The whole subject has been attacked from a new point of view by Herlant 1 and Brachet 2 who have pointed out that the agent em- ployed to provoke parthenogenesis does not exercise a specific chemical action on the egg but merely acts as a stimulus to which the egg as a living organism responds. Whether butyric acid or a needle be employed the response is the same; the egg " wakes up " so to speak, the nucleus emits something which acts as a centrosome and from this is developed a great series of radiating rays traversing the cytoplasm, a huge " monaster " in fact. The chromatin of the nucleus becomes resolved into chromosomes which are split longitu- dinally and which become adherent to the rays of the " monaster." In the case of the egg of the sea-urchin it is only extremely rarely that the monaster becomes changed into an ordinary mitotic spindle by the division of the centrosome. In most cases after persisting for about an hour the monaster disappears; the nucleus returns to the resting condition and then after a short interval it passes through the same phases, a monaster being again formed. After this process has been repeated about six times over a period lasting twelve hours the egg dies and cytolysis supervenes. If, however, after the egg has been exposed to the action of the butyric acid and then washed in sea -water it is placed in hypertonic sea-water, and then after a limited period of immersion in this fluid replaced in ordinary sea-water, additional asters are formed in the cytoplasm. When the egg forms a monaster this becomes connected with these other asters by longitudinal fibres so as to form a complex spindle. By properly choosing the period of immersion in hypertonic water it is possible to arrange that only one additional aster should be formed; this then joins with the monaster to form a normal mitotic spindle on to which the egg chromosomes migrate; a regular division of the nucleus follows and thereafter a division of the whole egg into two cells and so parthenogenetic development is initiated.
The course of events in the frog's egg is fundamentally similar to the process which we have just described, although there are differences in detail. A prick with a sterilized needle induces the formation of a huge monaster, which then divides into two forming a short mitotic spindle on to which the chromosomes of the egg migrate. Since, however, the length of the spindle stands in relation to the number of chromosomes in the nucleus and as these chromo- somes are only present in half the number found in the nucleus of the fertilized egg, the spindle which is formed is only four-fifths of the length of the first spindle formed in the fertilized egg. The length of spindle in turn determines the length of the actual rays from its poles, and if these are too short to reach the periphery of the egg the spindle is unable to bring about the division of the egg into two cells. This is the case with the spindle formed in the parthenogenetic egg, and although abortive and transitory furrows on the egg's surface are formed no division into cells results; the nucleus, it is true, divides and a multiplication of nuclei fojlows in which the numerous short spindles formed interfere with one another and make orderly development impossible and so after a short time the egg dies.
If , however, the needle be " infected " by being dipped into frog's blood before being used to prick the egg, then the foreign substance thus introduced produces additional asters in the cytoplasm just as did the hypertonic water in the sea-urchin's egg. These asters have a tendency, as their rays develop, to repel one another, and they push the mitotic spindle developed around the egg nucleus over to the one side. // this side happens to be the side of the egg at which the cytoplasm is concentrated, then the spindle is able to start the formation of a furrow which cuts right through the egg and divides it into two cells, and so parthenogenetic development is begun. We see then that the difficulty of initiating parthenogenesis de- pends on two factors, viz. (i) the quiescent condition of the egg and (2) the small amount of chromatin present in the nucleus. If we choose the unripe eggs of the sea-urchin as the subjects of our experiment then it is sometimes possible to induce them to develop by the use of one reagent alone, such as hypertonic sea-water; since in these eggs the " reducing " division of the nucleus has not occurred (see CYTOLOGY) and the chromatin is consequently present in un- diminished quantity.
Parthenogenetic development is closely related to the problem of heterogeneous fertilization. It has been shown that under certain circumstances it is possible to fertilize the eggs of the sea- urchin with the sperm of creatures so diverse in zoological affinity as the annelid worm (Chaetopterus) and the sea-mussel (Mytilus). In the first case the male and female pronuclei fuse but the male chromatin falls out of the zygote nucleus before the first division takes place. In the second case the male pronucleus refuses to enter into union with the female pronucleus at all, but the sperm- aster brings about the division of the egg. When the eggs of the sea-urchin (Echinus) are fertilized with the sperm of the
1 M. Herlant, " Ld Mecanisme de la Parthenogenese Experi- mentale." Bull. Scientifique de la France el de la Belgique. 7th Series, vol. 1., 1917.
2 Brachet, " L'CEuf et les facteurs de 1'Ontogenese." Encyclo- pedie Scientifique, Paris, 1916.
heart-urchin (Echinocardium) , in the vast majority of cases cytolysis results exactly as it does after the exposure of the echinus eggs to the action of butyric acid, but in some few cases the egg develops and produces a hybrid. We conclude that in most cases the sperm of Echinocardium is so alien to the cytoplasm of the egg of Echinus that it is not even able to bring about the formation of a spermaster.
Under certain circumstances (slight staleness of the egg, excess of sperm, etc.) more than one spermatozoon may enter the egg. In large eggs such as those of cephalopoda, reptiles and birds this seems to be a normal occurrence ; only one of these nuclei unites with the female pronucleus and forms the zygote nucleus from which begins the cell-division which initiates development ; but the other spermatozoa also form centres for cell-division which gives rise to the so-called free cells which are characteristic of these eggs. These free cells are gradually crushed out and destroyed by the developing cells produced by the activity of the zygote nucleus.
Brachet, however, has shown 3 that when the frog's egg is entered by spermatozoa in moderate numbers, whereas only one fuses with the female pronucleus, the others form centres for the formation of cells which are built up into the body of the embryo. As these sperm-heads, however, contain only half the quantity of chromatin contained in the zygote nucleus, the cells to which they give rise are markedly smaller than those which contain nuclei descended from the zygote nucleus, and so it is possible to distinguish in the growing tadpole the regions which contain cells which have nuclei derived from the zygote nucleus from those which contain cells having nuclei derived from the supernumerary spermatozoa.
Brachet's observations prove in the clearest manner that the differentiation of organs in the frog's egg is due to the differentiation of regions in the cytoplasm and not to the differentiation of the nuclei produced by the division of the zygote nucleus as Weismann* had supposed, for some of these nuclei can be replaced by sperm- nuclei each of which carries in it the potentiality of producing the whole organism not a mere region of it and yet no dislocation of development results.
The entry of two or more spermatozoa into small eggs such as those of the sea-urchin usually produces abnormal development followed by early death. The reason is that the centrosomes which are carried into the egg by these spermatozoa are so near each other that instead of leading to the formation of separate spindles they give rise to three- (triaster) or four-poled (tetraster) spindles along which the chromosomes are arranged in an irregular manner. This causes the formation of abnormal nuclei incapable of properly fulfilling their functions and the embryo dies.
Development of the Egg. If we now turn to consider the normal development of the egg we find that this can be divided into three stages which in primitive forms are sharply delimited, but which in more modified forms tend to overlap one another. These stages are (i) segmentation, or the division of the egg into a number of indifferent cells or blastomeres; (2) the formation of the so-called germ layers, i.e. the differentiation of the blasto- meres into the primitive organs viz: (a) the ectoderm (or epiblast) which is the primitive skin, (b) the endoderm (or hypoblast) which is the primitive lining of the gut, and (c) the mesoderm (or mesoblast) which is the primitive peritoneum or lining of the body-cavity; (3) organogeny, i.e. the formation of the separate organs of the body, such as brain, liver, kidneys, etc., from the germ-layers.
Segmentation of the Egg. -Considering first the process of seg- mentation, we find, as Balfour 6 pointed out long ago, that the effect of the accumulation of yolk in the egg is to impede cell-division. It acts exactly as if it were a dilutant of the cytoplasm in lowering sur- face tension. Cell-division is accompanied by a great increase in surface tension as is obvious from the way each daughter cell rounds itself off from its sister. This is particularly evident in the segmentation of alecithal eggs, for in them, in the early stages of segmentation, all the blastomeres divide simultaneously, and just after each period of division these take on the appearance of a pile of balls only touching each other in points; whereas during the interval between two such periods the surface tension diminishes and the blastomeres become flattened out against each other.
In all alecithal and telolecithal eggs there is a pole (see above) from which the polar bodies are given off which is termed the animal pole of the egg. This pole is the region of the egg which contains least yolk; here cell-division is most rapid and the smallest blasto- meres are produced, whereas as we pass towards the vegetative pole of the egg, where the yolk is concentrated, the blastomeres be- come fewer and larger.
3 Brachet, loc. cit.
4 A. Weismann, The Germ-Plasm. A Theory of Heredity (1893).
6 F. M. Balfour, Treatise on Comparative Embryology, vol i., p. 95.
