2-氨基乙基联苯基硼酸酯对玻璃化冷冻牛成熟卵母细胞发育能力的影响  

Effect of 2-APB on Development Capacity of Vitrified Bovine Mature Oocytes

作　　者：莫显红 岳凯平 孙丽瑶 李冰 赵冰 郭成 徐振军 MO Xianhong;YUE Kaiping;SUN Liyao;LI Bing;ZHAO Bing;GUO Cheng;XU Zhenjun(College of Chemistry and Life Science,Chifeng University,Chifeng 024000,China)

机构地区：[1]赤峰学院化学与生命科学学院,赤峰024000

出　　处：《中国畜牧兽医》2022年第7期2669-2676,共8页China Animal Husbandry & Veterinary Medicine

基　　金：内蒙古自治区高等学校科学研究项目(NJZY22130);赤峰学院大学生创新创业训练计划项目(S202010138017);赤峰学院服务赤峰市经济社会发展应用项目(cfxyfc201847);赤峰学院一流学科建设项目(CFXYYLXKB202102);蒙东地区生物资源开发利用创新团队(cfxykycxtd202008)。

摘　　要：【目的】研究内质网IP3受体(IP3R)抑制剂2-氨基乙基联苯基硼酸酯(2-APB)对玻璃化冷冻-解冻牛MⅡ期卵母细胞发育能力的影响。【方法】将体外成熟24 h的卵丘-卵母细胞复合体(COCs)用0.1%透明质酸酶消化去除卵丘颗粒细胞,获得的卵母细胞分为对照组和2-APB组,采用OPS法进行玻璃化冷冻,对照组直接进行玻璃化冷冻,2-APB组在玻璃化冷冻前先用10μmol/L 2-APB预处理10 min。2 d后解冻卵母细胞,统计解冻后(0 h)及恢复培养2 h时卵母细胞的存活率,用FITC-PNA荧光探针检测恢复培养2 h时卵母细胞皮质颗粒(CG)的分布,用2′,7′-DCHFDA和Cell Tracker Blue CMF2HC分别检测胞内活性氧(ROS)和谷胱甘肽(GSH)含量。将成熟培养24 h未冷冻卵母细胞(Fresh组),与解冻后恢复2 h的对照组和2-APB组卵母细胞同时进行孤雌激活,于培养的第2、7天分别统计孤雌胚胎的卵裂率和囊胚率。【结果】与对照组相比,2-APB组冷冻-解冻卵母细胞0和2 h的存活率均显著增加(P<0.05);2-APB组皮质颗粒皮质区分布比例显著提高(P<0.05),损伤型分布比例显著降低(P<0.05);2-APB组卵母细胞内GSH含量显著增加(P<0.05),ROS含量显著降低(P<0.05);2-APB组卵母细胞孤雌激活胚胎的卵裂率、囊胚率均显著增加(P<0.05),且与Fresh组无显著差异(P>0.05)。【结论】2-APB预处理可通过增加卵母细胞内GSH含量,降低卵母细胞皮质颗粒损伤型分布比例和胞内ROS含量,提高玻璃化冷冻保存牛MⅡ期卵母细胞质量及早期胚胎发育能力。【Objective】The study was aimed to investigate the effects of endoplasmic reticulum IP3 receptor(IP3R)inhibitor 2-aminoethyl diphenylborinate(2-APB)on the developmental capacity of frozen-thawed bovine MⅡoocytes.【Method】Oocytes matured in vitro for 24 h were digested with 0.1%hyaluronidase to remove cumulus cells,and were randomly divided into control group and 2-APB group,the open pulled straw(OPS)method was used for vitrification.The control group was vitrified directly,2-APB group was pretreated with 10μmol/L 2-APB for 10 min before vitrification.Oocytes were thawed after 2 d,the survival rate of oocytes after thawed(0 h)and warmed for 2 h were counted.After recovering for 2 h,cortical granules(CG)were labeled with FITC-PNA fluorescent probe to detect distribution,intracellular reactive oxygen species(ROS)and glutathione(GSH)contents were deteced by 2′,7′-DCHFDA and Cell Tracker Blue CMF2HC respectively.The non-vitrified oocytes(Fresh group)matured in vitro for 24 h were parthenogenetic activated as well as those of the control and 2-APB groups,the cleavage and blastocyst rates were counted at 2 and 7 d,respectively.【Result】Compared with control group,the survival rate of frozen-thawed oocytes at 0 and 2 h in 2-APB group was significantly increased(P<0.05),the proportion of CG distributed in cortical area in 2-APB group was significantly increased(P<0.05),the proportion of CG injury type was significantly decreased(P<0.05),the content of GSH was significantly increased(P<0.05),and the content of ROS was significantly decreased(P<0.05),the cleavage and blastocyst rates of parthenogenetic activated embryos in 2-APB group were significantly increased(P<0.05),and there was no significant difference with the Fresh group(P>0.05).【Conclusion】Pretreatment of bovine MⅡoocytes with 2-APB before vitrification could increase GSH content,decrease the proportion of CG injury type and ROS content,improve the frozen-thawed oocyte quality and early embryo developmental capacity.

关 键 词：玻璃化冷冻 2-氨基乙基联苯基硼酸酯 卵母细胞 发育能力 牛 

分 类 号：S823[农业科学—畜牧学]