Sox9a and amh indirectly cooperate to promote spermatogonia differentiation and gonadal development in Cyprinus carpio var. koi(koi carp).

In teleost fish, sex differentiation is co-regulated by genetic factors and environmental signals. Genetically, Wnt4, Rspo1, Foxl2, and cyp19a dominate female differentiation, while dmrt1, Sox9, SF-1, and amh govern male differentiation. Among these, amh (TGF-βsuperfamily domain) and Sox9a (HMG-box domain) are conserved male-specific genes critical for testicular development in zebrafish and goldfish. Notably,their functional interactions under long-term artificial selection in koi carp remains unelucidated at the molecular level. Additionally, female koi carp have higher market value in aquaculture due to unique body morphology and growth advantages, and boosting their proportion significantly improves farmers' economic benefits.In this study, we detected the gonadal expression patterns of amh and Sox9a genes in koi carp via quantitative real-time PCR (RT-qPCR), and investigated their functions and interaction using targeted RNA interference (RNAi) and yeast two-hybrid assays. Both genes showed male-biased gonadal expression, and exhibited higher expression in the testes during spermatogonial differentiation at 160 days post-hatching. RNAi-mediated knockdown of amh or Sox9a inhibited testicular growth and spermatogonial maturation, and altered the expression of female-related genes cyp19a and Foxl2. Mechanistically, Sox9a interacted indirectly with amh to activate the AMHR-mediated signaling pathway, promoting Müllerian duct regression and testicular somatic cell proliferation. In addition, RNAi-mediated inhibition of Sox9a delayed testicular development.This study identifies amh and Sox9a as key interactive regulators of koi carp testicular development, reveals male gonadal differentiation mechanisms, and highlights amh as a promising gene target to boost female proportions and aquaculture economic benefits.