Proteomics Analysis of Porcine Endometrial Cell-Derived Extracellular Vesicles Involved in Embryo Attachment

Maternal-embryo interactions play a critical role in early mammalian development, with extracellular vesicles (EVs) playing a key role in intercellular communication. Recent studies have focused on the mechanisms by which maternal-derived factors, such as RNA, proteins, and metabolites influence gap junctions, EVs, and direct cell-to-cell interactions, contributing to embryonic development. In this study, using a proteomics approach, we investigated the impact of EVs secreted from porcine endometrial cells (pEECs) and their protein cargoes on embryonic development. We characterized EVs isolated from pEECs (pEEC-EVs) during the diestrus stage using a nanoparticle tracking analysis and cryo-transmission electron microscopy. Furthermore, the effects of pEEC-EVs with or without hormone treatment on the in vitro attachment of hatched blastocysts were evaluated. The attachment rate of porcine embryos was significantly higher for pEEC-EVs in the hormone treatment group than the control group (23.0 ± 1.7% versus 36.9 ± 1.9% for control and pEEC-EVs, respectively). Furthermore, hormone treatment altered the expression of proteins involved in cellular organization, protein transport, and immunity. Proteomic analysis revealed distinct biological processes between groups: control EVs supported cytoskeletal organization and adhesion, while hormone-treated EVs were enriched in protein transport, immune regulation, and stress response pathways. Key signaling pathways, including VEGFA-VEGFR2, focal adhesion, and TGF-β, were modulated, influencing implantation and embryogenesis. EVs play a crucial role in maternal-embryo interactions, optimizing implantation conditions and supporting embryo-derived stem cell establishment. These findings enhance our understanding of EV-mediated communication and suggest potential applications for improving reproductive health and assisted reproductive technologies.