The evolution and possible role of two Sox8 genes during sex differentiation in Japanese flounder (Paralichthys olivaceus)

Abstract

Sox8 genes, as members of the Sox family, have been studied widely in mammals. However, regulation of sox8 genes in teleosts has rarely been studied, and functional analysis of these genes in teleosts has rarely been performed. Here, two duplicates of sox8 genes were identified in Japanese flounder, Posox8a and Posox8b. The analysis of expression showed that Posox8a and Posox8b were expressed in Sertoli cells of the testis, indicating that they play important roles in development and functional maintenance of the testis. Positive selection and phylogenetic analysis found that both Posox8a and Posox8b underwent the purification selection during evolutionary and that sox8 was most likely to be the ancestor sox8a. These results suggested that both Posox8a and Posox8b had important biological functions after generation from three rounds of whole-genome duplication in Japanese flounder. The functional differentiation of Posox8a and Posox8b was verified using cell transfection and dual-luciferase reporter assays; Posox8a overexpression-promoted 3β-hydroxysteroid dehydrogenase expression and Posox8b overexpression-promoted cytochrome P450 aromatase (cyp19a1; P450arom) expression. Finally, combined with Posox8a and Posox8b expression analysis from 30 to 100 days after hatch, we speculated that Posox8a and Posox8b might participate in the process of sex differentiation and gonadogenesis by regulating sex hormone biosynthesis in the Japanese flounder. Our study is the first to demonstrate the possible mechanism of Posox8a and Posox8b in Japanese flounder sex differentiation and gonadogenesis, laying a solid foundation for functional studies of sox8 genes in teleosts. K E Y W O R D S dimorphic expression, evolutionary analysis, Japanese flounder, Posox8a and Posox8b, sex differentiation

A review on sox genes in fish

“…2011) and P. olivaceus (Yu et al . 2019), suggesting that sox8 plays an important role in testicular differentiation rather than in ovary. Many reports have also suggested that sox9 plays an important role in sex differentiation during sex reversal of fish.…”

“…Many studies have proved that the expression of sox8 is higher in the testis than in the ovary of fish, such as P. dabryanus (Xia et al 2010), M. anguillicaudatus (Xia et al 2011) and P. olivaceus (Yu et al 2019), suggesting that sox8 plays an important role in testicular differentiation rather than in ovary. Many reports have also suggested that sox9 plays an important role in sex differentiation during sex reversal of fish.…”

“…Sox8 was expressed in Sertoli cells of the testis of P. olivaceus , suggesting that sox8 plays an important role in development and functional maintenance of the testis (Yu et al . 2019). There are two versions of sox9 , sox9a and sox9b , which are found in many fish species.…”

Roles of two cyp11 genes in sex hormone biosynthesis in Japanese flounder (Paralichthys olivaceus)

“…On the 5th day after the first injection, the testis and ovaries were immediately frozen in liquid nitrogen and then stored at −80°C until RNA extraction. The injection doses and times were chosen according to our previous studies in Japanese flounder (Yu et al, ).…”

“…The Japanese flounder testis cell line used in this experiment was constructed by Yu et al (). Transient transfection assay of testis cells was performed using LipoGene™ 2000 Plus Transfection Reagent (US Everbright, Inc., Suzhou, China) following the manufacturer's protocol.…”