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 Agonist1

Abstract

Evidence has been accumulated indicating that GnRH-like peptides are present in a variety of extrabrain areas of mammalian and nonmammalian 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 one or more testicular GnRH peptides 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-pGlu(1),D-Phe(2),D-Trp(3,6)-GnRH). Cytoplasmic and nuclear Fos-like protein localization has been found by Western blot analysis 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 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. The GnRH antagonist counteracted the effects of GnRHa. Furthermore, in vivo treatments showed that GnRHa acted negatively on a 43-kDa nuclear Fos-related signal and that gonadotropins caused the decrease of 52-kDa cytoplasmic signal. In conclusion, we show, to our knowledge 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.

Increased Activity of Activator Protein-1 Transcription Factor Components ATF2, c-Jun, and c-Fos in Human and Mouse Autosomal Dominant Polycystic Kidney Disease

“…However, Ser73-phosphorylated c-Jun was also detected in the cytoplasm of collecting duct epithelial cells. Cytoplasmic localization of c-Jun has been demonstrated previously (22)(23)(24)(25). Expression of Thr69/71-phosphorylated ATF2 and c-Fos was not detected by immunohistochemistry in normal renal tissue.…”

Endocannabinoids and Kisspeptins: Two Modulators in Fight for the Regulation of GnRH Activity

“…During the frog annual sexual cycle, FOS protein appeared inside the cytoplasm of spermatogonia before the proliferative period, whereas it appeared inside the nucleus as soon as spermatogonia proliferation resumes . Estradiol, produced by Leydig cells, induces the transcription of c-fos inside the spermatogonia and the protein is stored in cytoplasmic compartment ; then, at the end of the winter stasis, GnRH, produced by Sertoli cells, induces FOS activity by means of FOS protein traslocation from cytoplasmic to nuclear compartment (Cobellis et a., 2003) with a consequent increase of spermatogonial mitotic index. The involvement of c-fos as well as of estradiol in spermatogonial proliferation has recently been confirmed also in cell lines (He et al, 2008;Sirianni et al, 2008).…”

“…Interestingly, in zebrafish male, high levels of both ligand and receptor expression have been observed during the first stages of spermatogenesis, when testis is mainly populated by type A spermatogonia to decrease after puberty (Biran et al, 2008;Filby et al, 2008). Kisspeptin expression is modulated by estradiol (Clarkson & Herbison, 2009) and estradiol cooperates with GnRH in order to induce proliferation of spermatogonia (Cobellis et al, 2003), thus raising the possibility of kisspeptin involvement in such a process. The presence of CB2 protein in mouse differentiating spermatogonia (Grimaldi et al, 2009) makes such an item an interesting issue for future investigations.…”

Fra1 Activity in the Frog, Rana esculenta, Testis: A New Potential Role in Sperm Transport1

“…In Rana esculenta testis, the phosphorylation of c-Fos is involved in spermatogonial proliferation through the control of its cytoplasmic storage [14], nuclear translocation [15], and functional activation [14,16]. Similarly, Fra1 undergoes posttranslational modifications that cause a significant shift in its gel mobility from 36 to 46 kDa or more [12].…”

“…In serum-stimulated fibroblasts, Fra1 undergoes extensive phosphorylation [7,12,17] and shows both nuclear and cytoplasmic localization [17]. Cytoplasmic and nuclear localizations of Fos family proteins, described in vitro [17,18], have also been reported recently in in vivo models of amphibians [14][15][16][19][20][21], reptiles [22], and mammals [23][24][25]. In particular, Fra1 has been localized in cytoplasmic and nuclear compartments of ovarian and testicular cells of pigs [25] and foxes [23], respectively.…”