卵巢颗粒细胞对卵子老化的影响及抑制晚期糖基化终末产物对卵子质量的干预研究

The development of a competent oocyte intimately depends on the maintenance of energetic homeostasis in the ovarian and follicular microenvironment.Starting from the molecular evidence for energy perturbations in the whole ovary, we discuss about the involvement of endogenous highly reactive metabolites in follicle aging. On this basis, the process of ovarian aging can be viewed as the progressive and irreversible accumulation of damage to macromolecular integrity leading to loss of metabolic homeostasis and decrease of primary functions. Granulosa cells perform a continuous crosstalk between the somatic and oocyte,and increase production of toxic metabolites might be relevant to age-related oxidative stress in oocyte.More than half a century after the proposal of the free radical theory of aging, research on female reproductive aging has provided compelling evidence for the key role of oxidative stress in the age-related decline of ovarian function.Oxidative stress in the ovarian follicle was obtained by research on stress signaling pathways in older granulosa cells. The molecular marker of oxidative stress on gradulosa cells have been proved to work as a effecive predictive indicator for ART clincal outcome.Reactive carbonyl species (RCS) is a stable and highly reactive endogenous metabolites and providing a more deleterious insult to the macromolecular integrity of the cell and extracellular microenvironment. The aged cumulus cells exhibited a decreased ability to protect the oocyte from carbonyl stress. RCS in follicular fluid and in serum correlated negatively with follicular growth, fertilization, and embryonic development. There is a the positive feedback loop between Reactive Oxidative Species(ROS)and RCS and which can promote the accumulation of toxic byproducts and oocyte aging. Here we hypothesize that ROS and RCS in the aged granulosa cells are the key factors in the pathogenesis of aging oocyte. The coculture system of aged gradulosa cells and young oocyte will be perform in CD-1 mice and Polycystic ovary syndrome（PCOS）women undergo the Assisted Reproductive Technology(ART). We test the activity of ROS and RCS systems between the aged gradulosa/young oocyte and the yong gradulosa/young oocyte. The mitochondrial membrane potential, apoptosis analysis, ADP/ATP are utilized to evaluate the vitality and function of oocyte. The Aging associated alteration of oocyte-specific gene expressions will be examined by RT-RCR to assess the effect of aged gradulosa cells on the process of oocyte aging. Finally, we will detect whether the aminoguanidine and metformin,the potential blocker of ROS and Advanced Glycation End Product(AGE), could protected the oocyte from the endogenous highly reactive stress of aged gradulosa cells.

卵子老化的氧化应激与近年来提出的羰基毒化学说为改善卵子老化导致的生育力下降提供了新的治疗思路。已证实高龄妇女颗粒细胞中活性氧簇（ROS）与活性羰基类物质（RCS）堆积明显增多，我们选择在小鼠与多囊卵巢患者样本中，通过卵子与颗粒细胞的共培养模型，研究老化颗粒细胞对正常卵子老化进程的影响：通过激光共聚焦检测线粒体膜电位（JC-1）、TUNEL凋亡率、还原型与氧化型的谷胱甘肽含量以及ADP/ATP来评价卵子的质量和代谢功能；采用ELISA检测ROS活性与乙二醛酶活性来评估卵子氧化应激与羰基毒化代谢产物堆积情况；应用聚合酶链式反应（PCR）研究端粒酶活性，卵子老化相关分子标志物来直接评价卵子老化的进程。并通过在体外成熟培养体系中添加晚期糖基化终末产物（AGE）的抑制剂氨基胍或二甲双胍来抑制或逆转卵子老化的病理过程，揭示卵子老化的机制，以期提高高龄妇女辅助生殖技术（ART）的临床结局。

卵子老化的氧化应激与近年来提出的羰基毒化学说为改善卵子老化导致的生育力下降提供了新的治疗思路。已证实高龄妇女颗粒细胞中活性氧簇（ROS）与活性羰基类物质（RCS）堆积明显增多，我们将老化卵泡液作用于体外成熟的小鼠卵母细胞，另外将NMN添加入体外成熟培养液中，观察其对小鼠卵母细胞体外成熟及对线粒体功能的影响。高龄患者的卵泡液中ROS增加。我们发现说明年轻患者卵泡液的添加并未影响小鼠卵母细胞体外成熟率，高龄患者的卵泡液添加则降低了小鼠卵母细胞体外成熟率。添加老化卵泡液添加NMN后ROS含量均降低，氧化应激反应均下降。我们的研究发现sRAGE随龄减低，对高龄患者而言，sRAGE与卵子质量，受精率，胚胎发育能力及良好的妊娠结局呈正比。