85 Extracellular vesicles secreted by bovine embryos during the hatching period induce the expression of nonclassical interferon-stimulated genes in endometrial cells

Extracellular vesicles (EVs) are nanoparticles that carry molecules that participate in cellular communication. Recently, the EVs have taken importance in embryo-maternal communication, which is crucial for normal embryo development. Furthermore, it is known that in ruminants, the conceptus-derived EVs are capable of inducing pathways associated with interferon tau (IFNT) in epithelial endometrial cells during in vitro culture. However, it seems that this embryonic effect starts earlier, at the blastocyst stage. This study aims to evaluate the response of epithelial endometrial cells to EVs released by preimplantation bovine embryos to determine whether similar communication occurs before the formation of conceptus. For this purpose, the EVs released by in vitro-produced bovine embryos from Day 7–9 of development (after embryo hatching), considering day 0 as IVF, were used. The embryos were individually cultured in EVs depleted SOF (oviducal synthetic fluid) medium. Once the culture medium was collected, the EVs from 50 embryos were isolated using ExoLutE^® Exosome Isolation Kit and characterised through flow cytometry and transmission electron microscopy. Quantification was performed using nanoparticle tracking (NTA). Endometrial epithelial bovine cells (bEECs), previously characterised, were cultured in 12 well culture plate and supplemented with 1 × 10⁸ embryo-derived EVs (E-EVs) or similar volume of Phosphate-buffered saline (PBS) as negative control. Three replicates were performed per group. EV internalisation was assessed 24 h after the addition of PKH67-labelled EVs, while the cell nucleus was identified by DAPI staining. 48 h after supplementation, cells were lysed and total RNA was extracted using E.Z.N.A Total RNA Kit I (OMEGA Biotek). RT-qPCR was performed to evaluate genes associated with IFNT pathways in bEECs with or without embryonic EVs. The nanoparticles released by preimplantation bovine embryos were positive to surface markers CD9, CD63, and CD81. By NTA, the mean size of the particles was 179.7 ± 7.7 nm while the average size of EVs identified by TEM was 234 nm. The Evs were successfully internalised by the endometrial epithelial cells and nonclassical interferon stimulated genes (IGS) such as CST6, WNT7A, and CTSL were activated. Furthermore, OXTR, a gene associated with sex steroid signalling was strongly induced by the EVs released by embryos. It is concluded that, after hatching and before elongation, bovine embryos secrete extracellular vesicles capable of modifying the mRNA level of genes associated with IFNT response in epithelial endometrial cells.

This research was supported by Fondecyt 1210334 and ANID 21201060.