The Organism as a Whole, from a Physicochemical Viewpoint

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 spermatozoon — we should assent to such an assumption — but that the "fertilizin" acts like an "amboceptor" between egg and spermatozoon, 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 sidechain theory which, besides, can only be applied in a metaphorical sense to the mechanism of the entrance of the spermatozoon into the egg.1

The writer may be permitted to illustrate by a special case his reason for declining to accept Ehrlich's side-chain theory. Ehrlich and Sachs found that if to a given mass of toxin small quantities of antitoxin are added successively the first fraction added neutralized more than the later fractions; and on the basis of this reasoning Ehrlich concluded that ten different toxins were contained in the diphtheria toxin. Ar- rhenius showed that the same phenomenon can be obtained when a weak base like NH4OH is neutralized by a weak acid (e. g., boric acid) ; hence we should assume that NH 4OH consists of ten different forms of ammonia. Both cases, the saturation of toxin with antitoxin and ammonia with boric acid are equilibrium phenomena. (Arrhenius, S.f Quantitative Laws in Biological Chemistry, London,

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 role in the theories of evolution. We are here only concerned with the mechanism which determines the block to the entrance of a spermatozoon into an egg of the same hermaphroditic individual.

Castle1 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. Morgan2 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. Fuchs1 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 monoecious (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.2

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. 3 He fertilized two individuals of Cardamine crosswise and raised sixty plants of the first generation. He compared the fertility of these Fx 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: (B) fertile with one (B), sterile with the other parent (G). (b) fertile with G, sterile with B. Type Bg (O 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 spermatozoon is important. Should it be possible that the block created by the entrance of a spermatozoon into the egg is also due to an inhibitory substance carried by a spermatozoon into the egg; and furthermore that the effect of the inhibitory substance should be the prevention of further agglutination of the spermatozoon 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 nermaphrodite and a spermatozoon 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 autoagglutinins (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 o.oi per cent, malic acid if put into a capillary tube will attract the spermatozoa of ferns.

When the liquid in the tube contains only o.oi 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.1 These experiments were continued and amplified by Shibata. Bruchmann2 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 ob-

served 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 r61e; 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.

I. The majority of eggs cannot develop unless they are fertilized, that is to say, unless a spermatozoon enters into the egg. The question arises: How does the spermatozoon cause the egg to develop into a new organism? The spermatozoon 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 spermatozoon. No progress was possible in this field until ways were found to replace the action of the living spermatozoon by wellknown physicochemical agencies.1 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 larvae. In 1899 tne writer succeeded in causing the

1 The substitution of well-known physicochemical agencies for the mysterious action of the spermatozoon was the task the writer set himself in this work and not the explanation of natural parthenogenesis, as the author of a recent text-book seems to assume. unfertilized eggs of the sea urchin Arbacia to develop into swimming larvae, blastulse, gastrulae, 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 larvae, blastulae, gastrulae, and plutei. r 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 spermatozoon enters. These experiments furnished proof that the activating effect of the spermatozoon upon the egg can be replaced by a purely physicochemical agency. 2

The first method used in the production of larvae from the unfertilized eggs did not lend itself to an analysis of the activating effect of the spermatozoon 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 spermatozoon causes the egg to lose water. No further progress was possible until another method of artificial parthenogenesis was found. When a spermatozoon 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). 0. and R. Hertwig and Herbst had flagellum is omitted in the drawing). FIG. 5. The same egg after a spermatozoon has entered. The 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 spermatozoon. 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/io 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 blastulas1; 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 spermatozoon 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. 1 The reader will find a description of the development of this egg in the next chapter. ment with hypertonic sea water or with lack of oxygen or with KCN they developed into normal larvae. 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/io 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. 2j^ 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 17^ minutes to 22^ 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 spermatozoon upon the egg in every detail and eggs treated in this way will develop in large numbers into perfectly normal larvae. We shall see later that they can also be raised to the adult state.

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 larvae; 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 spermatozoon 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 : (i) 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.j Bad 2 and SrCl2 in the case of the egg of purpuratus, and according to R. Lillie, Nal or NaCXS 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

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 I 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

1 The reader is referred for details to the writer's book on the subject. 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. BaG 2 acts similarly. The fact that SrCla 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 spermatozoon produces this effect; and as a rule no previous sensitizing action of SrCh 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 spermatozoon causes membrane formation before the spermatozoon 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 spermatozoon, only the living spermatozoon 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 spermatozoon is insoluble in water but soluble in the e£g (or m Hpoids). If this assumption is correct the two substances are essentially different. , Robertson1 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 spermatozoon 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 wrhole 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 spermatozoon 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.1 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.2

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 1 Loeb, J., Uber den chemischen Charakter des Befruchtungsvor gangs, etc., Leipzig, 1908. 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.