The Role of Amh in Gonadal Development and Its Response to Exogenous Hormones in Blotched Snakehead (Channa maculata).

Anti-Müllerian hormone (Amh) is a key regulator of sex determination and differentiation in fish. In this study, we identified and characterized a single-copy Amh ortholog, designated CmAmh, in blotched snakehead (Channa maculata), an economically important species in China. The cDNA spans 2340 bp and encodes a 545-amino-acid protein with conserved AMH-N and TGF-β domains. CmAmh exhibited pronounced male-biased gonadal expression, with transcript levels in testes markedly exceeding those in ovaries across all developmental stages (45~365 days post-hatching (dph)). Cellular localization revealed CmAmh transcripts in the Sertoli cells and spermatogonia of the testis and, more weakly, in the oogonia and primary oocytes of the ovary, suggesting a primary role in spermatogenesis and a potential modulatory function in early oogenesis. A strong inverse correlation between promoter CpG methylation and transcriptional activity, hypermethylation in ovaries (82.5~89.7%) versus hypomethylation (15.8~19.1%) in testes, indicated epigenetic regulation, which functionally integrates with hormonal signaling: 17β-estradiol (E2) suppressed, whereas methyltestosterone (MT) upregulated CmAmh expression. Long-term E2 exposure induced male-to-female secondary sex reversal (SSR) in 26.7% of XY individuals, whereas MT failed to reverse XX fish, indicating that CmAmh modulation alone is insufficient to override ovarian fate. Furthermore, CRISPR/Cas9-mediated mutagenesis achieved 40~45% editing efficiency, predominantly generating frameshift alleles, providing a platform for future functional studies. Collectively, these findings position CmAmh as a hormone-responsive nodal point within a broader sex-differentiation network. This work provides a theoretical foundation for sex-control strategies in C. maculata aquaculture, while future studies should focus on homozygous mutant generation to definitively establish its loss-of-function phenotype and regulatory networks.