COUP-TFII regulates early bipotential gonad signaling and commitment to ovarian progenitors

The absence of expression of the Y-chromosome linked testis-determining gene SRY in early supporting gonadal cells (ESGC) of bipotential gonads leads to ovarian development. However, genetic variants in NR2F2 /COUP-TFII represent a novel cause of SRY -negative 46,XX testicular/ovotesticular differences of sex development (T/OT-DSD). Thus, we hy-pothesized that COUP-TFII is part of the ovarian developmental network. We examined NR2F2 /COUP-TFII expression and the genetic network under its regulation in human gonadal cells by analyzing single cell RNA-sequencing datasets of fetal gonads, differentiating induced pluripotent stem cells into bipotential gonad-like cells in vitro , and generating a NR2F2 knockout (KO) in the human granulosa-like cell line COV434. NR2F2 expression is highly upregulated during the bipotential gonad development, being detected in ESGCs. We identified that NR2F2 ablation in COV434 cells downregulated markers of ESGC and pre-granulosa cells, suggesting that COUP-TFII has a role in maintaining a multipotent state necessary for commitment to the ovarian development. We propose that impairment of COUP-TFII function may disrupt the transcriptional plasticity of ESGCs and instead drive them into commitment to the testicular pathway.