Ovarian follicular fluids (FFs) contain several kinds of regulatory factors that maintain a suitable microenvironment for oocyte development. Extracellular vesicles (EVs) are among the factors that play essential roles in regulating follicle and oocyte development through their cargo molecules that include microRNAs (miRNAs). This study aimed to investigate small-EV (s-EV) miRNAs in porcine FFs and their potential association with oocyte quality. Individual aspirated oocytes were stained with lissamine green B stain (LB), a vital stain for oocyte quality, and each oocyte was classified as high-quality (unstained; HQ) or low-quality (stained; LQ). FFs corresponding to oocytes were pooled together into HQ and LQ groups. Small-EVs were isolated from FFs, characterized, and their miRNA cargo was identified using the Illumina NovaSeq sequencing platform. Additionally, s-EVs from the HQ and LQ groups were utilized to investigate their effect on oocyte development after co-incubation during in vitro maturation. A total of 19 miRNAs (including miR-125b, miR-193a-5p, and miR-320) were significantly upregulated, while 23 (including miR-9, miR-206, and miR-6516) were downregulated in the HQ compared to the LQ group. Apoptosis, p53 signaling, and cAMP signaling were among the top pathways targeted by the elevated miRNAs in the HQ group while oocyte meiosis, gap junction, and TGF-beta signaling were among the top pathways targeted by the elevated miRNAs in the LQ group. The supplementation of small-EVs during maturation does not affect the oocyte developmental rates. However, LQ s-EVs increase the proportion of oocytes with homogeneous mitochondrial distribution and decrease the proportion of heterogeneous distribution. Our findings indicated that FF-EVs contain different miRNA cargos associated with oocyte quality and could affect the mitochondrial distribution patterns during oocyte maturation.

中文翻译：

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来自猪卵泡液的小细胞外囊泡及其 microRNA 货物：与卵母细胞质量的潜在关联

卵巢卵泡液 (FFs) 含有多种调节因子，可维持适合卵母细胞发育的微环境。细胞外囊泡 (EV) 是通过其包括 microRNA (miRNA) 在内的货物分子在调节卵泡和卵母细胞发育中发挥重要作用的因素之一。本研究旨在调查猪 FF 中的小 EV (s-EV) miRNA 及其与卵母细胞质量的潜在关联。用丽丝胺绿 B 染色剂 (LB) 对单个吸出的卵母细胞进行染色，该染色剂是卵母细胞质量的重要染色剂，每个卵母​​细胞被分类为高质量（未染色；HQ）或低质量（染色；LQ）。将对应于卵母细胞的 FF 汇集到 HQ 和 LQ 组中。小型电动汽车从 FF 中分离出来，其特点是，他们的 miRNA 货物是使用 Illumina NovaSeq 测序平台鉴定的。此外，来自 HQ 和 LQ 组的 s-EV 被用来研究它们在体外成熟期间共同孵化后对卵母细胞发育的影响。与对照组相比，总部共有 19 个 miRNA（包括 miR-125b、miR-193a-5p 和 miR-320）显着上调，而 23 个（包括 miR-9、miR-206 和 miR-6516）下调LQ组。细胞凋亡、p53 信号和 cAMP 信号是 HQ 组中升高的 miRNA 靶向的主要途径，而卵母细胞减数分裂、间隙连接和 TGF-β 信号是 LQ 组中升高的 miRNA 靶向的主要途径。在成熟过程中补充小型电动汽车不会影响卵母细胞的发育速度。然而，LQ s-EVs增加了线粒体分布均匀的卵母细胞比例，降低了分布不均匀的卵母细胞比例。我们的研究结果表明，FF-EV 含有与卵母细胞质量相关的不同 miRNA 货物，并可能影响卵母细胞成熟过程中的线粒体分布模式。