ABSTRACT: This study employed single-cell RNA sequencing (scRNA-seq) to systematically map the cellular landscape of Hu sheep placental tissues across three gestational stages (50, 80, and 120 days), uncovering dynamic cell fate transitions and regulatory mechanisms. Analysis of 51,808 high-quality single-cell transcriptomes identified 14 cellular clusters, including uninucleate trophoblasts (UNCs, 58.83%), binucleate trophoblasts (BNCs, 21.82%), endothelial cells, fibroblasts, and immune cells. UNCs were subdivided into five subtypes expressing PAG2 and GATA3, while BNCs formed four subtypes marked by CSH2, PAG4, and PRP2. Functional enrichment revealed stage-specific activation of hypoxia-inducible factor 1 (HIF-1), vascular endothelial growth factor (VEGF), and MAPK signaling pathways, alongside shifts in proliferation, adhesion, and autophagy. Pseudotemporal trajectory analysis delineated a developmental continuum from UNCs to BNCs, highlighting dynamically expressed genes linked to MAPK signaling and HIF-1 responses. Immunofluorescence validated the spatial expression of key regulators, such as GCM1 and GATA3, in trophoblasts. This comprehensive atlas provides novel insights into molecular programs driving ovine placental maturation, offering potential targets to enhance reproductive efficiency and fetal development.
