Insulin-like receptor signaling pathways regulate spermatogenesis in the mud crab Scylla paramamosain

The insulin-like androgenic gland hormone (IAG) plays a crucial role in male sexual differentiation. Although the putative IAG receptor has been identified in several crustacean species, IAG receptor in brachyuran crabs has yet to be determined. This study identified a new insulin-like receptor ( Sp-IR2 ) in the mud crab Scylla paramamosain , and explored its possible role in spermatogenesis. RT-PCR results suggested that Sp-IR2 was highly expressed in the androgenic gland (AG) and testes. During testicular development, the levels of Sp-IR2 transcript in the AG and testes gradually increased. In addition, bovine insulin significantly up-regulated the expression of Sp-IR2 , Sp-vasa and Sp-sox9 in the testes in vitro . Injection of bovine insulin induced Sp-vasa and Sp-dsx expression in the testes, and increased hemolymph testosterone titer. In vitro silencing of Sp-IR2 significantly increased the expression levels of Sp-IR1 and Sp-vasa transcripts in the testes. To further investigate the function of Sp- IR2 in testicular development, we performed short- and long-term RNA interference (RNAi) on male crabs. Short-term knockdown of Sp-IR2 led to the significant up-regulation of Sp-IR1 in the testes and AG, and Sp-vasa in AG. Prolonged silence of Sp-IR2 markedly reduced the expression of Sp-sox9 in testes, and arrested spermatogenesis at the secondary spermatocyte stage, suggesting Sp- IR2 plays an important role in transitioning secondary spermatocytes to spermatids. In combination with our previous study, our results indicated that insulin-like receptor signaling pathways are important for spermatogenesis in S. paramamosain , in which Sp- IR1 is involved in transitioning of primary spermatocytes to secondary spermatocytes while Sp- IR2 is essential for the transition of secondary spermatocytes to spermatids. • A new insulin-like receptor that is highly expressed in the testes and AG was isolated from the mud crab ​ S. paramamosain. • Testosterone may involve in the IR signaling pathways-regulated testicular development and spermatogenesis of ​ S. paramamosain. • A cross talking may exist between ​ Sp- IR1 and ​ Sp- IR2 signaling to regulate spermatogenesis in ​ S. paramamosain . • Sp-IR2 ​plays an important role in regulating the transition of secondary spermatocytes to spermatids during spermatogenesis.