解冻液I温度设定对小鼠卵母细胞玻璃化冻融效果的影响

Effects of Setting Temperature of Thawing Medium I on Mouse Oocytes Vitrification

目的探讨预热至37℃的解冻液I和预热至室温(22℃)的解冻液I对小鼠卵母细胞玻璃化冻融效果的影响。方法对6周龄昆明系雌鼠进行促排卵,脱去颗粒细胞后选取形态良好的MII期卵母细胞,用含不同浓度DMSO+EG的磷酸盐缓冲液(5%DMSO+5%EG,10%DMSO+10%EG,20%DMSO+20%EG+30%蔗糖)对其进行玻璃化冷冻,储存2天后对小鼠卵子进行解冻复温。将卵子随机分为两组,一组投入预热至37℃的解冻液I(含30%蔗糖的磷酸盐溶液),另一组投入预热至室温(22℃)的相同浓度的解冻液I中,再分别移至预热至室温(22℃)的解冻液II、III和IV(分别含20%、10%、5%蔗糖的磷酸盐溶液),随后对两组卵母细胞的复苏率进行统计比较。结果共计对119枚小鼠卵子进行玻璃化冻融,其中61枚置于预热至37℃的解冻液I,有52枚存活良好,复苏率为82.25%(52/61),其余58枚置于预热至室温(22℃)的解冻液I进行解冻,有23枚存活良好,复苏率为39.66%(23/58),37℃组的复苏率显著高于室温(22℃)组(P<0.01)。结论虽然降低平衡时的温度可以减少高浓度玻璃化冷冻保护剂对卵子的毒性作用,但较快的升温速度可以帮助卵子快速通过-120℃^-35℃的危险阶段,而37℃的解冻液I环境较之室温(22℃)能更好地加速这一过程,避免重结晶对细胞的致命损伤。

Objective To investigate the effect of thawing medium I preheating to 37 ℃ and 22 ℃（room tem-perature） on mouse oocytes vitrification. Methods Six-week old Kunming female mice were induced to superovu-late. After granulosa cells of cumulus-oocyte-comlexes （COC） were taken off in hyaluronic acid, the mature oocytes were frozen by using phosphate buffer containing different concentrations of DMSO＋EG （5% DMSO＋5%EG,10% DMSO＋10% EG,20% DMSO＋20% EG＋30% sucrose）. After 2 d, the frozen oocytes were randomly allo-cated to two thawing groups： thawing medium I （phosphate buffer containing 30% of sucrose） preheating to 37℃group and room temperature （22 ℃） group, respectively, then both to thawing mediumⅡ, Ⅲ and Ⅳ（phosphate buffer containing 20%, 10%, and 5% of sucrose, respectively）. The survival rates of two groups were calculated. Results A total of 119 oocytes were frozen, 61 out of which were allocated to thawing medium I preheating to 37℃, and the survival rate was 82.25%（52/61）; the rest 58 oocytes were in thawing medium I preheating to room temperature group, and 23 of 58 （39.66%） were survived. A significant difference was found between two groups （P〈0.01）. Conclusion Though balance in lower temperature can reduce the toxic effects of high concentrations of vitrification cryoprotectants on the oocytes, a faster heating rate can help the oocytes quickly pass the dangerous phase of - 120 ℃ ~ -35 ℃. 37 ℃ thawing medium I environment could speed up this process better than the room temperature （22 ℃） one, and could avoid the recrystallization which cause fatal damage to the cells.