Single nucleotide insertion within a remote enhancer causes XX mice to be born as males

In this study, we show that mice carrying a 3 bp deletion or 1 bp insertion within the SOX9 BS of Enh13 are born as XX males. At adult stages, mice appear externally and internally as males but their testes are small and devoid of sperm due to the lack of the Y chromosomes. At embryonic stages, XX homozygous gonads appear as ovotestis at various severities with the 1 bp insertion appearing more as a testis. Transcriptomic analysis of E12.5 gonads shows that indeed the XX homozygous gonads are positioned in between the testis and ovary while XY homozygous gonads are indistinguishable of wild type (WT) XY gonads. These findings implicate Enh13 as a dual regulatory element essential for both testicular and ovarian development, placing it at the critical intersection of sexual differentiation pathways.