Abstract

Extracellular vesicles (EVs), including exosomes and microvesicles, are secreted by different cell types and participate in cellular communication by carrying molecules as microRNAs (miRNAs) that can interfere with gene expression of target cells. Extracellular vesicles have become relevant as a mechanism of embryo-maternal communication. The aim of this study was to evaluate miRNA content in EVs secreted after embryonic genome activation, by bovine embryos with different developmental potential. Bovine embryos were produced in vitro and cultured in group until Day 3.5 in synthetic oviductal fluid (SOF) medium. Only 8-16-cell embryos were cultured individually in EVs-depleted SOF until Day 5. The SOF was EV depleted by ultrafiltration. Culture media (CM) were collected at Day 5 and embryos continued in culture until Day 7 with fresh SOF. Collected media were conserved individually and identified with the corresponding embryo. Then, CM were classified according to capacity of its embryo to reach blastocyst stage at Day 7: G1-CM (blocked embryos in 8-16 cell) and G2-CM (embryos that reach blastocyst stage). The EV isolation was carried out using the protocol described by Mellisho et al. (2017). Recovered EVs were evaluated by nanoparticle tracking analysis (NTA), Transmission electron microscopy and the presence of surface markers (CD9, CD63, CD81, and CD40L). After NTA, individual CM were pooled to organise 3 replicates of 10 CM each, for G1 and G2. The whole miRNA isolation, library preparation, and sequencing was performed by Norgen Biotek facilities (Canada). The quality of libraries was analysed using the FastQC program platform followed by Trimmomatic to remove remnant adapters. For the miRNA library it accepted reads with value above 30 Phreads and 22 to 30 bp length. The reads were mapped against the reference genome ARS-UCD1.2 using Bowtie2 software and miRDeep2 mapper, and the gene counts were calculated using HTSeq. Differential expression analysis was performed in EdgeR package. To expand this information, principal component analysis, Heatmap, and Volcano plot were plotted and pathway enrichment analysis was conducted. The NTA, transmission electron microscopy, and flow cytometry confirmed the presence of exosomes and microvesicles in isolated EVs. According to NTA, the mean size of EVs was 102.1-176.2 nm and concentration of 8.4 × 107-8.6 × 108 particles mL−1 in G1 and G2, respectively. We identified 96 miRNAs significantly expressed across the samples. Only eight miRNAs in EVs were differentially expressed between groups (G2 vs. G1). The bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were up-regulated (Log2 fold-change > 1), whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were down-regulated (Log2 fold-change < 1). The more significant (P-value < 0.01) up-regulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The EVs secreted by embryos to culture environment carry miRNAs that can reflect the molecular state of their parental cell. This lets us suggest culture media derived-EVs and their miRNA cargo as early biomarkers to select more competent bovine embryos.