Expression pattern and potential role of Nanos3 in regulating testosterone biosynthesis in Leydig cells of sheep

Nanos3 is from a highly conserved family of genes with putative RNA-binding activity and plays an important role in germ cell development. Yet, its function in Leydig cells (LCs) for supporting germ cell development, is still poorly understood. In this study, we investigated the expression pattern and function of Nanos3 in the sheep testis. The Nanos3 mRNA and protein were highly expressed in the ram testis. The sheep Nanos3 amino acid sequence was 93.6%-88.6% homologous with those of other mammalian species. The expression of Nanos3 in the ram testis was significantly lower in 9 and 24-month-old sheep than in at 3-month-old sheep (P < 0.05). Immunohistochemistry analysis revealed that sheep Nanos3 was not only localized at spermatogenesis cell but also at LCs. Thus the functions of Nanos3 in LCs were further investigated by using siRNA-Nanos3 in vitro. The secretion of T hormone and expression of testosterone synthesis-related enzymes in LCs were significantly inhibited (P < 0.05) by siRNA-Nanos3. Moreover, the BAX/Bcl-2 mRNA and protein levels were significantly increased (P < 0.05) by siRNA-Nanos3. In addition, we examined the expression levels of Nanos3 mRNA and protein in energy-restricted(ER) rams and in vitro ER LCs. Their levels were also significantly decreased (P < 0.05) and were accompanied by an increased BAX/Bcl-2 mRNA expression compared with the control group (P < 0.05). To the best of our knowledge, our study is the first to show that Nanos3 is an important regulator of testosterone biosynthesis in rams for supporting germ cell development.