In the Helly fixed tissue, stained with Giemsa, the granules of the blood granulocytes stain either blue or red, depending upon the phase of development of the cells. The majority of the cells at this stage contain red-staining granules; a few contain only unripe blue staining granules; and a certain number have mixed granules, including all phases of the ripening process of an eosinophil granule. The nucleus also stains a deep blue. The primordial germ-cell of this tissue, on the contrary, has a light bluish-pink-staining nucleus, with a deep purple nucleolus. The cytoplasm of the germ-cell is vacuolated, the vacuoles being the negative of the dissolved yolk-globules; and it has a coarsely reticular character in consequence, and stains only faintly pink.
Already, from a study of only this stage of development, much information can be gained concerning the fact, route, and method of the migration of the germ-cells from the gut entoderm to the peritoneal epithelium of the gonad. It may be stated here that at no stages studied was any primordial germ-cell, with two exceptions, ever seen in a blood-vessel. The blood-channels in this form evidently do not greatly assist in the transfer of the germ-cells to the sex-gland, contrary to what Swift has demonstrated for the chick; nor has such a route been advocated for any other form except duck (von Berenberg Gossler) ; but that the cells do migrate is indicated by the different locations occupied, progressively nearer the genital ridge, corresponding with successively later developmental stages, and that the migration is the result of an inherent amceboid capacity is shown by the various shapes assumed by these cells (figs. 3 aa, 3 ab, plate 1); and the path of migration is quite clearly indicated by the pointed condition of the forward end, combined with the altered (flattened and distorted) condition of the cells lying directly in the path of progression. For instance, the primordial germ-cells among the entodermal cells of the closed hind-gut in the 11-day embryo are surrounded by entodermal cells which have become crowded, flattened, and distorted on either side as if through pressure by a cell which enlarged and assumed a spherical shape in situ. The similarity in structure and staining reac tion between the germ-cells and the entodermal cells, a similarity which becomes progressively closer as earlier stages are approached, seems to speak in favor of an entodermal origin of the germ-cells or a close genetic relationship between entoderm and germ-cells. When
the germ-cell lies near the basement membrane of the primitive gut entoderm, the cellular distortion is such as would be caused by a cell migrating from the entoderm into the surrounding mesenchyma. Occasionally a germ-cell can be seen partially within the entoderm and partially within the mesenchyma, an observation which proves a migratory process; and the manner of the distortion of the mesen chymal cells shows that this migration is in a peripheral direction.
Within the mesenchyma of the gut and the mesentery the distortion and flattening of the cells are generally in the direction of the root of the mesentery, thus showing a migratory progression in that direction. Occasional cells, judged by this criterion, are moving towards the mesothelial layer of the mesentery (figs. 3 aa, 3 ab, plate 1). Occasional cells are also found among the cells of the peritoneal epithelium of the genital ridge. In the region of the angle between the root of the mesentery and the gonad, and again within the mesenchyma of the sexual gland, the shape of cell and the distortion of the adjacent mesen chymal nuclei are such as to indicate a ventro-medial progression to the surface of the gonad, where the germ-cells come to rest among the peritoneal cells (fig. 2, plate 1). Figure 3ac, plate 1, shows a germ-cell similarly located, but almost completely filled with a huge yolk-mass.
The germ-cells are apparently most abundant at about the 11-day stage. This increase is probably merely apparent, due to the segrega tion of the cells within relatively more restricted and conspicuous limits, viz, gut and mesentery. During earlier stages (to the seventh day), when the gut is still entirely open over the yolk, their distribution is scattered over a wider area; also in later stages, when they have taken their definite position within the enlarged gonad. The number of germ-cells in the 11-day embryo, all situated behind the level of the cephalic tip of the pronephric duct, is approximately 400. Only an occasional cell may be seen at this stage in mitosis. Similarly, during the later stages mitosis is a very rare occurrence. In the stages of 2 to 4 days a few of the germ-cells are in process of division among the entodermal cells. Cells in division are apparently of smaller size and contain relatively little yolk.