DNA双链断裂对牛卵丘细胞生长发育、卵母细胞成熟的影响及其关键分子通路解析

DNA-Double strand breaks, in which both strands in the double helix are severed, are particularly hazardous to the cell because they can lead to genome rearrangements. Regulations of Oocyte and Cumulus Cell Interactions are important for ovarian follicular development and oocyte maturation. But the effects and mechanisms of DNA-DSBs in cumulus cells growth, development and oocyte maturation are still unclear. Our previous studies found that DNA double-strand breaks increased with age in bovine cumulus cells and oocytes. Similar patterns were also found in Bleomycin-induced primary cumulus cell injury model, however, it was not observed in passage cells. These are a strong indication that there is a specific DNA-DSBs repair mechanisms in bovine cumulus cells. Gene expression and transcriptome analysis were also carried out to elucidate the pathways and mechanisms of bovine cumulus cell growth and development and oocyte maturation. Critical gene expression and transcriptome analysis were also carried out to elucidate the pathways and mechanisms of bovine cumulus cell growth, development and oocyte maturation. Our researches will not only provide a good basis for understanding the DNA damage and repair mechanisms, but also provide theoretical supports to improve fertility in females.

DNA双链断裂（DNA-DSBs）是DNA双螺旋结构中的双链异常断开并可导致基因组重排的一种严重影响细胞生理状态的DNA损伤。卵巢中卵丘细胞、卵母细胞间的相互调控作用对于卵泡发育及卵子成熟具有重要作用，但卵丘细胞中DNA-DSBs的产生及修复对于自身生长发育及卵母细胞成熟有何影响、具体机制如何尚不明确。项目组研究发现卵丘细胞、卵母细胞中DNA-DSBs程度随年龄逐渐增大；而博来霉素诱导的原代卵丘细胞损伤模型中DNA-DSBs情况与之类似，但传代细胞中却不明显，表明在牛卵丘细胞中存在特定的DNA-DSBs修复机制。据此，本项目将深入研究DNA-DSBs对牛卵丘细胞生长发育、卵母细胞成熟的影响，从全转录组水平解析其在卵丘细胞增殖与凋亡、卵母细胞成熟相关分子通路中的作用、探求其中未知的关键基因及调控机制。研究结果将为揭示DNA损伤的产生、修复机制提供理论基础，并为提高雌性动物繁殖力提供理论依据。

随着年龄的增加,雌性哺乳动物的卵巢储备功能逐渐降低，卵丘细胞凋亡程度增大、卵母细胞的损失明显增高。而在此过程中，卵巢内DNA双链断裂（DNA-DSB）程度不断增大。尽管DNA-DSB影响卵子质量与胚胎发育的研究已有报道，但多集中于对卵子中特定修复通路里的几个关键基因的表达，对于细胞中分子水平上有关如何引发DNA-DSB发生尚不明确。因此，本研究首先利用博来霉素（BLM）创建牛卵丘细胞、卵母细胞DNA-DSB模型，通过高通量测序技术构建DNA-DSB相关基因表达谱。随后，通过RNA干扰方法敲减候选基因的表达，明确其对卵丘细胞的生理状态、功能及卵母细胞成熟及早期胚胎发育的影响。结果表明，200μM 的BLM可稳定诱导卵丘细胞和卵母细胞发生DNA-DSB，而BLM处理前后，卵丘细胞和卵母细胞中有700余个基因（转录本）差异表达，涉及Single-organism cellular process、Chromatin binding、intrinsic component of membrane、anconi anemia pathway、cell cycle、RNA degradation等生物学通路；sulfatase-modifying factor 2 precursor、high mobility group nucleosome-binding domain-containing protein 4、transmembrane protein 246、leukotriene B4 receptor 2、storkhead-box protein 1等基因表达变化最大。最后，本项目明确了COL1A1、DUSP1、Imperatorin、Laminarin、miRNA-29b、miR-375-BMPR2-BMP15/GDF9-Smad 1/2/3/5/8 可通过调控线粒体功能、ROS积累、自噬水平等潜在影响卵丘/卵母细胞的DNA-DSB，进而调控卵丘/卵母细胞的生理状态及功能。研究结果不但有助于明确DNA双链断裂对卵丘细胞与卵母细胞之间相互调控作用的影响，更为深入了解DNA 双链断裂及DNA 损伤的产生、修复机制，通过控制或减少DNA 损伤进一步充分利用雌雄动物种用性能、提高动物繁殖力及人类生殖缺陷模型的建立、相关疾病的治疗。