Within the follicular fluid, extracellular vesicles (EVs) guide oocyte growth through their cargo microRNAs (miRNAs). Here, we investigated the role of EVs and their cargo miRNAs by linking the miRNAs found in EVs, derived from the fluid of an individual follicle, to the ability of its oocyte to become a blastocyst (competent) or not (non-competent). Bovine antral follicles were dissected, categorized as small (2–4 mm) or large (5–8 mm) and the corresponding oocytes were subjected to individual maturation, fertilization and embryo culture to the blastocyst stage. Follicular fluid was pooled in 4 groups (4 replicates) based on follicle size and competence of the corresponding oocyte to produce a blastocyst. Follicular fluid-derived EVs were isolated, characterized, and subjected to miRNA-sequencing (Illumina Miseq) to assess differential expression (DE) in the 4 groups. Functional validation of the effect of miR-34c on embryo development was performed by supplementation of mimics and inhibitors during in vitro maturation (IVM). We identified 16 DE miRNAs linked to oocyte competence when follicular size was not considered. Within the large and small follicles, 46 DE miRNAs were driving blastocyst formation in each group. Comparison of EVs from competent small and large follicles revealed 90 DE miRNAs. Cell regulation, cell differentiation, cell cycle, and metabolic process regulation were the most enriched pathways targeted by the DE miRNAs from competent oocytes. We identified bta-miR-34c as the most abundant in follicular fluid containing competent oocytes. Supplementation of miR-34c mimic and inhibitor during IVM did not affect embryo development. However, blastocyst quality, as evidenced by higher cell numbers, was significantly improved following oocyte IVM in the presence of miR-34c mimics, while miR-34c inhibitors resulted in the opposite effect. This study demonstrates the regulatory effect of miRNAs from follicular fluid-derived EVs on oocyte competence acquisition, providing a further basis for understanding the significance of miRNAs in oocyte maturation and embryonic development. Up-regulation of miR-34c in EVs from follicular fluid containing competent oocytes and the positive impact of miR-34c mimics added during IVM on the resulting blastocysts indicate its pivotal role in oocyte competence.

中文翻译：

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从卵泡到囊胚：来自卵泡液来源的细胞外囊泡的 microRNA-34c 调节囊胚质量


在卵泡液中，细胞外囊泡 (EV) 通过其负载 microRNA (miRNA) 引导卵母细胞生长。在这里，我们通过将来自单个卵泡液体的 EV 中发现的 miRNA 与其卵母细胞成为囊胚（有能力）或没有（无能力）的能力联系起来，研究了 EV 及其货物 miRNA 的作用。牛窦卵泡被解剖，分为小（2-4毫米）或大（5-8毫米），相应的卵母细胞进行个体成熟、受精和胚胎培养至囊胚阶段。根据卵泡大小和相应卵母细胞产生囊胚的能力，将卵泡液分为 4 组（4 个重复）。分离、表征卵泡液来源的 EV，并进行 miRNA 测序 (Illumina Miseq) 以评估 4 组中的差异表达 (DE)。通过在体外成熟 (IVM) 过程中补充模拟物和抑制剂来对 miR-34c 对胚胎发育的影响进行功能验证。在不考虑卵泡大小的情况下，我们鉴定了 16 个与卵母细胞能力相关的 DE miRNA。在大卵泡和小卵泡内，每组都有 46 个 DE miRNA 驱动囊胚形成。比较来自有能力的小和大卵泡的 EV 发现了 90 个 DE miRNA。细胞调节、细胞分化、细胞周期和代谢过程调节是来自感受态卵母细胞的 DE miRNA 靶向的最丰富的途径。我们确定 bta-miR-34c 在含有活性卵母细胞的卵泡液中含量最丰富。 IVM 期间补充 miR-34c 模拟物和抑制剂不会影响胚胎发育。 然而，在 miR-34c 模拟物存在的情况下，卵母细胞 IVM 后，囊胚质量（细胞数量增加）显着改善，而 miR-34c 抑制剂则产生相反的效果。本研究证明了卵泡液来源的EVs中的miRNA对卵母细胞能力获得的调节作用，为理解miRNA在卵母细胞成熟和胚胎发育中的重要性提供了进一步的基础。来自含有活性卵母细胞的卵泡液中 miR-34c 的上调以及 IVM 期间添加的 miR-34c 模拟物对所得囊胚的积极影响表明其在卵母细胞活性中的关键作用。