Expression profile and gap-junctional transfer of microRNAs in the bovine cumulus-oocyte complex. Expression profile and gap-junctional transfer of microRNAs in the bovine cumulus-oocyte complex

MicroRNAs (miRNA) are important regulators of oocyte maturation, playing a key role in modulating gene expression both in a temporal- and spatial-specific manner. These small non-coding RNAs are involved in important processes during oocyte maturation, acting as messengers between the oocyte and its surrounding cumulus cells. Despite its significance, the bidirectional communication mechanism is still unknown. To test miRNA communication between oocyte and surrounding cumulus cells through the gap junctions the gap junctions were either blocked with carbenoxolone or not. MiRNA sequencing of oocytes at 1, 6, and 22 hours of in vitro maturation was then performed. Among the differentially expressed miRNAs, bta-miR-21-5p, a regulator of cumulus cell viability and oocyte maturation, was the only previously known miRNA. Furthermore, by labeling a bta-miR-21-5p mimic with FAM, crossing of this miRNA through the gap junctions within the cumulus-oocyte complex could be visualized and internalization in the oocyte was confirmed by RT-qPCR. In conclusion, this study provides, for the first time, evidence that miRNA communication within the bovine cumulus-oocyte complex is enabled through the gap junctional network. Overall design: To investigate the miRNA communication in the bovine COC through the gap-junctional network, we blocked the gap-junctions using carbenoxolone. Together with three control groups (normal maturation conditions, denuded state maturation, denuded stat maturation with COCs in co-culture) oocytes were collected after 1, 6, and 22 hours of maturation for miRNAseq on each sample in triplicate.

微小核糖核酸（microRNA，miRNA）是卵母细胞成熟的重要调控因子，以时空特异性方式调控基因表达并发挥关键作用。这类小型非编码RNA参与卵母细胞成熟过程中的诸多重要生理过程，充当卵母细胞与其周围卵丘细胞之间的信号信使。尽管该过程意义重大，但这种双向通讯的具体机制目前仍未明确。为探究卵母细胞与周围卵丘细胞通过间隙连接进行的miRNA通讯，研究人员分别采用羧苄酮（carbenoxolone）阻断间隙连接，或不进行任何阻断处理。随后对体外成熟1、6和22小时的卵母细胞开展miRNA测序。在差异表达的miRNA中，bta-miR-21-5p——一种可调控卵丘细胞活性与卵母细胞成熟的miRNA——是此前仅有的已知相关miRNA。此外，研究人员用羧基荧光素（FAM）标记bta-miR-21-5p模拟物，可观测到该miRNA穿过牛卵丘-卵母细胞复合体（cumulus-oocyte complex，COC）内的间隙连接，并通过实时定量聚合酶链式反应（RT-qPCR）证实其在卵母细胞内的内化过程。综上，本研究首次提供直接证据，证实牛卵丘-卵母细胞复合体内部的miRNA通讯可通过间隙连接网络实现。总体实验设计：为探究牛卵丘-卵母细胞复合体（cumulus-oocyte complex，COC）内通过间隙连接网络实现的miRNA通讯，研究人员使用羧苄酮阻断间隙连接。同时设置三组对照组：正常成熟条件组、裸卵成熟组、与卵丘-卵母细胞复合体共培养的裸卵成熟组。分别在体外成熟1、6、22小时后收集卵母细胞，对每份样本进行三次重复的miRNA测序。