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. 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RNA（MicroRNAs, miRNA）是卵母细胞成熟的重要调控因子，以时间特异性与空间特异性的模式调控基因表达，发挥关键调控作用。这类小型非编码RNA参与卵母细胞成熟过程中的多项关键生理过程，充当卵母细胞与其周围卵丘细胞之间的信号信使。尽管该过程具有重要生物学意义，二者间的双向通信机制至今仍未明确。为验证miRNA通过间隙连接（gap junctions）在卵母细胞与周围卵丘细胞间的通信作用，研究人员分别使用甘珀酸（carbenoxolone）阻断间隙连接，设置未阻断的对照组。随后，对体外成熟1、6及22小时的卵母细胞开展miRNA测序（miRNA sequencing）。在鉴定得到的差异表达miRNA（differentially expressed miRNAs）中，bta-miR-21-5p是目前已知唯一可调控卵丘细胞活性与卵母细胞成熟的miRNA。此外，研究人员利用FAM标记bta-miR-21-5p模拟物，可视化观察到该miRNA通过卵丘-卵母细胞复合体（cumulus-oocyte complex, COC）内的间隙连接进行跨膜转运，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，本研究首次证实，牛源卵丘-卵母细胞复合体（COC）内的miRNA通信可通过间隙连接网络实现。为进一步探究牛COC中基于间隙连接网络的miRNA通信机制，研究人员使用甘珀酸阻断间隙连接，并设置三组对照：正常成熟条件组、裸卵成熟组、裸卵与COC共培养成熟组。在体外成熟1、6、22小时后收集卵母细胞，每份样本设置三次生物学重复，进行miRNA测序。