The Project Gutenberg eBook, The Organism as a Whole, by Jacques Loeb

Transcriber’s note:

In this transcription a black dotted underline indicates a hyperlink to a specific page or illustration; hyperlinks also show aqua highlighting when the cursor hovers over them. Page numbers appear in the right margin.

The Organism as a Whole

From a Physicochemical Viewpoint

By

Jacques Loeb, M.D., Ph.D., Sc.D.

Member of the Rockefeller Institute for Medical Research

With 51 Illustrations

G. P. Putnam’s Sons New York and London

Copyright, 1916 by JACQUES LOEB

To THE MEMORY OF DENIS DIDEROT Of the Encyclopédie and the Système de la nature

“He was one of those simple, disinterested, and intellectually sterling workers to whom their own personality is as nothing in the presence of the vast subjects that engage the thoughts of their lives.”
John Morley.
(Article Diderot, Encyclopædia Britannica.)

PREFACE

It is generally admitted that the individual physio­logical processes, such as diges­tion, metabolism, the produc­tion of heat or of electricity, are of a purely physico­chemical character; and it is also conceded that the func­tions of individual organs, such as the eye or the ear, are to be analysed from the viewpoint of the physicist. When, however, the biologist is confronted with the fact that in the organism the parts are so adapted to each other as to give rise to a harmonious whole; and that the organisms are endowed with structures and instincts calculated to prolong their life and perpetuate their race, doubts as to the adequacy of a purely physico­chemical viewpoint in biology may arise. The difficulties besetting the biologist in this problem have been rather increased than diminished by the discovery of Mendelian heredity, according to which each character is transmitted independently of any other character. Since the number of Mendelian characters in each organism is large, the possibility must be faced that the organism is merely a mosaic of independent hereditary characters. If this be the case the ques­tion arises: What moulds these independent characters into a harmonious whole?

The vitalist settles this ques­tion by assuming the existence of a pre-established design for each organism and of a guiding “force” or “principle” which directs the working out of this design. Such assump­tions remove the problem of accounting for the harmonious character of the organism from the field of physics or chemistry. The theory of natural selec­tion invokes neither design nor purpose, but it is incomplete since it disregards the physico­chemical constitu­tion of living matter about which little was known until recently.

In this book an attempt is made to show that the unity of the organism is due to the fact that the egg (or rather its cytoplasm) is the future embryo upon which the Mendelian factors in the chromo­somes can impress only individual characteristics, probably by giving rise to special hormones and enzymes. We can cause an egg to develop into an organism without a spermato­zoön, but apparently we cannot make a spermato­zoön develop into an organism without the cytoplasm of an egg, although sperm and egg nucleus transmit equally the Mendelian characters. The concep­tion that the cytoplasm of the egg is already the embryo in the rough may be of importance also for the problem of evolu­tion since it suggests the possibility that the genus- and species-heredity are determined by the cytoplasm of the egg, while the Mendelian hereditary characters cannot contribute at all or only to a limited extent to the forma­tion of new species. Such an idea is supported by the work on immunity, which shows that genus- and probably species-specificity are due to specific proteins, while the Mendelian characters may be determined by hormones which need neither be proteins nor specific or by enzymes which also need not be specific for the species or genus. Such a concep­tion would remove the difficulties which the work on Mendelian heredity has seemingly created not only for the problem of evolu­tion but also for the problem of the harmonious character of the organism as a whole.

Since the book is intended as a companion volume to the writer’s former treatise on The Comparative Physiology of the Brain a discussion of the func­tions of the central nervous system is omitted.

Completeness in regard to quota­tion of literature was out of the ques­tion, but the writer notices with regret, that he has failed to refer in the text to so important a contribu­tion to the subject as Sir E. A. Schäfer’s masterly presidential address on “Life” or the addresses of Correns and Goldschmidt on the determina­tion of sex. Credit should also have been given to Professor Raymond Pearl for the discrimina­tion between species and individual inheritance.

The writer wishes to acknowledge his indebtedness to his friends Professor E. G. Conklin of Princeton, Professor Richard Goldschmidt of the Kaiser Wilhelm Institut of Berlin, Dr. P. A. Levene of the Rockefeller Institute, Professor T. H. Morgan of Columbia University, and Professor Hardolph Wasteneys of the University of California who kindly read one or more chapters of the book and offered valuable sugges­tions; and he wishes especially to thank his wife for suggesting many correc­tions in the manuscript and the proof.

The book is dedicated to that group of freethinkers, including d’Alembert, Diderot, Holbach, and Voltaire, who first dared to follow the consequences of a mechanistic science—incomplete as it then was—to the rules of human conduct and who thereby laid the founda­tion of that spirit of tolerance, justice, and gentleness which was the hope of our civiliza­tion until it was buried under the wave of homicidal emo­tion which has swept through the world. Diderot was singled out, since to him the words of Lord Morley are devoted, which, however, are more or less characteristic of the whole group.

J. L.

The Rockefeller Institute
for Medical Research,
August, 1916

CONTENTS

The Organism as a Whole

CHAPTER I

INTRODUCTORY REMARKS

1. The physical researches of the last ten years have put the atomistic theory of matter and electricity on a definite and in all probability permanent basis. We know the exact number of molecules in a given mass of any substance whose molecular weight is known to us, and we know the exact charge of a single electron. This permits us to state as the ultimate aim of the physical sciences the visualiza­tion of all phenomena in terms of groupings and displacements of ultimate particles, and since there is no discontinuity between the matter constituting the living and non-living world the goal of biology can be expressed in the same way.

This idea has more or less consciously prevailed for some time in the explana­tion of the single processes occurring in the animal body or in the explana­tion of the func­tions of the individual organs. Nobody, not even a scientific vitalist, would think of treating the process of diges­tion, metabolism, produc­tion of heat, and electricity or even secre­tion or muscular contrac­tion in any other than a purely chemical or physico­chemical way; nor would anybody think of explaining the func­tions of the eye or the ear from any other standpoint than that of physics.

When the actions of the organism as a whole are concerned, we find a totally different situa­tion. The same physiologists who in the explana­tion of the individual processes would follow the strictly physico­chemical viewpoint and method would consider the reac­tions of the organism as a whole as the expression of non-physical agencies. Thus Claude Bernard,[1] who in the investiga­tion of the individual life processes was a strict mechanist, declares that the making of a harmonious organism from the egg cannot be explained on a mechanistic basis but only on the assump­tion of a “directive force.” Bernard assumes, as Bichat and others had done before him, that there are two opposite processes going on in the living organism: (1) the phenomena of vital crea­tion or organizing synthesis; (2) the phenomena of death or organic destruc­tion. It is only the destructive processes which give rise to the physical manifesta­tions by which we judge life, such as respira­tion and circula­tion or the activity of glands, and so on. The work of crea­tion takes place unseen by us in the egg when the embryo or organism is formed. This vital crea­tion occurs always according to a definite plan, and in the opinion of Bernard it is impossible to account for this plan on a purely physico­chemical basis.

There is so to speak a pre-established design of each being and of each organ of such a kind that each phenomenon by itself depends upon the general forces of nature, but when taken in connec­tion with the others it seems directed by some invisible guide on the road it follows and led to the place it occupies. . . .

We admit that the life phenomena are attached to physico­chemical manifesta­tions, but it is true that the essential is not explained thereby; for no fortuitous coming together of physico­chemical phenomena constructs each organism after a plan and a fixed design (which are foreseen in advance) and arouses the admirable subordina­tion and harmonious agreement of the acts of life. . . .

We can only know the material conditions and not the intimate nature of life phenomena. We have therefore only to deal with matter and not with the first causes or the vital force derived therefrom. These causes are inaccessible to us, and if we believe anything else we commit an error and become the dupes of metaphors and take figurative language as real. . . . Determinism can never be but physico­chemical determinism. The vital force and life belong to the metaphysical world.

In other words, Bernard thinks it his task to account for individual life phenomena on a purely physico­chemical basis—but the harmonious character of the organism as a whole is in his opinion not produced by the same forces and he considers it impossible and hopeless to investigate the “design.” This attitude of Bernard would be incomprehensible were it not for the fact that, when he made these statements, the phenomena of specificity, the physi­ology of development and regenera­tion, the Mendelian laws of heredity, the animal tropisms and their bearing on the theory of adapta­tion were unknown.

This explanation of Bernard’s attitude is apparently contradicted by the fact that Driesch[2] and v. Uexküll,[3] both brilliant biologists, occupy today a standpoint not very different from that of Claude Bernard. Driesch assumes that there is an Aristotelian “entelechy” acting as directing guide in each organism; and v. Uexküll suggests a kind of Platonic “idea” as a peculiar characteristic of life which accounts for the purposeful character of the organism.

v. Uexküll supposes as did Claude Bernard and as does Driesch that in an organism or an egg the ultimate processes are purely physico­chemical. In an egg these processes are guided into definite parts of the future embryo by the Mendelian factors of heredity—the so-called genes. These genes he compares to the foremen for the different types of work to be done in a building. But there must be something that makes of the work of the single genes a harmonious whole, and for this purpose he assumes the existence of “supergenes.”[4] v. Uexküll’s ideas concerning the nature of a Mendelian factor and of the “supergenes” are expressed in metaphorical terms and the assump­tion of the “supergenes” begs the ques­tion. The writer is under the impression that this author was led to his views by the belief that the egg is entirely undifferentiated. But the unfertilized egg is not homogeneous, on the contrary, it has a simple but definite physico­chemical structure which suffices to determine the first steps in the differentia­tion of the organism. Of course, if we suppose as do v. Uexküll and Driesch that the egg has no structure, the development of structure becomes a difficult problem—but this is not the real situa­tion.

2. Claude Bernard does not mention the possibility of explaining the harmony or apparent design in the organism on the basis of the theory of evolu­tion, he simply considers the problem as outside of biology. It was probably clear to him as it must be to everyone with an adequate training in physics that natural selec­tion does not explain the origin of varia­tion. Driesch and v. Uexküll consider the Darwinian theory a failure. We may admit that the theory of a forma­tion of new species by the cumulative effect of aimless fluctuating varia­tions is not tenable because fluctuating varia­tion is not hereditary; but this would only demand a slight change in the theory; namely a replacement of the influence of fluctuating varia­tion by that of equally aimless muta­tions. With this slight modifica­tion which is proposed by de Vries,[5] Darwin’s theory still serves the purpose of explaining how without any pre-established plan only purposeful and harmonious organisms should have survived. It must be said, however, that any theory of life phenomena must be based on our knowledge of the physico­chemical constitu­tion of living matter, and neither Darwin nor Lamarck was concerned with this. Moreover, we cannot consider any theory of evolu­tion as proved unless it permits us to trans­form at desire one species into another, and this has not yet been accomplished.

It may be of some interest to point out that we do not need to make any definite assump­tion concerning the mechanism of evolu­tion and that we may yet be able to account for the fact that the surviving organisms are to all appearances harmonious. The writer pointed out that of all the 100,000,000 conceivable crosses of teleost fish (many of which are possible) not many more than 10,000, i. e., about one-hundredth of one per cent., are able to live and propagate. Those that live and develop are free from the grosser type of disharmonies, the rest are doomed on account of a gross lack of harmony of the parts. These latter we never see and this gives us the erroneous concep­tion that harmony or “design” is a general character of living matter. If anybody wishes to call the non-viability of 9999/100 per cent. of possible teleosts a process of weeding out by “natural selec­tion” we shall raise no objec­tion, but only wish to point out that our way of explaining the lack of design in living nature would be valid even if there were no theory of evolu­tion or if there had never been any evolu­tion.

3. v. Uexküll is perfectly right in connecting the problem of design in an organism with Mendelian heredity. The work on Mendelian heredity has shown that an extremely large number of independently transmissible Mendelian factors help to shape the individual. It is not yet proven that the organism is nothing but a mosaic of Mendelian factors, but no writer can be blamed for considering such a possibility. If we assume that the organism is nothing but a mosaic of Mendelian characters it is difficult indeed to understand how they can force each other into a harmonious whole[6]; even if we make ample allowance for the law of chance and the corresponding wastefulness in the world of the living. But it is doubtful whether this idea of the rôle of Mendelian factors is correct. The facts of experi­mental embryology strongly indicate the possibility that the cytoplasm of the egg is the future embryo (in the rough) and that the Mendelian factors only impress the individual (and variety) characters upon this rough block. This idea is supported by the fact that the first development—in the sea urchin to the gastrula stage inclusive—is independent of the nucleus, which is the bearer of the Mendelian factors. Not before the skeleton or mesenchyme is formed in the sea urchin egg is the influence of the nucleus noticeable. This has been shown in the experi­ments of Boveri in which an enucleated fragment of an egg was fertilized with a spermato­zoön of a foreign species. If this is generally true, it is conceivable that the generic and possibly also the species characters of organisms are determined by the cytoplasm of the egg and not by the Mendelian factors.

In any case, we can state today that the cytoplasm contains the rough preforma­tion of the future embryo. This would show then that the idea of the organism being a mosaic of Mendelian characters which have to be put into place by “supergenes” is unnecessary. If the egg is already the embryo in the rough we can imagine the Mendelian factors as giving rise to specific substances which go into the circula­tion and start or accelerate different chemical reac­tions in different parts of the embryo, and thereby call forth the finer details characteristic of the variety and the individual. The idea that the egg is the future embryo is supported by the fact that we can call forth a normal organism from an unfertilized egg by artificial means; while it is apparently impossible to cause the spermato­zoön to develop into an organism outside the egg.

4. The influence of the whole on the parts is nowhere shown more strikingly than in the field of regenera­tion. It is known that pieces cut from the plant or animal may give rise to new growth which in many cases will restore somewhat the original organism. Instead of asking what is the cause of this so-called regenera­tion we may ask, why the same pieces do not regenerate as long as they are parts of the whole. In this form the mysterious influence of the whole over its parts is put into the foreground. We shall see that growth takes place in certain cells when certain substances in the circula­tion can collect there. The mysterious influence of the whole on these parts consists often merely of the fact that the circulating specific or non-specific substances—we cannot yet decide which—will in the whole be attracted by certain spots and that this will prevent them from acting on other parts of the organism. If such parts are isolated the substances can no longer flow away from these parts and the parts will begin to grow. It thus becomes utterly unnecessary to endow such organisms with a “directing force” which has to elaborate the isolated parts into a whole.

5. The same difficulty which we have discussed in regard to morphogenesis exists also in connec­tion with those instincts which preserve the life of the organism and of the race. The reader need only be reminded of all the complicated instincts of mating by which sperm and eggs are brought together; or those by which the young are prevented from starva­tion to realize the apparently desperate problems in store for a mechanist, to whom the assump­tion of design is meaningless. And yet we are better off in regard to our knowledge of the instincts than we are in regard to morphogenesis, as in the former we can show that the apparent instincts in some cases obey simple physico­chemical laws with almost mathematical accuracy. Since the validity of the law of gravita­tion has been proved for the solar system the idea of design in the motion of the planets has lost its usefulness, and this fact must serve us as a guide wherever we attempt to put science beyond the possibility of mysticism. As soon as we can show that a life phenomenon obeys a simple physical law there is no longer any need for assuming the action of non-physical agencies. We shall see that this has been accomplished for one group of animal instincts; namely those which determine the rela­tion of animals to light, since these are being gradually reduced to the law of Bunsen and Roscoe. This law states that the chemical effect of light equals the product of intensity into dura­tion of illumina­tion. Some authors object to the tendency toward reducing everything in biology to mathematical laws or figures; but where would the theory of heredity be without figures? Figures have been responsible for showing that the laws of chance and not of design rule in heredity. Biology will be scientific only to the extent that it succeeds in reducing life phenomena to quantitative laws.

Those familiar with the theories of evolution know the extensive rôle ascribed to the adapta­tions of organisms. The writer in 1889 called atten­tion to the fact that reac­tions to light—e. g., positive helio­tropism—are found in organisms that never by any chance make use of them; and later that a great many organisms show definite instinctive reac­tions towards a galvanic current—galvano­tropism—although no organism has ever had or ever will have a chance to be exposed to such a current except in laboratory experi­ments. This throws a different light upon the seemingly purposeful character of animal reac­tions. Heliotropism depends primarily upon the presence of photo­sensitive substances in the eye or the epidermis of the organism, and these substances are inherited regardless of whether they are useful or not. It is only a metaphor to call reac­tions resulting from the presence of photo­sensitive substances “adapta­tion.” In this book other examples are given which show that authors have too often spoken of adapta­tion to environ­ment where the environ­ment was not responsible for the phenomena. The blindness of cave animals and the resistance of certain marine animals to higher concentra­tions of sea water are such cases. Cuénot speaks of “preadapta­tion” to express this rela­tion. The fact is that the “adapta­tions” often existed before the animal was exposed to surroundings where they were of use. This relieves us also of the necessity of postulating the existence of the inheritance of acquired characters, although it is quite possible that the future may furnish proof that such a mode of inheritance exists.

6. We have mentioned that according to Claude Bernard two groups of phenomena occur in the living organism: (1) the phenomena of vital crea­tion or organizing synthesis (especially in the egg and during development); (2) the phenomena of death or organic destruc­tion. These two processes are briefly discussed in the first and last chapters.

These introductory remarks may perhaps make it easier for the reader to retain the thread of the main ideas in the details of experi­ments and tables given in this book.

CHAPTER II

THE SPECIFIC DIFFERENCE BETWEEN LIVING AND DEAD MATTER AND THE QUESTION OF THE ORIGIN OF LIFE

1. Each organism is characterized by a definite form and we shall see in the next chapter that this form is determined by definite chemical substances. The same is true for crystals, where substance and form are definitely connected and there are further analogies between organisms and crystals. Crystals can grow in a proper solu­tion, and can regenerate their form in such a solu­tion when broken or injured; it is even possible to prevent or retard the forma­tion of crystals in a supersaturated solu­tion by preventing “germs” in the air from getting into the solu­tion, an observa­tion which was later utilized by Schroeder and Pasteur in their experi­ments on spontaneous genera­tion. However, the analogies between a living organism and a crystal are merely superficial and it is by pointing out the fundamental differences between the behaviour of crystals and that of living organisms that we can best understand the specific difference between non-living and living matter. It is true that a crystal can grow, but it will do so only in a supersaturated solu­tion of its own substance. Just the reverse is true for living organisms. In order to make bacteria or the cells of our body grow, solu­tions of the split products of the substances composing them and not the substances themselves must be available to the cells; second, these solu­tions must not be supersaturated, on the contrary, they must be dilute; and third, growth leads in living organisms to cell division as soon as the mass of the cell reaches a certain limit. This process of cell division cannot be claimed even metaphorically to exist in a crystal. A correct apprecia­tion of these facts will give us an insight into the specific difference between non-living and living matter. The forma­tion of living matter consists in the synthesis of the proteins, nucleins, fats, and carbohydrates of the cells, from the split products. To give an historical example, Pasteur showed that yeast cells and other fungi could be raised on the following sterilized solu­tion: water, 100 gm., crystallized sugar, 10 gm., ammonium tartrate, 0.2 gm. to 0.5 gm., and fused ash from yeast, 0.1 gm.[7] He undertook this experi­ment to disprove the idea that protein or organic matter in a state of decomposi­tion was needed for the origin of new organisms as the defenders of the idea of spontaneous genera­tion had maintained.

2. That such a solu­tion can serve for the synthesis of all the compounds of living yeast cells is due to the fact that it contains the sugars. From the sugars organic acids can be formed and these with ammonia (which was offered in the form of ammonium tartrate) may give rise to the forma­tion of amino acids, the “building stones” of the proteins. It is thus obvious that the synthesis of living matter centres around the sugar molecule. The phosphates are required for the forma­tion of the nucleins, and the work of Harden and Young suggests that they play also a rôle in the alcoholic fermenta­tion of sugar.

Chlorophyll, under the influence of the red rays of light, manufactures the sugars from the CO₂ of the air. This makes it appear as though life on our planet should have been preceded by the existence of chlorophyll, a fact difficult to understand since it seems more natural to conceive of chlorophyll as a part or a product of living organisms rather than the reverse. Where then should the sugar come from, which is a constituent of the majority of culture media and which seems a prerequisite for the synthesis of proteins in living organisms?

The investiga­tions of Winogradsky on nitrifying,[8] sulphur and perhaps also on iron bacteria have to all appearances pointed a way out of this difficulty. It seemed probable that there were specific micro-organisms which oxidized the ammonia formed in sewage or in the putrefac­tion of living matter, but the attempts to prove this assump­tion by raising such a nitrifying micro-organism on one of the usual culture media, all of which contained organic compounds, failed. Led by the results of his observa­tions on sulphur bacteria it occurred to Winogradsky that the presence of organic compounds stood in the way of raising these bacteria, and this idea proved correct. The bacteria oxidizing ammonia to nitrites were grown on the following medium; 1 gm. ammonium sulphate, 1 gm. potassium phosphate, 1 gm. magnesium carbonate, to 1 litre of water. From this medium, which is free from sugar and contains only constituents which could exist on the planet before the appearance of life, the nitrifying bacteria were able to form sugars, fatty acids, proteins, and the other specific constituents of living matter. Winogradsky proved, by quantitative determina­tion, that with the nitrifica­tion an increase in the amount of carbon compounds takes place. “Since this bound carbon in the cultures can have no other source than the CO₂ and since the process itself can have no other cause than the activity of the nitrifying organism, no other alternative was left but to ascribe to it the power of assimilating CO₂.”[9] “Since the oxida­tion of NH₃ is the only source of chemical energy which the nitrifying organism can use it was clear a priori that the yield in assimila­tion must correspond to the quantity of oxidized nitrogen. It turned out that an approximately constant ratio exists between the values of assimilated carbon and those of oxidized nitrogen.” This is illustrated by the results of various experi­ments as shown in Table I.

TABLE I

It is obvious that 1 part of assimilated carbon corresponds to about 35.4 parts oxidized nitrogen or 96 parts of nitrous acid.

These results of Winogradsky were confirmed in very careful experi­ments by E. Godlewski, Sr.[10]

The nitrites are further oxidized by another kind of micro-organisms into nitrates and they also can be raised without organic material.

Winogradsky had already previously discovered that the hydrogen sulphide which is formed as a reduc­tion product from CaSO₄ or in putrefac­tion by the activity of certain bacteria can be oxidized by certain groups of bacteria, the sulphur bacteria. Such bacteria, e. g., Beggiatoa, are also commonly found at the outlet of sulphur springs. They utilize the hydrogen sulphide which they oxidize to sulphur and afterwards to sulphates, according to the scheme:

(1) 2H₂S + O₂ = 2H₂O + S₂

(2) S₂ + 3O₂ + 2H₂O = 2H₂SO₄

The sulphuric acid is at once neutralized by carbonates.

Winogradsky assumes that the oxida­tion of H₂S by the sulphur bacteria is the source of energy which plays the same rôle as the oxida­tion of NH₃ plays in the nitrifying bacteria, or the oxida­tion of carbon compounds—sugar and others—in the case of the other lower and higher organisms. Winogradsky has made it very probable that sulphur bacteria do not need any organic compounds and that their nutri­tion may be accomplished with a purely mineral culture medium, like that of the nitrite bacteria. On the basis of this assump­tion they should also be able to form sugars from the CO₂ of the air.

Nathanson[11] discovered in the sea water the existence of bacteria which oxidize thiosulphate to sulphuric acid. They will develop if some Na₂S₂O₃, is added to sea water. These bacteria can only develop if CO₂ from the air is admitted or when carbonates are present. For these organisms the CO₂ cannot be replaced by glucose, urea, or other organic substances. Such bacteria must therefore possess the power of producing sugar and starch from CO₂ without the aid of chlorophyll. Similar observa­tions were made by Beijerinck on a species of fresh-water bacteria.[12]

Finally the case of iron bacteria may briefly be mentioned though Winogradsky’s views are not accepted by Molisch.

We may, therefore, consider it an established fact that there are a number of organisms which could have lived on this planet at a time when only mineral constituents, such as phosphates, K, Mg, SO₄, CO₂, and O₂ besides NH₃, or SH₂, existed. This would lead us to consider it possible that the first organisms on this planet may have belonged to that world of micro-organisms which was discovered by Winogradsky.

If we can conceive of this group of organisms as producing sugar, which in fact they do, they could have served as a basis for the development of other forms which require organic material for their development.

In 1883 the small island of Krakatau was destroyed by the most violent volcanic erup­tion on record. A visit to the islands two months after the erup­tion showed that “the three islands were covered with pumice and layers of ash reaching on an average a thickness of thirty metres and frequently sixty metres.”[13] Of course all life on the islands was extinct. When Treub in 1886 first visited the island, he found that blue-green algæ were the first colonists on the pumice and on the exposed blocks of rock in the ravines on the mountain slopes. Investiga­tions made during subsequent expedi­tions demonstrated the associa­tion of diatoms and bacteria. All of these were probably carried by the wind. The algæ referred to were according to Euler of the nostoc type. Nostoc does not require sugar, since it can produce that compound from the CO₂ of the air by the activity of its chlorophyll. This organism possesses also the power of assimilating the free nitrogen of the air. From these observa­tions and because the Nostocaceæ generally appear as the first settlers on sand the conclusion has been drawn that they or the group of Schizophyceæ to which they belong formed the first settlers of our planet.[14] This conclusion is not quite safe since in the settlement of Krakatau as well as in the first colonizing of sand areas the nature of the first settler is determined chiefly by the carrying power of wind (or waves and birds).

We may now return from this digression to the real object of our discussion, namely that the nutritive solu­tions of organisms must be very dilute and consist of the split products of the complicated compounds of which the organisms consist. The examples given sufficiently illustrate this statement.

The nutritive medium of our body cells is the blood, and while we take up as food the complicated compounds of plants or animals, these substances undergo a diges­tion, i. e., a splitting up into small constituents before they can diffuse from the intestine into the blood. Thus the proteins are digested down to the amino acids and these diffuse into the blood as demonstrated by Folin and by Van Slyke. From here the cells take them up. The different proteins differ in regard to the different types of amino acids which they contain. While the bacteria and fungi and apparently the higher plants can build up all their different amino acids from ammonia, this power is no longer found in the mammals which can form only certain amino acids in their body and must receive the others through their food. As a consequence it is usually necessary to feed young animals on more than one protein in order to make them grow, since one protein, as a rule, does not contain all the amino acids needed for the manufacture of all the proteins required for the forma­tion of the material of a growing animal.[15]

3. The essential difference between living and non-living matter consists then in this: the living cell synthetizes its own complicated specific material from indifferent or non-specific simple compounds of the surrounding medium, while the crystal simply adds the molecules found in its supersaturated solu­tion. This synthetic power of trans­forming small “building stones” into the complicated compounds specific for each organism is the “secret of life” or rather one of the secrets of life.

What clew have we in regard to the nature of this synthetic power? We know that the comparatively great velocity of chemical reac­tions in a living organism is due to the presence of enzymes (ferments) or to catalytic agencies in general. Some of these catalytic agencies are specific in the sense that a given catalyzer can accelerate the reac­tion of only one step in a complicated chemical reac­tion. While these enzymes are formed by the action of the body they can be separated from the body without losing their catalytic efficiency. It was a long time before scientists succeeded in isolating the enzyme of the yeast cell which causes the alcoholic fermenta­tion of sugar; and this gave rise to the premature statement that it was not possible to isolate this enzyme since it was bound up with the life of the yeast cell. Such a statement was even made by a man like Pasteur, who was usually a model of restraint in his utterances, and yet the work of Buchner proved him to be wrong.

The general mechanism of the action of the hydrolyzing enzymes is known. The old idea of de la Rive, that a molecule of enzyme combines transitorily with a molecule of substrate; the further idea, which may possibly go back to Engler, that the molecule of substrate is disrupted in the “strain” of the new combina­tion and that the broken fragments fall off or are easily knocked off by collision from the ferment molecule which is now ready to repeat the process, seems to be correct. On the assump­tion that the velocity of enzyme reac­tion is propor­tional to the mass of the enzyme and that de la Rive’s idea was correct, Van Slyke and Cullen were able to calculate the coefficients of the velocity of enzyme reac­tions for the fermenta­tion of urea and other substances, and the agreement between calculated and observed values was remarkable.[16]

While the hydrolytic action of enzymes is thus clear the synthesis in the cell is still a riddle. An interesting sugges­tion was made by van’t Hoff, who in 1898 expressed the idea that the hydrolytic enzymes should also act in the opposite direc­tion, namely synthetically. Thus it should not only be possible to digest proteins with pepsin but also to synthetize them from the products of diges­tion with the aid of the same enzyme. This expecta­tion was based on the idea that the enzyme did not alter the equilibrium between the hydrolyzed and non-hydrolyzed part of the substrate but only accelerated the rate with which the equilibrium was reached. Van’t Hoff’s idea omitted, however, the possibility that in the transitory combina­tion between enzyme molecule and substrate a change in the molecular configura­tion of the substrate or in the distribu­tion of intramolecular strain may take place. The first apparently complete confirma­tion of van’t Hoff’s sugges­tion appeared in the form of the synthesis of maltose from grape sugar by the enzyme maltase, which decomposes maltose into grape sugar. By adding the enzyme maltase from yeast to a forty per cent. solu­tion of glucose Croft Hill[17] obtained a good yield of maltose. It turned out, however, that what he took for maltose was not this compound but an isomer, namely isomaltose, which has a different molecular configura­tion and cannot be hydrolyzed by the enzyme maltase.

Lactose is hydrolyzed from kephyr by an enzyme lactase into galactose and glucose; by adding this enzyme to galactose and glucose a synthesis was obtained not of lactose but of isolactose; the latter, however, is not decomposed by the enzyme lactase.

E. F. Armstrong has worked out a theory which tries to account for this striking phenomenon by assuming “that the enzyme has a specific influence in promoting the forma­tion of the biose which it cannot hydrolyze.”[18] The theory is very ingenious and seems supported by fact. This then would lead to the result that certain hydrolytic enzymes may have a synthetic action but not in the manner suggested by van’t Hoff.

The principle enunciated by Armstrong, that in the synthetic action of hydrolytic enzymes not the original compound but an isomer is formed which can not be hydrolyzed by the enzyme, may possibly be of great importance in the understanding of life phenomena. It shows us how the cell can grow in the presence of hydrolytic enzymes and why in hunger the disintegra­tion of the cell material is so slow. It was at first thought that the forma­tion of isomers contradicted the idea of the reversible action of enzymes, but this is not the case; on the contrary, it supports it but makes an addi­tion which may solve the riddle of what Claude Bernard called the creative action of living matter. We shall come back to this problem in the last chapter.

Kastle and Loevenhart demonstrated the synthesis of a trace of ethylbutyrate by lipase if the latter enzyme was added to the products of the hydrolysis of ethylbutyrate, ethyl alcohol, and butyric acid by the same enzyme.[19] Taylor[20] obtained the synthesis of a slight amount of triolein

by the addi­tion of the dried fat-free residue of the castor bean to a mixture of oleinic acid and glycerine. . . . No synthesis occurred with acetic, butyric, palmitic, and stearic acids with glycerine, mannite, and dulcite, and the experi­ments with the last two alcohols and oleinic acid likewise yielded no synthesis.

This suggests possibly a specific action of the enzyme. If this slight reversible action had any biological significance (which might be possible, since in the organism secondary favourable condi­tions might be at work which are lacking in vitro) there should be a parallelism between masses of lipase in different kinds of tissues and fat synthesis. Loevenhart indicated that this might be a fact, but a more extensive investiga­tion by H. C. Bradley has made this very dubious.[21]

Very little is known concerning the reversible action of the hydrolytic protein enzymes. A. E. Taylor digested protamine sulphate with trypsin and found that after adding trypsin to the products of diges­tion a precipitate was formed after long standing; and we may also refer to experi­ments of Robertson with pepsin on the products of caseinogen to which we shall return in the next chapter. It therefore looks at present as if van’t Hoff’s idea of reversible enzyme action might hold in the modifica­tion offered by Armstrong. It remains doubtful, however, whether this reversibility can explain all the synthetic processes in the cell. No objec­tion can be offered at present if any one makes the assump­tion that each cell has specific synthetic enzymes or some other synthetic mechanisms which are still unknown.

The mechanisms for the synthesis of proteins must have one other peculiarity: they must be specific in their action. We shall see in the next chapter that each species seems to possess one or more proteins not found in any other but closely related species. Each organism develops from a tiny microscopic germ and grows by synthetizing the non-specific building stones (amino acids) into the specific proteins of the species. This must be the work of the yet unknown synthetic enzymes or mechanisms. The elucida­tion of their character would seem one of the main problems of biology. Needless to say crystallography is not confronted with problems of such a nature.

The fact that the living cell grows after taking up food has given rise to curious misunderstandings. Traube has shown that drops of a liquid surrounded with a semipermeable membrane may increase in volume when put into a solu­tion of lower osmotic pressure. This has led and is possibly still leading to the statement that the process of growth by a living cell has been imitated artificially. Only one feature has been imitated, the increase in volume; but the essential feature of the process in the living cell, i. e., the forma­tion of the specific constituents of the living cell from non-specific products, has of course not been imitated.

4. The constant synthesis then of specific material from simple compounds of a non-specific character is the chief feature by which living matter differs from non-living matter. With this character is correlated another one; namely, when the mass of a cell reaches a certain limit the cell divides. This is perhaps most obvious in bacteria which on the proper nutritive medium take up food, grow, and divide into two bacteria, each of which takes up food, divides, and grows ad infinitum, as long as the food lasts, provided the harmful products of metabolism are removed. If it be true that specific synthetic ferments exist in each cell it follows that the cell must synthetize these also,[22] as otherwise the synthesis of specific proteins would have to come to a standstill.

This problem of synthesis leads to the assump­tion of immortality of the living cell, since there is no a priori reason why this synthesis should ever come to a standstill of its own accord as long as enough food is available and the proper outside physical condi­tions are guaranteed. It is well known that Weismann has claimed immortality for all unicellular organisms and for the sex cells of metazoa, while he claimed the necessity of death for the body cells of the latter. Leo Loeb was led by his investiga­tions on the transplanta­tion of cancer to assume immortality not only for the cancer cell but also for the body cell of the organism. He had found in transplanting a malignant tumor from one individual to another that the tumor grew; that it was not the cells of the host but the transplanted tumor cells of the graft which grew and multiplied, and that this process could be repeated apparently indefinitely so that it was obvious that the transplanted tumor cells outlived the original animal. Such experi­ments have since been carried on so long that we may now say that an individual cancer cell taken from an animal and transplanted from time to time on a new host lives apparently indefinitely. Leo Loeb had found that these tumor cells are simply modified somatic cells. He therefore suggested that the somatic cells might be considered immortal with the same right as we speak of the immortality of the germ cells of such animals.[23]

This view receives its support first from the fact that certain trees like the Sequoia live several thousand years and may therefore be considered immortal and second, from the method of tissue culture. The method of cultivating tissue cells in a test tube, in the same way as is done for bacteria, was first proposed and carried out by Leo Loeb, in 1897,[24] but his test-tube method did not permit the observa­tion of the transplanted cell under the microscope. This was made possible by a modifica­tion of the method by Harrison, who established the fact that the axis cylinder grows out from the ganglionic cell. Harrison and Burrows then perfected the method for the cultiva­tion of the cells of warm-blooded, animals, and with the aid of these methods Carrel succeeded in keeping connective-tissue cells of the heart of an early chick embryo alive more than four years, and these cells are still growing and dividing.[25] Only very tiny masses of cells can be kept alive in this way since all the cells in the centre of a piece die on account of lack of oxygen; and every two days a few cells from the margin of the piece have to be transferred to a new culture medium.

This effect of lack of oxygen explains also why the immortality of the somatic cells is not obvious. Death in a human being consists in the stopping of heart beat and respira­tion, which also terminates the action of the brain or at least of consciousness. Immediately after the cessa­tion of heart beat and respira­tion the cells of muscle and of the skin and probably many or most other organs are still alive and might continue to live if transferred to another body with circula­tion and respira­tion. As a consequence of the lack of oxygen supply in the dead body they will, however, die comparatively rapidly. It may be stated that hearts taken out of the body after a number of hours can still beat again when put into the proper solu­tions and upon receiving an adequate oxygen supply.

The idea that the body cells are naturally immortal and die only if exposed to extreme injuries such as prolonged lack of oxygen or too high a temperature helps to make one problem more intelligible. The medical student, who for the first time realizes that life depends upon that one organ, the heart, doing its duty incessantly for the seventy years or so allotted to man, is amazed at the precariousness of our existence. It seems indeed uncanny that so delicate a mechanism should func­tion so regularly for so many years. The mysticism connected with this and other phenomena of adapta­tion would disappear if we could be certain that all cells are really immortal and that the fact which demands an explana­tion is not the continued activity but the cessa­tion of activity in death. Thus we see that the idea of the immortality of the body cell if it can be generalized may be destined to become one of the main supports for a complete physico­chemical analysis of life phenomena since it makes the durability of organisms intelligible.

5. This generalized idea of the immortality of some or possibly most or all somatic cells has a bearing upon the problem of the origin of life on our planet. The experi­ments of Spallanzani, Schwann, Schroeder, Pasteur, Tyndall, and all those who have worked with pure cultures of micro-organisms, have proved that no spontaneous genera­tion of living from non-living matter can be demonstrated; and the statements to the contrary were due to experi­mental errors inasmuch as the new organisms formed were the offspring of others which had entered into the culture medium by mistake.

In the last chapter of that most fascinating book Worlds in the Making,[26] Arrhenius discusses the possibility of life being eternal and of living germs of very small dimensions—e. g., the spores of micro-organisms—being carried through space from one planet to another or even from one solar system to another. If it be true that there is no spontaneous genera­tion; if it be true that all cells are potentially immortal, we may indeed seriously raise the ques­tion: May not life after all be eternal? Such ideas were advocated by Richter in a rather phantastic way and more definitely by Helmholtz as well as Kelvin. The latter authors assumed that in the collision of planets or worlds on which there is life, fragments containing living organisms will be torn off and these fragments will move as seed-bearing stones through space. “If at the present instant no life existed upon this earth, one such stone falling upon it might . . . lead to its becoming covered with vegeta­tion.” Arrhenius points out the difficulties which oppose such a view, as, e. g., the fact “that the meteorite in its fall towards the earth becomes incandescent all over its surface and any seeds on it would therefore be deprived of their germinating power.”

Arrhenius suggests another and much more ingenious idea based on the fact that for particles below a certain size the mechanical pressure produced by light waves—the radia­tion pressure—can overcome the attractive force of gravita­tion.

Bodies which according to Schwarzschild would undergo the strongest influence of solar radia­tion must have a diameter of 0.00016 mm. supposing them to be spherical. The first ques­tion is therefore: Are there any living seeds of such extraordinary minuteness? The reply of the botanist is that spores of many bacteria have a size of 0.0003 or 0.0002 mm., and there are no doubt much smaller germs which our microscopes fail to disclose.

This assumption is undoubtedly correct.

We will, in the first instance, make a rough calcula­tion of what would happen if such an organism were detached from the earth and pushed out into space by the radia­tion pressure of our sun. The organism would first of all have to cross the orbit of Mars; then the orbits of the smaller and of the outer planets. . . . The organisms would cross the orbit of Mars after twenty days, the Jupiter orbit after eighty days, and the orbit of Neptune after fourteen months. Our nearest solar system would be reached in nine thousand years.

For the assump­tion of eternity of life only the transference of germs from one solar system to another would have to be considered and the ques­tion arises whether or not germs can keep their vitality so many thousands of years. Arrhenius thinks that this is possible on account of the low temperature (which must be below -220° C.) at which no chemical reac­tion and hence no decomposi­tion and deteriora­tion are possible in the spores; and on account of the absence of water vapour.

The ques­tion then arises: Have we any facts to warrant the assump­tion that spores may remain alive for thousands of years under such condi­tions and retain their power of germina­tion? We know that seeds have a very limited vitality, and the statement that grain found in the Egyptian tombs was still able to germinate has long been recognized as a myth. Miss White[27] found that in wheat grains, there appeared a well-marked drop in their germinating power after about the fourth year, reaching zero in eleven to seventeen years. In a drier climate they last longer than in a moist climate. It is of importance that the hydrolyzing enzymes in the seeds, such as diastase, erepsin, remained unimpaired even after the germinating power of the seeds had disappeared. The seeds were able to resist for two days the temperature of liquid air, though the subsequent germina­tion was delayed by this treatment. Macfadyen[28] exposed non-sporing bacteria, viz., B. typhosus, B. coli communis, Staphylococcus pyogenes aureus, and a Saccharomyces to liquid air.

The experi­ments showed that a prolonged exposure of six months to a temperature of about -190° has no appreciable effect on the vitality of micro-organisms. To judge by the results there appeared no reason to doubt that the experi­ment might have been successfully prolonged for a still longer period.

Paul Becquerel[29] found that seeds which possess a very thick integument may live longer than the grain in Miss White’s experi­ments. The thickness of the integument prevents the exchange of gases between air and seed. Thus seeds of leguminoses (Cassia bicapsularis, Cytisus biflorus, Leucæna leucocephala, and Trifolium arvense) had retained their power of germina­tion for eighty-seven years. Becquerel has shown that the dryness of the membrane is very essential for such a dura­tion of life, since when dry it is impermeable for gases and the slow chemical reac­tions inside the grain become impossible.

In the cosmic space there is no water vapour, no atmosphere, and a low temperature, and there is hence no reason why spores should lose appreciably more of their germinating power in ten thousand years than in six months. We must therefore admit the possibility that spores may move for an almost infinite length of time through cosmic space and yet be ready for germina­tion when they fall upon a planet in which all the condi­tions for germina­tion and development exist, e. g., water, proper temperature, and the right nutritive substances dissolved in the water (inclusive of free oxygen).

While thus everything is favourable to Arrhenius’s hypothesis, Becquerel raises the objec­tion that the spores going through space would yet be destroyed by ultraviolet light. This danger would probably exist only as long as the germ is not too far from a sun. The difficulty is a real one since the ultraviolet rays have a destructive effect even in the absence of oxygen. It is possible, however, that there are spores which can resist this effect of ultraviolet light. Arrhenius’s theory can not of course be disproved and we must agree with him that it is consistent not only with the theories of cosmogony but also with the seeming potential immortality of certain or of all cells.

The alternative to Arrhenius’s theory is that living matter did originate and still originates from non-living matter. If this idea is correct it should one day be possible to discover synthetic enzymes which are capable of forming molecules of their own kind from a simple nutritive solu­tion. With such synthetic enzymes as a starting point the task might be undertaken of creating cells capable of growth and cell division, at least in the apparently simple form in which these phenomena occur in bacteria; viz., that after the mass has reached a certain (still microscopic) size it divides into two cells and so on. If Arrhenius is right that living matter has had no more beginning than matter in general, this hope of making living matter artificially appears at present as futile as the hope of making molecules out of electrons.

The problem of making living matter artificially has been compared to that of constructing a perpetuum mobile; this comparison is, however, not correct. The idea of a perpetuum mobile contradicts the first law of thermodynamics, while the making of living matter may be impossible though contradicting no natural law.

Pasteur’s proof that spontaneous genera­tion does not occur in the solu­tions used by him does not prove that a synthesis of living from dead matter is impossible under any condi­tions. It is at least not inconceivable that in an earlier period of the earth’s history radio-activity, electrical discharges, and possibly also the action of volcanoes might have furnished the combina­tion of circumstances under which living matter might have been formed. The staggering difficulties in imagining such a possibility are not merely on the chemical side—e. g., the produc­tion of proteins from CO₂, and N—but also on the physical side if the necessity of a definite cell structure is considered. We shall see in the sixth chapter that without a structure in the egg to begin with, no forma­tion of a complicated organism is imaginable; and while a bacterium may have a simple structure, such a structure as it possesses is as necessary for its existence as are its enzymes.

Attempts have repeatedly been made to imitate the structures in the cell and of living organisms by colloidal precipitates. It is needless to point out that such precipitates are of importance only for the study of the origin of structures in the living, but that they are not otherwise an imita­tion of the living since they are lacking the characteristic synthetic chemical processes.

CHAPTER III

THE CHEMICAL BASIS OF GENUS AND SPECIES

1. It is a truism that from an egg of a species an organism of this species only and of no other will arise. It is also a truism that the so-called protoplasm of an egg does not differ much from that of eggs of other species when looked at through a microscope. The ques­tion arises: What determines the species of the future organism? Is it a structure or a specific chemical or groups of chemicals? In a later chapter we shall show that the egg has a simple though definite structure, but in this chapter we shall see that the egg must contain specific substances and that these substances which determine the “species” and specificity in general are in all probability proteins. Since solu­tions of different proteins look alike under a microscope we need not wonder that it is impossible to discriminate microscopically between the protoplasm of different eggs.

The idea of definiteness and constancy of species, a matter of daily observa­tion in the case of man and higher animals in general, was not so readily accepted in the case of the micro-organisms, which on account of their minuteness and simplicity of structure are not so easy to differentiate. There existed for a long time serious doubt whether or not the simplest organisms, the bacteria, possessed a definite “specificity” like the higher organisms, or whether they were not endowed, as Warming put it, with an “unlimited plasticity,” which forbade classifying them according to their form into definite species as Cohn had done. An interesting episode in this discussion, which was settled about twenty-five years ago arose concerning the sulphur bacteria, which often develop in large masses on parts of decaying plants or animals along the shore. Sir E. Ray Lankester found collec­tions of red bacteria covering putrefying animal matter in a vessel and forming a continuous membrane along its wall. These red bacteria were of very different shape, size, and grouping, but they seemed to be connected by transi­tion forms. They had a common character, however, namely, their peach-coloured appearance. This common character, together with their associa­tion in the same habitat, led Lankester to the then justifiable belief that they all belonged to one species which was protean in character and that the different forms were only to be considered as phases of growth of this one species. The presence of the same red pigment “Bacterio-purpurin” seemed justly to indicate the existence of common chemical processes. Cohn, on the contrary, considered the different forms among these red bacteria (they are today called sulphur bacteria since they oxidize the hydrogen sulphide produced by bacteria of putrefac­tion to sulphur and sulphates) as definite and distinct species, in spite of their common colour and their associa­tion. Later observa­tions showed that Cohn was right. Winogradsky[30] succeeded in proving by pure culture experi­ments that each of these different forms of sulphur bacteria was specific and did not give rise to any of the other forms of the same colour found in the same condi­tions.

The method of pure line breeding inaugurated by Johannsen[31] has shown that the degree of definiteness goes so far that apparently identical forms with only slight differences in size may breed true to this size; but for reasons which will become clear later on we may doubt whether they are to be considered as definite species.

The fact of specificity is supported by the fact of constancy of forms. de Vries has pointed out that regardless of the possible origin of new species by muta­tion the old species may persevere. Walcott has found fossils of annelids, snails, crustaceans, and algæ in a precambrian forma­tion in British Columbia whose age (estimated on the rate of forma­tion of radium from uranium) may be about two hundred million years and estimated on the basis of sedimenta­tion sixty million years. And yet these invertebrates are so closely related to the forms existing today that the systematists have no difficulty in finding the genus among the modern forms into which each of these organisms belongs. W. M. Wheeler, in his investiga­tions of the ants enclosed in amber, was able to identify some of them with forms living today, though the ants observed in the amber must have been two million years old. The constancy of species, i. e., the permanence of specificity may therefore be considered as established as far back as two or possibly two hundred millions of years. The definiteness and constancy of each species must be determined by something equally definite and constant in the egg, since in the latter the species is already fixed irrevocably.

We shall show first that species if sufficiently separated are generally incompatible with each other and that any attempt at fusing or mixing them by grafting or cross-fertilizing is futile. In the second part of the chapter we shall take up the facts which seem destined to give a direct answer to the ques­tion as to the cause of specificity. It is needless to say that this latter ques­tion is of paramount importance for the problem of evolu­tion, as well as for that of the constitu­tion of living matter.

I. The Incompatibility of Species not closely Related

2. It is practically impossible to transplant organs or tissues from one species of higher animals to another, unless the two species are very closely related; and even then the transplanta­tion is uncertain and the graft may either fall off again or be destroyed. This specificity of tissues goes so far that surgeons prefer, when a transplanta­tion of skin in the human is intended, to use skin of the patient or of close blood rela­tions. The reason why the tissues of a foreign species in warm-blooded animals cannot grow well on a given host has been explained by the remarkable experi­ments of James B. Murphy of the Rockefeller Institute.[32] Murphy discovered that it is possible to transplant successfully any kind of foreign tissue upon the early embryo of the chick. Even human tissue transplanted upon the chick embryo will grow rapidly. This shows that at this early stage the chick embryo does not yet react against foreign tissue. This lack of reac­tion lasts until about the twenty-first day in the life of the embryo; then the growth of the graft not only ceases but the graft itself falls off or is destroyed. Murphy noticed that this critical period coincides with the development of the spleen and of lymphatic tissue in the chick and that a certain type of migrating cells, the so-called lymphocytes, which develop in the lymphatic tissue, gather at the edge of the graft in great numbers, and he suggested that these lymphocytes (by a secre­tion of some substance?) rid the host of the graft. He applied two tests both of which confirmed this idea. First he showed that when small fragments of the spleen of an adult chicken are transplanted into the embryo the latter loses its tolerance for foreign grafts. The second proof is still more interesting. It was known that by treatment with Roentgen rays the lymphocytes in an animal could be destroyed. It was to be expected that an animal so treated would have lost its specific resistance to foreign tissues. Murphy found that this was actually the case. On fully grown rats in which the lymphocytes had been destroyed by X-rays (as ascertained by blood counts) tissues of foreign species grew perfectly well. These experi­ments have assumed a great practical importance since they can also be applied to the immuniza­tion of an animal against transplanted cancer of its own species. Murphy found that by increasing the number of lymphocytes in an animal (which can be accomplished by a mild treatment with X-rays) the immunity against foreign grafts as well as against cancer from the same species can be increased. It is quite possible that the apparent immunity to a transplanta­tion of cancer produced by Jensen, Leo Loeb, and Ehrlich and Apolant through the previous transplanta­tion of tissue in such an animal was due to the fact that this previous tissue transplanta­tion led to an increase in the number of lymphocytes in the animal. The medical side, however, lies outside of our discussion, and we must satisfy ourselves with only a passing notice. The facts show that each warm-blooded animal seems to possess a specificity whereby its lymphocytes destroy transplanted tissue taken from a foreign species.

A lesser though still marked degree of incompatibility exists also in lower animals for grafts from a different species.[33] The graft may apparently take hold, but only for a few days, if the species are not closely related. Joest apparently succeeded in making a permanent union between the anterior and posterior ends of two species of earthworms, Lumbricus rubellus and Allolobophora terrestris. Born and later Harrison healed pieces of tadpoles of different species together. An individual made up of two species Rana virescens and Rana palustris lived a considerable time and went through metamorphosis. Each half regained the characteristic features of the species to which it belonged. It seems, however, that if species of tadpoles of two more distant species are grafted upon each other no lasting graft can be obtained, e. g., Rana esculenta and Bombinator igneus. These experi­ments were made at a time when the nature and bearing of the problem of specificity was not yet fully recognized. The rôle of lymphocytes in these cases has never been investigated. The grafted piece always retained the characteristics of the species from which it was taken.

Plants possess no leucocytes and we therefore see that they tolerate a graft of foreign tissues better than is the case in animals. As a matter of fact hetero­c grafting is a common practice in horticulture, although even here it is known that indiscriminate hetero­plastic grafting is not feasible and that therefore the specificity is not without influence. The host is supposed to furnish only nutritive sap to the graft and in this respect does not behave very differently from an artificial nutritive solu­tion for the raising of a plant. The law of specificity, however, remains true also for the grafted tissues: neither in animals nor in plants does the graft lose its specificity, and it never assumes the specific characters of the host, or vice versa. The apparent excep­tions which Winkler believed he had found in the case of grafts of nightshade on tomatoes turned out to be a further proof of the law of specificity. Winkler, after the graft had taken, cut through the place of grafting, after which opera­tion a callus forma­tion occurred on the wound. In most cases either a pure nightshade or a pure tomato grew out from this callus. In some cases he obtained shoots from the place where graft and host had united, which on one side were tomato, on the other side nightshade. What really happened was that the shoots had a growing point whose cells on the one side consisted of cells of nightshade, on the other side of tomato.[34] We know of no case in which the cell of a graft has lost its specificity and undergone a trans­forma­tion into the cell of the host.

3. Another manifesta­tion of the incompatibility of distant species is found in the domain of fertiliza­tion. The eggs of the majority of animals cannot develop unless a spermato­zoön enters. The entrance of a spermato­zoön into an egg seems also to fall under the law of specificity, inasmuch as in general only the sperm of the same or a closely related species is able to enter the egg. The writer[35] has found, however, that it is possible to overcome the limita­tion of specificity in certain cases by physico­chemical means, and by the knowledge of these means we may perhaps one day be able to more closely define the mechanism of specificity in this case. He found that the eggs of a certain Californian sea urchin, which cannot be fertilized by the sperm of starfish in normal sea water, will lose their specificity towards this type of foreign sperm if the sea water is rendered a little more alkaline, or if a little more Ca is added to the sea water, or if both these varia­tions are effected. Godlewski has confirmed the efficiency of this method for the fertiliza­tion of sea-urchin eggs with the sperm of crinoids.

Fig. 3. Five-days-old larvæ of two closely related forms of sea urchins (S. purpuratus ♀ and S. franciscanus ♂). In this case the larva has also paternal characters as shown by the skeleton.

If such hetero­geneous hybridiza­tions are carried out, two striking results are obtained. The one is that the resulting larva has only maternal characteristics (Figs. 1 and 2), as if the sperm had contributed no hereditary material to the developing embryo. This result could not have been predicted, for if we fertilize the egg of the same Californian sea urchin, Strongylo­centrotus purpuratus, with the sperm of a very closely related sea urchin, S. franciscanus, the hereditary effect of the spermato­zoön is seen very distinctly in the primitive skeleton formed by the larva.[36] (Fig. 3.) In the case of the hetero­geneous hybridiza­tion the spermato­zoön acts practically only as an activating agency upon the egg and not as a transmitter of paternal qualities.

The second striking fact is that while the sea-urchin eggs fertilized with starfish sperm develop at first perfectly normally they begin to die in large numbers on the second and third day of their development, and only a very small number live long enough to form a skeleton; and these are usually sickly and form the skeleton considerably later than the pure breed. It is not quite certain whether the sickliness of these hetero­geneous hybrids begins or assumes a severe character with the development of a certain type of wandering cells, the mesenchyme cells; it would perhaps be worth while to investigate this possibility. The writer was under the impression that this sickliness might have been brought about by a poison gradually formed in the hetero­geneous larvæ.

He investigated the effects of hetero­geneous hybridiza­tion also in fishes, which are a much more favourable object. The egg of the marine fish Fundulus hetero­clitus can be fertilized with the sperm of almost any other teleost fish, as Moenkhaus[37] first observed. This author did not succeed in keeping the hybrids alive more than a day, but the writer has kept many hetero­geneous hybrids alive for a month or longer,[38] and found the same two striking facts which he had already observed in the hetero­geneous cross between sea urchin and starfish: first, practically no transmission of paternal characters, and second, a sickly condi­tion of the embryo which begins early and which increases with further development. The hetero­geneous fish hybrids between, e. g., Fundulus hetero­clitus ♀ and Menidia ♂ have usually no circula­tion of blood, although the heart is formed and beats and blood-vessels and blood cells are formed; the eyes are often incomplete or abnormal though they may be normal at first; the growth of the embryo is mostly retarded. In excep­tional cases circula­tion may be established and in these a normal embryo may result, but such an embryo is chiefly maternal.

This incompatibility of two gametes from different species does not show itself in the case of hetero­geneous hybridiza­tion only, but also though less often in the case of crossing between two more closely related forms. The cross between the two related forms S. purpuratus ♀ and S. franciscanus ♂ is very sturdy and shows no abnormal mortality as far as the writer’s observa­tions go. If, however, the reciprocal crossing is carried out, namely that of S. franciscanus ♀ and S. purpuratus ♂, the development is at first normal, but beginning with the time of mesenchyme forma­tion the majority of larvæ become sickly and die; and again the ques­tion may be raised whether or not the beginning of sickliness coincides with the development of mesenchyme cells. If we assume that the sickliness and death are due to the forma­tion of a poison, we must assume that the poison is formed by the protoplasm of the egg, since otherwise we could not understand why the reciprocal cross should be healthy.

All of these data agree in this one point, that the fusion by grafting or fertiliza­tion of two distant species is impossible, although the mechanism of the incompatibility is not yet understood. It is quite possible that this mechanism is not the same in all the cases mentioned here, and that it may be different when two different species are mixed and when incompatibility exists between varieties, as is the case in the graft on mammals.

II. The Chemical Basis of Genus and Species and of Species Specificity

4. Fifty or sixty years ago surgeons did not hesitate to transfuse the blood of animals into human beings. The practice was a failure, and Landois[39] showed by experi­ment that if blood of a foreign species was introduced into an animal the blood corpuscles of the transfused blood were rapidly dissolved and the animal into which the transfusion was made was rendered ill and often died. The result was different when the animals whose blood was used for the purpose of transfusion belonged to the same species or a species closely related to the animal into which the blood was transfused. Thus when blood was exchanged between horse and donkey or between wolf and dog or between hare and rabbit no hemoglobin appeared in the urine and the animal into which the blood was transfused remained well.[40] This was the beginning of the investiga­tions in the field of serum specificity which were destined to play such a prominent rôle in the development of medicine. Friedenthal was able to show later that if to 10 c.c. of serum of a mammal three drops of defibrinated blood of a foreign species are added and the whole is exposed in a test tube to a temperature of 38°C. for fifteen minutes the blood cells contained in the added blood are all cytolyzed; that this, however, does not occur so rapidly when the blood of a related species is used. He could thus show that human blood serum dissolves the erythrocytes of the eel, the frog, pigeon, hen, horse, cat, and even that of the lower monkeys but not that of the anthropoid apes. The blood of the chimpanzee and of the human are no longer incompatible, and this discovery was justly considered by Friedenthal as a confirma­tion of the idea of the evolu­tionists that the anthropoid apes and the human are blood rela­tions.[41]

This line of investiga­tion had in the meanwhile entered upon a new stage when Kraus, Tchistowitch, and Bordet discovered and developed the precipitin reac­tion, which consists in the fact that if a foreign serum (or a foreign protein) is introduced into an animal the blood serum of the latter acquired after some time the power of causing a precipitate when mixed with the antigen, i. e., with the foreign substance originally introduced into the animal for the purpose of causing the produc­tion of antibodies in the latter; while, of course, no such precipita­tion occurs if the serum of a non-treated rabbit is mixed with the serum of the blood of the foreign species.

In 1897 Kraus discovered that if the filtrates from cultures of bacteria (e. g., typhoid bacillus) are mixed with the serum of an animal immunized with the same serum (e. g., typhoid serum) it causes a precipitate; and that this precipitin reac­tion is specific. This fact was confirmed and has been extended by the work of many authors.

Tchistowitch in 1899 observed that the serum of rabbits which had received injec­tions of horse or eel serum caused a precipitate when mixed with the serum of these latter animals.

Bordet found in 1899 that if milk is injected into a rabbit the serum of such a rabbit acquires the power of precipitating casein, and Fish found that this reac­tion is specific inasmuch as the lactoserum from cow’s milk can precipitate only the casein of cow’s milk but not that of human or goat milk. Wassermann and Schütze reached the same result independently of each other.

Myers and later Uhlenhuth showed that if white of egg from a hen’s egg is injected into a rabbit, precipitins for white of egg are found in the serum of the latter, and Uhlenhuth[42] found, by trying the white of egg of different species of birds, that the precipitin reac­tion called forth by the blood of the immunized animal is specific, inasmuch as the proteins from a hen’s egg will call forth the forma­tion of precipitins in the blood of the rabbit which will precipitate only the white of egg of the hen or of closely related birds.

To Nuttall[43] belongs the credit of having worked out a quantitative method for measuring the amount of precipitate formed, and in this way he made it possible to draw more valid conclusions concerning the degree of specificity of the precipitin reac­tion. He found by this method that when the immune serum is mixed with the serum or the protein solu­tion used for the immuniza­tion a maximum precipitate is formed, but if it is mixed with the serum of related forms a quantitatively smaller precipitate is produced. In this way the degree of blood rela­tionship could be ascertained. He thus was able to show that when the blood of one species, e. g., the human, was injected into the blood of a rabbit, after some time the serum of the rabbit was able to cause a precipitate not only with the serum of man, or chimpanzee, but also of some lower monkeys; with this difference, however, that the precipitate was much heavier when the immune serum was added to the serum of man. The method thus shows the existence of not an absolute but of a strong quantitative specificity of blood serum. This statement may be illustrated by the following table from Nuttall. The antiserum used for the precipitin reac­tion was obtained by treating a rabbit with human blood serum. The forty-five bloods tested had been preserved for various lengths of time in the refrigerator with the addi­tion of a small amount of chloroform.

TABLE II

Quantitative Tests with Anti-Primate Sera

Tests with Antihuman Serum

Blood of		Precipitum Amount	Percentage
Primates
Man		.0310	100
Chimpanzee		.0400	130 (loose precipitum)
Gorilla		.0210	064
Ourang		.0130	042
Cynocephalus mormon		.0130	042
Cynocephalus sphinx		.0090	029
Ateles geoffroyi		.0090	029
Insectivora
Centetes ecaudatus		.0000	000
Carnivora
Canis aureus		.0030	010 (loose precipitum)
Canis familiaris		.0010	003
Lutra vulgaris		.0030	010 (concentrated serum)
Ursus tibetanus		.0025	008
Genetta tigrina		.0010	003
Felis domesticus		.0010	003
Felis caracal		.0008	003
Felis tigris		.0005	002
Ungulata
Ox		.0030	010
Sheep		.0030	010
Cobus unctuosus		.0020	007
Cervus porcinus		.0020	007
Rangifer tarandus		.0020	007
Capra megaceros		.0005	002
Equus caballus		.0005	002
Sus scrofa		.0000	000
Rodentia
Dasyprocta cristata		.0020	007 (concentrated serum clots)
Guinea-pig		.0000	000
Rabbit		.0000	000
Marsupialia
Petrogale xanthopus
Petrogale penicillata
Onychogale frenata
Onychogale unguifera		.0000	000
Onychogale unguifera
Macropus bennetti
Thylacinus cynocephalus

Among the Primate bloods that of the Chimpanzee gave too high a figure, owing to the precipitum being flocculent and not settling well, for some reason which could not be determined. The figure given by the Ourang is somewhat too low, and the difference between Cynocephalus sphinx and Ateles is not as marked as might have been expected in view of the qualitative tests and the series following. The possibilities of error must be taken into account in judging of these figures; repeated tests should be made to obtain something like a constant. Other bloods than those of Primates give small reac­tions or no reac­tions at all. The high figures (10%) obtained with two Carnivore bloods can be explained by the fact that one gave a loose precipitum, and the other was a somewhat concentrated serum.[44]

We have mentioned that even the proteins of the egg are specific according to Uhlenhuth. Graham Smith, one of Nuttall’s collaborators, applied the latter’s quantitative method to this problem and confirmed the results of Nuttall. A few examples may serve as an illustra­tion.

TABLE III

Tests with Anti-Duck’s-Egg Serum

Frog, Amphiuma, and Dogfish sera, as well as Tortoise and Dogfish egg-albumins, were also tested, with negative results.

TABLE IV

Tests with Anti-Fowl’s-Egg Serum

Thrush, Emu, Greenfinch, and Hedge-sparrow egg-albumins were tested and gave traces of precipita, as also did Tortoise and Turtle sera. The egg-albumins of the Tortoise, Frog, Skate, and two species of Dogfish did not react. Alligator, Frog, Amphiuma, and Dogfish sera also yielded no results.[45]

By improving the quantitative method in various ways, Welsh and Chapman[46] were able to explain why the precipitin reac­tion with egg-white was not strictly specific but gave also, though quanti­tatively weaker, results with the egg-white of related birds. They found that by a new method devised by them “it is possible to indicate in an avian egg-white antiserum the presence of a general avian antisubstance (precipitin) together with the specific antisubstance.”

The Bordet reaction was not only useful in indicating the specificity and blood rela­tionship for animals but also among plants. Thus Magnus and Friedenthal[47] were able to demonstrate with Bordet’s method the rela­tionship between yeast (Saccharomyces cerevisiæ) and truffle (Tuber brumale).

5. We must not forget, while under the spell of the problem of immunity, that we are interested at the moment in the ques­tion of the nature of the specificity of living organisms. It is only logical to conclude that the fossil forms of invertebrate animals and of algæ and bacteria, which Walcott found in the Cambrian and which may be two hundred million years old, must have had the same specificity at that time as they or their close relatives have today; and this raises the ques­tion: What is the nature of the substances which are responsible for and transmit this specificity? It is obvious that a definite answer to this ques­tion brings us also to the very problem of evolu­tion as well as that of the constitu­tion of living matter.

There can be no doubt that on the basis of our present knowledge proteins are in most or practically all cases the bearers of this specificity. This has been found out not only with the aid of the precipitin reac­tion but also with the anaphylaxis reac­tion, by which, as the reader may know, is meant that when a small dose of a foreign substance is introduced into an animal a hypersensitiveness develops after a number of days or weeks, so that a new injec­tion of the same substance produces serious and in some cases fatal effects. This hypersensitiveness, which was first analysed by Richet,[48] is specific for the substance which has been injected. Now all these specific reac­tions, the precipitin reac­tion as well as the anaphylactic reac­tion, can be called forth by proteins. Thus Richet, in his earliest experi­ments, showed that only the protein-containing part of the extract of actinians, by which he called forth anaphylaxis, was able to produce this phenomenon, and later he showed that it was generally impossible to produce anything resembling anaphylaxis by non-protein substances, e. g., cocain or apomorphin.[49] Wells isolated from egg-white four different proteins (three coagulable proteins and one non-coagulable) which can be distinguished from each other by the anaphylaxis reac­tion, although all come from the same biological object.[50] Michaelis as well as Wells found that the split products of the protein molecule are no longer able to call forth the anaphylaxis reac­tion. Since peptic diges­tion has the effect of annihilating the power of proteins to call forth anaphylaxis, we are forced to the conclusion that the first cleavage products of proteins have already lost the power of calling forth immunity reac­tions.

A pretty experiment by Gay and Robertson[51] should be mentioned in this connec­tion. Robertson had shown

that a substance closely resembling paranucleins both in its properties and its C, H, and N content can be formed from the filtered products of the complete peptic hydrolysis of an approximately four per cent. neutral solu­tion of potassium caseinate by the action of pure pepsin at 36°C.

He considered this a case of a real synthesis of proteins from the products of its hydrolytic cleavage. This interpreta­tion was not generally accepted and received a different interpreta­tion by Bayliss and other workers. Gay and Robertson were able to show that paranuclein when injected into an animal will sensitize guinea-pigs for anaphylactic intoxica­tion for either paranuclein or casein and apparently indiscriminately. The products of complete peptic diges­tion of casein had no such effect, but the synthetic product of this diges­tion obtained by Robertson’s method has the same specific antigenic properties as paranuclein, thus making it appear that Robertson had indeed succeeded in causing a synthesis of paranuclein with the aid of pepsin from the products of diges­tion of casein by pepsin.

There are a few statements in the literature to the effect that the specificity of organisms might be due to other substances than proteins. Thus Bang and Forssmann claimed that the substances (antigens) responsible for the produc­tion of hemolysis were of a lipoid nature, but their statements have not been confirmed, and Fitzgerald and Leathes[52] reached the conclusion that lipoids are non-antigenic. Ford claims to have obtained proof that a glucoside contained in the poisonous mushroom Amanita phalloides can act as an antigen. But aside from this one fact we know that proteins and only proteins can act as antigens and are therefore the bearers of the specificity of living organisms.

Bradley and Sansum[53] found that guinea-pigs sensitized to beef or dog hemoglobin fail to react or react but slightly to hemoglobin of other origin. The hemoglobins tried were dog, beef, cat, rabbit, rat, turtle, pig, horse, calf, goat, sheep, pigeon, chicken, and man.

6. It would be of the greatest importance to show directly that the homologous proteins of different species are different. This has been done for hemoglobins of the blood by Reichert and Brown,[54] who have shown by crystallographic measurements that the hemoglobins of any species are definite substances for that species.

The crystals obtained from different species of a genus are characteristic of that species, but differ from those of other species of the genus in angles or axial ratio, in optical characters, and especially in those characters comprised under the general term of crystal habit, so that one species can usually be distinguished from another by its hemoglobin crystals. But these differences are not such as to preclude the crystals from all species of a genus being placed in an isomorphous series (p. 327).

As far as the genus is concerned it was found that the hemoglobin crystals of any genus are isomorphous.

In some cases this isomorphism may be extended to include several genera, but this is not usually the case, unless as in the case of dogs and foxes, for example, the genera are very closely related.

The most important ques­tion for us is the following: Are the differences between the corresponding hemoglobin crystals of different species of the same genus such as to warrant the statement that they indicate chemical differences? If this were the case we might say that blood reac­tions as well as hemoglobin crystals indicate that differences in the constitu­tion of proteins determine the species specificity and, perhaps, also species heredity. The following sentences by Reichert and Brown seem to indicate that this may be true for the crystals of hemoglobin.

The hemoglobins of any species are definite substances for that species. But upon comparing the corresponding substances (hemoglobins) in different species of a genus it is generally found that they differ the one from the other to a greater or less degree; the differences being such that when complete crystallographic data are available the different species can be distinguished by these differences in their hemoglobins. As the hemoglobins crystallize in isomorphous series the differences between the angles of the crystals of the species of a genus are not, as a rule, great; but they are as great as is usually found to be the case with minerals or chemical salts that belong to an isomorphous group (p. 326).

As Professor Brown writes me, the difficulty in answering the ques­tion definitely, whether or not the hemoglobins of different species are chemically different, lies in the fact that there is as yet no criterion which allows us to discriminate between a species and a Mendelian muta­tion except the morpho­logical differences. It is not impossible that while species differ by the constitu­tion of some or most of their proteins, Mendelian heredity has a different chemical basis.

It is regrettable that work like that of Reichert and Brown cannot be extended to other proteins, but it seems from anaphylaxis reac­tions that we might expect results similar to those in the case of the hemoglobins. The proteins of the lens are an excep­tion inasmuch as, according to Uhlenhuth, the proteins of the lens of mammals, birds, and amphibians cannot be discriminated from each other by the precipitin reac­tion.[55]

7. The serum of certain humans may cause the destruc­tion or agglutina­tion of blood corpuscles of certain other humans. This fact of the existence of “isoagglutinins” seems to have been established for man, but Hektoen states that he has not been able to find any isoagglutinins in the serum of rabbits, guinea-pigs, dogs, horses, and cattle. Landsteiner found the remarkable fact that the sera of certain individuals of humans could hemolyze the corpuscles of certain other individuals, but not those of all individuals. A systematic investiga­tion of this variability led him to the discovery of three distinct groups of individuals, the sera of each group acting in a definite way towards the corpuscles of the representatives of each other group. Later observers, for example Jansky and Moss, established four groups. These groups are, according to Moss,[56] as follows:

Group 1. Sera agglutinate no corpuscles.
Corpuscles agglutinated by sera of Groups 2, 3, 4.

Group 2. Sera agglutinate corpuscles of Groups 1, 3.
Corpuscles agglutinated by sera of Groups 3, 4.

Group 3. Sera agglutinate corpuscles of Groups 1, 2.
Corpuscles agglutinated by sera of Groups 2, 4.

Group 4. Sera agglutinate corpuscles of Groups 1, 2, 3.
Corpuscles agglutinated by no serum.

The relative frequency of the four groups follows from the following figures. Of one hundred bloods tested by Moss in series of twenty there were found:

10 belonging to Group 1.
40 belonging to Group 2.
7 belonging to Group 3.
43 belonging to Group 4.

Groups 2 and 4 are in the majority and in overwhelming numbers, which indicates that, as a rule, the sera agglutinate the blood corpuscles of individuals of the other groups, but not those of individuals belonging to the same group. The phenomenon that a serum agglutinates no corpuscles (Group 1), or that the corpuscles are agglutinated by no serum (Group 4), are the excep­tions. It is obvious that, as far as our problem is concerned, only Groups 2 and 3 are to be considered. There is no Mendelian character which refers only to one half of the individuals except sex. Since nothing is said about a rela­tion of Groups 2 and 3 to sex such a rela­tion probably does not exist.

8. The facts thus far reported imply the sugges­tion that the heredity of the genus is determined by proteins of a definite constitu­tion differing from the proteins of other genera. This constitu­tion of the proteins would therefore be responsible for the genus heredity. The different species of a genus have all the same genus proteins, but the proteins of each species of the same genus are apparently different again in chemical constitu­tion and hence may give rise to the specific biological or immunity reac­tions.

We may consider it as established by the work of McClung, Sutton, E. B. Wilson, Miss Stevens, Morgan, and many others, that the chromo­somes are the carriers of the Mendelian characters. These chromo­somes occur in the nucleus of the egg and in the head of the sperm. Now the latter consists, in certain fish, of lipoids and a combina­tion of nucleinic acid and protamine or histone, the latter a non-coagulable protein, more resembling a split product of one of the larger coagulable proteins.

A. E. Taylor[57] found that if the spermatozoa of the salmon are injected into a rabbit, the blood of the animal acquires the power of causing cytolysis of salmon spermatozoa. When, however, the isolated protamines or nucleinic acid or the lipoids prepared from the same sperm were injected into a rabbit no results of this kind were observed. H. G. Wells more recently tested the relative efficiency of the constituents of the testes of the cod (which in addi­tion to the constituents of the sperm contained the proteins of the testicle). From the testicle he prepared a histone (the protein body of the sperm nucleus), a sodium nucleinate, and from the sperm-free aqueous extract of the testicles a protein resembling albumin was separated by precipita­tion.[58]

The albumin behaved like ordinary serum albumin or egg albumin, producing typical and fatal anaphylactic reac­tions and being specific when tried against mammalian sera. The nucleinate did not produce any reac­tions when guinea-pigs were given small sensitizing and larger intoxicating doses (0.1 gm.) in a three weeks’ interval; a result to be expected, since no protein is present in the prepara­tion. The histone was so toxic that its anaphylactic properties could not be studied.

It is not impossible that protamines and histones might be found to act as specific antigens if they were not so toxic. The positive results which Taylor observed after injec­tion of the sperm might have been due to the proteins contained in the tail of the spermatozoa, which in certain animals at least does not enter the egg and hence can have no influence on heredity.

It is thus doubtful whether or not any of the constituents of the nucleus contribute to the determina­tion of the species. This in its ultimate consequences might lead to the idea that the Mendelian characters which are equally transmitted by egg and spermato­zoön, determine the individual or variety heredity, but not the genus or species heredity. It is, in our present state of knowledge, impossible to cause a spermato­zoön to develop into an embryo,[59] while we can induce the egg to develop into an embryo without a spermato­zoön. This may mean that the protoplasm of the egg is the future embryo, while the chromo­somes of both egg and sperm nuclei furnish only the individual characters.

CHAPTER IV

SPECIFICITY IN FERTILIZATION

1. We have become acquainted with two characteristics of living matter: the specificity due to the specific proteins characteristic for each genus and possibly species and the synthesis of living matter from the split products of their main constituents instead of from a supersaturated solu­tion of their own substance, as is the case in crystals. We are about to discuss in this and the next chapter a third characteristic, namely, the phenomenon of fertiliza­tion. While this is not found in all organisms it is found in an overwhelming majority and especially the higher organisms, and of all the mysteries of animated nature that of fertiliza­tion and sex seems to be the most captivating, to judge from the space it occupies in folklore, theology, and “literature.” Bacteria, when furnished the proper nutritive medium, will synthetize the specific material of their own body, will grow and divide, and this process will be repeated indefinitely as long as the food lasts and the temperature and other outside condi­tions are normal. It is purely due to limita­tion of food that bacteria or certain species of them do not cover the whole planet. But, as every layman knows, the majority of organisms grow only to a certain size, then die, and the propaga­tion takes place through sex cells or gametes: a female cell—the egg—containing a large bulk of protoplasm (the future embryo) and reserve material; and the male cell which in the case of the spermato­zoön contains only nuclear material and no cytoplasmic material except that contained in the tail which in some and possibly many species does not enter the egg. The male element—the spermato­zoön—enters the female gamete—the egg—and this starts the development. In the case of most animals the egg cannot develop unless the spermato­zoön enters. The ques­tion arises: How does the spermato­zoön activate the egg? And also how does it happen that the spermato­zoön enters the egg? We will first consider the latter ques­tion. These problems can be answered best from experi­ments on forms in which the egg and the sperm are fertilized in sea water. Many marine animals, from fishes down to lower forms, shed their eggs and sperm into the sea water where the fertiliza­tion of the egg takes place, outside the body of the female.

The first phenomenon which strikes us in this connec­tion is again a phenomenon of specificity. The spermato­zoön can, as a rule, only enter an egg of the same or a closely related species, but not that of one more distantly related. What is the character of this specificity? The writer was under the impression that a clue might be obtained if artificial means could be found by which the egg of one species might be fertilized with a distant species for which this egg is naturally immune. Such an experi­ment would mean that the lack of specificity had been compensated by the artificial means. It is well known that the egg of the sea urchin cannot as a rule be fertilized with the sperm of a starfish in normal sea water. The writer tried whether this hybridiza­tion could not be accomplished provided the constitu­tion of the sea water were changed. He succeeded in causing the fertiliza­tion of a large percentage of the eggs of the Californian sea urchin, Strongylo­centrotus purpuratus, with the sperm of various starfish (e. g., Asterias ochracea) and Holothurians by slightly raising the alkalinity of the sea water, through the addi­tion of some base (NaOH or tetra­ethyl­ammonium­hydroxide or various amines), the optimum being reached when 0.6 c.c. N/10 NaOH is added to 50 c.c. of sea water. It is a peculiar fact that this solu­tion is efficient only if both egg and sperm are together in the hyperalkaline sea water. If the eggs and sperm are treated separately with the hyperalkaline sea water and are then brought together in normal sea water no fertiliza­tion takes place as a rule, while with the same sperm and eggs the fertiliza­tion is successful again if both are mixed in the hyperalkaline solu­tion. From this the writer concluded that the fertilizing power depends on a rapidly reversible action of the alkali on the surface of the two gametes. It was found that an increase of the concentra­tion of calcium in the sea water also favoured the entrance of the Asterias sperm into the egg of purpuratus; and that if C_Ca was increased it was not necessary to add as much NaOH.

The spermato­zoön enters the egg through the so-called fertiliza­tion cone, i. e., a proto­plasmic process comparable to the pseudo­podium of an amœboid cell. The analogy of the process of phago­cytosis—i. e., the taking up of particles by an amœboid cell—and that of the engulfing of the spermato­zoön by the egg presents itself. We do not know definitely the nature of the forces which act in the case of phago­cytosis—although surface tension forces and agglutina­tion have been suggested; both are surface phenomena and are rapidly reversible.

We should then say that the specificity in the process of fertiliza­tion consists in a peculiarity of the surface of the egg and spermato­zoön which in the case of S. purpuratus ♀ and Asterias ♂ can be supplied by a slight increase in the C_OH or C_Ca.

By this method fifty per cent. or more of the eggs of purpuratus could be fertilized with the sperm of the starfish Asterias ochracea, capitata, Ophiurians, and Holothurians, while with the sperm of another starfish, Pycnopodia spuria, only five per cent., and with the sperm of Asterina only one per cent. could be fertilized.[60] Godlewski succeeded by the same method in fertilizing the eggs of a Naples starfish with the sperm of a crinoid.[61] The writer did not succeed in bringing about the fertiliza­tion of the egg of another sea urchin in California, Strongylo­centrotus franciscanus, with the sperm of a starfish. Although these eggs formed a membrane in contact with the sperm, the latter did not enter the egg; nor has the writer as yet succeeded in causing the sperm of Asterias to enter the egg of Arbacia.

Kupelwieser[62] observed that the spermato­zoön of molluscs may occasionally enter into the egg of S. pur­pur­atus in normal sea water and later, at Naples, he observed the same for the sperm of annelids. In these cases no development took place. In teleost fishes the spermato­zoön can enter the eggs of widely different species but with rare excep­tions all the embryos will die in an early stage of development.[63]

2. The fact that an increase in the alkalinity or in the concentra­tion of calcium allowed foreign sperm to enter the egg of the sea urchin, suggested the idea that a diminu­tion of alkalinity or calcium in the sea water might block the entrance of the sperm of sea urchin into eggs of their own species. This was found to be correct; when we put eggs and sperm of the same species of sea urchin into solu­tions whose concentra­tion of Ca or of OH is too small, the sperm, although it may be intensely active, cannot enter the egg.

For the purpose of these experi­ments the ovaries and testes of the sea urchins were not put into sea water, but instead into pure m/2 NaCl and after several washings in this solu­tion were kept in it (they remain alive for several days in pure m/2 NaCl). Several drops of such sperm and one drop of eggs were in one series of experi­ments put into 2.5 c.c. of a neutral mixture of m/2 NaCl and ³⁄₈ m MgCl₂ in the propor­tion in which these two salts exist in the sea water. In such a neutral solu­tion eggs of Arbacia or purpuratus are not fertilized no matter how long they remain in it, although the spermatozoa swim around the eggs very actively. That no spermato­zoön enters the eggs can be shown by the fact that the eggs do not divide (although they can segment in such a solu­tion if previously fertilized in sea water or some other efficient solu­tion). When, however, eggs and sperm are put into 2.5 c.c. of the same solu­tion of NaCl+MgCl₂, containing in addi­tion one drop of a N/100 solu­tion of NaOH (or NH₃ or benzylamine or butylamine) or eight drops of m/100 NaHCO₃, most, and often practically all of the eggs at once form fertiliza­tion membranes and segment at the proper time, indicating that fertiliza­tion has been accomplished. The same result can be obtained if the eggs are transferred into a neutral mixture of NaCl+MgCl₂+CaCl₂ (in the propor­tion in which these salts exist in the sea water) or into a neutral mixture of NaCl+MgCl₂+KCl+CaCl₂. In such neutral mixtures the eggs form fertiliza­tion membranes and begin to segment. The eggs are not fertilized in a neutral solu­tion of NaCl or of NaCl+KCl.[64]

It is, therefore, obvious that if we diminish the alkalinity of the solu­tion surrounding the egg and deprive this solu­tion of CaCl₂ we establish the same block to the entrance of the spermato­zoön of Arbacia into the egg of the same species as exists in normal sea water for the entrance of the sperm of the starfish into the egg of purpuratus.

The “block” created in this way, to the entrance of the sperm of Arbacia into the egg of the same species is also rapidly reversible.

We reach the conclusion, therefore, that the specificity which allows the sperm to enter an egg is a surface effect which can be increased or diminished by an increase or diminu­tion in the concentra­tion of OH as well as of Ca. The writer has shown that an increase in the concentra­tion of both substances may cause an agglutina­tion of the spermatozoa of starfish to the jelly which surrounds the egg of purpuratus.[65] It is thus not impossible that the specificity which favours the entrance of a spermato­zoön into an egg of its own species may consist in an agglutina­­tion between spermato­zoön and egg protoplasm (or its fertiliza­tion cone); and that this agglutina­tion is favoured if the C_OH or C_Ca or both are increased within certain limits.

Godlewski discovered a very interesting form of block to the entrance of the spermato­zoön into the egg which takes place if two different types of sperm are mixed. He had found that the sperm of the annelid Chætopterus is able to enter the egg of the sea urchin and that in so doing it causes membrane forma­tion. The egg, however, does not develop but dies rapidly, as is the case when we induce artificial membrane forma­tion, as we shall see in the next chapter.

Godlewski found that if the sperm of Chætopterus and the sperm of sea urchins are mixed the mixture is not able to induce development or membrane forma­tion, since now neither spermato­zoön can enter; blood has the same inhibiting effect as the foreign sperm. The mixture does not interfere with the development of the eggs if they are previously fertilized.[66]

The phenomenon was further investigated by Herlant[67] who found that if the sperm of a sea urchin is mixed with the sperm of certain annelids (Chætopterus) or molluscs, and if after some time the eggs of the sea urchin are added to the mixture of the two kinds of sperm no egg is fertilized. If, however, the solu­tion is subsequently diluted with sea water or if the egg that was in this mixture is washed in sea water, the same sperm mixture in which the egg previously remained unfertilized will now fertilize the egg. From these and similar observa­tions Herlant draws the conclusion that the block existed at the surface of the egg, inasmuch as a reac­tion product of the two types of sperm is formed after some time which alters the surface of the egg and thereby prevents the sperm from entering. This view is supported not only by all the experi­ments but also by the observa­tion of the writer that foreign sperm or blood is able to cause a real agglutina­tion after some time if mixed with the sperm of a sea urchin or a starfish.[68] We can imagine that the precipitate forms a film around the egg and acts as a block for the agglutina­tion between egg and spermato­zoön. The block can be removed mechanically by washing.

3. The fact has been mentioned that the most motile sperm will not be able to enter into the egg if certain other condi­tions (specificity or C_OH or C_Ca) are not fulfilled. On the other hand, living but immobile sperm cannot enter the egg under any condi­tions. If we add a trace of KCN to the sperm of Arbacia so that the spermato­zoön becomes immobile no egg is fertilized even if the eggs and the sperm are thoroughly shaken together; while the same spermatozoa will fertilize these eggs as soon as the HCN has evaporated and they again become motile. It was formerly thought that the spermato­zoön had to bore itself into the egg, being propelled by the movements of the flagellum. It is, however, more probable that only a certain energy of vibra­tion is needed on the part of the spermato­zoön to make the latter stick to the surface of the egg and agglutinate and that later forces of a different character bring the spermato­zoön into the egg. The fact that under normal condi­tions a very slight degree of motility on the part of the spermato­zoön allows it to enter the egg of its own species seems to favour such a view.

It is a common experience that spermatozoa become very active when they reach the neighbourhood of an egg. v. Dungern assumed that only foreign sperm became thus active, but F. R. Lillie[69] has pointed out that this may be a specific effect. The writer tested this idea on the sperm and eggs of two species of starfish and of sea urchins. It should be men­tioned that the eggs of the starfish used in this experi­ment were completely immature and could not be fertilized, while the eggs of the sea urchins were mature. The testicles and ovaries had been kept in NaCl and all the sperm was immotile. Eggs and sperm were mixed together in a pure m/2 NaCl solu­tion where the sperm was only rendered motile by the proximity of eggs. The following table gives the result.[70]

TABLE V

Specificity of Activation of Sperm by Eggs

The spermatozoa of starfish show a marked specificity inasmuch as they are strongly activated only by the eggs of their own species, although in this experi­ment these were immature, and to a slight degree only by the eggs of the sea urchin purpuratus. But it is also obvious that the specificity is far from exclusive since the immature eggs of Asterina activate the sperm of the sea urchin franciscanus as powerfully as is done by the mature eggs of the sea urchin purpuratus and franciscanus. In studying these results the reader must keep in mind first that all these experi­ments were made in a NaCl solu­tion and second that it requires a stronger influence to activate the spermatozoa of the starfish, which are not motile at first even in sea water, than the sea urchin spermatozoa which are from the first very active in such sea water, and which may therefore be considered as being at the threshold of activity in pure NaCl solu­tion.

Wasteneys and the writer (in experi­ments not yet published) did not succeed in demonstrating an activating effect of the eggs of various marine teleosts upon sperm of the same species.

4. F. R. Lillie[71] has studied the very striking phenomenon of transitory sperm agglutina­tion which takes place when the sperm of a sea urchin or of certain annelids is put into the supernatant sea water of eggs of the same species. If we put one or more drops of a very thick sperm suspension of the Californian sea urchin S. purpuratus carefully into the centre of a dish containing 3 c.c. of ordinary sea water and let the drop stand for one-half to one minute and then by gentle agita­tion mix the sperm with the sea water the mass of thick sperm which is at first rather viscous is distributed equally in sea water in a few seconds and the result is a homogeneous sperm suspension. When, however, the same experi­ment is made with the sea water which has been standing for a short time over a large mass of eggs of the same species, the thick drop of sperm seems to be less miscible and instead of a homogeneous suspension we get, as a result, the forma­tion of a large number of distinct clusters which are visible to the naked eye and which may possess a diameter of 1 or 2 mm. The rest of the sea water is almost free from sperm. These clusters of spermatozoa may last for from two to ten minutes and then dissolve by the gradual detachment of the spermatozoa from the periphery of the cluster.

This phenomenon seems to occur in sea urchins and annelids. The writer has vainly looked for it in different forms of the Californian starfish or molluscs and in fish at Woods Hole. Lillie failed to find it in the starfish at Woods Hole.

The writer found that the sperm of the Californian sea urchin Strongylo­centrotus purpuratus will form clusters with the egg sea water of purpuratus but not with that of franciscanus; while the sperm of franciscanus will agglutinate with the egg sea water of both species, but the clusters last a little longer with the eggs of its own species.

He also found that the clusters are more durable in a neutral than in a slightly alkaline solu­tion and that the agglutina­tion disappears the more rapidly the more alkaline the solu­tion. The presence of bivalent ca­tions, especially Ca, also favours the agglutina­tion.

It was also found that this agglutina­tion occurs only when the spermatozoa are very motile; thus if a trace of KCN is added to a mass of thick sea-urchin sperm so that the spermatozoa become immotile a drop of this sperm will not agglutinate when put in egg sea water of the same species; while later, after the HCN has evaporated, the same sperm will agglutinate when put into such sea water.

The writer suggests the following explana­tion of the phenomenon. The egg sea water contains a substance which forms a precipitate with a substance on the surface of the spermato­zoön whereby the latter becomes slightly sticky. This precipitate is slowly soluble in sea water and the more rapidly the more alkaline (within certain limits). Only when the spermatozoa run against each other with a certain impact will they stick together, as Lillie suggested. Lillie assumes that this agglutinating substance contained in egg sea water is required to bring about fertiliza­tion and he therefore calls it “fertilizin.”[72] But this assump­tion seems to go beyond the facts inasmuch as the existence of such an agglutinating substance can only be proved in a few species of animals (sea urchins and annelids); and as, moreover, sea-urchin sperm can fertilize eggs which will not cause the sperm to agglutinate, e. g., the egg of franciscanus can be fertilized by sperm of purpuratus, although the egg sea water of franciscanus causes no agglutina­tion of the sperm of purpuratus. When the jelly surrounding the egg of the Californian sea urchin S. purpuratus is dissolved with acid and the eggs are washed, the eggs will not cause any more sperm agglutina­tion; and yet one hundred per cent. of such eggs can be fertilized by sperm.[73]

5. It is well known that if an egg is once fertilized it becomes impermeable for other spermatozoa. This cannot well be due to the fact that the egg develops; for the writer found some time ago that eggs of Strongylo­centrotus purpuratus which are induced to develop by means of artificial parthenogenesis can be fertilized by sperm. The following observa­tion leaves no doubts in this respect. When the unfertilized eggs of purpuratus are put for two hours into hypertonic sea water (50 c.c. of sea water+8 c.c. 2¹⁄₂ m NaCl) and then transferred into sea water it occasionally happens that a certain percentage of the eggs will begin to divide into 2, 4, 8 or more cells, without developing any further. When to such eggs after they have remained in the resting stage for a number of hours or a day, sperm is added, some or all of the blasto­meres form a fertiliza­tion membrane and now begin to develop into larvæ; and if the spermato­zoön gets into a blastomere of the 2- or 4-cell stage normal plutei will result. When the sperm is added while the eggs are in active partheno­genetic cell division the individual blasto­meres into which a spermato­zoön enters will also form a fertiliza­tion membrane, but such blasto­meres perish very rapidly. It is not yet possible to state why it should make such a difference for the possibility of development whether the spermato­zoön enters into a blastomere when at rest or when it is in active nuclear division, although the idea presents itself that in the latter case an abnormal mix-up and separa­tion of chromo­somes and other constituents may be responsible for the fatal result. Whatever may be the explana­tion of this phenomenon it proves to us that it is not the process of development in itself which acts as a block to the entrance of a spermato­zoön into an egg which is already fertilized.[74]

When the spermato­zoön enters the egg of the sea urchin it calls forth the forma­tion of a membrane—the fertiliza­tion membrane. It might be considered possible that this membrane forma­tion or the altera­tion underlying or accompanying it is responsible for the fact that an egg once fertilized becomes immune against a spermato­zoön. We shall see in the next chapter that it is possible to call forth the membrane in an unfertilized sea-urchin egg by treating it with butyric acid. This membrane is so tough in the egg of Strongylo­centrotus that no spermato­zoön can get through it; in the egg of Arbacia the membrane is occasionally replaced by a soft gelatinous film. If no second treatment is given to such eggs they will disintegrate in a comparatively short time, but when sperm is added some or most of the eggs will develop in the way characteristic of fertilized eggs.[75] When the membrane is too tough to allow the spermato­zoön to enter the egg it can be shown that if the membrane is torn mechanically the egg can still be fertilized by sperm.

Should it be possible that the spermato­zoön can no longer agglutinate with the fertilized egg or that those phagocytotic reac­tions which we suppose to play a rôle in the entrance of the spermato­zoön into the egg are no longer possible after a spermato­zoön has entered? The mere fact of development is apparently not the cause which bars a spermato­zoön from entering an egg already fertilized by sperm.

Lillie assumes that the egg loses its “fertilizin” in the process of membrane forma­tion since the sea water containing such eggs no longer gives the agglutinin reac­tion with sperm, and he believes that the lack of “fertilizin” in the fertilized egg or in the egg after membrane forma­tion is the cause of the block in the fertilized egg. But we have seen that the artificial membrane forma­tion does not create such a block although it puts an end to the “fertilizin” reac­tion. In the egg of purpuratus the “fertilizin” reac­tion ceases when the jelly surrounding the egg is dissolved by an acid and the eggs are repeatedly washed; yet such eggs can easily be fertilized by sperm.

Lillie does not assume that the “fertilizin” causes an agglutina­tion between egg and spermato­zoön—we should assent to such an assump­tion—but that the “fertilizin” acts like an “amboceptor” between egg and spermato­zoön, the latter being the complement, the former the antigen. The pathologist would probably object to this interpreta­tion since no “amboceptor” is needed for agglutina­tion. The writer has had some doubts concerning the value of Ehrlich’s side-chain theory which, besides, can only be applied in a metaphorical sense to the mechanism of the entrance of the spermato­zoön into the egg.[76]

6. The reason that an egg once fertilized with sperm cannot be fertilized again may be found in a group of facts which we will now discuss, namely, the self-sterility of many hermaph­ro­dites. The fact that hermaph­ro­dites are often self-sterile, while their eggs can be fertilized with sperm from a different individual of the same species has played a great rôle in the theories of evolu­tion. We are here only concerned with the mechanism which determines the block to the entrance of a spermato­zoön into an egg of the same hermaph­ro­ditic individual.

Castle[77] observed and studied the phenomenon of self-sterility in an Ascidian, Ciona intestinalis, which is hermaph­ro­ditic. Animals which were kept isolated discharged both eggs and sperm into the surrounding sea water. Often no egg was fertilized, but in some cases five, ten, or as many as fifty per cent. of the eggs could be successfully fertilized with sperm from the same individual; while if several individuals were put into the same dish as a rule one hundred per cent. of the eggs which were discharged segmented. Morgan[78] found that the eggs of various females differ in their power of being fertilized by sperm of the same individual while one hundred per cent. could usually be fertilized with sperm of a different individual. He found in addi­tion that if the eggs of Ciona are put for about ten minutes into a two per cent. ether solu­tion in sea water in a number of cases the percentage of eggs fertilized by sperm of the same individual shows a slight increase. Fuchs[79] has reported results similar to those of Castle and Morgan.

A new point of attack has been introduced into the work of self-sterility in plants by the considera­tion of heredity. Darwin found that in Reseda which is monœcious (or hermaph­ro­ditic) certain individuals are either completely self-sterile or completely self-fertile; and Compton showed that apparently self-fertility is a Mendelian dominant to self-sterility.[80]

According to Jost this self-sterility in hermaph­ro­ditic plants is due to the fact that if pollen of the same plant is used the normal growth of the pollen tube is inhibited, while this inhibi­tion does not exist for pollen from a different individual. Correns calls these substances which prevent the adequate growth of pollen, “inhibitory” substances, and finds that they can apparently be transmitted to the offspring. He made experi­ments on Cardamine pratensis which is self-sterile.[81] He fertilized two individuals of Cardamine crosswise and raised sixty plants of the first genera­tion. He compared the fertility of these F₁ plants toward (a) their parents, and (b) foreign plants. All the fertiliza­tions with the foreign plants were successful, but the fertiliza­tions with the parents were only partly successful. According to their reac­tion they could be divided into four groups:

(A) fertile with both parents. Type bg
(B) fertile with one (B), sterile with the other parent (G).
(a) fertile with B, sterile with G. Type bG
(b) fertile with G, sterile with B. Type Bg
(C) sterile with both parents. Type BG

It was found that approximately fifteen of the sixty children belonged to each of the four groups. This should be expected if the inhibitory substance to each parent is transmitted to the children independently. Half of the children will thus inherit the inhibitory substance of one parent and the other half will inherit the inhibitory substance of the other parent. This agrees with the assump­tion that there are definite determiners for the inhibitory substances in the children which will be transmitted to half of the children. Rather complicated assump­tions are needed to explain all the facts observed by Correns on this basis and since the subject is still under investiga­tion we need not go further into the details.

To us the assump­tion and experi­mental support of the idea that self-sterility is caused by the presence of a substance inhibitory to the entrance of a spermato­zoön is important. Should it be possible that the block created by the entrance of a spermato­zoön into the egg is also due to an inhibitory substance carried by a spermato­zoön into the egg; and furthermore that the effect of the inhibitory substance should be the preven­tion of further agglutina­tion of the spermato­zoön with the egg or of the growth of the pollen tube in plants? On such an assump­tion self-sterility would be due to a lack of agglutina­tion between the egg of a hermaph­ro­dite and a spermato­zoön of the same individual. The experi­ments on the agglutinins have shown that while isoagglutinins (i. e., agglutinins for other individuals of the same species) are common auto-agglutinins (i. e., agglutinins for cells of the same individual) rarely if ever occur.

7. A positive chemotropism of the spermatozoa toward an egg of the same species has been demonstrated in a few cases, but it seems that this phenomenon is not determined by that type of substances which give rise to species specificity. The famous experi­ment of Pfeffer on the spermatozoa of ferns inaugurates this line of investiga­tion. He found that such spermatozoa when moving in a straight line through the water will be deviated in their course if they come near an archegonium; they will then turn toward it, enter it, and enter the egg. Pfeffer showed that 0.01 per cent. malic acid if put into a capillary tube will attract the spermatozoa of ferns.

When the liquid in the tube contains only 0.01 per cent. malic acid the spermatozoa of ferns very soon move toward the opening of the capillary tube and within from five to ten minutes many hundreds of spermatozoa may accumulate in the tube. The malic acid acts as well in the form of a free acid as in the form of salts.[82]

These experi­ments were continued and amplified by Shibata. Bruchmann[83] found that the spermatozoa of Lycopodium are positively chemotactic to citric acid and salts of this acid, although no citric acid could be shown in the contents of the archegonia. They are also positively chemotactic to the watery extract from archegonia.

Dewitz, Buller, and the writer have vainly tried to prove the existence of a positive chemotropism of spermatozoa to eggs of the same species. Lillie claims to have proved a positive chemotropism of the sperm of sea urchins to “fertilizin,” but such a conclusion is only justified if a method similar to that of Pfeffer’s with capillary tubes, gives positive results; such a method was not used in Lillie’s experi­ments. It seems that the fertiliza­tion of the egg by sperm is rendered possible by two facts; first that where fertiliza­tion takes place outside the body egg and sperm are shed simultaneously by the two sexes. This can be easily observed in the case of fish. But it is also the case in invertebrates. Thus the writer has observed that the sea urchins Strongylo­centrotus purpuratus at the shore of Pacific Grove all spawn simultaneously. The examina­tion extended over several miles of shore. At such spawning seasons the sea water becomes a suspension of sperm.

The second fact guaranteeing the fertiliza­tion of the eggs is the overwhelming excess of spermatozoa over eggs. The enormous waste in animated nature is in agreement with the idea of a lack of purpose; since in this case the laws of chance must play a great rôle; and the origin of durable organisms by laws of chance is only comprehensible on the basis of an enormous wastefulness, for which evidence is not lacking.

CHAPTER V

ARTIFICIAL PARTHENOGENESIS

1. The majority of eggs cannot develop unless they are fertilized, that is to say, unless a spermato­zoön enters into the egg. The ques­tion arises: How does the spermato­zoön cause the egg to develop into a new organism? The spermato­zoön is a living organism with a complicated structure and it is impossible to explain the causa­tion of the development of the egg from the structure of the spermato­zoön. No progress was possible in this field until ways were found to replace the action of the living spermato­zoön by well-known physico­chemical agencies.[84] Various observers such as Tichomiroff, R. Hertwig, and T. H. Morgan had found that unfertilized eggs may begin to segment under certain condi­tions, but such eggs always disintegrated in their experi­ments without giving rise to larvæ. In 1899 the writer succeeded in causing the unfertilized eggs of the sea urchin Arbacia to develop into swimming larvæ, blastulæ, gastrulæ, and plutei, by treating them with hypertonic sea water of a definite osmotic pressure for about two hours. When such eggs were then put back into normal sea water many segmented and a certain percentage developed into perfectly normal larvæ, blastulæ, gastrulæ, and plutei.[85] Soon afterward this was accomplished by other methods for the unfertilized eggs of a large number of marine animals, such as starfish, molluscs, and annelids. None of these eggs can develop under normal condi­tions unless a spermato­zoön enters. These experi­ments furnished proof that the activating effect of the spermato­zoön upon the egg can be replaced by a purely physico­chemical agency.[86]

The first method used in the produc­tion of larvæ from the unfertilized eggs did not lend itself to an analysis of the activating effect of the spermato­zoön upon the egg, since nothing was known about the action of a hypertonic solu­tion, except that it withdraws water from the egg; and there was no indica­tion that the entrance of the spermato­zoön causes the egg to lose water. No further progress was possible until another method of artificial parthenogenesis was found. When a spermato­zoön enters the egg of a sea urchin or starfish or certain annelids, the surface of the egg undergoes a change which is called membrane forma­tion; and which consists in the appearance of a fine membrane around the egg, separated from the latter by a liquid (Figs. 4 and 5). O. and R. Hertwig and Herbst had observed that such a membrane could be produced in an unfertilized egg if the latter was put into chloroform or xylol, but such eggs perished at once. It was generally assumed, moreover, that the process of membrane forma­tion was of no significance in the phenomenon of fertiliza­tion, except perhaps that the fertiliza­tion membrane guarded the fertilized egg against a further invasion by sperm. However, since the fertilized egg is protected against this possibility by other means the membrane is hardly needed for such a purpose.

In 1905 the writer found that membrane forma­tion, or rather the change of the surface of the egg underlying the membrane forma­tion, is the essential feature in the activa­tion of the egg by a spermato­zoön. He observed that when unfertilized eggs of the Californian sea urchin Strongylo­centrotus purpuratus are put for from one and a half to three minutes into a mixture of 50 c.c. of sea water+2.6 c.c. N/10 acetic or propionic or butyric or valerianic acid and are then put into normal sea water all or the majority of the eggs form membranes; and that such eggs when the temperature is very low will segment once or repeatedly and may even—if the temperature is as low as 4°C. or less—develop into swimming blastulæ[87]; but they will then disintegrate. On the other hand, if they are kept at room temperature they will develop only as far as the aster forma­tion and nuclear division and then begin to disintegrate. It should be men­tioned that the time which elapses between artificial membrane forma­tion and nuclear division is greater than that between the entrance of a spermato­zoön and nuclear division.

It was obvious, therefore, that artificial membrane forma­tion induced by butyric acid initiates the processes underlying development of the egg but that for some reason the egg is sickly and perishes rapidly.

When, however, such eggs were given a short treatment with hypertonic sea water or with lack of oxygen or with KCN they developed into normal larvæ. This new or improved method of artificial parthenogenesis is as follows: The eggs are put for from two to four minutes into 50 c.c. sea water containing a certain amount of N/10 butyric acid (2.6 c.c. in the case of S. purpuratus in California and 2.0 c.c. in the case of Arbacia in Woods Hole). Ten or fifteen minutes later the eggs are put into hypertonic sea water (50 c.c. sea water+8 c.c. 2¹⁄₂ m NaCl or Ringer solu­tion or cane sugar) in which they remain, at 15° C. from thirty-five to sixty minutes in the case of purpuratus, and from 17¹⁄₂ minutes to 22¹⁄₂ minutes at 23° in the case of Arbacia at Woods Hole. If the eggs are then transferred to normal sea water they will develop. In making these experi­ments, which have been repeated and confirmed by numerous investigators, it should be remembered that this effect of the hypertonic solu­tion has a high temperature coefficient (about two for 10° C.) and that a slight overexposure to the hypertonic sea water injures the eggs so that development is abnormal. By this method it is possible to imitate the activating effect of the living spermato­zoön upon the egg in every detail and eggs treated in this way will develop in large numbers into perfectly normal larvæ. We shall see later that they can also be raised to the adult state.

2. The next task was to find out the nature of the action of the two agencies upon the development of the egg. It soon became obvious that the membrane forma­tion (or the altera­tion underlying membrane forma­tion) was the more important of the two, since in the eggs of starfish and annelids this was sufficient for the produc­tion of larvæ, and that the second treatment had only the corrective effect, of overcoming the sickly condi­tion in which mere membrane forma­tion had left the eggs. It was, therefore, of great interest to ascertain what substances or agencies caused membrane forma­tion in the egg, since it now became clear that the spermato­zoön could only cause membrane forma­tion by carrying one such substance into the egg. These investiga­tions led the writer to the result that all those substances and agencies which are known to cause cytolysis or hemolysis (see Chapter III) will also induce membrane forma­tion, and that the essential feature in the causa­tion of development is a cytolysis of the superficial or cortical layer of the egg. As soon as this layer is destroyed the development of the egg can begin.

The substances and agencies which cause cytolysis and hence, if their action is restricted to the surface of the egg, will induce development are, besides the fatty acids: (1) saponin or solanin or bile salts; (2) the solvents of lipoids, benzol, toluol, amylene, chloroform, aldehyde, ether, alcohols, etc.; (3) bases; (4) hypertonic or hypotonic solu­tions; (5) rise in temperature, and (6) certain salts, e. g., BaCl₂ and SrCl₂ in the case of the egg of purpuratus, and according to R. Lillie, NaI or NaCNS in the egg of Arbacia. Whenever we submit an unfertilized sea-urchin egg to any of these agencies and restrict the cytolysis to the superficial or cortical layer of the egg (i. e., if we transfer the egg to normal sea water before the cytolytic agent has had time to diffuse into the main egg) the egg will form a membrane and behave as if the membrane forma­tion had been called forth by a fatty acid, with this difference only, that the various agencies are not all equally harmless for the egg.[88]

If the idea was correct that the change underlying membrane forma­tion was essentially a cytolysis of the cortical layer of the egg, it was to be expected (from the data contained in Chapter III) that the blood serum or the cell extracts of foreign species would also cause membrane forma­tion and thus induce the development of the unfertilized egg, while serum of animals of the same species or genus would have no such effects. This was found to be correct. In 1907 the writer showed that the blood serum of a Gephyrean worm, Dendrostoma, was able to cause membrane forma­tion in the egg of the sea urchin. When added in a dilu­tion of 1 c.c. of serum to 500 or 1000 c.c. of sea water to eggs of purpuratus a certain number formed fertiliza­tion membranes. It was found later that the serum and tissue extracts of a large number of animals, especially of mammals (rabbit, pig, ox, etc.), had the same effect, though it was necessary to use higher concentra­tions, one-half sea water and one-half isotonic blood serum. The eggs of every female sea urchin, however, did not give the reac­tion and not all the eggs even of sensitive females formed membranes. The writer found, however, that it was possible to increase the susceptibility of the eggs against foreign blood serum by putting them into a ³⁄₈ m solu­tion of SrCl₂ for from five to ten minutes (or possibly a little longer) before exposing them to the foreign blood serum. BaCl₂ acts similarly. The fact that SrCl₂ alone can cause membrane forma­tion in unfertilized eggs if they are left long enough in the solu­tion suggests that the sensitizing effect of the substance consists in a modifica­tion of the cortical layer similar to that underlying membrane forma­tion; and that the subliminal effect of a short treatment with SrCl₂ and the subliminal effect of the foreign serum when combined suffice to bring about the membrane forma­tion.

Not only the watery extract of foreign cells but also that of foreign sperm, induces membrane forma­tion in the sea-urchin egg. The watery extract of sperm of starfish is especially active, but the degree of activity varies considerably with the species of starfish from which the sperm is taken. The eggs of different species of sea urchins also show a different degree of susceptibility for the sperm of foreign species. Thus the eggs of Strongylo­centrotus purpuratus require a higher concentra­tion of sperm extract than the eggs of S. franciscanus. For the latter the amount of foreign cell constituents which suffices to call forth membrane forma­tion is so small that contact with almost any foreign living spermato­zoön produces this effect; and as a rule no previous sensitizing action of SrCl₂ is required. When we bring the unfertilized eggs of S. franciscanus into contact with the living sperm of starfish or shark or even of fowl, the eggs form a fertiliza­tion membrane without previous sensitiza­tion. A specific substance from the foreign spermato­zoön causes membrane forma­tion before the spermato­zoön has time to enter the egg. The effect is the same as if artificial membrane forma­tion had been called forth with butyric acid, i. e., they begin to develop and then disintegrate unless they receive a second short treatment.

When, however, we treat the eggs with the watery extracts from the cells of their own or closely related species we find that these extracts are utterly inactive, even if used in comparatively strong concentra­tions. This agrees with the results given in Chapter III.

These phenomena lead to a very paradoxical result; namely that while in the case of foreign sperm we can cause membrane forma­tion by both the living and the dead spermato­zoön, only the living spermato­zoön of the same species can induce membrane forma­tion. This might find its explana­tion on the assump­tion that the active substance contained in the foreign sperm or serum is water-soluble and a protein, while the activating or membrane-forming substance in the spermato­zoön is insoluble in water but soluble in the egg (or in lipoids). If this assump­tion is correct the two substances are essentially different.

Robertson[89] has succeeded in extracting a substance from the sperm of the sea urchin which causes membrane forma­tion of the sea-urchin egg after the latter has been sensitized by a treatment with SrCl₂. It seems to the writer that if the substance extracted by Robertson were the real fertilizing agent contained in the spermato­zoön it should fertilize the egg without a previous sensitiza­tion of the egg with SrCl₂ being required.

3. The action of acids in the mechanism of artificial parthenogenesis provides some interesting physio­logical problems. When unfertilized sea-urchin eggs are left in sea water containing any of the lower fatty acids up to capronic, the eggs will form no membranes, while in such sea water, and they will show no outer signs of cytolysis (swelling). When, however, the eggs are left in sea water containing any of the fatty acids from heptylic upward the eggs will form membranes while in the acid sea water and soon afterward will cytolyze completely and swell enormously. In solu­tions of the mineral acids no membranes are formed and none are formed as a rule when the eggs are transferred back to sea water. When both a mineral and a lower fatty acid, e. g., butyric, are added to sea water the mineral acid acts as if it were not present, i. e., the eggs form membranes when transferred back to sea water if the concentra­tion of the butyric acid is high enough. All these data are comprehensible if we assume that only that part of the acid causes membrane forma­tion which is lipoid soluble, while the water soluble part is not involved in the process of membrane forma­tion; and that the cytolysis or swelling of the whole egg can only take place in the higher fatty acids (heptylic or above) which are little soluble in water and very soluble in lipoids, while the lower fatty acids, whose water solubility is comparatively high, can only bring about a cytolysis and swelling in the cortical layer but not in the rest of the egg. This makes it appear as though the part undergoing an altera­tion in membrane forma­tion was a lipoid; and this would harmonize with the assump­tion that the specific membrane-inducing substance in the spermato­zoön is not soluble in water, but soluble in fat.

4. These and other observa­tions led the writer to the view that the essential process which causes development might be an altera­tion of the surface of the egg, in all probability an altera­tion of the superficial layer probably of the nature of a superficial cytolysis. The ques­tion remains: What could be the physico­chemical nature of this cytolysis? The writer had suggested in former papers that in the cytolysis underlying membrane forma­tion lipoids were dissolved, and he supposed that the substance to be dissolved might be a calcium-lipoid compound which might form a continuous layer under the surface of the egg.[90] v. Knaffl, working on the cytolysis of eggs in the writer’s laboratory, gave the following idea of the process:

Protoplasm is rich in lipoids; probably it is mainly an emulsion of these and proteins. Any physical or chemical stimulus which can liquefy the lipoids causes cytolysis of the egg. The protein of the egg can really only swell or be dissolved if the condi­tion of aggrega­tion of the lipoid is altered by chemical or physical agencies. The mechanism of cytolysis consists in the liquefac­tion of the lipoids and thereupon the lipoid-free protein swells or is dissolved by taking up water. . . . Hence this supports Loeb’s view that membrane forma­tion is induced by the liquefac­tion of lipoids.[91]

The writer suggested that the destruc­tion of an emulsion in the cortical layer might possibly be the essential feature of the altera­tion leading to membrane forma­tion and development. It had been long observed that unfertilized starfish eggs may begin to develop apparently without any outside “stimulus,” and A. P. Mathews found that slight mechanical agita­tion of these eggs in sea water increased the number which developed. It has been shown in numerous experi­ments by Delage, R. S. Lillie, and the writer, that the substances causing development in the starfish egg are identical or closely related to those which bring about this effect in the egg of the sea urchin and in both cases the development is preceded by a membrane forma­tion.

But how can membrane forma­tion be produced by mere agita­tion? It seems to me that this can be understood if we suppose that it depends upon the destruc­tion of an emulsion in the cortical layer of the egg. It is conceivable that in the egg of certain forms the stability of this emulsion is so small that mere shaking would be enough to destroy it and thus induce membrane forma­tion and development.[92]

The durability of emulsions varies, and where an emulsion is very durable shaking has no effect, while where it is at the critical point of separating into two continuous phases a slight shaking will bring about the separa­tion, and where the emulsion is still less durable we observe the phenomenon of a “spontaneous” parthenogenesis. Eggs like those of most sea urchins belong to the former, eggs like those of some starfish and annelids belong to the second or third type.

It is impossible to state at present whether the fertiliza­tion membrane is preformed in the fertilized egg and merely lifted off from the egg or whether its forma­tion is due to the hardening of a colloidal substance separated from the emulsion (or excreted) and hardened in touch with sea water. But we can be sure of one thing, namely, that the liquid between egg and membrane contains some colloidal substance which determines the tension and spherical shape of the membrane. The membrane is obviously permeable not only to water but also to dissolved crystalloids, while it is impermeable to colloids. When we add some colloidal solu­tion (e. g., white of egg, blood serum, or tannic acid) to the sea water containing fertilized eggs of purpuratus, the membrane collapses and lies close around the egg; while if the eggs are put back into sea water or a sugar solu­tion the membrane soon assumes its spherical shape. This is intelligible on the assump­tion that in the process of membrane forma­tion (or in the destruc­tion of the emulsion in the cortical layer) a colloidal substance goes into solu­tion which cannot diffuse into the sea water since the membrane is impermeable to the colloidal particles. The membrane is, however, permeable to the constituents of sea water or to sugar. Consequently sea water will diffuse into the space between membrane and egg until the tension of the membrane equals the osmotic pressure of the colloid dissolved in the space between egg and the membrane. If we add enough colloid to the outside solu­tion so that its osmotic pressure is higher than that of the colloidal solu­tion inside the membrane the latter will collapse.

It should also be stated that the unfertilized eggs of many marine animals are surrounded by a jelly (chorion) which is dissolved when the egg is fertilized.[93] The writer has shown that the same chemical substances which will induce membrane forma­tion and artificial parthenogenesis will as a rule also cause a swelling and liquefac­tion of the chorion.

We have devoted so much space to the mechanism of membrane forma­tion since it is likely to give a clearer insight into the physico­chemical nature of physio­logical processes than the phenomena of muscular stimula­tion and contrac­tion or nerve stimula­tion, upon which the majority of physiologists base their conclusions concerning the mechanism of life phenomena.

Before we come to the discussion of the second factor in the activa­tion of the egg it should be stated more definitely that for the eggs of some forms the first factor, the process underlying membrane forma­tion, suffices for the development of the egg into a larva and that no second factor is required in these cases. This is true for the eggs of starfish and certain annelids. Thus in 1901 Loeb[94] and Neilson showed that a short treatment with HCl and HNO₃ sufficed to cause some eggs of Asterias in Woods Hole to develop into larvæ without a second treatment being needed, and Delage[95] showed the same for CO₂; and in 1905 the writer found that the eggs of the Californian starfish Asterina can be induced to form a membrane by butyric acid treatment and that ten per cent. of these eggs developed into normal larvæ. Quite recently R. S. Lillie observed that the eggs of Asterias at Woods Hole can be caused to form membranes and develop into larvæ by a treatment with butyric acid and that the time of exposure required to get a maximal number of larvæ varies approximately inversely with the concentra­tion of the acid, within a range of 0.0005 to 0.006 N butyric acid. If the exposure is too short membrane forma­tion will occur without normal development.[96]

All this leads us to the conclusion that the main effect of the spermato­zoön in inducing the development of the egg consists in an altera­tion of the surface of the latter which is apparently of the nature of a cytolysis of the cortical layer. Anything that causes this altera­tion without endangering the rest of the egg may induce its development. The spermato­zoön, therefore, causes the development of the egg by carrying a substance into the latter which effects an altera­tion of its surface layer.

5. We will now discuss the action of the second, corrective factor, in the inducement of development. When we cause membrane forma­tion in a sea-urchin egg by the proper treatment with butyric acid it will commence to develop and segment but will disintegrate rapidly if kept at room temperature and the more rapidly the higher the temperature. If, however, the eggs are treated afterward for a certain length of time (from thirty-five to sixty minutes at 15° C. for purpuratus and 17¹⁄₂ to 22¹⁄₂ minutes for Arbacia at 23° C.) in a solu­tion which is isosmotic with 50 c.c. sea water+8 c.c. 2¹⁄₂ m NaCl,[97] they will develop into larvæ, many of which may be normal. Any hypertonic solu­tion of this osmotic pressure, sea water, sugar, or a single salt, will suffice provided the solu­tion does not contain substances that are too destructive for living matter. The hypertonic solu­tion produces its corrective effect only if the egg contains free oxygen; and in a slightly alkaline medium more rapidly than in a neutral medium. The time of exposure in the hypertonic solu­tion diminishes in certain limits with the concentra­tion of OH ions in the solu­tion.

It is strange that in the eggs of purpuratus the corrective effect can also be brought about by exposing the eggs after the artificial membrane forma­tion for about three hours to normal sea water free from oxygen; or to sea water in which the oxida­tions have been retarded by the addi­tion of KCN. This method is not so reliable as the treatment with hypertonic solu­tion.

What does the hypertonic solu­tion do to prevent the disintegra­tion of the egg after the artificial membrane forma­tion? The writer suggested in 1905 that the artificial membrane forma­tion alone starts the development but leaves the eggs usually in a sickly condi­tion and that the hypertonic solu­tion or the lack of oxygen allows them to recuperate from such a condi­tion. The second factor is, according to this view, merely a corrective or curative factor. The following observa­tions will explain the reasons for such an assump­tion.

The writer found that if we keep the unfertilized eggs after artificial membrane forma­tion in sea water deprived of oxygen the disintegra­tion of the egg following artificial membrane forma­tion is prevented for a day at least. The same result can be obtained by adding ten drops of ¹⁄₁₀ per cent. KCN to 50 c.c. of sea water, and certain narcotics, e. g., chloral hydrate, act in the same way. Wasteneys and the writer found that chloral hydrate (and other narcotics) in the concentra­tion required do not suppress or even lower the oxida­tions in the egg to any considerable extent,[98] but they prevent the processes of cell division. Hence it seems that the egg disintegrates so rapidly after artificial membrane forma­tion because it is killed by those processes leading to nuclear division or cell division which are induced by the artificial membrane forma­tion. If we suppress these phenomena of development (for not too long a time) we give the egg a chance to recover and if now the impulse to develop is still active we notice a perfectly normal development. If the egg is kept too long without oxygen it suffers for other reasons and cannot develop; the writer has shown that if eggs fertilized by sperm are kept for too long a time without oxygen they also will no longer be able to develop normally. The short treatment with a hypertonic solu­tion supplies the corrective factor required, so that the egg can then undergo cell division at room temperature without disintegrating.

The correctness of this interpreta­tion, which is in reality mainly a statement of observa­tions, is proved by the two following groups of facts. The older observers had already noticed that the unfertilized eggs of the sea urchin when lying in sea water will die after a day or more, and that occasionally such eggs show nuclear division or even the beginning of cell division shortly before disintegra­tion sets in. The writer has studied this phenomenon in the unfertilized eggs of purpuratus and found that only the eggs of certain females show this cell division before disintegra­tion and that the cell division is preceded by an atypical form of membrane forma­tion; the eggs surrounding themselves by a fine gelatinous film comparable to that produced in the egg of Arbacia by a treatment with butyric acid. It is difficult to state what induces the altera­tion of the surface in the eggs that lie so long in sea water. It may be due to the CO₂ formed by the eggs—since we know that CO₂ may induce membrane forma­tion—or it may be due to the alkalinity of the sea water or to a substance originating from the jelly surrounding the eggs. It was found that if such eggs are kept without oxygen their disintegra­tion (and cell division) will be delayed considerably. The presumable explana­tion for this is that the lack of oxygen prevents the internal changes underlying cell division and thus prevents the disintegra­tion of the egg. The direct proof that an egg in the process of cell division is more endangered by abnormal solu­tions than an egg at rest has been furnished by numerous observa­tions of the writer. He showed in 1906 that the fertilized egg of purpuratus dies rather rapidly in a pure m/2 NaCl or any other abnormal isotonic solu­tion, while the unfertilized egg can live for days in such solu­tions.[99] In a series of papers, beginning in 1905, he showed that the fertilized egg will live longer in hypertonic, hypotonic, and otherwise abnormally constituted solu­tions when the cell divisions are suppressed by lack of oxygen or by the addi­tion of KCN or of chloral hydrate.[100] It is thus obvious that coincident with the changes underlying nuclear division or cell division altera­tions occur in the sensitiveness of the egg to salt solu­tions of abnormal concentra­tion or constitu­tion, e. g., NaCl+CaCl₂ isotonic with sea water, hypertonic, or hypotonic solu­tions.

We must, therefore, conclude that artificial membrane forma­tion induces development but that it leaves the egg in a sickly condi­tion in which the very processes leading to cell division bring about its destruc­tion; that if it is given time it can recover from this condi­tion and that the treatment with the hypertonic solu­tion also brings about this recovery rapidly and reliably.

Herlant[101] suggested that the corrective effect of the hypertonic solu­tion consisted in the proper development of the astrospheres required for cell division. According to this author mere membrane forma­tion does not lead to the forma­tion of sufficiently large astrospheres and hence cell division may remain impossible.[102] The writer has no a priori objec­tion to this sugges­tion which agrees with earlier observa­tions by Morgan except that it is at present difficult to harmonize it with all the facts. Why should it be possible to replace the treatment with the hypertonic solu­tion by a suspension of the oxida­tions in the egg for three hours while we know that lack of oxygen suppresses the forma­tion of astrospheres in the fertilized eggs? What becomes of the astrospheres if the treatment with the hypertonic solu­tion precedes the membrane forma­tion by a number of hours or a day (which is possible as we shall see), and why do they not induce cell division, if Herlant’s idea is correct? Nevertheless the sugges­tion of Herlant deserves to be taken into serious considera­tion.

6. How can an altera­tion of the surface of the egg—e. g., a cytolytic or other destruc­tion of the cortical layer—lead to a beginning of development? The answer is possibly given in the rela­tion of oxida­tion to development. The writer found in 1895 that if oxygen is withdrawn from the fertilized sea-urchin egg it can not segment and this seems to be the case for eggs in general.[103] In 1906 he found that the rapid disintegra­tion of the eggs of the sea urchin which follows artificial membrane forma­tion could be prevented when the eggs were deprived of oxygen or when the oxida­tions were suppressed in the eggs by KCN. This suggested a connec­tion between the disintegra­tion of the egg after artificial membrane forma­tion and the increase in the rate of oxida­tions; and he found further that the forma­tion of acid is greater in the fertilized than in the unfertilized egg. He, therefore, expressed the view in 1906 that the essential feature (or possibly one of the essential features) of the process of fertiliza­tion was the increase of the rate of oxida­tions in the egg and that this increase was caused by the membrane forma­tion alone.[104] These conclusions have been since amply confirmed by the measurements of O. Warburg as well as those of Loeb and Wasteneys, both showing that the entrance of the spermato­zoön into the egg raises the rate of oxida­tions from 400 to 600 per cent., and that membrane forma­tion alone brings about an increase of similar magnitude. Loeb and Wasteneys found that the hypertonic solu­tion does not increase the rate of oxida­tions in a fertilized egg. It does do so, however, in an unfertilized egg without membrane forma­tion, but merely for the reason that in such an egg the hypertonic solu­tion brings about the cytolytic change in the cortex of the egg underlying membrane forma­tion.[105] According to Warburg it is probable that the oxida­tions occur mainly if not exclusively at the surface of the egg since NaOH, which does not diffuse into the egg, raises the rate of oxida­tions more than NH₄OH which does diffuse into the egg. And finally, the same author showed that the oxida­tions in the sea-urchin egg are due to a catalytic process in which iron acts as a catalyzer.[106] In view of all these facts and their harmony with the methods of artificial parthenogenesis the sugges­tion is justifiable that the altera­tion or cytolysis of the cortical layer of the egg is in some way connected with the increased rate of oxida­tions.

The question remains then: How can membrane forma­tion or the altera­tion of the cortical layer underlying membrane forma­tion cause an increase in the rate of oxida­tions? One possibility is that the iron (or whatever the nature of the catalyzer may be) exists in the cortex of the egg in a masked condi­tion—or in a condi­tion in which it is not able to act—while the altera­tion of the cortical layer makes the iron active. It might be that either the iron or the oxidizable substrate is contained in the lipoid layer in the unfertilized condi­tion of the egg and that the destruc­tion or cytolysis of the cortical layer brings both the iron and the oxidizable substrate into the watery phase in which they can interact.

Another possibility is that the act of fertiliza­tion increases the permeability of the egg. This idea, which seems attractive, was first suggested and discussed by the writer in 1906.[107] He had found that when fertilized and unfertilized eggs were put into abnormal salt solu­tions, e. g., pure solu­tions of NaCl, the fertilized eggs died more rapidly than the unfertilized eggs and he pointed out that these experi­ments suggested the possibility that fertiliza­tion increases the permeability of the egg for salts. The reason for his hesita­tion to accept this interpreta­tion was, that the fertilized egg is also more easily injured by lack of oxygen than the unfertilized egg and in this case the greater sensitiveness of the fertilized egg was obviously due to its greater rate of metabolism. Later experi­ments by the writer showed that the fertilized egg can be made more resistant to abnormal salt solu­tions if its development is suppressed by lack of oxygen or by KCN or by certain narcotics. With our present knowledge it does not seem very probable that lack of oxygen diminishes the permeability of the egg, but we know that it inhibits the developmental processes. Warburg has made it appear very probable that the fertilized egg is impermeable for NaOH and if this is the case it should also be impermeable for NaCl.[108]

The idea that fertiliza­tion and membrane forma­tion cause an increase in the permeability of the egg was later accepted and elaborated by R. Lillie. This author assumes that the unfertilized egg cannot develop because it contains too much CO₂ but that the CO₂ can escape from the egg as soon as its permeability is increased through the destruc­tion of the cortical layer of the egg.[109] After the CO₂ has escaped, the excessive permeability must be restored to its normal value and this is the rôle of the hypertonic treatment. It is, however, difficult to harmonize the assump­tion of an impermeability of the unfertilized egg for CO₂ with the fact that if the unfertilized sea-urchin egg is cut into two, as is done in merogony, no development takes place, while such pieces will develop when a spermato­zoön enters. The cortical layer is removed along the cut surface and there is no reason why the CO₂ should not escape. Besides, the experi­ments of Godlewski and the writer prove that the cortical layer of the unfertilized sea-urchin egg is apparently very permeable for CO₂ since the latter causes membrane forma­tion if contained in the sea water in sufficiently high concentra­tion.

Lillie assumes that the hypertonic treatment restores the permeability raised to excess by the butyric acid treatment, but this assump­tion is not in harmony with the following facts. The writer has shown that it is immaterial whether the eggs are treated first with the hypertonic solu­tion and then with butyric acid or the reverse, if only the eggs remain longer in the hypertonic solu­tion when the hypertonic treatment precedes the butyric acid treatment. It was stated in the beginning of this chapter that the development of the egg can be induced by hypertonic sea water, and we know the reason since hypertonic sea water is a cytolytic agency. The writer found that when we expose unfertilized eggs of purpuratus for from two to two and a half-hours to hypertonic sea water they will often not develop and only a few eggs will undergo the first cell divisions, then going into a condi­tion of rest. When these eggs, both the segmented and unsegmented, were treated twenty-four or thirty-six hours later with butyric acid, so that they formed a membrane, they all developed into larvæ without further treatment. It is impossible to apply Lillie’s theory to these facts, for the simple reason that the treatment with hypertonic sea water was just long enough to induce development in some eggs and hence according to Lillie’s ideas must have increased the permeability of these eggs. Yet these same eggs were induced to develop normally when subsequently treated with butyric acid, which according to Lillie also acts by increasing the permeability. Nothing indicates that the treatment of the eggs with a hypertonic solu­tion diminishes their permeability; the reverse would be much more probable.

Lillie’s theory also fails to explain that mere treatment of the eggs with a hypertonic solu­tion can bring about their development into larvæ. This, however, is intelligible on the assump­tion that the hypertonic solu­tion in this case has two different effects, first a cytolysis of the cortical layer of the egg and second an entirely different effect, possibly upon the interior of the egg, which represents the second or corrective effect.

McClendon[110] has shown that the electrical conductivity of the egg is increased after fertiliza­tion, and J. Gray[111] has found that this increase in conductivity is only transitory and disappears in fifteen minutes. This might indicate that the egg becomes transitorily more permeable for salts after the entrance of the spermato­zoön or after membrane forma­tion; although an increase in conductivity might be caused by other changes than a mere increase in permeability of the egg. The writer is of the opinion that it is necessary to meet all these and other difficulties before we can state that the altera­tion of the cortical layer, which is the essential feature of development, acts chiefly or exclusively by an increase in the permeability of the egg.[112]

7. When the experi­ments on artificial parthenogenesis were first published they aroused a good deal of antagonism not only among reac­tionaries in general but also among a certain group of biologists. O. Hertwig had defined fertiliza­tion as consisting in the fusion of two nuclei, the egg nucleus and the sperm nucleus. No such fusion of two nuclei takes place in artificial parthenogenesis since no spermato­zoön enters the egg, and it became necessary, therefore, to abandon Hertwig’s defini­tion as wrong. The objec­tion raised that the phenomena are limited to a few species soon became untenable since it has been possible to produce artificial parthenogenesis in the egg of plants (Fucus, according to Overton) as well as of animals, from echinoderms up to the frog; and it may possibly one day be accomplished also in warm-blooded animals. A second objec­tion was that the eggs caused to develop by the methods of artificial parthenogenesis could never reach the adult stage and that hence the phenomenon was merely pathological. There was no basis for such a statement, except that it is extremely difficult to raise marine invertebrates. Delage[113] was courageous enough to make an attempt to raise partheno­genetic larvæ of the sea urchin beyond the larval stage and he succeeded in one case in carrying the animal to the mature stage. It proved to be a male.

Better opportunities were offered when a method was discovered which induced the development of the unfertilized eggs of the frog. In 1907, Guyer made the surprising observa­tion that if he injected lymph or blood into the unfertilized eggs of frogs he succeeded in starting development and he even obtained two free-swimming tadpoles. “Apparently the white rather than the red corpuscles are the stimulating agents which bring about development, because injec­tions of lymph which contains only white corpuscles produce the same effects as injec­tions of blood.” Curiously enough, Guyer thought that probably the cells which he introduced and not the egg were developing. In 1910, Bataillon showed that a mere puncture of the egg with a needle could induce development but he believes that for the full development the introduc­tion of a fragment of a leucocyte is required. Bataillon has called atten­tion to the analogy with the writer’s results on lower forms, the puncturing of the egg corresponding to the cytolysis of the surface layer of the egg and the introduc­tion of a leucocyte as the analogue of the second or corrective factor. The method of producing artificial parthenogenesis by puncturing the egg has thus far been successful only in the egg of the frog. The writer has tried it in vain on the eggs of many other forms. He has at present seven partheno­genetic frogs over a year old, produced by merely puncturing the eggs with a fine needle (Fig. 6). These frogs have reached over half the size of the adult frog. They can in no way be distinguished from the frogs produced by fertiliza­tion with a spermato­zoön. This makes the proof conclusive that the methods of artificial parthenogenesis can result in the produc­tion of normal organisms which can reach the adult stage.