Molecular Characterization and Functional Analysis of Amhr2 in Sex Differentiation and Gonadal Development of Blotched Snakehead (Channa maculata)

The anti-Müllerian hormone type II receptor (Amhr2) is a critical component of the transforming growth factor-β (TGF-β) signaling pathway and plays essential roles in sex determination and gonadal development in teleosts. However, its function in the blotched snakehead (Channa maculata), an economically important fish in China, remains unexplored. In this study, we cloned and characterized the Amhr2 ortholog from C. maculata, designated CmAmhr2. The gene encodes a 443-amino acid protein containing a conserved STYKc kinase domain. Sequence and phylogenetic analyses revealed that CmAmhr2 is homologous to autosomal Amhr2 in other teleosts. Spatiotemporal expression analyses showed that CmAmhr2 was predominantly expressed in testes, particularly during critical windows of gonadal differentiation. In situ hybridization localized CmAmhr2 transcripts mainly in spermatogonia, with weaker signals in primary spermatocytes, Sertoli cells, and early-stage oocytes (oogonia and primary oocytes). Dietary administration of 30 mg/kg 17β-estradiol (E2) from 15 to 45 days post-fertilization (dpf) for 30 days induced male-to-female sex reversal, producing neofemales (XY-F) and intersex individuals (XY-I). CmAmhr2 expression levels progressively declined with the degree of gonadal feminization: highest in normal XY male (XY-M) testes, intermediate in XY-I ovotestes, and lowest in fully feminized XY-F and normal XX female (XX-F) ovaries. Furthermore, CRISPR/Cas9-mediated mutagenesis of CmAmhr2 generated frameshift mutations predicted to disrupt the kinase domain. These findings suggest that CmAmhr2 is involved in male sex differentiation and testis development in C. maculata, providing novel molecular insights and a foundation for future sex-control research in aquaculture.