BMP15/GDF9—miR-375—BMPR2循环负反馈通路在牛卵丘细胞增殖与凋亡中的作用与机制

BMP15 and GDF9, oocyte-secreted factors, play a key role in cross-dialogue between oocytes and cumulus cells in mammals. However, how BMPR2, type 2 receptor for both GDF9 and BMP15, is regulated in the cumulus cells and what is the mechanism for the synergy effects of BMP15 and GDF9 on cumulus cells are still unclear. Our previous studies found that BMPR2 is a direct target of miR-375 in bovine cumulus cells, while exogenous BMP15 can significantly stimulate miR-375 expression in cumulus cells and then significantly reduced BMPR2 level. The current project will continue to clarify miR-375-mediated the effects of BMP15 or/and GDF9 alone or cooperate on cumulus cell proliferation and apoptosis; Transcriptome analysis the synergy roles of BMP15 and GDF9 on cell proliferation and apoptosis-related signaling pathways in bovine cumulus cells, and identify the unknown genes which involve in this biological physiology; Systematic characterize of BMP15/GDF9-miR-375-BMPR2 negative feedback loop pathway, and illuminate its regulatory mechanism involved in cell proliferation and apoptosis in bovine cumulus cell. Our results will not only provide a theoretical basis for understanding the mechanism during oocyte development, but also provide theoretical support to establish better simulation environment for in vitro maturation of oocytes.

哺乳动物卵母细胞特异分泌的BMP15与GDF9在卵母细胞与卵丘细胞交叉对话中发挥关键作用。然而，卵丘细胞如何调节BMP15与GDF9共同受体BMPR2的表达，以及BMP15与GDF9协同作用于卵丘细胞而产生加性效应的分子机制都不清楚。我们前期发现牛卵丘细胞内源的miR-375可以靶向调控BMPR2；外源BMP15使卵丘细胞miR-375表达上调，BMPR2表达下调。据此，本项目将深入研究miR-375在BMP15与GDF9单独或协同介导的牛卵丘细胞增殖与凋亡中的关键功能；全转录组水平分析BMP15/GDF9协同作用对牛卵丘细胞增殖与凋亡相关信号通路的影响，并鉴定其中未知的关键基因；系统识别BMP15/GDF9-miR-375-BMPR2循环负反馈相关通路，解析其在牛卵丘细胞增殖与凋亡中的调控机制。研究结果将为揭示卵泡与卵母细胞发育机理提供理论基础，并为完善卵母细胞体外成熟体系提供理论支持。

哺乳动物卵母细胞特异分泌的BMP15与GDF9在卵母细胞与卵丘细胞交叉对话中发挥关键作用。然而，卵丘细胞如何调节BMP15与GDF9共同受体BMPR2的表达，以及BMP15与GDF9协同作用于卵丘细胞而产生加性效应的分子机制尚不清楚。因此，本项目深入研究miR-375在BMP15与GDF9单独及协同调控作用下对牛卵丘细胞增殖与凋亡中的关键功能，并通过全转录组水平分析BMP15/GDF9协同作用对牛卵丘细胞增殖与凋亡相关信号通路的影响，鉴定其中未知的关键基因。研究结果表明，在牛卵丘细胞体外培养液中添加100ng/mL的BMP15、200ng/mL的GDF9、联合添加50ng/mL的BMP15与100ng/mL的GDF9可负调控卵丘细胞中miR-375、正调控 BMPR2 的表达水平。而BMP15、GDF9可通过miR-375影响I型受体ALK4/5/6/7和II型受体BMPR2的表达水平，并激活Smad信号通路，进而调控PTX3、HAS2、PTGS2基因影响卵丘细胞的扩散、增殖及凋亡，并具有显著的协同效应。构建了BMP15和GDF9处理的卵丘细胞差异基因表达谱，明确了USP42、YWHAH、SMAD6和NOG在miRNA-375影响下通过调控CCND2、PCNA、CDC42、 SMAD6影响卵丘细胞增殖与凋亡的关键作用机制，从而系统识别了BMP15/GDF9-miR-375-BMPR2循环负反馈相关通路，解析了其在牛卵丘细胞增殖与凋亡中的调控机制。研究结果将为揭示卵泡与卵母细胞发育机理提供理论基础，并为完善卵母细胞体外成熟体系提供理论支持。