【作者】 梁洋； 杜文敬； 王春生； 李昊； 朴善花； 安铁洙；

【Author】 LIANG Yang,Du Wen-Jing,WANG Chunsheng,LI Hao,PIAO Shan-Hua,AN Tie-zhu (College of Life Science.Northeast Forestry University,harbin 150040,China)

【机构】 东北林业大学生命科学学院；

【摘要】 为了完善小鼠卵母细胞的冷冻方法和探明冷冻保护剂和冷冻解冻对卵母细胞的影响,本研究利用FSEA对小鼠成熟卵母细胞进行冷冻保存。与此H时,采用免疫组化、DAPI、FDA和PI法对卵母细胞进行染色,分别观察了FSEA处理和经冷冻解冻处理对卵母细胞纺锤体、染色体、细胞膜等的影响。结果显示:1.与未经处理的卵母细胞相比,经玻璃化冷冻解冻的卵母细胞的体外受精率显著降低(P<0.01),但是在卵母细胞透明带上作一切口后,其体外受精率显著提高(P<0.05),且其胚胎发育至4细胞期的能力与对照组相比差异不显著;2.与未经处理的卵母细胞相比,经FSEA处理和经冷冻解冻后的卵母细胞正常纺锤体和正常染色体的比率显著降低(P<0.01),但是两者间差异不显著;3.当用FDA染色后,未经处理、FSEA处理和经冷冻解冻的卵母细胞间发出均匀荧光(有活性)和无荧光或不均匀荧光(无活性)比率间差异不显著。与其相比,冷冻解冻组由PI诱发的橘红色荧光(细胞膜损伤)的卵母细胞的比率显著高于未经处理(P<0.01)和FSEA处理组(<0.05)。上述结果表明,FSEA玻璃化冷冻保护剂结合透明带切割法,可用于小鼠卵母细胞的冷冻保存;冷冻保护剂处理是造成卵母细胞纺锤体和染色体异常的主要原因,而冷冻解冻过程对其影响有限;冷冻保护剂处理和冷冻解冻过程并不影响卵母细胞的活力,但是,冷冻解冻过程可诱发卵母细胞膜的损伤。更多还原

【Abstract】 In the study,mouse mature oocytes vitrified in FSEA were used to explore the influence of cryoprotectant and vitri?cation-thawing scheme.After oocytes were treated with FSEA and vitri?cation-thawing,their spindle,chromosome and cell membrane were observed by immunohistochemical method using an antibody against microtube protein,combining with DAPI,FDA and PI staining.The results showed that fertilization rate of vitri?cation-thawing oocytes was significantly lower than those untreated oocytes(P<0.01),whereas it was significantly increased when pellucid zones were cut(P<0.05). Moreover,the capacity of vitri? cation-thawing oocytes developing to 4-cell embryos was not significant than control group. Ratios of normal spindle and chromosome of oocytes by FSEA or vitri?cation-thawing treatment were similar,but they were lower than untreatment oocytes(P<0.01).The ratios of normal(activity),abnormal,no fluorescence of oocytes by FDA stainning were not significant among untreated,FSEA and vitri?cation-thawing processing.But the rate of stainned oocytes by PI(membrane damage) in vitri?cation-thawing group was significantly higher than untreated(P<0.01) and FSEA treatment group(P<0.05).These results suggested that FSEA vitrification cryoprotectant combined with zona pellucida cut method can be used for mouse oocyte cryopreservation.Cryoprotectant was the main reason of oocyte spindle and chromosome abnormalities,whereas they were less influenced by freez-thawing process.The viability of oocytes was not affected by cryoprotectant treatment,nor freezing and thawing process,but oocyte membrane can be damaged by freezing and thawing process.更多还原