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Franca Raucci and Maria Maddalena Di Fiore

The green frog Rana esculenta is 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-kit receptor positive cells in the testis of this amphibian. The presence of c-kit receptor 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 for c-kit receptor, 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-kit expression showed a seasonal pattern connected with both testicular and plasma profiles of testosterone during the reproductive cycle. The highest expression of c-kit receptor 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 and c-kit receptor 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 the c-kit receptor may play a role in germ cell proliferation and provide a basis for future detailed investigation of regulatory factors of the proliferation of SPG.

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Alessandra Santillo, Massimo Venditti, Sergio Minucci, Gabriella Chieffi Baccari, Sara Falvo, Luigi Rosati and Maria Maddalena Di Fiore

D-Aspartate (D-Asp) is an endogenous amino acid that plays a central role in the development of the central nervous system (CNS) and functioning of the neuroendocrine system. In line with its functions, it is abundantly present in the CNS and reproductive systems of vertebrates and invertebrates. It has been implicated in the biosynthesis and/or secretion of hormones and factors that are involved in various reproductive functions, such as GnRH from the hypothalamus and testosterone from the testis. We conducted an in vivo study consisting of acute (i.p. injection of 2 µmol/g body weight) and chronic (15 days drinking solution) administration of D-Asp to adult rats to understand the signaling pathways elicited by D-Asp in the rat testis. We found that D-Asp upregulated the expression of prolyl endopeptidase (PREP), a serine protease having a pivotal role in the regulation of mammalian spermatogenesis and spermiogenesis. Immunofluorescence analysis revealed its overexpression in Leydig cells, Sertoli cells and spermatogonia. Moreover, PREP was found to co-localize with GluA2/3, an AMPA receptor subunit, whose protein expression also increased after D-Asp treatments. Finally, we found a significant increase in ERK and Akt activities in the testis of rats treated with D-Asp. Since PREP is known to be involved in regulating GnRH levels and in germ cell differentiation, we hypothesize D-Asp to play a pivotal role in regulating hormone homeostasis and spermatogenesis through activation of PREP, AMPAR, ERK and Akt.