149 Identification of microRNAs associated with bovine in vitro embryo development in extracellular vesicles from follicular fluid

The follicular fluid (FF) contains a variety of regulatory factors that support oocyte development. Among these factors, extracellular vesicles (EVs) play a crucial role in regulating oocyte and follicular growth through the transport of signalling molecules such as microRNAs (miRNAs). This work aimed to study the relationship between EV-derived miRNAs from differently sized follicles and oocyte developmental competence. To do so, individual follicles (IF) were dissected from slaughterhouse-derived bovine ovaries, measured, and categorised as small ([S] < 4 mm, n = 118) or large ([L] > 5 mm, n = 82). Oocytes (n = 200; 20 replicates) were liberated by rupturing the IF and collected individually. Immediately after, the FF from IF was collected separately in a 2-mL tube and centrifuged at 1000 × g at 4°C for 5 min to track back the individual oocyte to the original follicle, and stored at −80°C. Oocytes were cultured individually for in vitro maturation-fertilisation-culture in 20-µL droplets under paraffin oil. The blastocyst yield was measured at Day 8. Later, FFs from IF were pooled according to the blastocyst outcome of the original oocyte (Embryo Yes [EY] or Embryo No [EN]) and the follicle size (S or L) to have a total number of four FF groups (small follicle EY [SEY], small follicle EN [SEN], large follicle EY [LEY] and large follicle EN [LEN]). Each FF group consisted of four replicates of 200-µL each. All the FF groups were processed for EV isolation through qEV size-exclusion column and concentrated with Amicon filters (Merck). Recovered EVs were characterised by Western blot, nanoparticle tracking analysis, and electron microscopy. EVs from each group were used for total RNA extraction (Norgen Biotek) and a small RNA library was constructed (Qiagen). Sequencing was performed on the Illumina Miseq. The reads were mapped to the Bos Taurus genome using miRPro software with miRbase mapper. Differential expression analysis was performed using the edgeR package and pathway enrichment analysis was performed using the fgsea package. Differentially expressed EVs/FF miRNAs were identified in LEN versus LEY (n = 46; 30 down- and 16 upregulated; P < 0.05), SEN versus SEY (n = 46; 24 down- and 22 upregulated; P < 0.05), LEY versus SEY (n = 90, 44 down- and 46 upregulated; P < 0.05), and LEN versus SEN (n = 40, 26 down- and 14 upregulated; P < 0.05). Moreover, we compared EVs/FF miRNAs expression of blastocyst and non-blastocyst independent of follicle size resulting in 16 differential features, of which 11 were down- and 5 upregulated (P < 0.05). For this last comparison, the most enriched biological processes were signalling pathway response, cell differentiation, extracellular assembly, phosphorylation processes regulation, and apoptotic process regulation, suggesting a potential role of these miRNAs in oocyte competence acquisition. Furthermore, these results support the critical role of miRNAs from FF/EVs in maintaining a suitable environment for oocyte growth during folliculogenesis. The role of targeted miRNAs from EVs as biomarkers of oocyte quality will be studied in the future.