Testis

Cytoplasmic Versus Nuclear Localization of Fos-Related Proteins in the Frog, Rana esculenta, Testis: In Vivo and Direct In Vitro Effect of a Gonadotropin-Releasing Hormone Agonist

Abstract

Evidence has been accumulated indicating that GnRH-like peptides are present in a variety of extra-brain areas of mammalian and non-mammalian vertebrates. A pioneer study carried out in the frog, Rana esculenta, demonstrated that testicular GnRH induced spermatogonial proliferation. Recently, we have shown that in proliferating spermatogonia (SPG) of frogs a change of localization of the oncoprotein Fos, from the cytoplasm to the nucleus, occurs. This leads to the hypothesis that testicular GnRH peptide(s) may regulate SPG proliferation through Fos family proteins. Therefore, in vivo experiments in intact R. esculenta and in vitro incubations of testis fragments have been carried out using GnRH agonist (GnRHa, buserelin) and GnRH antagonist (D-pGlu¹,D-Phe²,D-Trp^3,6- GnRH). Cytoplasmic and nuclear Fos-like protein localization has been found by Western blot in testicular extracts. Immunocytochemistry confirmed that cytoplasmic immunostaining was restricted to SPG; change of localization into the nuclear compartment was observed after GnRHa treatment. Northern blot analysis showed that treatments of testis fragments with GnRHa did not modify testicular c-fos mRNA expression. On the contrary, a Fos-like protein of 52 kDa, while it is not affected in vivo, disappeared from testicular cytosolic extracts after in vitro treatment with GnRHa. Contemporaneously, a 55 kDa Fos-related signal appeared in nuclear extracts. GnRH antagonist counteracted GnRHa effects. Furthermore, in vivo treatments showed that GnRHa acted negatively on a 43-kDa nuclear Fos-related signal and gonadotropins caused the decrease of 52-kDa cytoplasmic signal. In conclusion, we show, for the first time, that Fos is regulated by GnRHa directly (not through the pituitary) at the testicular level. The main effect appears to be related to Fos translocation from cytoplasmic to nuclear compartments of SPG.