies is formed by the specific fat solving hydrocarbons like amylen, benzol, toluol, and in a much lesser degree chloroform, etc. Hert^vig had alreadv observed that chloroform calls forth the membrane formation and Herbst had seen the same effect brought about by benzol and toluol. But these substances act so violently that the membrane formation is followed almost immediately by a cytolysis of the egg, and for this reason these authors could not notice that the membrane formation was followed by the development of the egg. I have, however, been able to convince myself that if amylen or benzol are allowed to act only for one moment and if the eggs are then quickly transferred into normal sea-water a membrane formation can be produced in some of them ^vithout subsequent c}i:olysis. If such eggs were afterward treated ^^'ith hypertonic sea-water they developed into larvae.

A further group of cytolytic agencies is ether or alcohols. Cytolysis of the eggs by these agencies is also preceded by a membrane formation. If the eggs are taken out from such solutions immediately after membrane formation they can be saved from cji^ol^-tic destruction (Figs. 44-47). Bases can also call forth membrane formation, but their action is rather slow and depends on the presence of free ox>'gen. One gains the impression as if the alkali acted in this case only as an accelerator of oxidations and as if a product of oxidation was the proper cause for the membrane formation. The membrane formation usually becomes manifest only if one treats the eggs afterward for a short time with a hypertonic solution ; such a treatment causing them to develop into larvae.

An increase in temperature can also produce a cytolytic effect. I have observed that at 34° or 35° the eggs of Strongylocentrotus purpuratus form often but not always a membrane Figs. 44-47. — Membrane formation and subsequent cytolysis of the egg under the influence of the addition of a minute quantity of salicylaldehyde to sea-water. Camera drawings. Fig. 44, unfertilized egg at the beginning of the experiment. Fig. 45, membrane formation in the salicylaldehyde-sea-water. Fig. 46, beginning of the cytolysis. Fig. 47, cytolysis completed. The cytolyzed egg has in this case an entirely different appearance from that of an egg cytolyzed in saponin.

of fertilization. Such a temperature kills these eggs almost instantly and consequently they are no longer able to develop after this treatment. The eggs of the star-fish Asterias forhesii are, however, not killed so rapidly after the membrane formation, and R. Lillie was able to show that such eggs can develop into larvae if the membrane formation is called forth by raising the temperature. Von Knaffl has shown that if a high temperature acts for some time on these eggs they perish by c}i:olysis and are transformed into ^'ghosts."

We have been able to convince ourselves, therefore, that all the agencies which cause c>i:olysis also call forth the membrane formation; while the agencies which do not call forth cytolysis do not cause a membrane formation. We find in addition that the cytolytic power of these agencies runs parallel with their power of causing membrane formation. From this we draw the inference that the membrane formation depends upon the c\i:ol3"sis of the surface layer of the egg. We shall see later on that we must discriminate between a cortical layer and the core of the unfertilized egg. This superficial cortical layer of the egg is very thin. The essential feature of the developmental stimulus consists in the cj^tolysis of this cortical layer of the egg and this cytolysis is caused by the spermatozoon.

We have already mentioned that the c5i:olysis which underlies the membrane formation causes the development of the egg, but that the egg is as a rule sickly after this membrane formation. To fix our ideas provisionally we assume that through the membrane formation a substance is formed which must be abolished or destroyed before the egg is able to develop normally. If we permit the egg to begin its development while it still contains this hypothetical detrimental substance in a sufficient quantity it is sickly and dies prematurely. The destruction of this hj^^othetical substance can be brought about in two ways: first, by treating the egg for a short time wdth a hypertonic solution. When I discovered this fact there was no analogue known which allowed us to draw an inference concerning the mode of action of a hypertonic solution. I succeeded

in showing that such a solution is only effective in artificial parthenogenesis if it contains free oxygen. If the hypertonic solution is deprived of oxygen it remains without any effect. It remains also inefficient if a trace of KCN is added to it. Since KCN inhibits the oxidations in the cell it is obvious that the hypertonic solution only acts by a modification of the process of oxidation. The second method of saving the life of the egg consists in putting it after the membrane formation for about three hours into sea-water which is practically free from oxygen, or contains a trace of KCN whereby the oxidations in the egg are suppressed. If these eggs are transferred after this time into normal seawater containing free oxygen they are often able to develop normally.^

Thus far we have dealt only with artificial parthenogenesis. We are now about to take up the causation of development by a spermatozoon. Is the formative stimulation of the egg by spermatozoon of the same character as that in artificial parthenogenesis? This question can be answered in the affirmative. It is possible to show that the spermatozoon also calls forth the normal development of the egg by at least two substances and that one of these substances acts like butyric acid or saponin in artificial parthenogenesis, inasmuch as it causes the cytolysis of the thin cortical layer of the egg; while the second substance has an effect similar to that of the hypertonic solution. The correctness for this view is proved by the fact that I succeeded in separating these two effects of the spermatozoon.

If we wish to bring about a separation of these two agencies in the spermatozoon we cannot use the spermatozoa of the same species of sea-urchins from which the egg is taken; for in this case the spermatozoon penetrates at once into the 1 A further discussion of the facts in this chapter is contained in the next paper on " The Prevention of the Death of the Egg through the Act of Fertilization. " protoplasm of the egg as soon as it comes in contact with it. In this way almost simultaneously both substances of the spermatozoon become effective, the cytolytic substance, which causes the membrane formation, and the ''corrective" substance. The experiments, however, result differently when we add the spermatozoa of a foreign species, e.g., star-fish, to the egg of the sea-urchin. Under ordinary conditions sperm of the star-fish cannot cause the egg of the sea-urchin to develop; it becomes effective, however, in seawater which has been rendered a little more alkaline through the addition of some NaHO. If 0.6 c.c. n/10 NaHO is added to 50 c.c. of seawater all the eggs of the sea-urchin form fertilization membranes in such a mixture if only a trace of living sperm of a star-fish {Asterias ochracea) is added. It takes, however, some time, mostly from ten to fifty minutes, until the living star-fish sperm brings about this effect; while after the addition of sea-urchin sperm this result is obtained in one minute.

If the sea-urchin eggs, all of which have formed membranes upon the addition of living star-fish sperm, are put back into normal sea-water and if we watch their further fate, we soon notice that we are dealing with two groups of eggs. The one group acts as if only one of the two agencies, namely, the cytolytic one, had taken effect. These eggs show at room temperature only the beginning of nuclear division and then disintegrate, while at a lower temperature they may develop a little farther. If we treat them, however, after the membrane formation by star-fish sperm for from thirty to fifty minutes with a hypertonic solution they all develop at room temperature mostly into normal larvae. The other eggs develop without any subsequent treatment with a hypertonic solution into normal larvae.

What causes this difference in the behavior of both groups of eggs ? A histological examination of these eggs decides this point. My assistant, Mr. Elder, found that a spermatozoon had entered into those eggs which develop after the addition of star-fish sperm without subsequent treatment \\'ith a hypertonic solution into normal larvae; while the eggs which behave as if only an artificial membrane formation had taken place do not contain any spermatozoon.