99 Including extracellular vesicles from follicular fluid of competent oocytes during in vitro maturation improves blastocyst yield in cattle

Bovine oocyte competence can be improved by simulated physiological oocyte maturation (SPOM), which extends the communication between the somatic compartment and the oocyte (Sugimura et al. 2018 J. Reprod. Dev. 64, 233–241). The extracellular vesicles (EVs) and the microRNAs (miRNAs) present in the follicular fluid (FF) play a crucial role in this bidirectional communication (Macaulay et al. 2015 Biol. Reprod. 94, 16). This work aimed to evaluate whether including EVs isolated from follicles enclosing competent oocytes into an SPOM system would improve oocyte competence in cattle. FF was collected from individual follicles of abattoir-derived ovaries, diluted 1:20 in PBS, centrifuged, and stored at −80°C. At the same time, the corresponding oocytes were cultured in a Well of the Well culture system (VivaVitro) to track individual developmental capacity. Three pools of FF from 20 follicles corresponding to three categories of oocytes were collected: cleaved oocytes that reached the blastocyst stage (B), cleaved oocytes with arrested development (AC), and uncleaved oocytes (UN). From each FF pool, EVs were isolated through ultracentrifugation and stored at −20°C. NanoSight (Malvern Panalytical) determined EV size and concentration. After RNA isolation, small RNA libraries were obtained using the TruSeq kit (Illumina). The differentially expressed miRNAs were identified by EdgeR and quantified using the miRDeep2 quantifier module. Finally, abattoir-derived bovine oocytes (n = 258 in four replicates) were matured in vitro using the SPOM system (Sugimura et al. 2018) in the presence of either 0 (control) or 1 × 10⁷ EVs derived from group B. The EV concentration was chosen after a preliminary dose-response trial. After IVM, oocytes were fertilized and cultured by standard procedures (Di Francesco et al. 2011 Anim. Reprod. Sci. 123, 48–53). Cleavage and blastocyst rates were evaluated after 8 days of culture. Analysis of variance revealed that the EV size and concentration were higher in the B group than in the AC group (P < 0.05), with no differences compared with the UN group (213.4 ± 3.1, 202.1 ± 2.3, and 208.9 ± 1.2 nm, and 4.0 ± 0.2 e+11, 2.8 ± 0.1 e+11, and 3.9 ± 0.6 e+11 particles mL⁻¹, respectively). Concerning miRNA cargo, EVs from the B group had higher expression of bta-miR-143 and lower expression of bta-miR-21–5p than group UN. In the B group EVs, bta-miR-19a was overexpressed compared with the AC group. Finally, the EVs from the AC group showed a higher expression of bta-miR-19a and a lower expression of bta-let-7f than the UN group. Notably, the inclusion of group B-derived EVs during IVM did not affect the cleavage rate (82.7 ± 2.7% vs. 79.4 ± 2.8%) but increased blastocyst rates (59.1 ± 7.1% vs. 41.8 ± 7.0%; P < 0.05). In conclusion, including EVs isolated from FF of competent oocytes into an SPOM system was shown to improve in vitro bovine embryo development. The different miRNA cargo of EVs isolated from FFs enclosing oocytes of contrasting competence suggests a potential role of these miRNAs in regulating the expression of competence-associated genes, which requires further investigations.