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

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. Author summaryIn this study, we investigated the cell fate trajectory and key gene expression profile of goat fetus gonads using scRNA-seq. The analysis revealed cellular heterogeneity among normal male, normal female, and intersex gonadal somatic and germ cells, alongside their dynamic fate transitions across various developmental states. Pseudo-time trajectory analysis suggested that stromal cells might act as the initiation signal for female and intersexual gonad development, while epithelial cells could serve as the primary source of gonadal cells in male fetal goats. GO functional enrichment and KEGG analyses confirmed that abnormal expression of FOXL2, SOX9, AMH, DMRT1 and RSPO1 was linked to the emergence of intersexual gonads. Newly identified candidate genes including LRP2, PITX2, ARX, LHX9, PHLDB2, ZNRF3 and TEAD1 may contribute to sex-specific differences, while INSL3 and TCF21 could be linked to intersexual gonads. We identified TREM2+ macrophages analogous to those in humans, which could regulate immune privilege mechanisms. Circadian rhythmic changes were observed during early goat gonadal development.