Background: Heterozygous loss-of-function variants in NR2F2/COUP-TFII cause SRY-negative 46,XX ovo/testicular difference of sex development (DSD), characterized by the presence of both ovarian and testicular tissue in genetically female individuals. Although mouse studies have linked NR2F2 to gonadal interstitial cell fate, the mechanism underlying testicular development in affected individuals remains unclear, as gonadal differentiation is orchestrated by supporting cells.
Methods: To investigate the role of NR2F2 during human gonadal supporting cell differentiation, we differentiated female NR2F2-reporter embryonic stem cells (ESCs) and NR2F2-knockout (KO) induced pluripotent stem cells (iPSCs) into fetal gonadal cells.
Results: At the bipotential stage of differentiation, NR2F2-expressing cells were FACS-sorted and replated. This enriched population exhibited increased expression of supporting cell markers at later stages, suggesting that NR2F2-positive progenitors contribute to the supporting cell lineage. To determine whether NR2F2 dosage regulates supporting cell fate, we differentiated heterozygous (Het) and homozygous (Hom) NR2F2-KO female hiPSC lines into testis-like cells. RNA sequencing at the bipotential stage revealed that Het, but not Hom, NR2F2-KO cells exhibited a transcriptional shift from a mesenchymal progenitor state toward supporting cell lineage commitment, suggesting that reduced, but not absent, NR2F2 expression primes gonadal progenitors for differentiation. At later stages, Het NR2F2-KO cultures upregulated testis differentiation genes and exhibited an increased area of SOX9-positive testis cord-like structures. In contrast to control cultures, which formed discrete FOXL2-positive clusters resembling ovarian development, FOXL2-positive cells in Het NR2F2-KO cultures were dispersed and disorganized. These findings recapitulate testicular development in a 46,XX genetic background following partial loss of NR2F2 and suggest that dysregulation of molecular pathways governing gonadal supporting cell lineage commitment impacts the balance between ovarian and testicular cell fate.
Conclusion: We identified a previously unrecognized dose-dependent role for NR2F2 in regulating human gonadal supporting cell differentiation, thereby providing mechanistic insight into 46,XX ovo/testicular DSD.