不同浓度保护剂及渗透平衡时间对慢速冷冻小鼠卵母细胞活力的影响

Effects of Different Concentration Cryoprotective Agents and Osmotic Equilibration Time on Biological Activation of Ultrarapid Freezing Mice Oocytes

目的探讨不同的冷冻保护剂浓度、渗透平衡时间对小鼠卵母细胞冷冻后存活率及存活卵母细胞ICSI受精后胚胎发育潜能的影响。方法小鼠卵母细胞来自用HMG／HCG刺激的性成熟小鼠，依据预平衡液浓度，即1．0M或1．5M丙二醇（PROH），将卵母细胞分为A、B组。依据装载液蔗糖浓度（0．1、0．2、0．3M）的不同将A、B两组分为A1、A2、A3、B1、B2、B3 6组；依卵母细胞在平衡液中的渗透平衡时间不同（〈10min、〉20min）分为C、D两组；依装载液中的渗透平衡时间不同（〈10min、〉30min）分为E、F两组。卵母细胞在预平衡液中渗透平衡10—20min，在装载液中渗透平衡5～30min后进入慢速程序化冷冻并保存，快速解冻后观察卵母细胞存活率、ICSI受精率和优质胚胎形成率。结果A、B组间冻存卵母细胞存活率差异有非常显著性统计学意义（43．7％vs23．5％）；A2组的卵母细胞存活率最高（52．5％），如组次之（42．9％）；C、D两组间冻存卵母细胞存活率、透明带破裂率差异无统计学意义。E、F两组间存活率、透明带破损率差异有统计学意义（52．5％vs6．3％；6．6％vs75．0％）；新鲜组和冷冻组卵母细胞受精率、卵裂率、优质胚胎率差异均有统计学意义（81．8％vs40％；77．3％vs100％；90．9％vs83．3％）。结论（1）在慢速冷冻中，平衡液较低的PROH浓度（1．0M）和装载液较高的蔗糖浓度（0．2M）有益于卵母细胞冻存存活率的提高。（2）小鼠卵在平衡液中的渗透平衡时间以10min为宜；在装载液中室温下的渗透平衡时间以5～10min为宜，最好不要超过15min。（3）冻存复苏后的存活卵经ICSI受精后受精率下降，但受精后胚胎的发育潜能似乎未受多大影响。

Objective To study effects of different cryopmtective solution concentration and osmotic equilibration time on the survival rate of frozen-thawed mice oocytes and the developmental potential of the embryos that the survival oocytes were fertilized by ICSI. Methods Mice oocytes were obtained from the mice that were treated by ovarian stimulation protocol with HMG/HCG. By equilibration solution concentration, oocytes were divided into A（1.0M PROH） and B（ 1.5M PROH） group. Then by loading solutions（sucrose） of different concentration （0.1M, 0.2 M, 0.3M）, A group and B group were respectively divided into A1,A2, A3 group and B1 ,B2,B3 group. By different osmotic equilibration time of equilibration solutions, oocytes were divided into C （ 〈 10min） and D（ 〉 20min） group. By different osmotic equilibration time of loading solutions, oocytes were divided into E（ 〈 10min） and F（ 〉 30min） group. Mice oocytes were slowly frozen by an automated biological freezer and stored in liquid nitrogen tanks after the mice oocytes were osmotically equilibrated within 10 - 20min in the equilibration solutions and within 5 - 30min in the loading solutions. The survival rate, fertilization rate and high quality embryos rate of oocytes post-thawed rapidly by ICSI were observed. Results The survival rate of frozen oocytes in A group was significantly higher than that in B group. No difference of survival rate of frozen oocytes and broken rate of zona between C and D group was found. The survival rate of frozen oocytes in E group was significantly higher than that in F group and the broken rate of zona in E group was significantly lower than that in F group. The fertilization rate, cleavage mice and high quality embryo mice in fresh group were significantly higher than that in frozen group. Conclusion （1）As slow frozen, lower concentration PROH （ 1.0M） of the equilibration solution and higher concentration sucrose （0.2M） of the loading solution were benetidal for increasing the survival rate of cryopreservation mouse oocytes. （2） The osmotic equilibration time of oocytes in the equilibration solution was suitable in 10 minutes, and its time in the loading solution in the room temperature was also suitable in 5 - 10 minutes and had better not to exceed 15 minutes. （3） Although the fertilization rates of oocytes post- thawed by ICSI was low, the developmental potential of embryos seemed not to be influenced.