氧化应激对猪有腔卵泡闭锁的影响及机制

Many previous researches have proved that apoptosis of granulosa cells in antral follicles and follicular atresia could be induced by artificial oxidative stress. However, the factors underlying follicular atresia when antral follicles under the natural state of oxidative stress is not still clear. Follicular fluid provides the micro-environment for granulosa cell growth and development, but whether the hormones and growth factors in follicular fluid will affect oxidative stress response of granulosa cells still needs further exploration. Based on the early signal of follicular atresia, follicles of different sizes, different stages and diameters, redox states, levels of hormones and growth factors in follicular fluids, the correlationship among these indicators will be analyzed to determine the occurrence of oxidative stress under natural state, and its influence on the oxidation of antral follicular granulosa cell apoptosis. Subsequently, combined with follicle and granulosa cell culture in vitro, to study roles of the factors in follicular fluids on cellular oxidative stress tolerance and their mechanisms. In terms of molecular mechanisms underlying the oxidative stress-induced apoptosis in porcine granulosa cells, we will focus on the regulatory roles of JNK/14-3-3/FoxO1 signaling pathway. Finally, influence of dietary antioxidants on follicular oxidative status and reproductive performance will be investigated. The results will be helpful for improvement of animal breeding, raising pig fertility, and treatment of human reproductive diseases.

人为造成卵泡氧化应激能够诱导有腔卵泡颗粒细胞凋亡与卵泡闭锁已为研究所证实，然而自然状态下氧化应激是否是有腔卵泡闭锁的启动因素缺少研究；另一方面，卵泡液是卵泡颗粒细胞生长发育的微环境，其所含激素与生长因子是否影响颗粒细胞对氧化应激的反应有待深入探索。本试验选取猪不同大小、不同卵泡期阶段以及不同闭锁程度卵泡，在测定卵泡氧化还原状态、卵泡液激素与生长因子水平、卵泡凋亡早期信号的基础上，通过各指标间的关联性分析明确自然状态下猪有腔卵泡氧化应激的发生状况及其对卵泡凋亡的影响，并结合卵泡与颗粒细胞体外培养试验探索卵泡液因子对颗粒细胞氧化应激耐受性的影响及机制。在氧化应激诱导猪卵泡颗粒细胞凋亡的分子机制方面，重点探讨JNK/14-3-3/FoxO1信号通路的功能。最后，研究日粮中添加抗氧化剂对母猪卵泡氧化应激以及繁殖性能的影响。研究结果对于丰富繁殖理论、提高猪繁殖力、治疗人类生育类疾病都将具有重要意义。

猪卵泡生长发育伴随氧化应激的频繁发生，氧化应激对卵泡颗粒细胞凋亡以及卵泡闭锁的影响及机制值得深入研究。本项目开展了猪有腔卵泡凋亡早期信号与卵泡氧化/还原状态的关联性分析、卵泡液激素与生长因子影响卵泡凋亡的机制、氧化应激诱导猪颗粒细胞凋亡的分子机制以及卵巢保护性抗氧化剂的筛选与应用四个方面的研究，研究结果表明：（1）猪有腔卵泡的闭锁伴随卵泡液ROS水平的显著升高；健康卵泡中ROS水平变异大，并且ROS水平与死亡-受体途径的颗粒细胞凋亡显著关联。（2）卵泡液FSH与TGFα水平与卵泡液ROS水平极显著负相关，提示这两种卵泡液因子可能调控卵泡ROS的生成。（3）健康卵泡间颗粒细胞增殖活性存在巨大差异，转录组测序表明DNA损伤修复、低氧环境以及氧化应激相关的FoxO信号通路是影响颗粒细胞增殖活性的主要因素，该发现为研究卵泡闭锁的启动因素提供了新的方向。（4）氧化应激通过PKCδ/JNK/14-3-3/FoxO1信号通路诱导卵泡颗粒细胞凋亡，新发现PKCδ和14-3-3蛋白参与了氧化应激对颗粒细胞凋亡的诱导作用。（5）FSH、褪黑素通过抑制自噬活性保护卵泡颗粒细胞免受氧化损伤，明确了FSH、褪黑素调控卵泡颗粒细胞氧化损伤的新机制。（6）卵泡生长的低氧环境能够引起颗粒细胞G1/S期阻滞，卵泡液因子IGF1能够通过HIF1α/FOXO1/Cdkn1b信号通路解除这种抑制作用；该结果为调控卵泡发育提供了新的靶点。（7）FSH能够显著促进低氧条件下颗粒细胞HIF1α的表达，并通过HIF1α/PINK-Parkin信号通路促进颗粒细胞线粒体自噬，从而减少因线粒体损伤导致的细胞凋亡，与此同时FSH促进了线粒体的新生合成，避免了线粒体数量的大幅度下降；研究结果揭示了FSH调控卵泡发育的新机制。（8）研发成功一种提高母猪繁殖力的技术方法。