Abstract

Extracellular vesicles (EVs) are released to the extracellular environment by different cell types, including embryonic cells. The characteristics and content of EVs might vary according to embryo competence, so they have been proposed as markers for embryo selection to improve pregnancy outcomes. Moreover, it seems that EVs secretion is modified during the preimplantation period due to molecular and cellular changes that take place during embryo development. The aim of this work was to compare the morphological characteristics and microRNA (miRNA) content of EVs secreted by competent bovine embryos during the period from compaction to blastulation (Day 3–7). In vitro-produced embryos were individually cultured in EV-depleted synthetic oviductal fluid (SOF) (80 μL) from 8 cells (Day 3) to morula stage (Day 5); culture media were individually collected and assigned to group W2. Morulae were kept in culture up to blastocyst stage to determine the developmental competence. In a separate experiment, embryos were cultured up to morula stage (Day 5). Then morulae were collected and cultured individually in fresh EV-depleted SOF (80 μL) up to blastocyst stage (Day 7); culture media were assigned to group W3. For W2, embryos were classified as competent if they reached the blastocyst stage at Day 7, while for W3, embryo competence was defined by their linear in vitro growth up to Day 11. Only individual culture media from competent embryos were used for the analyses. EVs were separated from culture medium of W2 and W3 using a protocol that combines ultrafiltration and ultracentrifugation. EVs characteristics (size and concentration) from individual culture medium were determined by nanoparticle tracking analysis (NTA), analysed according its respective experimental group and then compared with zona pellucida pore size determined by scanning electron microscopy. The miRNA content of EVs was evaluated by RNA-sequencing. The mean size of EVs was 109.4 and 106.6 nm in W2 and W3, respectively (P > 0.05). Concentration was lower (P < 0.05) in W2 (9.4 × 108 particles mL−1) compared to W3 (2.4 × 109 particles mL−1). The zona pellucida of embryos from W2 had fewer pores and these were larger in contrast to the W3 embryos. A total of 140 miRNAs were identified, 79 of which were differentially expressed (P < 0.05): 28 were up-regulated (log2 fold change > 0.5) and 51 down-regulated (log2 fold change < -0.5) in EVs secreted during compaction (W2) compared with those secreted during blastulation (W3). Twenty miRNAs were equally expressed. It seems that the concentration and content of miRNAs of EVs secreted by bovine embryos depends on the developmental period during which the vesicles were released. This could be explained by the size and number of pores in the zona pellucida and the molecular changes that take place in the embryos during the preimplantation period. EVs secreted to culture medium could be useful as early biomarkers for embryo selection; however, the day of culture media collection must be considered.