The Mechanistic Conception of Life

1 Reprinted from the Harvey Lectures, 1911, by courtesy of Messrs. J. B. Lippincott & Co. oxygen can be shown in various ways. The maturation of the egg itself depends upon oxidations. If the ox;y^gen is withheld from the immature eggs, or if the oxidations in the immature eggs are inhibited by potassium cyanide, the process of maturation does not take place. Maturation is, therefore, also a function of oxidations. The eggs of a female, which were unripe, were divided into two groups: the one group remained in sea-water in contact with oxygen; the other was put into sea-water whose oxygen had been removed by a current of hydrogen. The eggs of the second group remained alive; the eggs of the first group perished in a few hours.

It is not even necessary to drive out the air by hydrogen; the life of the unfertilized eggs can also be preserved by putting large masses of them into a narrow glass tube which is sealed at the bottom. The eggs sink to the bottom of the tube, and those which are lying near the bottom receive no oxygen, since the oxygen which diffuses from the air through the sea-water is consumed by the uppermost layer of the eggs. On account of this lack of ox^^gen the eggs at the bottom of the tube do not mature and do not perish; hence by withholding ox^^gen from the immature eggs their maturation and death are prevented.

If the oxygen is withheld from the eggs immediately after they become mature their life is also saved. A. P. Mathews has repeated this experiment and obtained the same results. This proves that the death of the mature but unfertilized egg is determined by oxidations. If these oxidations are inhibited death does not occur. When these experiments were first published they caused opposition. This opposition was based on the fact that potassium cyanide was used in part of the experiments. The objection was raised that the potassium cyanide in these experiments acted only by preventing the development of bacteria. The authors, however, who raised this objection, overlooked the fact that lack of oxygen acts in exactly the same w^ay as the addition of potassium cyanide,

and that it is entirely immaterial how lack of oxygen is produced, whether the oxygen is driven out by carefully purified hydrogen or whether the eggs are put together in a large heap, whereby only those lying on the surface of the heap receive sufficient oxygen. It is, however, easy to show directly that the abovementioned objection is incorrect. The eggs of the star-fish can easily be put into sterilized sea-water without bacterial infection. The following experiment was tried. The eggs of a star-fish were separated into three parts: one part was put aseptically into a series of flasks with sterilized seawater; the second part was put into ordinary seawater without asepsis; the third part was put into sea-water to which a large quantity of a putrid culture of bacteria had been added that had developed on the dead eggs of the star-fish. It was found that in all three cases the mature eggs died within the same period of time. The sterilization of the eggs of the first group was complete, as was shown by the fact that the eggs although dead preserved their form for two months, while the dead eggs in the normal sea-water were completely destroyed in a few days by the action of the bacteria.

It is, therefore, certain that the death of the star-fish eggs which are not fertilized is not caused by bacteria, but by the process of oxidation in the egg. If no spermatozoon enters the egg, or if the egg is not caused to develop by chemical treatment it perishes very rapidly. If, however, a spermatozoon enters the egg, the latter remains alive in spite of the fact that the entrance of the spermatozoon causes an acceleration of the oxidations in the egg. Warburg found for the eggs of the seaurchin at Naples that fertilization raises the velocity of the process of oxidations to six times their original value, while Wasteneys and I found that fertilization caused an increase in the velocity of oxidations of Arbacia in Woods Hole to three or four times the rate found in the unfertilized eggs.

How can we explain the fact that fertiHzation saves the life of the egg ? Let us make the following preliminary assumption : The unfertilized egg contains a poison, or some faulty combination of conditions which, if oxidations take place, causes the death of the egg. In the unfertilized but mature egg oxidations take place. The spermatozoon carries into the egg among other substances something which protects the egg against the fatal effects of the oxidations, and allows them even to carry on oxidations at an increased rate without suffering. We might say that the mature but unfertilized egg is comparable to an anaerobic being for which oxidations are fatal, and that the spermatozoon transforms the egg into an aerobic organism.

If we compare the eggs of different animals, w^e j&nd great differences in regard to the above-mentioned conditions. The eggs of certain annelids (Polynoe) also perish rapidly if they become mature without being caused to develop, while the eggs of the sea-urchin remain alive for a longer period of time after they have become mature. The problem as to what determines this difference has not yet been investigated. The analysis of the process of fertilization by the spermatozoon shows that we must discriminate between two kinds of effects, the hereditary effect and the activating or developmental effect. The experiments on artificial parthenogenesis make it very probable that the two groups of substances, the substances which determine the heredity of paternal characters and the substances which cause the egg to develop, are entirely different. In this paper we are concerned only with the second group of substances, namely, those which cause the development of the egg.

The analysis of the causation of development of the egg by a spermatozoon has shown that the latter acts by carrying at least two substances or groups of substances into the egg. The first of these substances causes the formation of a membrane; the second serves the purpose of rendermg the egg immune against the fatal action of oxidations. I have shown in a number of papers that the essential feature in the causation of the development of the egg is a

Figs. 48-50. — Disintegration of the sea-urchin egg after the membrane formation with butjTic acid or with foreign serum or with the extract of sperm or of foreign cells; if the eggs are not treated after the membrane formation with a hypertonic solution or a suppression of oxidations. A indicates the area where the disintegration begins. modification of its surface, which in many cases leads to the formation of a membrane. If we cause membrane formation in an unfertilized sea-urchin egg by artificial means, it begins to develop, but very soon perishes; much more rapidly than if it is not exposed to any treatment. I was able to show that

this rapid death of the sea-urchin egg, after artificial membrane formation, can be prevented either by withdrawing the oxygen from the egg or by inhibiting the oxidations in the egg by the addition of a trace of potassium cyanide. The membrane formation, therefore, causes the rapid death of the egg through an acceleration of oxidations. Warburg has recently shown that the artificial membrane formation in the unfertilized seaurchin egg causes the same increase in the rapidity of oxidations as the entrance of a spermatozoon.

If we wish to cause the unfertilized eggs to develop to the pluteus stage after the membrane formation, we have to subject them to a second treatment. This may consist in putting them about fifteen minutes after the membrane formation into a hypertonic solution of a certain osmotic pressure (for instance, 50 c.c. of sea-water +8 c.c. n/2j NaCl) for one-half to one hour. If, after this time, they are put back into normal seawater they no longer perish, but develop into normal larvae. I ventured the hypothesis that the artificial membrane formation causes a rapid increase of the oxidations in the egg and in this way causes it to develop, but that these oxidations lead to the rapid decay of the eggs at room temperature for the reason that the egg contains a toxic substance, or a toxic complex of conditions, which in the presence of oxidations leads to the rapid death of the egg. The second treatment serves the purpose of rendering the egg immune against the toxic effects of the oxidations.

If we first cause the artificial membrane formation in the unfertilized egg by any of the various means which I have described in former papers, and if we afterward treat the eggs for a short time with a hypertonic solution, they develop after being transferred to normal sea-water in the same way as if a spermatozoon had entered them. They reach the successive larval stages, develop into a blastula, gastrula, and pluteus, and live as long as the larvae produced from eggs fertilized by a spermatozoon.

Hence the physico-chemical activation of the unfertiUzed egg of the sea-urchin consists of two kinds of treatment. The one is a change in the surface of the egg which may or may not result in the so-called formation of the membrane. This change causes the acceleration of oxidations which in my opinion is the essential feature of the process of fertilization. The second treatment consists in abolishing the faulty condition which makes oxidations fatal to the egg. This second treatment may consist in exposing the eggs for about half an hour or a little more to a hypertonic solution. We can substitute, however, for this treatment another treatment, namely, the deprivation of the egg for three hours from oxidations, either by removing the oxygen from the solution or by adding a trace of potassium cyanide to the solution. If, after the treatment with the hypertonic solution for half an hour, or the treatment with lack of oxygen for about three hours, the eggs are put back into normal sea-water they can develop into normal larvae.

We can show that the spermatozoon also causes the development of the egg by two different agencies comparable in their action to the agencies used in the methods of chemical fertilization which we have just described. For this purpose we must fertilize the egg of the sea-urchin with a sperm different from its own, and for the following reason: The spermatozoon of the sea-urchin enters so rapidly into the egg that it is impossible to show that it causes the development of the egg by two different substances.

If, however, we fertilize the sea-urchin egg with the sperm of star-fish, it takes from ten to fifty minutes to cause the membrane formation in the eggs, the reason bemg that the star-fish sperm can penetrate only very slowly into the egg of the sea-urchin. It is, as a rule, not possible to fertilize the egg of the seaurchin by star-fish sperm in normal sea-water. But I found eight years ago that if we make the seawater slightly more alkaline than it naturally is the eggs of the sea-urchin can be fertilized by the sperm of the star-fish. For the fertilization of the Californian sea-urchin, Strongylocentrotus purpuratus, with the sperm of Asterias, the best results were obtained when 0.6 c.c. of n/10 NaOH were added to 50 c.c. of sea-water. In this case, with active sperm, in about fifty minutes all the eggs form the typical fertilization membrane.

If we watch the further development of sea-urchin eggs fertilized by star-fish sperm we notice very soon that there are two different kinds of eggs present; the one kind of eggs behave as if they had been fertilized with sperm of their own kind. That is to say, they segment regularly and develop into swimming blastulae and gastrulae. The other kind of eggs, however, act as if they had been treated with one of the agencies which cause the membrane formation in the unfertilized seaurchin egg; these eggs begin to segment, but at room temperature they slowly perish by cytolysis. If, however, these eggs are treated for half an hour with a hypertonic solution they develop into larvae.

If we examine the eggs of a sea-urchin which have been treated in an alkaline medium with the sperm of the star-fish, we find that only a certain percentage of these eggs contain the sperm nucleus, and this percentage seems to be identical with the percentage of the eggs which develop into larvae. As far as the other eggs are concerned, which form only a membrane and then disintegrate, no sperm nucleus can be found inside of them. I am inclined to draw the following conclusion from these observations: The spermatozoon of the star-fish penetrates very slowly through the surface film of the sea-urchin egg. When it lingers for some time partially imbedded in the surface film, one of the substances of the spermatozoon is dissolved in the superficial layer of the egg and causes the membrane formation. Through the act of

membrane formation the further entrance of the spermatozoon mto the egg is prevented, since the fertilization membrane is impermeable to sperm. This membrane formation leads to an increase in the rate of oxidations and the beginning of the development of the egg. The latter, however, contains a toxic substance, or a faulty complex of conditions which has to be abolished, before the oxidations necessary for development can take place T\dthout the egg being destroyed by them. The spermatozoon carries a second substance into the egg which renders it immune against the fatal actions of the oxidations. While the membrane-forming substance of the spermatozoon may be situated at its surface, or superficially at least, the second substance which transforms the egg from an anaerobe into an aerobe must be situated in the interior of the spermatozoon; since it can only act if the spermatozoon penetrates into the egg. We see in these observations, concerning the fertilization of the sea-urchin egg by the star-fish sperm, a proof that the activation of the egg by the spermatozoon is also caused by two different substances, one of which causes the membrane formation, while the second renders the egg immune against the toxic action of the oxidations. These data support the assumption made above that the life-saving action of the spermatozoon is due to the fact that it carries a substance into the egg which renders the latter immmie against the toxic action of oxidations.

Seven years ago I fomid that a number of agencies destroy the fertilized egg much more rapidly than the unfertilized egg. Thus, for instance, while in a pure sodium chloride solution the unfertilized egg of the Californian sea-urchin may be kept alive for several days, the fertilized egg is destroyed in such a solution in less than twenty-four hours. If we use slightly alkaline solutions of sodium chloride the greater resistance of the unfertilized egg is perhaps still more striking. The egg of the Atlantic form of sea-urchin, Arbacia, is cytolyzed in a neutral sodium chloride solution in a few hours, while the unfertilized egg may live for a considerably longer period of time. When we put fertilized and unfertilized eggs into hypertonic solutions, we find also that the fertilized eggs suffer much more than the unfertilized. What causes this difference of sensitiveness between fertilized and unfertilized eggs? It is possible that the permeabihty of the fertilized eggs is greater than that of the unfertilized. While this is probably to some extent true, yet it is not the whole explanation of the difference in the behavior of the two kinds of eggs. I have been able to show for a number of toxic solutions that their effect can be either completely annihilated or at least diminished if we take the oxygen away from the solution. Thus, for instance, fertilized eggs of the sea-urchin which perish very rapidly in pure salt solutions, or a solution of sodium + calcium, or a solution of sodium + barium, can be kept alive for a considerable period of time in the same solutions if we either carefully remove the oxygen from the solutions, or if we diminish the rate of the oxidations in the eggs by adding a trace of sodium cyanide. In this case we have the direct proof that solutions which are fatal for the egg when the oxidations are allowed to go on are rendered completely, or at least partially, harmless if we stop the oxidations in the egg. Not only the toxic action of salt solutions upon the fertilized egg could be inhibited by the suppression of the oxidations in the egg, but also the toxic action of sugar solutions, or of solutions of alcohol in the sea-water, or of a solution of chloral hydrate.^

These observations prove directly that in the presence of certain toxic substances or mixtures of substances the oxidations in the egg lead to its rapid destruction; while a suppression of the oxidation saves the life of the egg. 1 Or of phenylurethane. This observation does not agree very well with the assumption that the narcotic action of these substances is due to a retardation of oxidation. We therefore believe that we may conclude that the rapid death of the unfertilized egg of certain species is caused by the oxidations which take place in these eggs; and that the lifesaving action of the spermatozoon consists in the fact that the latter, in addition to the membrane-forming substance, carries a second substance, or group of substances, into the egg which renders it immune against the harmful effect or consequences of oxidations.

Less is known of the role of the salts in the animal body than of the role of the three other main food-stuffs, namely, carbohydrates, fats, and proteins. As far as the latter are concerned, we know at least that through oxidation they are capable of furnishing heat and other forms of energy. The neutral salts, however, are not oxidizable. Yet it seems to be a fact that no animal can live on an ash-free diet indefinitely, although no one can say why this should be so. We have a point of attack for the investigation of the role of the salts in the fact that the cells of our body live longest in a liquid which contains the three salts, NaCl, KCl, and CaCU in a definite proportion, namely, 100 molecules NaCl, 2.2 molecules KCl, and 1 . 5 molecules of CaCl.^. This proportion is identical with the proportion in which these salts are contained in sea-water; but the concentration of the three salts is not the same in both cases. It is about three times as high in the sea-water as in our blood serum.

Biologists have long been aware of the fact that the ocean has an incomparably richer fauna than fresh-water lakes or streams and it is often assumed that life on our planet originated in the ocean. The fact that the salts of Na, Ca, and K exist in about the same proportion in our blood serum as in the ocean has led some authors to the conclusion that our ancestors were marine animals, and that, as a kind of inheritance, we still carry diluted sea-water in our blood. Statements of this kind have

1 Carpenter lecture delivered at the Academy of Medicine of New York, October 19. 1911. Reprinted from Science, N.S., XXXIV, No. 881, 653-65, November 17, 1911, by courtesy of Professor James McKeen Cattell. mainly a metaphorical value, but they serve to emphasize the two facts, that the three salts, NaCl, KCl, and CaCl,, exist in our blood in the same relative proportion as in the ocean and that they seem to play an important role in the maintenance of life. I intend to put before you a series of experiments which seem to throw some light on the mechanism by which the solutions surrounding living cells influence their duration of life.

In order to give a picture of the extent to which the life of many animals depends upon the cooperation of the three salts I may mention experiments made on a small marine crustacean, Gammarus, of the Bay of San Francisco. If these animals are suddenly thrown into distilled water, their respiration stops (at a temperature of 20° C.) in about half an hour. If they are put back immediately after the cessation of respiration into seawater, they can recuperate. If ten minutes or more are allowed to elapse before bringing them back into the sea-water, no recuperation is possible. Since in this case death is caused obviously through the entrance of distilled water into the tissues of the animals, one would expect that the deadly effect of distilled water would be inhibited if enough cane sugar were added to the distilled water to make the osmotic pressure of the solution equal to that of the sea-water. If, however, the animals are put into cane-sugar solution, the osmotic pressure of which is equal to that of sea-water, the animals die just about as rapidly as in distilled water. The same is true if the osmotic pressure of the sugar solution is higher or lower than that of the sea-water. The sugar solution is, therefore, about as toxic for these animals as the distilled water, although in the latter case water enters into the tissues of the animal, while in the former case it does not.

If the sea-water is diluted with an equal quantity of distilled water in one case, and of isotonic cane-sugar solution in the other, in both cases the duration of life is shortened by practically the same amount. If the crustaceans are brought into a pure solution of NaCl, of the same osmotic pressure as the sea-water, they also die in about half an hour. If to this solution a little calcium chloride be added in the proportion in which it is contained in the seawater the animals die as rapidly as without it. If, however, both CaClo and KCl are added to the sodium chloride solution, the animals can live for several days. The addition of KCl alone to the NaCl prolongs their life but little.

If KCl and CaCl, are added to a cane-sugar solution isotonic with sea-water, the animals die as quickly or more so than in the pure cane-sugar solution. If other salts be substituted for the three salts the animals die. The only substitution possible is that of SrCl, for CaCl.,. We find also that the proportion in which the three salts of sodium, calcium, and potassium have to exist in the solution cannot be altered to any extent. All this leads us to the conclusion, that in order to preserve the life of the crustacean Gammarus, the solution must not only have a definite concentration or osmotic pressure but that this osmotic pressure must be furnished by definite salts, namely, sodium chloride, calcium chloride, and potassium chloride in the proportion in which these three salts exist in the sea-water (and in the blood) ; this fact could also be demonstrated for many other marine animals. The relative tolerance of various cells and animals for abnormal salt solutions is, however, not the same, a point which we shall

What is the role of the salts in these cases ? The botanists have always considered salt solutions as nutritive solutions. It is a well-kno^\^l fact that plants require definite salts, e.g., nitrates and potassium salts, for their nutrition, and the question now arises whether the three salts XaCl, KCl, and CaCl,, which are needed for the preservation of animal life, play the role of nutritive salts. Experiments which I made on a small marine fish, Fundulus, proved beyond question that this is not the case. If the young, newly hatched fish are put into a pure solution of sodium chloride of the concentration in which this salt is contained in sea-water, the animals very soon die. If, however, KCl and CaCl, be added to the solution in the right proportion, the animals can live indefinitely. These fish, therefore, behave in this respect like Gammarus and the tissues of the higher animals, but they differ from Gammarus and the majority of marine animals inasmuch as the fish can live long, and in some cases, indefinitely, in distilled and fresh water, and certainly in a very dilute solution of sodium chloride. From this fact I drew the conclusion that KCl and CaClg do not act as nutritive substances for these animals, that they only serve to render NaCl harmless if the concentration of the latter salt is too high. I succeeded in showing that as long as the sodium chloride solution is very dilute and does not exceed the concentration of m/8, the addition of KCl and CaCl, is not required. Only when the solution of NaCl has a concentration above m/8 does it become harmful and require the addition of KCl and CaCl.

The experiments on Fitndi^ZiiS, therefore, prove that a mixture of NaCl + KCl +CaCl2 does not act as a nutritive solution, but as a -protective solution. KCl and CaCl,^ are only necessary in order to prevent the harmful effects which NaCl produces if it is alone in solution and if its concentration is too high. We are dealing, in other words, with a case of antagonistic salt action ; an antagonism between NaCl on the one hand and KCl and CaClo on the other. The discovery of antagonistic salt action was made by Ringer, who found that there is a certain antagonism between K and Ca in their action on the heart. When he put the heart of a frog into a mixture of NaCl + KCl he found that the contractions of the heart were not normal,

but they were rendered normal b}' the addition of a Uttle CaClo. A mixture of NaCl+CaCl. also caused abnormal contractions of the heart, but these were rendered normal by the addition of KCl. Ringer drew the conclusion that there existed an antagonism between potassium and calcium, similar to that which Schmiedeberg had found between different heart poisons, e.g., atropin and muscarin. Biedermann had found that alkaline salt solutions cause twitchings in the muscle and Ringer found that the addition of Ca inhibited these twitchings. Since these experiments were made many examples of the antagonistic action of salts have become kno^^Ti.

It had generally been assumed that the antagonistic action of two salts was based on the fact that each salt, when applied singly, acted in the opposite way from that of its antagonist. We shall see that in certain cases of antagonistic salt action at least this view is not supported bj' fact. What is the mechanism of antagonistic salt action? I believe that an answer to this question lies in the following observations on the eggs of Fundidus, If these eggs are put immediately after fertilization into a pure sodium chloride solution which is isotonic with sea-water, they usuall}' die without forming an embryo. If, however, only a trace of a calcium salt, or of any other salt with a bivalent metal (with the exception of Hg, Cu, or Ag) is added to the m/2 NaCl solution, the toxicity of the solution is diminished or even abolished. Even salts which are very poisonous, namely, salts of Ba, Zn, Pb, Ko, Ni, JVIn, and other bivalent metals, are able to render the pure solution of sodium chloride harmless, at least to the extent that the eggs can live long enough to form an embryo. The fact that a substance as poisonous as Zn or lead can render harmless a substance as indifferent as sodium chloride seems so paradoxical that it demanded an explanation,