CHAPTER V

ARTIFICIAL PARTHENOGENESIS

1. The majority of eggs cannot develop unless they are fertilized, that is to say, unless a spermatozoön enters into the egg. The question arises: How does the spermatozoon cause the egg to develop into a new organism? The spermatozoon is a living organism with a complicated structure and it is impossible to explain the causation of the development of the egg from the structure of the spermatozoön. No progress was possible in this field until ways were found to replace the action of the living spermatozoön by wellknown physicochemical agencies. Various observers such as Tichomiroff, R. Hertwig, and T. H. Morgan had found that unfertilized eggs may begin to segment under certain conditions, but such eggs always disintegrated in their experiments without giving rise to larvæ. In 1899 the writer succeeded in causing the

I

The substitution of well-known physicochemical agencies for the mysterious action of the spermatozoon was the task the writer set himself in this work and not the explanation of natural parthenogenesis, as the author of a recent text-book seems to assume.

unfertilized eggs of the sea urchin Arbacia to develop into swimming larvæ, blastulæ, gastrulæ, and plutei, by treating them with hypertonic sea water of a definite osmotic pressure for about two hours. When such eggs were then put back into normal sea water many segmented and a certain percentage developed into perfectly normal larvæ, blastulæ, gastrulæ, and plutei.1 Soon afterward this was accomplished by other methods for the unfertilized eggs of a large number of marine animals, such as starfish, molluscs, and annelids. None of these eggs can develop under normal conditions unless a spermatozoön enters. These experiments furnished proof that the activating effect of the spermatozoon upon the egg can be replaced by a purely physicochemical agency.2

The first method used in the production of larvæ from the unfertilized eggs did not lend itself to an analysis of the activating effect of the spermatozoön upon the egg, since nothing was known about the action of a hypertonic solution, except that it withdraws water from the egg; and there was no indication that the entrance of the spermatozoon causes the egg to lose water. No further progress was possible until another method of artificial parthenogenesis was found. When a spermatozoön enters the egg of a sea urchin or starfish

Loeb. J., Am. Jour. Physiol., 1899, iii., 135; 1900, iii., 434.

1 Loeb, J., Artificial Parthenogenesis and Fertilization, Chicago, 1913. The reader is referred to this bock for the literature on the subject.

or certain annelids, the surface of the egg undergoes a change which is called membrane formation; and which consists in the appearance of a fine membrane around the egg, separated from the latter by a liquid (Figs. 4 and 5). O. and R. Hertwig and Herbst had

FIG. 4. Unfertilized egg surrounded by spermatozoa (whose flagellum is omitted in the drawing).

FIG. 5. The same egg after a spermatozoon has entered. The fertilization membrane is separated from the egg by a clear space.

observed that such a membrane could be produced in an unfertilized egg if the latter was put into chloroform or xylol, but such eggs perished at once. It was generally assumed, moreover, that the process of membrane formation was of no significance in the phenomenon of fertilization, except perhaps that the fertilization membrane guarded the fertilized egg against a further invasion by sperm. However, since the fertilized egg is protected against this possibility by other means the membrane is hardly needed for such a purpose.

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 lessdevelop into swimming blastulæ1; 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 treat

The reader will find a description of the development of this egg in the next chapter.

ment 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. 22 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 172 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