Abstract
The anti-Müllerian hormone (Amh) is a protein belonging to the TGF-β superfamily, the function of which has been considered important for male sex differentiation in vertebrates. The Japanese flounder (Paralichthys olivaceus) is a teleost fish that has an XX/XY sex determination system and temperature-dependent sex determination. In this species, amh expression is up-regulated in genetic males and in temperature-induced masculinization during the sex differentiation period. However, to the best of our knowledge, no reports on the Amh receptor (Amhr2) in flounder have been published, and the details of Amh signaling remain unclear. In this study, we produced amhr2-deficient mutants using the CRISPR/Cas9 system and analyzed the gonadal phenotypes and sex-related genes. The results revealed that the gonads of genetically male amhr2 mutants featured typical ovaries, and the sex differentiation-related genes showed a female expression pattern. Thus, the loss of Amhr2 function causes male-to-female sex reversal in Japanese flounder. Moreover, the treatment of genetically male amhr2 mutants with an aromatase inhibitor fadrozole, which inhibits estrogen synthesis, resulted in testicular formation. These results strongly suggest that Amh/Amhr2 signaling causes masculinization by inhibiting estrogen synthesis during gonadal sex differentiation in the flounder.