Roles of Two Sox9 Genes during Gonadal Development in Japanese Flounder: Sex Differentiation, Spermatogenesis and Gonadal Function Maintenance

Abstract

The transcription factor <i>sox9</i> has been implicated in cartilage formation and testis determination in mammals. Here, two duplicates of <i>sox9</i> were found in Japanese flounder (<i>Paralichthys olivaceus</i>) named <i>Posox9a</i> and <i>Posox9b</i>, respectively. Phylogenetic and gene structure analyses revealed that <i>Posox9a</i> and <i>Posox9b</i> were homologous to that of teleosts and tetrapods. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that both <i>Posox9a</i> and <i>Posox9b</i> expressed higher in testis than in ovary of adult tissues. The in situ hybridization (ISH) analysis of gonads showed that <i>Posox9a</i> and <i>Posox9b</i> mRNA were both detected in oocytes, Sertoli cells and spermatocytes. During sex differentiation, the expression of <i>Posox9a</i> exhibited obvious sexual dimorphic expression from 60 days after hatch (dah) with higher expression in male preferred individuals than female preferred individuals and increased gradually from 30 to 100 dah. A similar pattern was detected in <i>Posox9b</i> expression. After injection of androgen (17α-methyltestosterone) of different concentrations, the expression level of <i>Posox9b</i> increased significantly, whereas <i>Posox9a</i> did not change obviously. These results indicated that the two <i>sox9</i> genes of Japanese flounder had converse functions in sex differentiation, whereas their differences in 17α-methyltestosterone administration were obvious and worthwhile for exploring evolutionary and adaptive significance. This study provided a foundation for further exploration of the roles of <i>Posox9</i> genes during the sex determination and differentiation, spermatogenesis and gonadal function maintenance of Japanese flounder.