Submicroscopical observations on the differentiation of chick gonads

By ROBERTO 1 0 Authors' address: Instituto de Anatomia General y Embriologfa, Facultad de Medicina , Paraguay 2155, Buenos Aires , Argentina 1 From the Faculty of Medicine , Buenos Aires The role of hormones in gonadal differentiation has not been fully elucidated. One of the main problems consists in determining the exact moment in which steroid synthesis begins. If, as has been claimed, sex hormones act as organizers and are responsible for the morphological changes which characterize gonadal differentiation, then they should appear before these changes take place. Although the morphological differentiation of chick gonads is evident only after the eighth day of incubation small differences in epithelium height permit sex identification on the seventh day. Biological (Wolff, 1946), biochemical (Gallien & Le Foulgoc, 1961) and histochemical (Scheib, 1959; Narbaitz & Sabatini, 1963; Narbaitz & Kolodny, 1964; Chieffi, Manelli, Botte & Mastrolia, 1964) evidence suggests that estrogen synthesis takes place in embryonic ovaries after the eighth day. On the other hand, steroid production by embryonic testes has not been proven. Wolff (1950) showed that a testicular secretion is responsible for the atrophy of Mullerian ducts on the ninth day; however histochemical techniques have given contradictory results. Thus, lipids are present after the eighth day but cholesterol does not appear until the tenth day (Narbaitz & Sabatini, 1963); Chieffi et al. (1964) claim that 3/?-hydroxysteroid dehydrogenase activity is present in 8-day testes, but Narbaitz & Kolodny (1964) obtained negative results with the same material. Regarding the undifferentiated gonads, the evidence for steroid synthesis is still scarce. Weniger (1961) claims that the feminizing action of embryonic ovaries described by Wolff (1946) is already present in 5-day gonads belonging to genetically female embryos, but no biochemical nor histochemical facts have yet supported this claim. - MATERIALS AND METHODS White Leghorn embryos were used in all cases. The left gonads from 6-, 7-, 8-, 9-, 11-, 13- and 16-day embryos were dissected. Macroscopic examination did not permit sex identification at 6 and 7 days; to ensure that gonads of both sexes were examined 25 embryos of these ages were used. At the other ages at least four gonads of each sex were studied. Immediately after dissection the gonads were fixed in a 2 % osmium tetroxide solution in Periston (Bayer) (Polyvinylpyrrolidone, 6 g ; NaCl, 0-55 g; KC1, 0-042 g; CaCl2, 0-05 g; MgCl2, 0-0005 g; dist. water, 100 ml) at pH 7-4 and 4C. After 2 h fixation they were transferred to 2 % uranyl acetate aqueous solution for 90 min, dehydrated by bringing through 50, 70, 80 and 96 % ethanol solutions for 15 min each, and allowed to remain in 100 % ethanol for 2 h with two changes. The gonads were then transferred to a solution consisting of equal volumes of propylene oxide and Epon 812 mixture, After 90 min the gonads were transferred to Epon 812 mixture. Embedding was performed in the usual way. Sections were made with a Porter Blum microtome and stained with lead citrate as described by Reynolds (1963). They were studied with a Siemens Elmiskop I. Electron microscopic observation with low magnifications permitted the identification of the diverse histological structures described in classical studies on chick gonads differentiation (see Hamilton, 1952). Therefore, no special scanning procedure was needed in order to identify the different parts of the gonads. Undifferentiated gonads. Plate 1, fig. A shows the aspect of a typical sex cord of a gonad of a 6-day embryo. The cells have an oval nucleus and their cytoplasm contains a large number of ribosomes and a fair number of mitochondria with well-developed cristae. In some of these cord cells characteristic vesicles are seen (a, Plate 1, fig. B). These are limited by agranular membranes and are irregularly distributed throughout the cytoplasm. Some of the vesicles contain a material of low electronic density (,'Plate 1, fig. B). The number of vesicles varies greatly from cell to cell. Some lipid droplets with wavy contour may be observed in some of the cells. In the sex cords, gonocytes can be identified by their large, rounded nucleus with finely divided chromatin. The cytoplasm is similar to that of the other cells Fig. A. Gonad of 6-day embryo: several cells in a sex cord are seen. Fig. B. Same gonad at higher magnification, a, Vesicle surrounded by agranular membrane; b, vesicle with contents of low electron density. Fig. C. Same gonad. Germ cell in a sex cord; several lipid droplets are seen (/). /. Embryol. exp. Morph., Vol. 16, Part 1 Fig. D. Nine-day testis. Testicular cord cells show many vesicles. Basement membrane is seen limiting the cord (b.m.). Fig. E. Nine-day testis. Interstitial cell showing many vesicles. of the cord but with a larger content of lipid droplets (Plate 1, fig. C). Among the sex cords there are mesenchymal cells with an elongated nucleus and very sparse cytoplasm. In the cortical zone of 6-day gonad two types of cells are recognized: gonocytes and epithelial cells. Their structure is similar to that described for gonocytes and sex cord cells in the medullary region. Mitoses are seen in all parts of the gonad. Seven-day gonads are very similar to those of 6-day embryos, but in some of them some cord cells show an intense proliferation of vesicles. Testes. Plate 2, fig. D shows the submicroscopical aspect of the cord in 9-day testis. There is a basement membrane separating the cord from the interstitium. A large number of vesicles are now seen in the cytoplasm of all the sex cords. In the 8-day testis vesiculization is found only in few cord cells but on the ninth day it is present in nearly all of them. After the ninth day many vesicles also appear in some cells of the interstitium (Plate 2, fig. E). Lipid droplets of wavy contour are seen in some of the cord and interstitial cells. At the eleventh and thirteenth days the testes are very similar to those at the ninth day. The only difference observed is in the width of the basement membrane, which increases with age. In the 16-day testis some of the cord and interstitial cells appear filled with lipid deposits (Plate 3, fig. F). These deposits appear to have an empty vacuole in the center, the lipid being restricted to the peripheral zone. Ovaries. In the 8-day ovary the medullary region shows a striking difference from that of the 7-day gonad. Sex cords have disappeared as such; parts of them have acquired a lumen and form lacunae, while other parts remain in closely packed cell groups. Mesenchymal cells appear intermingled between the lacunae and the groups of cord cells. The cytoplasm of the cord cells undergoes striking changes. On the eighth day vesicles increase in size and number in some of them (Plate 3, fig. G) while in others large lipid deposits appear. On the ninth day the lipid deposits show a central vacuole (Pl (...truncated)