Progesterone modulates protein cargo in extracellular vesicles released from bovine uterine epithelial cells.

ABSTRACT: Extracellular vesicles (EVs) are membrane-bound particles that play an important role in cell-to-cell communication. Embryo-maternal communications are vital for early embryo development and implantation. Uterus-derived EVs play a crucial role in embryo-maternal crosstalk. The current study investigated how progesterone regulates protein cargoes in EVs derived from bovine uterine epithelial cells (BUTE cell-EVs). After isolation, BUTE cell-EVs were characterized using scanning transmission electron microscopy, nanoparticle tracking analysis, western blotting, and mass spectrometry. BUTE cells were treated with 0 or 300 nM progesterone to investigate potential regulation of protein cargoes in BUTE cell-EVs. We determined that BUTE cell-EVs have the characteristic cup shape structure, ranging in size from 20 to 550 nm, and expressed CD9 and CD81, as well as other known proteins for EV biogenesis. The number of BUTE cell-EVs released was not affected by progesterone. Proteomic analysis showed that BUTE cell-EVs contained proteins with functions related to implantation, uterine receptivity, and trophoblast differentiation. Progesterone altered the EV protein cargo, with 45 proteins differentially expressed compared to control. Differentially expressed proteins related to implantation and adhesion included galectin-3 and galectin-3-binding protein. The actin-binding protein gelsolin was also found to be differentially expressed in progesterone-treated BUTE cell-EVs. We also showed that BUTE cell-EVs were successfully internalized into bovine embryos. In conclusion, the proteomic profile of BUTE cell-EVs was altered by progesterone, and these EVs are internalized into the bovine embryos, delivering proteins into bovine embryos that are important for implantation, embryo adhesion, and proper embryo metabolism and development. LAY SUMMARY: Communication between the womb (uterus) and the developing embryo is essential for successful pregnancy. Tiny particles called EVs are released from cells in the uterus and carry proteins and other molecules that help the embryo grow and attach to the uterus. In this study, we investigated how the hormone progesterone influences the contents of EVs released from the cells (epithelial cells) that line the cow's uterus. We found that progesterone alters the amount of specific proteins in these vesicles without affecting the number of vesicles released. Many of the altered proteins are involved in preparing the uterus for implantation and supporting embryo development. We also demonstrated that these vesicles can be taken up by early-stage cow embryos. Our findings suggest that progesterone changes proteins that are packed in these vesicles, helping create a receptive environment for implantation and early development.