A longitudinal single-nucleus transcriptomic atlas of the bovine placenta reveals dynamic cellular hierarchies and regulatory networks during gestation

Elucidation of the cellular hierarchy and the underlying regulatory mechanisms for placentas during gestation can tremendously contribute to healthy pregnancy outcomes in mammalian species including cows. Here, we constructed a single-nucleus transcriptomic atlas of 281,257 placental nuclei spanning as many as 13 developmental stages (E12, E14, E16, E18, E24, E30, E50, E60, E85, E110, E180, E240, and E280) in cows, identifying 13 major cell types and 14 trophoblast subtypes. Bioinformatic analyses revealed that bovine placental trophoblast cells exhibited stage-specific expression patterns and functional characteristics during gestation, with HAND1 and DLX5 identified as novel key regulators for maternal recognition of pregnancy. We also found that binucleate cells (BNCs) arose from specific uninucleate cell (UNC) subpopulations on E24, with their differentiation trajectories co-regulated by imprinted genes and metabolic reprogramming. The subsequent integrated genome-wide association study (GWAS) analysis disclosed eight early trophoblast subtypes significantly associated with gestation length (GL), as well as the involved key pathways and risk genes (e.g., CYCS , HMGA1 , and VDAC1 ), with these signals under a strong evolutionary constraint. Furthermore, we found that placental macrophages originated from E30 in cows and were significantly associated with pregnancy loss in both cows and humans, sharing common risk pathways. Collectively, this study delineates the longitudinal cellular and molecular profiling of bovine placentas, uncovers the key regulatory networks at the maternal-fetal interface, and also provides a theoretical framework and potential molecular targets for understanding of pregnancy maintenance and improvement of reproductive traits in ruminants.