All this leads us to the conclusion that the main effect of the spermato­zoön in inducing the development of the egg consists in an altera­tion of the surface of the latter which is apparently of the nature of a cytolysis of the cortical layer. Anything that causes this altera­tion without endangering the rest of the egg may induce its development. The spermato­zoön, therefore, causes the development of the egg by carrying a substance into the latter which effects an altera­tion of its surface layer.

5. We will now discuss the action of the second, corrective factor, in the inducement of development. When we cause membrane forma­tion in a sea-urchin egg by the proper treatment with butyric acid it will commence to develop and segment but will disintegrate rapidly if kept at room temperature and the more rapidly the higher the temperature. If, however, the eggs are treated afterward for a certain length of time (from thirty-five to sixty minutes at 15° C. for purpuratus and 171⁄2 to 221⁄2 minutes for Arbacia at 23° C.) in a solu­tion which is isosmotic with 50 c.c. sea water+8 c.c. 21⁄2 m NaCl,[97] they will develop into larvæ, many of which may be normal. Any hypertonic solu­tion of this osmotic pressure, sea water, sugar, or a single salt, will suffice provided the solu­tion does not contain substances that are too destructive for living matter. The hypertonic solu­tion produces its corrective effect only if the egg contains free oxygen; and in a slightly alkaline medium more rapidly than in a neutral medium. The time of exposure in the hypertonic solu­tion diminishes in certain limits with the concentra­tion of OH ions in the solu­tion.

It is strange that in the eggs of purpuratus the corrective effect can also be brought about by exposing the eggs after the artificial membrane forma­tion for about three hours to normal sea water free from oxygen; or to sea water in which the oxida­tions have been retarded by the addi­tion of KCN. This method is not so reliable as the treatment with hypertonic solu­tion.

What does the hypertonic solu­tion do to prevent the disintegra­tion of the egg after the artificial membrane forma­tion? The writer suggested in 1905 that the artificial membrane forma­tion alone starts the development but leaves the eggs usually in a sickly condi­tion and that the hypertonic solu­tion or the lack of oxygen allows them to recuperate from such a condi­tion. The second factor is, according to this view, merely a corrective or curative factor. The following observa­tions will explain the reasons for such an assump­tion.

The writer found that if we keep the unfertilized eggs after artificial membrane forma­tion in sea water deprived of oxygen the disintegra­tion of the egg following artificial membrane forma­tion is prevented for a day at least. The same result can be obtained by adding ten drops of 1⁄10 per cent. KCN to 50 c.c. of sea water, and certain narcotics, e. g., chloral hydrate, act in the same way. Wasteneys and the writer found that chloral hydrate (and other narcotics) in the concentra­tion required do not suppress or even lower the oxida­tions in the egg to any considerable extent,[98] but they prevent the processes of cell division. Hence it seems that the egg disintegrates so rapidly after artificial membrane forma­tion because it is killed by those processes leading to nuclear division or cell division which are induced by the artificial membrane forma­tion. If we suppress these phenomena of development (for not too long a time) we give the egg a chance to recover and if now the impulse to develop is still active we notice a perfectly normal development. If the egg is kept too long without oxygen it suffers for other reasons and cannot develop; the writer has shown that if eggs fertilized by sperm are kept for too long a time without oxygen they also will no longer be able to develop normally. The short treatment with a hypertonic solu­tion supplies the corrective factor required, so that the egg can then undergo cell division at room temperature without disintegrating.

The correctness of this interpreta­tion, which is in reality mainly a statement of observa­tions, is proved by the two following groups of facts. The older observers had already noticed that the unfertilized eggs of the sea urchin when lying in sea water will die after a day or more, and that occasionally such eggs show nuclear division or even the beginning of cell division shortly before disintegra­tion sets in. The writer has studied this phenomenon in the unfertilized eggs of purpuratus and found that only the eggs of certain females show this cell division before disintegra­tion and that the cell division is preceded by an atypical form of membrane forma­tion; the eggs surrounding themselves by a fine gelatinous film comparable to that produced in the egg of Arbacia by a treatment with butyric acid. It is difficult to state what induces the altera­tion of the surface in the eggs that lie so long in sea water. It may be due to the CO2 formed by the eggs—since we know that CO2 may induce membrane forma­tion—or it may be due to the alkalinity of the sea water or to a substance originating from the jelly surrounding the eggs. It was found that if such eggs are kept without oxygen their disintegra­tion (and cell division) will be delayed considerably. The presumable explana­tion for this is that the lack of oxygen prevents the internal changes underlying cell division and thus prevents the disintegra­tion of the egg. The direct proof that an egg in the process of cell division is more endangered by abnormal solu­tions than an egg at rest has been furnished by numerous observa­tions of the writer. He showed in 1906 that the fertilized egg of purpuratus dies rather rapidly in a pure m/2 NaCl or any other abnormal isotonic solu­tion, while the unfertilized egg can live for days in such solu­tions.[99] In a series of papers, beginning in 1905, he showed that the fertilized egg will live longer in hypertonic, hypotonic, and otherwise abnormally constituted solu­tions when the cell divisions are suppressed by lack of oxygen or by the addi­tion of KCN or of chloral hydrate.[100] It is thus obvious that coincident with the changes underlying nuclear division or cell division altera­tions occur in the sensitiveness of the egg to salt solu­tions of abnormal concentra­tion or constitu­tion, e. g., NaCl+CaCl2 isotonic with sea water, hypertonic, or hypotonic solu­tions.

We must, therefore, conclude that artificial membrane forma­tion induces development but that it leaves the egg in a sickly condi­tion in which the very processes leading to cell division bring about its destruc­tion; that if it is given time it can recover from this condi­tion and that the treatment with the hypertonic solu­tion also brings about this recovery rapidly and reliably.

Herlant[101] suggested that the corrective effect of the hypertonic solu­tion consisted in the proper development of the astrospheres required for cell division. According to this author mere membrane forma­tion does not lead to the forma­tion of sufficiently large astrospheres and hence cell division may remain impossible.[102] The writer has no a priori objec­tion to this sugges­tion which agrees with earlier observa­tions by Morgan except that it is at present difficult to harmonize it with all the facts. Why should it be possible to replace the treatment with the hypertonic solu­tion by a suspension of the oxida­tions in the egg for three hours while we know that lack of oxygen suppresses the forma­tion of astrospheres in the fertilized eggs? What becomes of the astrospheres if the treatment with the hypertonic solu­tion precedes the membrane forma­tion by a number of hours or a day (which is possible as we shall see), and why do they not induce cell division, if Herlant’s idea is correct? Nevertheless the sugges­tion of Herlant deserves to be taken into serious considera­tion.

6. How can an altera­tion of the surface of the egg—e. g., a cytolytic or other destruc­tion of the cortical layer—lead to a beginning of development? The answer is possibly given in the rela­tion of oxida­tion to development. The writer found in 1895 that if oxygen is withdrawn from the fertilized sea-urchin egg it can not segment and this seems to be the case for eggs in general.[103] In 1906 he found that the rapid disintegra­tion of the eggs of the sea urchin which follows artificial membrane forma­tion could be prevented when the eggs were deprived of oxygen or when the oxida­tions were suppressed in the eggs by KCN. This suggested a connec­tion between the disintegra­tion of the egg after artificial membrane forma­tion and the increase in the rate of oxida­tions; and he found further that the forma­tion of acid is greater in the fertilized than in the unfertilized egg. He, therefore, expressed the view in 1906 that the essential feature (or possibly one of the essential features) of the process of fertiliza­tion was the increase of the rate of oxida­tions in the egg and that this increase was caused by the membrane forma­tion alone.[104] These conclusions have been since amply confirmed by the measurements of O. Warburg as well as those of Loeb and Wasteneys, both showing that the entrance of the spermato­zoön into the egg raises the rate of oxida­tions from 400 to 600 per cent., and that membrane forma­tion alone brings about an increase of similar magnitude. Loeb and Wasteneys found that the hypertonic solu­tion does not increase the rate of oxida­tions in a fertilized egg. It does do so, however, in an unfertilized egg without membrane forma­tion, but merely for the reason that in such an egg the hypertonic solu­tion brings about the cytolytic change in the cortex of the egg underlying membrane forma­tion.[105] According to Warburg it is probable that the oxida­tions occur mainly if not exclusively at the surface of the egg since NaOH, which does not diffuse into the egg, raises the rate of oxida­tions more than NH4OH which does diffuse into the egg. And finally, the same author showed that the oxida­tions in the sea-urchin egg are due to a catalytic process in which iron acts as a catalyzer.[106] In view of all these facts and their harmony with the methods of artificial parthenogenesis the sugges­tion is justifiable that the altera­tion or cytolysis of the cortical layer of the egg is in some way connected with the increased rate of oxida­tions.

The question remains then: How can membrane forma­tion or the altera­tion of the cortical layer underlying membrane forma­tion cause an increase in the rate of oxida­tions? One possibility is that the iron (or whatever the nature of the catalyzer may be) exists in the cortex of the egg in a masked condi­tion—or in a condi­tion in which it is not able to act—while the altera­tion of the cortical layer makes the iron active. It might be that either the iron or the oxidizable substrate is contained in the lipoid layer in the unfertilized condi­tion of the egg and that the destruc­tion or cytolysis of the cortical layer brings both the iron and the oxidizable substrate into the watery phase in which they can interact.