Single-cell RNA sequencing analysis reveals new insights for intersex gonad initiation and early sex differentiation of dairy goats

Polled intersex syndrome in goats impairs productivity and economic benefits, and serves as a valuable model for human reproductive defects, yet its underlying molecular and cellular mechanisms remain elusive. This study integrated single-cell RNA-sequencing, tissue morphology and immunofluorescence to characterize cellular heterogeneity, pseudo-time differentiation trajectories, differentially expressed genes and regulatory networks in female, intersex and male gonads of 55-65-day goat embryos. Significant heterogeneity and cell fate transitions were observed in somatic and germ cells across groups. Stromal cells may initiate female/intersex gonad development, while epithelial cells likely serve as the origin of male gonadal cells. Aberrant expression of SOX9 , AMH , DMRT1 and other key genes drives female-to-male sex reversal, with PHLDB2 , ZNRF3 and TEAD1 identified as novel candidate regulators. TREM2 + macrophages were detected in goat gonads, and multiple genes may modulate sex reversal via gonadal microenvironment regulation. Circadian rhythm-related genes also participate in early gonadal development and intersex trait formation. These findings provide critical references for deciphering the mechanisms of goat embryonic intersex and establishing human reproductive defect models.