卵母细胞膜蛋白JUNO的冻融性损伤机制及其对受精的影响

卵母细胞在超低温冷冻过程中会产生氧化应激反应，能够引起卵母细胞膜上蛋白发生异常变化，导致受精率显著下降。卵母细胞膜蛋白JUNO作为受精的质膜融合关键蛋白被广泛关注，但其冻融损伤是否影响受精能力尚不清楚。我们前期实验数据表明超低温冷冻影响JUNO在卵母细胞质膜上的连续分布。因此，本研究拟采用荧光染色、免疫印迹、亚硫酸氢盐测序和荧光定量PCR等方法，检测冷冻后卵母细胞JUNO的空间分布和表达、Juno基因甲基化水平、F-actin聚合、氧化应激及相关蛋白或基因表达，明确JUNO冻融损伤的主要因素；通过体外受精，探讨JUNO表达量对受精能力的影响；通过抗氧化剂褪黑素和姜黄素预处理或冷冻过程添加，采用前述方法阐明抗氧化剂对JUNO冻融损伤的保护作用机制。通过上述研究，阐明超低温冷冻对卵母细胞JUNO冻融损伤的分子机制及干预措施、完善受精理论基础，对提高冷冻卵母细胞受精效率具有重要的现实意义。

Oxidative Stress occurs in oocytes during cryopreservation, and induces disorders of proteins on oocyte membranes, and finally causes decreased fertilization rates. JUNO, an oocyte membrane protein, has been well known as a critical protein for sperm-oocyte membrane fusion, however, effects of its freeze-thaw damage on fertilization is unknown so far. Our previous data indicated that cryopreservation readily interfered with JUNO distribution on oocyte membrane. In this study, using mouse oocytes, fluorescent staining, western blot, sulfite sequencing assays and qRT-PCR will be performed to determine JUNO distribution and its expression patterns, methylation changes of Juno gene promoter region, F-actin polymerization as well as associated proteins or genes with oxidative stress on cryopreserved oocytes, which will assess if oxidative stress is the main factor for JUNO freeze-thaw damage. And then the effects of JUNO expression changes on oocyte fertilization potential will be examined with in vitro fertilization. Finally, mouse oocytes will be treated with antioxidant melatonin (MT) or Curcumin (Cur) before or during cryopreservation to clarify antioxidant protective effects on JUNO freeze-thaw damage. These data above will help to further understand the freeze-thaw damage mechanisms fertilization of oocyte membrane protein JUNO after cryopreservation and its potential interventions, which might contribute to improving theory of sperm-egg adhesion and fusion in fertilization and increasing the fertilization rates of cryopreserved oocytes.