玻璃化冻存对斑马鱼卵母细胞的影响

Effects of Vitrified Cryopreservation on GSH Content and Mitochondrial ATPase Activity in Oocytes of Zebrafish Danio rerio

采用比色法测定了在不同玻璃化冻存液中冻存1、2、4、8d时斑马鱼卵母细胞中谷胱甘肽含量和三磷酸腺苷酶的活性,以研究玻璃化冻存对卵母细胞谷胱甘肽含量和线粒体三磷酸腺苷酶活性的影响。试验结果显示,V4组斑马鱼(玻璃化液组分是:1.5mol/L甲醇、6.0mol/L乙二醇、0.25mol/L蔗糖)卵母细胞冻存1、2、4、8d后存活率最高[分别为(48.89±2.58)%、(39.35±1.34)%、(24.18±2.32)%和(14.56±1.64)%],显著高于其他组。经玻璃化冻存后,斑马鱼卵母细胞谷胱甘肽含量和线粒体三磷酸腺苷酶活性均随冻存天数的增加而显著下降(P<0.05)。V4组的谷胱甘肽含量[(13.37±0.59)mg/g]和线粒体三磷酸腺苷酶活性较高,线粒体Ca^(2+)-三磷酸腺苷酶[(3.823±0.144)U/mg]、Mg^(2+)-三磷酸腺苷酶[(4.503±0.144)U/mg]和Na^+/K^+-三磷酸腺苷酶[(7.520±0.198)U/mg]活性最高。各组细胞的活性随着冻存天数的增加而逐渐下降。试验结果表明,玻璃化冻存不能完全避免低温和冷冻保护剂的毒性和对卵母细胞的损伤。

The effects of vitrified cryopreservation on glutathione（GSH） content and mitochondrial AT- Pase activity in oocytes were investigated in zebrafish（Danio rerio） cryopreservated in different vitrifica- tion solutions for 1 d, 2 d, 4 d and 8 d by a chromatometry method. The results showed that the maximal survival rate was observed in the oocytes cryopreservated in group V4 in which the vitrification solution was consisted of 1.5 mol/L methanol, 6.0 mol/L ethylene glycol and 0. 25 mol/L sucrose, with survival rate of （48. 89±2.58）% in 1 d, （39. 35±1.34）% in 2 d, （24. 18±2.32）G in 4 d, and （14. 56±1.64）% in 8 d, significantly higher than in other groups （P〈0. 05）. The GSH contents and the activities of mito- chondrial ATPase in zebrafish oocytes were found to be significantly decreased with increase in elapse of vitrified cryopreservation （P%0.05）, with the maximal GSH contents and activities of mitochondrial AT- Pase in group V4, including Ca2＋-ATpase of （3. 823±0. 144） U/mg, Mg2＋-ATPase of （4. 503± 0. 144） U/mg and Na＋/K＋-ATPase of（7. 520±0. 198） U/mg. The oocytes exposed to the vitrification solutions showed lower cytoactive as the increase in vitrification time, indicating that vitrification did not completely avoid the damages to zebrafish oocytes caused by the low temperature and the toxicity of cryoprotectants.