Spermatogonial cell proliferation in organ culture of immature rat testis.

Regulatory mechanisms of male germ cell proliferation in mammals were investigated by using in vitro organ culture of immature rat testis. Nutritional and hormonal requirements for maintenance and differentiation of germ cells in vitro were first characterized by testing different culture conditions. FSH was essential for the progression of type A spermatogonia up to the stage of pachytene spermatocytes after 3 wk of in vitro culture, while vitamins A, C, and E, LH, and testosterone were not effective. The proliferative activity of Sertoli cells markedly declined after 1 wk of in vitro culture, irrespectively of the presence of FSH in the medium. In addition, basal testosterone production by Leydig cells was maintained after 1 wk of culture, provided that FSH was present in the medium. The appearance of differentiating type I and type B spermatogonia and meiotic cells in the seminiferous cords throughout culture was accompanied by a significant reduction in the number of undifferentiated spermatogonia. Moreover, a similar labeling index of undifferentiated spermatogonia was observed in both unstimulated and FSH-stimulated testis fragments at all culture times considered. Therefore, FSH did not influence the mitotic activity of undifferentiated spermatogonia, suggesting a differential role of this gonadotropin during the mitotic phase of spermatogenesis. These results indicate that the organ culture system of immature rat testis represents a useful experimental model for studying regulatory mechanisms of spermatogonial cell proliferation.