Of the earlier observations on this subject there need perhaps only be cited one of E. van Beneden, on the rabbit’s ovum, shewing the presence of two nuclei before the commencement of segmentation. Bütschli was the earliest to state from observations on Rhabditis dolichura that the first segmentation nucleus arose from the fusion of two nuclei, and this was subsequently shewn with greater detail for Ascaris nigrovenosa, by Auerbach ([76]). Neither of these authors gave at the first the correct interpretation of their results. At a later period Bütschli ([80]) arrived at the conclusion that in a large number of instances (Lymnæus, Nephelis, Cucullanus, &c.), the nucleus in question was formed by the fusion of two or more nuclei, and Strasburger at first made a similar statement for Phallusia, though he has since withdrawn it. Though Bütschli’s statements depend, as it seems, upon a false interpretation of appearances, he nevertheless arrived at a correct view with reference to what occurs in impregnation. Van Beneden [(78)] described in the rabbit the formation of the original segmentation nucleus from two nuclei, one peripheral and the other central, and deduced from his observations that the peripheral nucleus was derived from the spermatic element. It was reserved for Oscar Hertwig [(89)] to describe in Echinus lividus the entrance of a spermatozoon into the egg and the formation from it of the male pronucleus.
The general fact that impregnation consists in the fusion of the spermatozoon and ovum has now been established for some forms in the majority of invertebrate groups (Arthropoda and Rotifera excepted). Amongst Vertebrata also it has been shewn by E. van Beneden that the first segmentation nucleus is formed by the coalescence of the male and female pronucleus. Calberla, and Kupffer and Benecke have demonstrated that a single spermatozoon enters at first the ovum of Petromyzon.
The contact of the spermatozoon with the egg membrane causes in Petromyzon active movements of the protoplasm of the ovum, and a retreat of the protoplasm from the membrane.
In Amphibia the appearance of a peculiar pigmented streak extending inwards from the surface of the pigmented pole of the ovum, and containing in a clear space at its inner extremity a nucleus, has been demonstrated as the result of impregnation by Bambeke ([77]) and Hertwig ([90]). There can be little doubt that this nucleus is the male pronucleus, and that the pigmented streak indicates its path inwards. Close to it Hertwig has shewn that another nucleus is to be found, the female pronucleus, and that eventually the two join together. In Amphibia the phenomena accompanying impregnation are clearly of the same nature as in the Invertebrata. A precisely similar series of phenomena to those in Amphibia has been shewn by Salensky to take place in the Sturgeon.
Although there is a general agreement between the most recent observers, Hertwig, Fol, Selenka, Strasburger, &c., as to the main facts connected with the entrance of one spermatozoon into the egg, the formation of the male pronucleus, and its fusion with the female pronucleus, there still exist differences of detail in the different descriptions, which partly, no doubt, depend upon the difficulties of observation, but partly also upon the observations not having all been made upon the same species. Hertwig does not enter into details with reference to the actual entrance of the spermatozoon into the egg, but in his latest paper points out that considerable differences may be observed in the occurrences which succeed impregnation, according to the relative period at which this takes place. When, in Asterias, the impregnation is effected about an hour after the egg is laid, and previously to the formation of the polar cells, the male pronucleus appears at first to exert but little influence on the protoplasm, but after the formation of the second polar cell, the radial striæ around it become very marked, and the pronucleus rapidly grows in size. When it finally unites with the female pronucleus it is equal in size to the latter. In the case when the impregnation is deferred for four hours the male pronucleus never becomes so large as the female pronucleus. With reference to the effect of the time at which impregnation takes place, Asterias would seem to serve as a type. Thus in Hirudinea, Mollusca, and Nematoidea impregnation normally takes place before the formation of the polar bodies is completed, and the male pronucleus is accordingly as large as the female. In Echinus, on the other hand, where the polar bodies are formed in the ovary, the male pronucleus is always small.
Selenka, who has investigated the formation of the male pronucleus in Toxopneustes variegatus, differs in certain points from Fol. He finds that usually, though not always, a single spermatozoon enters the egg, and that though the entrance may be effected at any part of the surface it generally occurs at the point marked by a small prominence where the polar cells are formed. The spermatozoon first makes its way through the mucous envelope of the egg, within which it swims about, and then bores with its head into the polar prominence.
One important point has been so far only indirectly alluded to, viz. the number of spermatozoa required to effect impregnation.
The concurrent testimony of almost all observers tends to shew that one only is required for this purpose. But the number of cases tested is too small to admit of satisfactory generalization.
Both Hertwig and Fol have made observations on the result of the entrance into the egg of several spermatozoa. Fol finds that when the impregnation has been too long delayed the vitelline membrane is formed with comparative slowness, and several spermatozoa are thus enabled to penetrate. Each spermatozoon forms a separate pronucleus with a surrounding star; and several male pronuclei usually fuse with the female pronucleus. Each male pronucleus appears to exercise a repulsive influence on other male pronuclei, but to be attracted by the female pronucleus. When there are several male pronuclei the segmentation is irregular and the resulting larva a monstrosity. These statements of Fol and Hertwig are up to a certain point in contradiction with the more recent results of Selenka. In Toxopneustes variegatus Selenka finds that though impregnation is usually effected by a single spermatozoon yet several may be concerned in the act. The development continues, however, to be normal up to the gastrula stage, at any rate, if three or even four spermatozoa enter the egg almost simultaneously. Under such circumstances each spermatozoon forms a separate pronucleus and star. Selenka is of opinion (apparently rather on a priori grounds than as a result of direct observation) that normal development cannot occur when more than one male pronucleus fuses with the female pronucleus; and holds that, where he has observed such normal development after the entrance of more than one spermatozoon, the majority of male pronuclei become absorbed.
It may be noticed that, while the observations of Fol and Hertwig were admittedly made upon eggs in which the impregnation was delayed till they no longer displayed their pristine activity, Selenka’s were made upon quite fresh eggs; and it seems not impossible that the pathological symptoms in the embryos reared by the two former authors may have been due to the imperfection of the egg, and not to the entrance of more than one spermatozoon. This, of course, is merely a suggestion which requires to be tested by fresh observations.