AUTHOR=Liao Jiaqian , Zhang Ziping , Jia Xiwei , Zou Zhihua , Liang Keying , Wang Yilei 

TITLE=Transcriptional Regulation of Vih by Oct4 and Sox9 in Scylla paramamosain

JOURNAL=Frontiers in Endocrinology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2020.00650

DOI=10.3389/fendo.2020.00650

ISSN=1664-2392

ABSTRACT=<p>Mud crab (<italic>Scylla paramamosain</italic>) 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 <italic>Vih</italic> transcription in crabs are relatively rare. Our previous studies on the transcriptional regulation of mud crab <italic>Vih</italic> (<italic>SpVih</italic>) have proved that the binding site of Oct4/Sox9 transcription factor may be the key region for positively regulating the expression of <italic>SpVih</italic>. 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 <italic>SpVih</italic> 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 <italic>SpVih</italic>. <italic>In vitro, Oct4</italic> and <italic>Sox9</italic> gene overexpression vectors and <italic>SpVih</italic> core promoter fragment vector were constructed and co-transfected into HEK293T cells. As a result, <italic>SpVih</italic> activity increased with the concentration of transcription factors. <italic>In vivo</italic>, when <italic>Oct4</italic> and <italic>Sox9</italic> dsRNA were injected into the eyestalks of mud crab, respectively, the expression level of <italic>SpVih</italic> decreased significantly after interference with <italic>Oct4</italic> or <italic>Sox9</italic>, and the expression level of <italic>SpVtg</italic> in the ovary and hepatopancreatic increased. Both <italic>in vitro</italic> and <italic>in vivo</italic> experiments showed that Oct4 and Sox9 had a positive regulatory effect on <italic>SpVih</italic>. 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 <italic>SpVih</italic> through the interaction.</p>