Potential Involvement of ewsr1-w Gene in Ovarian Development of Chinese Tongue Sole, Cynoglossus semilaevis

SIMPLE SUMMARY: Sexual dimorphism is a phenomenon commonly existing in animals. Chinese tongue sole Cynoglossus semilaevis is an economical marine fish with obvious female-biased size dimorphism. So, it is important to explore the molecular mechanism beyond gonadal development for sex control in aquaculture industry. RNA-binding protein Ewing Sarcoma protein-like (ewsr1) gene is important for mouse gonadal development and reproduction, however there are limited studies on this gene in teleost. In this study, two ewsr1 genes were cloned and characterized from C. semilaevis. The ewsr1-w gene, located in W chromosomes, showed female-biased expression during C. semilaevis gonadal development. In addition, knock-down effect and transcriptional regulation of Cs-ewsr1-w further suggested its essential role in ovarian development. This study broadened our understanding on ewsr1 function in teleost, and provided genetic resources for the further development of sex control breeding techniques in C. semilaevis aquaculture. ABSTRACT: Ewsr1 encodes a protein that acts as a multifunctional molecule in a variety of cellular processes. The full-length of Cs-ewsr1-w and Cs-ewsr1-z were cloned in Chinese tongue sole (Cynoglossus semilaevis). The open reading frame (ORF) of Cs-ewsr1-w was 1,767 bp that encoded 589 amino acids, while Cs-ewsr1-z was 1,794 bp that encoded 598 amino acids. Real-time PCR assays showed that Cs-ewsr1-w exhibited significant female-biased expression and could be hardly detected in male. It has the most abundant expression in ovaries among eight healthy tissues. Its expression in ovary increased gradually from 90 d to 3 y with C. semilaevis ovarian development and reached the peak at 3 y. After Cs-ewsr1-w knockdown with siRNA interference, several genes related to gonadal development including foxl2, sox9b and pou5f1 were down-regulated in ovarian cell line, suggesting the possible participation of Cs-ewsr1-w in C. semilaevis ovarian development. The dual-luciferase reporter assay revealed that the -733/-154 bp Cs-ewsr1-w promoter fragment exhibited strong transcription activity human embryonic kidney (HEK) 293T cell line. The mutation of a MAF BZIP Transcription Factor K (Mafk) binding site located in this fragment suggested that transcription factor Mafk might play an important role in Cs-ewsr1-w basal transcription. Our results will provide clues on the gene expression level, transcriptional regulation and knock-down effect of ewsr1 gene during ovarian development in teleost.