Читать книгу The Organism as a Whole, from a Physicochemical Viewpoint - Jacques Loeb - Страница 15

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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.

Kupelwieser62 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 Herlant67 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. Lillie69 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

	Asterias♂	Asterina♂	Franciscanus♂	Purpuratus♂
Asterias♀ (immature)	Immediately very motile.	No activa­tion.	Moderately active.	Slight effect in immediate contact with egg.
Asterina♀ (immature)	Not motile.	Violent activity.	Violent activity.	Slight effect only near the egg.
Franciscanus♀ (mature)	Slightly motile.	No motility.	Immediately active.	Immediately active.
Purpuratus♀ (mature)	Slightly motile after some time.	Slight effect in immediate contact with eggs.	Immediately active.	Immediately active.

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. Lillie71 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.

Castle77 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. Morgan78 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. Fuchs79 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: