抗着丝粒抗体对卵母细胞减数分裂的干扰及机制研究

The relationship between antinuclear antibody (ANA) and female fertility is still controversial and recently, we found women with positive anticentromere antibody (ACA, a member of the ANA family) showed a reduced rate of matured oocyte (Fertility and sterility, 2013). This suggests that ACA may interfere with oocyte maturation. Then more findings in our pre-experiment showed: the presence of ACA in human follicular fluid; the developmental potential of mouse embryo was significantly impaired after co-cultured with ACA; and antibody localized on nucleus was detected in embryos from ACA positive mice, which was speculated to be ACA. Thus, we propose the hypothesis that ACA penetrates into the oocyte and interferes with the oocyte meiosis, which leads to oocyte maturation arrest and embryo development suffocate. So this project aims to explore evidence of ACA interfering with meiosis by examining the chromosome arrangement and kinetochore-microtubule attachment in the metaphase of meiosis, and to investigate the molecular mechanism of ACA interfering with meiosis via determination of localization and expression of Mad2 and Bub3 in the metaphase of meiosis, and through the investigation of the association between ACA and production of aneuploid oocytes to verify the interference of ACA on meiosis. This project is expected to gain evidence that ACA interferes with oocyte meiosis, to seek a new breakthrough for mechanism study associated with ANA and female fertility, and to provide scientific basis for exploring and optimizing assisted reproductive treatment for ACA positive patients.

抗核抗体（ANA）对女性生殖功能的影响尚不明确。我们前期研究发现ANA家族成员抗着丝粒抗体（ACA）阳性妇女的成熟卵率显著下降（Fertility and sterility，2013），提示ACA可能干扰卵母细胞成熟。进一步预实验发现：人类卵泡液存在ACA；小鼠胚胎与ACA共培养后发育能力明显受损；ACA可定位于早期胚胎细胞核。由此我们提出科学问题：ACA进入卵母细胞后干扰其减数分裂过程，导致卵母细胞成熟和胚胎发育受阻。本项目拟通过减数分裂中期染色体排列和动粒-微管附着状态检测以及Mad2和Bub3的定位与表达分析，探索ACA干扰卵母细胞减数分裂的直接证据和潜在机制；再通过ACA与非整倍体卵子发生的关联研究，事实验证ACA对减数分裂的干扰。本项目将获取ACA干扰卵母细胞减数分裂的有力证据，为ANA与女性生殖的机制研究找到新的突破点，也为探索和优化ACA阳性妇女的助孕方案提供理论依据。

我们的前期研究发现抗着丝粒抗体（ACA）阳性妇女的成熟卵率显著下降，提示ACA可能干扰卵母细胞成熟。本项目通过：（1）小鼠体外实验发现：与ACA共培养的小鼠早期胚胎细胞内可检测到定位于细胞核的抗体荧光信号，此外，与ACA共培养的小鼠胚胎发育能力明显受损；（2）小鼠体内研究采用人重组着丝粒蛋白A（CENPA）联合弗氏完全佐剂（CFA）成功诱导外周血ACA阳性小鼠模型，模型鉴定采用间接免疫荧光方法联合ELISA方法同时定性和定量检测小鼠外周血ACA。卵母细胞免疫荧光实验发现：ACA阳性小鼠的卵母细胞在减数分裂中期出现染色体排列紊乱、中期赤道板明显增宽等现象，这一结果与我们预期结果一致。然而，小鼠卵母细胞内ACA定位实验未发现外周血ACA阳性小鼠的卵母细胞内存在定位于细胞核的抗体荧光信号，这与我们的预期结果不一致。以上结果表明ACA对卵母细胞减数分裂过程存在干扰作用，但可能不是通过ACA直接进入卵母细胞内并结合于着丝粒来实现，推翻了我们前期的机制设想，暗藏着背后更多的可能性亟待我们思考和探索。