65 Deciphering the dialogue between the bovine blastocyst and the uterus: comparison of extracellular vesicle proteins from an ex vivo model and an in vivo environment

Bovine uterine explants represent an ex vivo model to study the interaction between embryos and maternal tissues, which is partly facilitated by extracellular vesicles (EVs). We analyzed the protein content of EVs derived from (1) conditioned medium (CM) of uterine explants cultured alone (EXPL) versus uterine fluid (UF) from cyclic heifers and (2) uterine explants co-cultured with blastocysts (EXPL+BL) versus UF from pregnant heifers. Heifers were synchronized, artificially inseminated or not, and slaughtered 7 days later. The uterine horn ipsilateral to the corpus luteum was flushed, and pregnancy was confirmed by the presence of a blastocyst. From each cyclic heifer, four 8-mm circular uterine explants were taken and individually cultured in 750 μL of protein-free synthetic oviduct fluid: two were cultured alone, and two in the presence of five in vitro-produced bovine blastocysts each. After 6 h, CM was collected for EV isolation. UF from five cyclic and five pregnant heifers and five replicates of CM from pools of the two EXPL and pools of the two EXPL+BL were used. EVs from UF and CM were isolated by size exclusion chromatography and concentrated by ultrafiltration. EV presence was confirmed by detecting CD63, CD81, and CD44 EV markers by flow cytometry. Proteomic analysis was performed with nano LC-MS/MS and spectral counting for protein identification and quantification. Statistical analysis was done using the student’s t-test with a P-value threshold of 0.05. Bioinformatic analysis was done using the PANTHER tool. We identified 125 unique proteins to UF-cyclic, 106 unique to CM-EXPL, and 1019 common proteins. Of these, 32.2% were differentially abundant proteins (DAPs) mainly associated with the integrin signaling pathway and inflammation. Common proteins were mainly protein-modifying enzymes and cytoskeletal proteins related to cellular processes, metabolic processes, and biological regulation. We identified 108 proteins unique to UF-pregnant, 195 unique to CM-EXPL+BL, and 1373 common proteins. Of these, 40.8% were DAPs mainly associated with cytoskeletal regulation and inflammation. Common proteins were predominantly involved in protein modification, membrane trafficking, and the cytoskeleton, all of which are essential for developmental and reproductive processes. These proteins are linked to key signaling pathways such as MAPK, Wnt, and PI3K. Additionally, several proteins associated with bovine embryo development (TRIM28, CAPN2, BIRC6, A2M, and HDAC1) and cell lineage segregation during preimplantation (ITGA3, SOX17, KRT8, RACK1, ROCK1, and ROCK2) were identified. Notably, SOX17, a transcriptional factor, promotes differentiation in embryonic stem cells. In conclusion, although there are limitations compared with the in vivo environment, both ex vivo and in vivo uterine EVs share common proteins involved in early embryo development. This supports embryo-maternal communication via EVs and highlights the utility of ex vivo models for studying this process.

Funding was provided by ES-MICIN PID2019-111641RB-I00 and PRE2020-094452.