Strain‐Dependent Functional Variation of a Sex Specific <scp> <i>Sox9</i> </scp> Enhancer

In mammalian sex differentiation, Sox9 expression in undifferentiated gonads determines male development. Sox9 is regulated by multiple enhancers, of which Enh13/mXYSRa is the most critical. Disruption of the GATA4 binding site in Enh13/mXYSRa causes XY sex reversal in the C57BL/6J (B6J) strain, but not in the mixed genetic background of B6J × DBA/2. To clarify the cause of these differences, mice with the same mutation were generated in DBA/2. Homozygous mutants developed as XY males. We hypothesized that the phenotypic differences between strains were due to functional differences of another enhancer. Mice lacking TESCO and carrying a mutation in the GATA4 binding site were generated and analyzed. The mutants developed according to their chromosomal sex despite reduced Sox9 expression. These findings imply that the DBA/2 genetic background contains factors preventing Sox9 expression from falling below the threshold required for male development. Bulk RNA-seq analysis of fetal gonads during sex determination identified strain-dependent differences in intermediate mesoderm development and protease inhibition pathways; the study also revealed that the expression of Sox8 has a redundant role in Sertoli cell differentiation to that of Sox9 in male embryonic gonads. These differences may contribute to the phenotypic variation observed between strains.