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Mud crab ( Scylla paramamosain ) is one of the most economically-important marine crabs in China. However, research on mechanisms of reproductive regulation is not sufficient. Vitellogenesis-inhibiting hormone (VIH) is a member of the crustacean hyperglycemia hormones (CHH) family, which plays an essential role in the regulation of gonadal development and maturation in crustaceans, and current studies on the regulation of  Vih  transcription in crabs are relatively rare. Our previous studies on the transcriptional regulation of mud crab  Vih  ( SpVih ) have proved that the binding site of Oct4/Sox9 transcription factor may be the key region for positively regulating the expression of  SpVih . In this study, the electrophoretic mobility shift assay (EMSA) experiment confirmed that the nuclear protein extracted from the eyestalk could bind to the key region of  SpVih  promoter, and these specific bindings were dependent on the presence of Oct4/Sox9 binding sites. Two specific binding complex bands were detected in the supershift group of EMSA supershift experiments by Oct4 and Sox9 antibodies, further confirming the specific recognition of these two transcription factors on the key regulatory region of  SpVih .  In vitro, Oct4  and  Sox9  gene overexpression vectors and  SpVih  core promoter fragment vector were constructed and co-transfected into HEK293T cells. As a result,  SpVih  activity increased with the concentration of transcription factors.  In vivo , when  Oct4  and  Sox9  dsRNA were injected into the eyestalks of mud crab, respectively, the expression level of  SpVih  decreased significantly after interference with  Oct4  or  Sox9 , and the expression level of  SpVtg  in the ovary and hepatopancreatic increased. Both  in vitro  and  in vivo  experiments showed that Oct4 and Sox9 had a positive regulatory effect on  SpVih . The GST pull-down experiment was carried out by purified Oct4 and Sox9 proteins, and the results showed that there was an interaction between them. It was speculated that they regulated the expression of  SpVih  through the interaction.

Transcriptional Regulation of Vih by Oct4 and Sox9 in Scylla paramamosain