The Organism as a Whole, from a Physicochemical Viewpoint

A. E. Taylor1 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. 2

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 (o.i gin.) 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 spermatozoon, 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 spermatozoon to develop into an embryo,1 while we can induce the egg to develop into an embryo without a spermatozoon. 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.

I . 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

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 spermatozoon 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 spermatozoon — enters the female gamete — the egg — and this starts the development. In the case of most animals the egg cannot develop unless the spermatozoon enters. The question arises: How does the spermatozoon activate the egg? And also how does it happen that the spermatozoon 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 spermatozoon 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/io 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 CCa was increased it was not necessary to add as muchlTaOH. The spermatozoon enters the egg through the socalled fertilization cone, i. e.t a protoplasmic process comparable to the pseudopodium of an amoeboid cell. The analogy of the process of phagocytosis — i. e., the taking up of particles by an amoeboid cell — and that of the engulfing of the spermatozoon 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 spermatozoon which in the case of 5. purpuratus 9 and Asterias cf can be supplied by a slight increase in the CQH or CCa. 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.1 Godlewski succeeded by the same method in fertilizing the eggs of a Naples starfish with the sperm of a crinoid. 3 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.

Kupelwieser3 observed that the spermatozoon 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 spermatozoon can enter the eggs of widely different species but with rare exceptions all the embryos will die in an early stage of development. 4 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/2NaCl 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 spermatozoon 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/ioo solution of NaOH (or NH3 or benzylamine or butylamine) or eight drops of m/ioo NaHC03, 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+MgCla+KQ+CaCla. 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+KCL1 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 spermatozoon 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.* It is thus not impossible that the specificity which favours the entrance of a spermatozoon into an egg of its own species may consist in an agglutination between spermatozoon and egg protoplasm (or its fertilization cone) ; and that this agglutination is favoured if the COH or Cca or both are increased within certain limits. Godlewski discovered a very interesting form of block to the entrance of the spermatozoon into the egg which takes place if two different types of sperm are mixed. He had found that the sperm of the annelid Chcetopterus 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 Chcetopterus and the sperm of sea urchins are mixed the mixture is not able to induce development or membrane formation, since now neither spermatozoon 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. 2 The phenomenon was further investigated by Herlant3 who found that if the sperm of a sea urchin is

mixed with the sperm of certain annelids (Chcztopterus) 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.1 We can imagine that the precipitate forms a film around the egg and acts as a block for the agglutination between egg and spermatozoon. 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 COH or Cca) 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 spermatozoon 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 spermatozoon 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 spermatozoon to make the latter stick to the surface of the egg and agglutinate and that later forces of a different character bring the spermatozoon into the egg. The fact that under normal conditions a very slight degree of motility on the part of the spermatozoon 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. Lillie1 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.1 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. Lillie1 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 I 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 offranciscanus; 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 spermatozoon 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." x 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 agglu-

tinate, 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.1 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. 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 blast omeres form a fertilization membrane and now begin to develop into larvae; and if the spermatozoon 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 eell division the individual blastomeres into which a spermatozoon 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 spermatozoon 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 spermatozoon into an egg which is already fertilized. x

When the spermatozoon 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 spermatozoon. 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 spermatozoon 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.1 When the membrane is too tough to allow the spermatozoon 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 spermatozoon can no longer agglutinate with the fertilized egg or that those phagocytotic reactions which we suppose to play a role in the entrance of the spermatozoon into the egg are no longer possible after a spermatozoon has entered? The mere fact of development is apparently not the cause which bars a spermatozoon 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