67 Deciphering the dialogue between the early bovine embryo and the oviduct: comparison of extracellular vesicle proteins from an ex vivo model and an in vivo environment

Bovine oviducal explants provide an alternative ex vivo approach to study embryo-maternal communication, which is partly mediated by extracellular vesicles (EVs). We investigated the protein content of EVs from (1) oviducal explants cultured alone (EXP) with oviducal fluid (OF) of cyclic heifers (CY) and (2) oviducal explants co-cultured with embryos (EXP+EMB) with OF of pregnant (PG) heifers. Heifers were synchronized, artificially inseminated (PG) or not (CY), and slaughtered 3.5 days after insemination. Oviducts ipsilateral to the corpus luteum were flushed and pregnancy was confirmed by an embryo presence. Six 0.25-mm² oviducal explants were obtained from each cyclic heifer and cultured individually in 750 μL of protein-free synthetic oviduct fluid: three were cultured alone, and three co-cultured with 10 in vitro-produced 8-cell bovine embryos each. After 6 h, conditioned medium (CM) was collected for EV isolation. The OF- and CM-EVs were isolated using size exclusion chromatography and concentrated by ultrafiltration. The EV presence was confirmed by flow cytometry using CD63, CD81, and CD44 markers. Proteomic analysis was carried out using nanoLC-MS/MS with spectral counting for protein identification and quantification. The OF-EVs from 5 cyclic and 5 pregnant heifers and 5 replicates (representing each cyclic heifer) of CM-EVs from pools of three explants cultured alone and pools of three explants cultured with embryos were used. Statistical analysis was carried out using Student’s t-test, with a P-value threshold of 0.05. Bioinformatic analysis was performed with DAVID and STRING tools. Comparing OF-CY and EXP, we identified 576 proteins: 60 unique to OF-CY, 62 unique to EXP, and 454 common. Of these, 65.4% were equally abundant (EAPs), while 34.6% were differentially abundant (DAPs). Common proteins were related to cell adhesion and communication such as endocytosis, gap junction, and tight junction. Comparing OF-PG and EXP+EMB, we identified 613 proteins: 53 unique to OF-PG, 16 unique to EXP+EMB, and 544 common. Of these, 78.5% were EAPs, while 21.5% were DAPs. Common proteins were involved in cell adhesion and communication (e.g. ITGA2, ITGB1, and ADAM10). The extracellular matrix-related protein A2M, as well as proteins linked to early embryo development and cell differentiation, such as CLTC, MACROH2A1, HSP90AA1, and HSP90AB1, were also found in both models. Furthermore, YBX1, a transcription factor that mediates maternal mRNA decay during pre-implantation development, was also common to both models. On the other hand, OVGP1, involved in gamete/embryo-oviduct interactions, was abundant in OF-CY and OF-PG, indicating that though the ex vivo model is a valid alternative, it has limitations compared to the in vivo environment. In conclusion, both ex vivo and in vivo oviducal EVs share common proteins involved in early embryo development, supporting embryo-maternal communication via EVs and highlighting the utility of ex vivo models for studying this process.

The study was funded by Spanish MINECO PID2019–111641RB-I00.