The c-kit receptor protein in the testis of green frog Rana esculenta: seasonal changes in relationship to testosterone titres and spermatogonial proliferation

Abstract

The green frogRana esculentais a seasonal breeder. The cyclic changes between almost arrested and highly activated spermatogenesis offer an ideal model to study basic mechanisms of spermatogenesis. In this study, we demonstrated, to our knowledge for the first time,c-kitreceptor positive cells in the testis of this amphibian. The presence ofc-kitreceptor protein was confirmed by western blotting (Wb) analyses carried out in the testis during all the three main phases of the sexual cycle. The antibody recognized a band of about 150 kDa that was correlated with the positive staining in the germinal epithelium. The immunolabelling forc-kitreceptor, evaluated by immunohistochemistry (IHC), was localized in I and II spermatogonia (SPG), in I and II spermatocytes, in both elongating spermatids and spermatozoa and in the Leydig cells. Furthermore,c-kitexpression showed a seasonal pattern connected with both testicular and plasma profiles of testosterone during the reproductive cycle. The highest expression ofc-kitreceptor occurred during the reproductive period, when the testis exhibited the maximum concentration of testosterone. In this period, the mitotic activity of germ cell, assessed by both Wb and IHC analyses for proliferating cell nuclear antigen (PCNA), was intensive. Indeed, during the post-reproductive period, testosterone titres were the lowest and the expression of both PCNA andc-kitreceptor protein in the testis, although present, is minor when compared with the reproductive phase. This evidence suggests that cell division can continue sufficiently to accumulate SPG for the next spring, when new germinal cells undergo multiplication. Finally, during the pre-reproductive period, testosterone levels begin to increase and mitotic activity of germinal epithelium is comparably enhanced. These events seem to precede the period of maximum stimulated spermatogonial proliferation, i.e. the reproductive period. These results suggest that thec-kitreceptor may play a role in germ cell proliferation and provide a basis for future detailed investigation of regulatory factors of the proliferation of SPG.