PGE₂ Modulates Uterine Luminal Fluid Composition and Endometrial Function in Dairy Heifers During Diestrus

Prostaglandin E₂ (PGE₂) plays a critical role in regulating uterine endometrial function and supporting embryonic development during early pregnancy in ruminants. However, its precise roles in shaping the uterine microenvironment remain unclear. Herein, 1 mg PGE₂ was infused daily into the uterus of dairy heifers from days 12 to 14 of the estrus cycle. ULF was subsequently collected for integrated proteomic, metabolomic, and targeted lipidomic analyses. In addition, bovine endometrial epithelial cells were used to evaluate the effects of PGE₂ on epithelial adhesion and responsiveness to interferon tau (IFNT). PGE₂ infusion resulted in 909 differentially abundant proteins (DAPs), which are primarily associated with early embryonic development, immune regulation, and cell adhesion. Untargeted metabolomics analysis identified 587 altered metabolites, which were enriched in sphingolipid, arachidonic acid, phenylalanine, and tryptophan metabolism. Proteomic-metabolomic analyses showed that these alterations were primarily associated with early embryonic development, immune regulation, and cell adhesion. Targeted lipidomic analysis showed a global reduction in lipid accumulation, with glycerophospholipid metabolism and choline metabolism most significantly affected. In vitro, PGE₂ reduced epithelial microvilli density, increased osteopontin (OPN) expression, and decreased the expression of junctional proteins (zona occludens-1 (ZO-1), E-cadherin (CDH1), and fibronectin 1 (FN1)). Moreover, PGE₂ enhanced the responsiveness of bEECs to IFNT by interferon alpha/beta receptor 1 (IFNAR1) and IFNAR2, and prostaglandin E receptor 4 (PTGER4) was identified as the primary receptor mediating this response. Collectively, these findings suggest that PGE₂ may modulate lipid metabolism and adhesion-related processes in the endometrium and influence endometrial responsiveness to IFNT, providing insights into molecular mechanisms associated with pregnancy establishment in dairy cows.