Abstract: Polled intersex syndrome in goats severely impacts productivity and economic benefits, serving as a potential model for human reproductive defects. However, its specific molecular and cellular regulatory mechanisms remain unclear. This study combined single-cell RNA-sequencing (scRNA-seq) with tissue morphology and immunofluorescence detection to investigate the cellular heterogeneity, pseudo-time differentiation trajectory, differentially expressed genes, and regulatory networks of female, intersex, and male gonads during the 55-65 days embryonic stage of goats. The results indicated significant heterogeneity and cell fate transition in the somatic and germ cells of female, intersex, and male gonads at different developmental stages. Stromal cells may initiate female and intersexual gonad development, and epithelial cells may be the source of gonad cells in male fetuses. The abnormal expression of FOXL2, SOX9, AMH, DMRT1, RSPO1, INSL3, TCF21 and CSF1R may cause female-to-male sex reversal. The new candidate genes like PHLDB2, ZNRF3 and TEAD1 may contribute to the gender differentiation. Additionally, TREM2+ macrophages analogous to those in humans were identified in male gonads, while genes such as CSF1R and TYROBP potentially regulate macrophage immune privilege mechanisms. Early gonadal development in goats may also be regulated by circadian rhythm mechanisms similar to humans, with genes such as NR1D1, LGR4, and ATG5 involved in granulosa cell development, while genes such as ATG5, NRIP1, and FBXW11 may affect intersex trait development. In general, this study provided an important reference for exploring molecular and cellular mechanisms of intersex initiation during early embryonic development in goats and also for modelling human reproductive defects.
