四种冷冻保护剂对小鼠2-细胞胚胎渗透性和毒性作用的比较

Permeability and toxicity of four cryoprotectants on 2-cell embryos in the mouse

作为胚胎冷冻保存的基础性研究,冷冻保护剂的渗透性和毒性研究非常重要。本试验选用1,2-丙二醇、甘油、乙二醇和二甲基亚砜4种常用冷冻保护剂,对小鼠2-细胞胚胎进行渗透性和毒性研究。结果显示:1.5mol/L的1,2-丙二醇、乙二醇和二甲基亚砜冷冻保护剂对2-细胞胚胎的渗透性显著高于甘油保护剂;4种冷冻保护剂对细胞膜的完整性没有影响;1.5mol/L的乙二醇、1,2-丙二醇和甘油保护剂处理后的2-细胞胚胎的囊胚发育率和孵化率与对照组胚胎比较差异不显著(P>0.05),但显著高于二甲基亚砜处理后的2-细胞囊胚发育率和孵化率(P<0.01)。结果表明:在4种冷冻保护剂中,乙二醇和1,2-丙二醇适合于小鼠2-细胞胚胎冷冻保存。

The permeability and the toxicity of cryoprotectants on early embryos are of paramount importance for basic research of embryo cryopreservation. In this study, four cryoprotectants （1, 2-propanediol, PROH; ethylene glycol, ETG; glycerol; dimethyl sulfoxide, DMSO） were compared for their permeability and toxicity on 2-cell mouse embryo cryopreservation. In Experiment 1, 2- cell mouse embryos were placed in four cryoprotectant solutions at concentration 1.5 mol/L for 10 min at room temperature （25 ℃）. The relative changes in volume of embryos and the integrity of cell membranes were analyzed to assess the permeability of cryoprotectants. In Experiment 2, four cryoprotectants were tested by exposing embryos to 1.5 mol/L cryoprotectants for 10 min, and then by washing out cryoprotectants in three steps with 1.0, 0. 5 and 0 mol/L cyroprotectant. Finally, embryos were placed in culture medium for 96 h. The rates of blastocyst formation were investigated as an index to determine the toxicity of cyroprotectant. In addition, the total cell number per blastocyst at the final 96 h observation point was counted. Results indicated that volume showed little change and recovered faster in PROH and in ETG, but glycerol showed the slowest permeation for 2-cell embryo. No difference was observed in blastocyst percentage and hatched percentage of 1.5 mol/L PROH, ETG in glycerol exposed compared with non-exposed embryos （ P 〉 0.05） , but significantly better than with 1.5 mol/L DMSO （ P 〈 0.01 and P 〈 0.05, respectively）. Our results demonstrate that PROH and ETG are suitable cryoprotectants for use in slow freezing procedure for 2-cell mouse embryo