小鼠卵子胞膜在显微穿刺中存活的膜特性因素研究

A Research on the Membrane Characteristics of the Mouse Alive Oolema in Microinjection

目的:拟通过特定穿刺实验来分析小鼠卵子胞膜在显微穿刺中存活的膜特性因素。方法:采用尖口注射针及37℃恒温台的显微条件下,①单纯穿刺实验:即单纯对卵子穿刺不同时间长度(1 s或60 s)或不同深度(50μm和90μm)共4个组合组(每组n=20),比较膜凹陷的回复时间;②显微授精(ICSI)实验:延时组(n=91),在穿刺时延缓对小鼠卵子抽吸破膜的时间;加深组(n=140),改良显微持卵管,使呈喇叭形开口,可在穿刺持卵时将卵子部分吸入该开口内,以增加注射针穿刺小鼠卵子的深度;对照组(n=115),即相同于传统的人ICSI操作。结果:①穿刺时间更长或更深可显著延长膜凹陷的恢复时间;②与对照组相比,延缓对卵子的抽吸破膜时间使得小鼠卵存活率显著提高(22%vs 48%,P<0.01);而应用改良的新型持卵管后,小鼠卵存活率更加显著地提高(22%vs 82%,P<0.01),且其正常受精率>70%。结论:小鼠卵子胞膜并非易脆;在显微穿刺时小鼠卵子膜凹陷柔顺性随时间和深度增加而增加,从而延缓膜凹陷回复时间,有利卵子胞膜破口修复而存活。

Objective： To investigate the membrane characteristics of the mouse alive oolema in microinjection by designing special injection factors.Methods： Sharp injection pipette was used in all microinjections and the micromanipulation was maintained in 37℃ constant temperature.In the puncturing experiment,mouse oocytes were only punctured with different insertion time length（1 s or 60 s） or different insertion depth（50 mm or 90 mm） in totally 4 groups（n=20 for each group）.The time of oolemma cave reverting in different groups was compared.In intracytoplasmic sperm injection（ICSI） experiment,there were 3 groups follows： C-ICSI group（n=115,the control）,conventional injection was performed according to the procedure for human introcytoplasmic sperm injection;M-ICSI group（n=91）,the conventional injection was modified by making the oolemma sucking slower during oolemma puncture;N-ICSI group（n=140）,the holding pipette was remodeled with a trumpet-shaped opening.Some part of oocyte was sucked into the holding pipette,which could prolong the depth of injection pipette insertion.Results： In puncturing experiment,increasing time length or depth for oolemma puncturing could prolong the time of oolemma cave reverting.In ICSI experiment,compared with the conventional injection,improved survival rate was obtained with slower oolemma sucking（22% vs 48%,P0.01）.Furthermore,significantly higher survival rate was achieved（82%,P0.01） by using modified holding pipette,with comparatively high normal fertilization rate（70%）.Conclusion： The mouse oolemma is not fragile.Increasing the insertion time length or insertion depth for oolemma puncturing may prolong the time of oolemma cave reverting,which is beneficial to the healing of the wounded oolemm and make oocyte survive.