Supplementary Material for: Evidence for sult1st6y as a major candidate sex-determining gene in Pacific bluefin tuna

Introduction: The Y chromosome-linked gene sult1st6y (a homolog of the estrogen sulfotransferase gene) has recently been identified in Thunnus, a genus characterized by an XX/XY sex determination system. This study examined whether sult1st6y is a sex-determining gene in bluefin tuna (T. orientalis). Methods: The expression of sult1st6y was examined using polymerase chain reaction analyses and in situ hybridization. Sult1st6y expression in masculinized XX testes produced by aromatase inhibitor administration and the expression profile of the sult1st6y mutant, which was produced using CRISPR/Cas9, were examined. Results: Sult1st6y was specifically expressed in XY gonads during sex differentiation. The onset of sult1st6y expression preceded that of other genes promoting sex differentiation. Sult1st6y expression was not detected in masculinized XX testes, indicating that gonads can differentiate into testes without sult1st6y if estrogens are depleted. The Sult1st6y mutant XY gonad showed a gene expression pattern similar to that of wild-type XX gonads. Discussion/Conclusion: Our results collectively suggest that sult1st6y is at the top of the molecular cascade that regulates gonadal sex differentiation. Sult1st6y may trigger testicular differentiation by deactivating estrogens, although its biochemical activity should be examined. This study provides evidence that sult1st6y is a major candidate sex-determining gene in tuna.