Male fertility is independent of Enh13 control of Sox9 testicular expression

Testis development relies on precise temporal control of Sox9 expression, which is rapidly activated to initiate testis development and subsequently maintained to preserve Sertoli cell identity and male fertility. The distal enhancer, Enh13, is essential for Sox9 upregulation as its constitutive deletion results in complete XY male-to-female sex reversal. Enh13s requirement across distinct developmental stages remains unexplored. We show that early gonadal deletion of Enh13 fully recapitulates XY sex reversal, demonstrating that Enh13 activity is strictly required to initiate the male pathway. In contrast, Sertoli cell-specific deletion of Enh13 after sex determination has no effect on Sox9 expression, testis architecture, or male fertility, revealing that Enh13 is dispensable for Sox9 maintenance in the differentiated testis. Chromatin accessibility identifies candidate enhancers gaining activity after sex determination, suggesting a regulatory handoff within the Sox9 locus. Finally, we show that duplicating Enh13 in the endogenous mouse locus fails to recapitulate the human XX sex reversal, suggesting that it may not be solely Enh13 sequence duplication that led to XX sex reversal in humans. To the best of our knowledge, this study represents the first in vivo conditional knockout of a developmental enhancer, allowing for temporally controlled interrogation of regulatory logic.