1 Preimplantation bovine embryos secrete extracellular vesicles that participate in embryo-maternal communication

The embryo–maternal interaction occurs during early stages of embryo development and is essential for implantation and full-term development of the embryo. In bovine, the secretion of interferon-tau (IFNT) during elongation is the main signal for pregnancy recognition. During this period, embryos also release extracellular vesicles (EVs) as an alternative mechanism of embryo–maternal communication. EVs are nanoparticles that carry bioactive molecules (proteins, lipids, mRNA, and miRNA) and play a role in intercellular communication. The aim of the study was to determine if EVs secreted by bovine embryos during blastulation (Days 5–7) could induce transcriptomic modifications, activating IFNT signalling in endometrial cells. Bovine embryos were produced by in vitro fertilisation and cultured in groups in synthetic oviducal fluid medium (SOF) supplemented with 2% fetal bovine serum and 3 mg mL⁻¹ bovine serum albumin for 5 days. Then, morulae were selected and individually cultured for 48 h in 96-well plates in EV-depleted SOF. The culture medium was collected and embryonic EVs were separated using ExoLutE® Exosome Isolation Kit (Rosetta Exosome). EVs were characterised by flow cytometry, transmission electron microscopy, and nanoparticle tracking analysis. Embryo-derived EVs (E-EVs) and and equivalent volume of phosphate-buffered saline were subjected to PKH67 staining. Endometrial bovine cells were cultured in 35-mm dishes and supplemented with 1 × 10⁸ labelled E-EVs or PBS as negative control. Internalisation of EVs was evaluated 24 h after supplementation. 4′,6-Diamidino-2-phenylindole (DAPI) staining was used to visualise the cellular nucleus. Forty-eight hours after supplementation, cells were lysed, and total RNA was extracted using E.Z.N.A Total RNA Kit (Omega Bio-Tek). Sequencing was obtained using NextSEqn 500/550 High Output kit in the Illumina NextSEqn 500 equipment (Illumina Inc.). The sequences were mapped against the Bos taurus reference genome (ARS-VCD1.2) using the HISAT2 program. Differential expression analysis was performed in EdgeR package. Genes were considered differentially expressed with Log2 fold change (FC) >1 or < −1 and false discovery rate (FDR) <0.05. E-EVs were internalised by endometrial cells, as demonstrated by the presence of green dots in the cell cytoplasm. From the RNA-seq data, 4547 genes were identified as a differentially expressed in endometrial cells exposed to E-EVs. Of those, 1879 genes were considered downregulated and 2668 upregulated. Within upregulated genes, interferon stimulated genes (ISGs) were detected such as OAS1Y (logFC = 2.65), MX1 (logFC = 2.45), ISG15 (logFC = 1.2) as well as other upregulated genes related to IFNT stimulation such as IRF6 (logFC = 2.6), CTSL (logFC = 3.0), and CST6 (logFC = 2.9). Key pathways related to endometrial function were also upregulated, including interferon signalling, eicosanoid metabolic process, and solute and water transport. From Gene Ontology, the upregulated pathways associated with biological process were biological adhesion, tissue development, epithelium development, and epithelial cell differentiation. In conclusion, EVs released by bovine blastocysts are internalised by endometrial cells inducing transcriptional modifications and stimulating genes associated with IFNT signalling.

This research was supported by Fondecyt 1210334 and ANID 21201060.