Transcriptional Inhibition of Sp-IAG by Crustacean Female Sex Hormone in the Mud Crab, Scylla paramamosain
Qingling Jiang, Bei Lu, Guizhong Wang, Haihui Ye
Abstract
In crustaceans, the regulation of sex differentiation is mediated by insulin-like androgenic hormone (IAG) and crustacean female sex hormone (CFSH). CFSH is reported to inhibit IAG gene (Sp-IAG) expression in the mud crab Scylla paramamosain, but the regulatory mechanism is not well understood. A 2674 bp 5′ flanking Sp-IAG contains many potential transcription factor binding sites. In this study, analysis of serially deleted 5′ flanking Sp-IAG and site-directed mutation (SDM) of transcription factor binding sites of the same gene showed that the promoter activity of reporter vectors with Sox-5-binding site, signal transducers and activators of transcription (STAT)-binding site and activator protein 1 (AP-1)-binding site were significantly higher than that of vectors without these regions, suggesting that they were involved in transcriptional regulation of Sp-IAG expression. The expression analysis of these transcription factor showed that there was no difference in the level of mRNA in Sox-5 and AP-1 in androgenic gland treated with recombinant CFSH, but expression of Sp-STAT was significantly reduced, suggesting that CFSH regulates the expression of Sp-STAT, inhibiting its function to regulate Sp-IAG. Further experiment revealed that RNAi mediated Sp-STAT gene knockdown reduced the expression of Sp-IAG. These results suggested that Sp-CFSH regulates Sp-IAG by inhibiting STAT. This is a pioneering finding on the transcriptional mechanism of IAG gene in crustaceans.