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Abstract：
BACKGROUND: 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).
METHODS: 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).
RESULTS: 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.
CONCLUSIONS: 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.
METHODS: 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).
RESULTS: 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.
CONCLUSIONS: 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.
摘要：
背景：在卵泡液中，细胞外囊泡（EV）通过其货物microRNAs（miRNA）引导卵母细胞生长。这里,我们通过连接在电动汽车中发现的miRNA来研究电动汽车及其货物miRNA的作用，来自单个卵泡的液体，其卵母细胞成为胚泡（有能力）或不（无能力）的能力。
方法：解剖牛窦卵泡，分类为小（2-4毫米）或大（5-8毫米）和相应的卵母细胞进行个体成熟，受精和胚胎培养到囊胚期。根据卵泡大小和相应卵母细胞产生胚泡的能力，将卵泡液分为4组（4个重复）。分离卵泡液来源的电动汽车，characterized,并进行miRNA测序(IlluminaMiseq)以评估4组中的差异表达(DE)。通过在体外成熟(IVM)期间补充模拟物和抑制剂来进行miR-34c对胚胎发育的作用的功能验证。
结果：当不考虑卵泡大小时，我们鉴定了与卵母细胞能力相关的16个DEmiRNA。在大卵泡和小卵泡中，每组46个DEmiRNAs驱动胚泡形成。来自感受态小卵泡和大卵泡的电动汽车的比较揭示了90个DEmiRNA。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DEmiRNA靶向的最富集的途径。我们确定bta-miR-34c在含有感受态卵母细胞的卵泡液中含量最高。在IVM期间补充miR-34c模拟物和抑制剂并不影响胚胎发育。然而,囊胚质量,更高的细胞数量证明了这一点，在存在miR-34c模拟物的情况下，卵母细胞IVM后显著改善，而miR-34c抑制剂导致相反的效果。
结论：本研究证明了来自卵泡液来源的EV的miRNA对卵母细胞能力获取的调节作用，为进一步理解miRNAs在卵母细胞成熟和胚胎发育中的意义提供了依据。来自含有感受态卵母细胞的卵泡液中的EV中miR-34c的上调以及在IVM期间添加的miR-34c模拟物对所得胚泡的积极影响表明其在卵母细胞能力中的关键作用。
方法：解剖牛窦卵泡，分类为小（2-4毫米）或大（5-8毫米）和相应的卵母细胞进行个体成熟，受精和胚胎培养到囊胚期。根据卵泡大小和相应卵母细胞产生胚泡的能力，将卵泡液分为4组（4个重复）。分离卵泡液来源的电动汽车，characterized,并进行miRNA测序(IlluminaMiseq)以评估4组中的差异表达(DE)。通过在体外成熟(IVM)期间补充模拟物和抑制剂来进行miR-34c对胚胎发育的作用的功能验证。
结果：当不考虑卵泡大小时，我们鉴定了与卵母细胞能力相关的16个DEmiRNA。在大卵泡和小卵泡中，每组46个DEmiRNAs驱动胚泡形成。来自感受态小卵泡和大卵泡的电动汽车的比较揭示了90个DEmiRNA。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DEmiRNA靶向的最富集的途径。我们确定bta-miR-34c在含有感受态卵母细胞的卵泡液中含量最高。在IVM期间补充miR-34c模拟物和抑制剂并不影响胚胎发育。然而,囊胚质量,更高的细胞数量证明了这一点，在存在miR-34c模拟物的情况下，卵母细胞IVM后显著改善，而miR-34c抑制剂导致相反的效果。
结论：本研究证明了来自卵泡液来源的EV的miRNA对卵母细胞能力获取的调节作用，为进一步理解miRNAs在卵母细胞成熟和胚胎发育中的意义提供了依据。来自含有感受态卵母细胞的卵泡液中的EV中miR-34c的上调以及在IVM期间添加的miR-34c模拟物对所得胚泡的积极影响表明其在卵母细胞能力中的关键作用。
