RNA interference-mediated silencing of amh reveals its regulatory role in gonadal development and sex hormone synthesis in grass carp (Ctenopharyngodon idella)
摘要
Anti-Müllerian hormone, a glycoprotein hormone encoded by the amh gene, induces Müllerian duct regression during male embryonic development and contributes to gonadal maintenance in adults. Recent studies indicate that amh also directly regulates sex determination in various fish species. Grass carp (Ctenopharyngodon idella), an important aquaculture species, exhibits a sexual maturation period of 4–5 years, yet the molecular mechanisms underlying its sex differentiation remain unclear. To explore the function of amh in the sexual development of grass carp, this study cloned the full-length sequence of the amh gene, detected the expression characteristics of the amh gene in different ages, and conducted amh gene interference experiments. The results are as follows: (1) The full-length cDNA of the grass carp amh gene is 2080 bp, including an open reading frame (ORF) of 1668 bp, a 5’ untranslated region (5’UTR) of 39 bp, and a 3’ untranslated region (3’UTR) of 373 bp, which encodes 555 amino acids. (2) The gene amh expression was consistently higher in male than in female gonads, with a sharp increase in 3–4-year-old males, whereas expression in females showed only a modest rise; (3) After amh silencing interference was performed on male fish, the expression level of amh decreased significantly, 1 day after interference, the expression levels of foxl2a, foxl2b, and dmrt1 were significantly upregulated; 3 days after interference, the expression level of cyp19a began to be significantly upregulated; 4 to 5 days after interference, the expression levels of foxl2a, foxl2b, cyp19a, and dmrt1 gradually returned to levels close to the control group. In addition, compared with the control group, the testosterone (T) content in male fish of the interference group decreased significantly, while the estradiol (E2) content increased significantly. Collectively, these findings suggest that amh inhibits foxl2a/foxl2b expression, thereby preventing premature cyp19a activation and estrogen production, which indirectly maintains androgen levels and supports testicular differentiation and spermatogenesis.