Putative Role of CFSH in the Eyestalk-AG-Testicular Endocrine Axis of the Swimming Crab Portunus trituberculatus

SIMPLE SUMMARY: The crustacean female sex hormone (CFSH) plays a crucial role in the development of secondary sexual characteristics in female Decapoda crustaceans, but its physiological function in males is less reported. In the present study, the CFSH of P. trituberculatus (PtCFSH) was identified, cloned and expressed in the recombinant form. The physiological functions of PtCFSH on the AG and testis were investigated using the RNAi and recombinant protein injection in a series of in vivo and in vitro experiments. It was confirmed that PtCFSH can negatively regulate the spermatogenesis and testicular development via a CFSH-IAG-testis endocrine axis. PtCFSH may also act directly on the testis, dependent or independent of IAG. The signaling system of PtCFSH involves the participation of cAMP, cGMP, and NO, which resembles the MIH signaling pathway. In summary, our results demonstrated the mode of CFSH action in P. trituberculatus. ABSTRACT: It has been shown in recent studies that the crustacean female sex hormone (CFSH) plays a crucial role in the development of secondary sexual characteristics in Decapoda crustaceans. However, research on the function of CFSH in the eyestalk-AG-testicular endocrine axis has been inadequate. We cloned and identified a homolog of CFSH, PtCFSH, in this study. RT-PCR showed that PtCFSH was mainly expressed in the eyestalk. A long-term injection of dsPtCFSH and recombinant PtCFSH (rPtCFSH) in vivo showed opposite effects on spermatogenesis-related gene expression and histological features in the testis of P. trituberculatus, and was accompanied by changes in AG morphological characteristics and PtIAG expression. In addition, the phosphorylated-MAPK levels and the expression of several IIS pathway genes in the testis was changed accordingly in two treatments, suggesting that PtCFSH may regulate the testicular development via IAG. The hypothesis was further validated by a mixed injection of both dsPtCFSH and dsPtIAG in vivo. The following in vitro studies confirmed the negatively effects of PtCFSH on AG, and revealed that the PtCFSH can also act directly on the testis. Treatment with rPtCFSH reduced the cAMP and cGMP levels as well as the nitric oxide synthetase activity. These findings provide vital clues to the mechanisms of CFSH action in both the eyestalk-AG-testis endocrinal axis and its direct effects on the testis.