Embryo Biotechnology and Reproduction Laboratory, Institute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences (CAAS), Beijing, China.
Shijiazhuang Tianquan Elite Dairy Lt.D., Shijiazhuang, China.
Reprod Domest Anim. 2021 Mar;56(3):519-530. doi: 10.1111/rda.13890. Epub 2021 Jan 18.
Many experiments show that vitrification significantly reduces the fertilization capacity of mammalian oocytes, restricting the application of vitrified oocytes. It has been proven that the JUNO protein plays a vital role in mammalian oocytes fertilization. However, little information is available about the effects of vitrification on the JUNO protein and the procedure to protect it in bovine oocytes. Here, the present study was designed to investigate the effect of vitrification on the JUNO protein level in bovine oocytes. In this study, MII oocytes were treated with cholesterol-loaded methyl-β-cyclodextrin (CLC; 0, 10, 15, 20 mM) for 45 min before vitrification and methyl-β-cyclodextrin (MβCD; 0, 2.25, 4.25, 6.25 mM) for 45 min after thawing (38-39°C). Then, the expression level and function of JUNO protein, cholesterol level in the membrane, the externalization of phosphatidylserine, sperm binding capacity and the developmental ability of vitrified bovine oocytes were examined. Our results showed that vitrification significantly decreased the JUNO protein level, cholesterol level, sperm binding capacity, development ability, and increased the promoter methylation level of the JUNO gene and apoptosis level of bovine oocytes. Furthermore, 15 mM CLC + 4.25 mM MβCD treatment significantly improved the cholesterol level and increased sperm binding and development ability of vitrified bovine oocytes. In conclusion, the combination treatment of cholesterol-loaded methyl-β-cyclodextrin and methyl-β-cyclodextrin significantly improves the fertilization capacity of vitrified bovine oocytes by protecting fertilization protein JUNO.
许多实验表明，玻璃化显著降低了哺乳动物卵母细胞的受精能力，限制了玻璃化卵母细胞的应用。已经证明 JUNO 蛋白在哺乳动物卵母细胞受精中起着至关重要的作用。然而，关于玻璃化对 JUNO 蛋白的影响以及在牛卵母细胞中保护它的程序知之甚少。在这里，本研究旨在研究玻璃化对牛卵母细胞中 JUNO 蛋白水平的影响。在这项研究中，MII 期卵母细胞在玻璃化前用胆固醇负载的甲基-β-环糊精（CLC；0、10、15、20mM）处理 45 分钟，在解冻后（38-39°C）用甲基-β-环糊精（MβCD；0、2.25、4.25、6.25mM）处理 45 分钟。然后，检查 JUNO 蛋白的表达水平和功能、膜中的胆固醇水平、磷脂酰丝氨酸的外排、精子结合能力以及玻璃化牛卵母细胞的发育能力。我们的结果表明，玻璃化显著降低了 JUNO 蛋白水平、胆固醇水平、精子结合能力、发育能力，增加了 JUNO 基因启动子甲基化水平和牛卵母细胞的凋亡水平。此外，15mM CLC+4.25mM MβCD 处理显著改善了胆固醇水平，并增加了玻璃化牛卵母细胞的精子结合和发育能力。总之，胆固醇负载的甲基-β-环糊精和甲基-β-环糊精的联合处理通过保护受精蛋白 JUNO 显著提高了玻璃化牛卵母细胞的受精能力。
