Identification and ovarian developmental regulation of ribosomal protein S6 kinase in Macrobrachium nipponense

Ribosomal protein S6 kinase (RPS6K) is a member of the Ser/Thr kinase family. To elucidate the role of RPS6K from Macrobrachium nipponense (MnRPS6K) in ovarian development, we cloned the full-length cDNA sequence of MnRPS6K gene using rapid amplification of cDNA ends (RACE)-PCR and analyzed its expression pattern using quantitative real-time PCR (qRT-PCR). Immunohistochemistry (IHC) was used to determine the subcellular localization of the MnRPS6K protein in ovarian tissues. Furthermore, the function of MnRPS6K in ovarian development was investigated via recombinant protein and neurotransmitter injection, as well as RNA interference (RNAi) experiments. The results indicated that the MnRPS6K gene's full-length cDNA sequence measures 2993 bp and encodes 932 amino acids. The MnRPS6K protein possesses two conserved ser/thr protein kinase catalytic (S_TKc) domains. MnRPS6K expression was highest in adult females among six developmental stages, and was most abundantly expressed in the ovary among nine tissues. During ovarian development, MnRPS6K expression peaked at stage V. IHC revealed that the MnRPS6K protein is predominantly localized in the cytoplasm of oogonia, oocytes, and follicle cells. Administration of recombinant MnRPS6K (rMnRPS6K) protein and 5-hydroxytryptamine (5-HT) upregulated the expression of MnRPS6K, and several key ovarian development-related genes, such as vitellogenin (MnVtg), vitellogenin receptor (MnVtgr), cyclin B (MnCyclin B), and cell division cycle protein 2 (MnCdc2). However, dopamine (DA) and MnRPS6K-siRNA injections downregulated these genes. Moreover, MnRPS6K silencing induced follicular cell apoptosis and delayed oocyte and ovarian development. These findings demonstrate that MnRPS6K can promote ovarian development by regulating oogenesis, vitellogenesis, and oocyte maturation in M. nipponense.