卵母细胞玻璃化冷冻对于家畜胚胎体外生产和优良、濒危动物遗传资源保存具有重要意义。然而，玻璃化冷冻卵母细胞精子融合能力显著降低，严重影响其应用潜力，亟待解决。JUNO蛋白是精子膜蛋白IZUMO1在卵母细胞质膜的唯一受体，IZUMO1-JUNO蛋白识别是精卵绑定、融合的关键环节，该环节阻断会导致受精完全失败。然而，玻璃化冷冻对牛卵母细胞JUNO蛋白的影响机制尚无报道，更缺乏调控措施。针对该问题，本项目拟从蛋白表达（空间分布、表达量等）、蛋白特性（分子量、序列、结构）、蛋白功能（二硫键、糖基化、磷酸化、精子融合）等角度，揭示玻璃化冷冻对牛卵母细胞JUNO蛋白的影响机制，并通过Juno mRNA注射、β-环糊精胆固醇包合物处理等措施，增强玻璃化冷冻牛卵母细胞JUNO蛋白水平和功能，进而提高其精子融合能力和体外受精效率。本研究成果，对于促进玻璃化冷冻卵母细胞的广泛应用，具有重要的科学意义和应用价值。

Oocyte vitrifcation plays an important role in the in vitro production of embryos and genetic resources preservation of excellent and endangered animals. However, vitrification significantly decreases the sperm fusion capacity of oocytes, which seriously inhibits the wide application of vitrified oocytes. Recently, JUNO protien located on the plasma of oocytes has been identified as the counter-receptor of fertilization protein IZUMO1 located on sperm. Many studies have proven that JUNO-IZUMO1 recognition and binding are critical for the fertilization progress, and the blocking of this recognition and binding will lead to the complete failure of fertilization. However, little information is still available about the effect of vitrification on the JUNO of oocytes, less is about the efficient approaches to positively regulate the JUNO level and function in vitrified oocytes. Therefore, the present study is designed to utilize vitrified bovine oocytes as the model to investigate the effect of vitrification on JUNO protein expression (spatial distribution and protein expression level), characteristics (molecular weight, protein sequence and structure) and function (disulfide bond, glycosylation, phosphorylation and sperm fusion). Furthermore, Juno mRNA microinjection and cholesterol-loaded methyl-β-cyclodextrin treatment are used to positively regulate the JUNO expression level and function to improve the fertilization capacity of vitrified bovine oocytes. The findings of the present study will contribute greatly to illustrating the mechanism via which vitrification decreases the fertilization capacity of oocytes and developing the rescuing methods, which ehances the wide application of vitrified oocytes.