Etablierung eines in vitro-Systems zur Analyse der Differenzierung und Apoptose immaturer testikulärer Keimzellen

We established a culture system for immature rat germ cells, which allows the analysis of differentiation and apoptosis of germ cells. For this purpose, we used testicular tissue from 20 day-old Wistar rats. By mechanical dissociation we obtained testicular cells and tissue fragments of well defined size. We examined the differentiation of germ cells in different culture systems: the culture of whole testes, fragments of tubuli seminiferi and of individual cells. In fragments of tubuli seminiferi pachytene spermatocytes differentiated into round spermatids. This development took place within a period of two weeks under the influence of testosterone and follicle stimulating hormone. The stage of differentiation was determined by the expression of stage specific markers, e.g. transition protein 1, transition protein 2 and protamine 1. We analysed the apoptosis of germ cells after stimulation with transforming growth factors-beta (beta-1 and beta-2) and used activation of caspase-3 as a measure of apoptosis. There were indications that TGF-betas induce germ cell apoptosis, but we could not definitely determine this.