Estrous cycle impacts microRNA content in extracellular vesicles that modulate bovine cumulus cell transcripts during in vitro maturation. Estrous cycle impacts microRNA content in extracellular vesicles that modulate bovine cumulus cell transcripts during in vitro maturation

Purpose: Extracellular vesicles (EVs) are nanoparticles that can be secreted by different cells, including cells found within the ovarian follicle. Currently, EVs are considered an important form of intercellular communication, since they carry biological contents. The goal of this study was to survey the effects of small EVs from follicles at different estrous cycle stage in bovine cumulus cells. Methods: We used an established model to obtain follicular fluid (FF) at early and late estrous cycle stage according to corpus luteum appearance, corresponding to low and high progesterone (P4) levels, respectively. We collected FF from 3-6 mm follicles and isolated small EVs, which were used as a supplement during in vitro maturation (IVM). Cumulus cells were collected from cumulus-oocyte complexes (COCs) pools and the RNAs were obtained and subjected to RNA sequencing. Results: The results showed that small EVs from different estrous cycle stage are capable to affect transcripts in cumulus cells and modulate different pathways and biological processes related to oocyte maturation, ovulation and immune response. Conclusion: This study demonstrated that small EVs from low and high P4 group impact the RNA profile in cumulus cells after 9 hours of in vitro maturation. Overall design: Cumulus-oocyte complexes (COCs; n=20) were maintained in maturation media without small EVs (EVs free FCS) or with small EVs from low or high P4 follicles for 9 hours. Six replicates of IVM were performed. Cumulus cells from 2 replicates were pooled to form 3 pools of which supplementation group, which were used for RNA sequencing.

研究目的
细胞外囊泡（Extracellular vesicles, EVs）是一类可由多种细胞分泌的纳米颗粒，其中也包括卵巢卵泡内的细胞。目前，细胞外囊泡被视为细胞间通讯的重要载体，因其可携带生物活性物质。本研究旨在探究不同发情周期阶段的牛卵泡来源小细胞外囊泡对卵丘细胞的影响。

研究方法
我们采用已建立的实验模型，根据黄体形态区分早期与晚期发情周期的卵泡液（follicular fluid, FF），分别对应孕酮（progesterone, P4）水平较低和较高的组别。收集3~6 mm卵泡的卵泡液，分离得到小细胞外囊泡，将其作为添加物用于卵母细胞体外成熟（in vitro maturation, IVM）培养体系。从卵丘-卵母细胞复合体（cumulus-oocyte complexes, COCs）池中分离卵丘细胞，提取总RNA并进行RNA测序。

研究结果
结果表明，不同发情周期阶段来源的小细胞外囊泡可调控卵丘细胞的转录组表达，显著影响与卵母细胞成熟、排卵及免疫应答相关的多条信号通路与生物学过程。

研究结论
本研究证实，经9小时体外成熟培养后，低孕酮组与高孕酮组来源的小细胞外囊泡均可改变卵丘细胞的RNA表达谱。

整体实验设计
将20枚卵丘-卵母细胞复合体（COCs）分为三组，分别置于不含小细胞外囊泡的胎牛血清（EVs free FCS）成熟培养基、添加低孕酮组卵泡来源小细胞外囊泡的成熟培养基，以及添加高孕酮组卵泡来源小细胞外囊泡的成熟培养基中培养9小时。本实验共开展6次体外成熟重复培养，每2次重复的卵丘细胞混合为1个样本池，最终每个处理组获得3个样本池用于RNA测序。