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.