Transcription factors foxl2 and foxl3 regulate cyp19a1a and cyp11b in orange-spotted grouper (Epinephelus coioides)

Abstract

In orange-spotted grouper (E. coioides), the transcription factors foxl2 and foxl3 are thought to play an important role in ovarian and testicular development, but how they do so remains unclear. In this study, we investigated the regulatory effect of foxl2 and foxl3 on the sex steroid genes cyp19a1a and cyp11b, and then we explored the function of foxl2 and foxl3 in the sex hormone synthesis pathway in teleost fish. First, we used FOXL2 and FOXL3 recombinant proteins to investigate the regulatory mechanism of foxl2/foxl3 on aromatase in E. coioides and found that the recombinant proteins can enter gonad cells and be expressed in the nucleus. The expression levels of cyp19a1a and cyp11b were detected by real-time fluorescent quantitative PCR when FOXL2 or FOXL3 recombinant proteins were used to stimulate primary ovarian cells of orange-spotted grouper. The FOXL2 recombinant protein significantly increased the expression of cyp19a1a mRNA, which is related to estrogen production, in a dose-dependent manner. The FOXL3 recombinant protein significantly down-regulated the expression of cyp19a1a mRNA and significantly up-regulated the expression of cyp11b mRNA, which is related to androgen synthesis. Dual luciferase analysis in vitro revealed that foxl2 directly acted on the promoter region of cyp19a1a and foxl3 directly acted on the promoter regions of both cyp19a1a and cyp11b. The FOXL2 and FOXL3 recombinant protein injection experiments in vivo by surgery further showed that foxl2 promoted ovarian differentiation by directly up-regulating the expression of the aromatase gene cyp19a1a, whereas foxl3 directly inhibited the expression of the aromatase gene cyp19a1a and up-regulated the expression of cyp11b to promote testicular differentiation.