amh alleles on XY sex chromosomes regulate male differentiation in amphidiploid Carassius auratus.

The amh (anti-Müllerian hormone) gene, residing on the sex chromosomes, has been hypothesized to be a potential master sex gene in amphidiploid Carassius auratus (crucian carp/goldfish). However, its role in male determination or differentiation remains unclear. Here, we identified the heteromorphic X and Y chromosomes and confirmed the localization of amh on the sex chromosome by fluorescence in situ hybridization in amphidiploid C. auratus. The transcriptional expressions of X-linked and Y-linked amh alleles showed no significant difference during the period of sex determination and differentiation. The mutation of either the X-linked or Y-linked amh allele did not result in sex reversal, while the disruption of both amh alleles resulted in male-to-female sex reversal in a proportion of genotypic males. This finding suggests that neither the X-linked nor the Y-linked amh allele is responsible for male determination, but rather, amh alleles contribute to male differentiation in amphidiploid C. auratus. Subsequent research found that Amh could repress cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a) transcription in vitro, and the inhibition of cyp19a1a partially rescued the sex reversal phenotype caused by amh knockout. Besides, the amh null mutation in artificial amphitriploid C. auratus, which possesses an XXX/XXY sex determination system, also led to male-to-female sex reversal in some genotypic males. This study illustrates that the sex chromosome-residing amh is not a male-determining gene but regulates male differentiation in C. auratus, confirming the conserved function of amh in male differentiation in vertebrates.