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.