激光打孔技术在小鼠卵子冷冻保存上的运用

目的比较卵子冷冻复苏后、以及复苏卵子经过激光打孔后,与新鲜精子、冷冻复苏精子体外受精,受精率的变化。方法 （1）通过免疫荧光染色技术,判断卵子冷冻前后透明带糖蛋白-2、微丝以及细胞核的变化;（2）冷冻C57BL/6J小鼠卵子,复苏后一部分卵子打孔,一部分不打孔,然后与9个品系的新鲜精子和冷冻精子体外受精,比较各组受精率的变化。结果 （1）新鲜卵子组透明带糖蛋白-2、微丝及细胞核结构清晰,而冷冻组以及冷冻剂处理组,微丝结构略有变化,透明带糖蛋白-2受到不同程度的损伤,而细胞核在冷冻前后无明显变化;（2）9个品系的新鲜精子与C57BL/6J复苏卵子受精率为17.6%～42.9%,平均为29.6%;新鲜精子与复苏打孔卵子受精率为29.1%～72.3%,平均为49.7%,差异显著（P〈0.05）;9个品系复苏精子与复苏卵子体外受精率为5.4%～23%,平均为15.5%;复苏精子与复苏打孔卵子受精率为16.7%～48.6%,平均为28.8%,差异显著（P〈0.05）。结论 （1）冷冻对卵子的透明带糖蛋白-2有较大的损伤,对微丝有一定的影响,但是对染色体没有影响;（2）冷冻卵子复苏后,体外受精率下降明显,但是复苏卵子经过激光打孔后,可以显著提高体外受精率。

Studying laser-punched system on freezed and post-thawed mouse oocytes

Objective To evaluate the IVF （in vitro fertilization） rate of the post-thawed oocytes and the post- thawed laser-punched oocytes of C57BL/6J fertilized with fresh or frozen sperms of 9 different strains. Methods （ 1 ） The changes of nuclear, microfilament and zona pellucida glycoprotein-2 of C57BL/6J oocytes before and after cryopreservation were studied by immunofluorescence technique. （2） The IVF rate of C57BL/6J post-thawed oocytes or laser-punched oocytes fertilized with fresh or frozen sperms of 9 strains were compared. Results （ 1 ） there were no obvious changes of nuclear before or after cryopreservation, microfilament were changed slightly when handled with cryopretectant or after cryopreservation. Zona pellueida glycoprotein-2 were hurted after cryopreservation or handled with eryopreservant ; （2） the IVF rate of fresh sperm of 9 strains with post-thawed oocytes were 17. 6 - 42.9% （ averaged 29.6% ）, which were significantly lower than the IVF rate of fresh sperm with laser-punched oocytes, 29.1 -72. 3% （average 49.7% , P 〈 0. 05 ） ; the 1VF rate of post-thawed sperms of 9 strains with post-thawed oocytes were 5.4 - 23% （ averaged 15.5% ）, also significantly lower than the IVF rate of post-thawed sperms fertilized with laser-punched oocytes, 16.7 - 48.6% （ average 28.8%, P 〈 0. 05）. Conclusions （1） Cryopreservation would have a remarkable effect on microfilament and zona pullucida glycoprotein-2, but have nut obvious effect on nuclear; （2） The IVF rate of oocytes would decreased remarkably after cryopreservation, but if the laser-punched system were used, the IVF rate would increased apparently. It showed that the laser-punched system would be useful for oocytes cryopreservation.