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1、小鼠精子和球形精子细胞显微注射受精研究解剖学报 1999年第2期第0卷 论著作者：李子义谭景和孙兴参刘忠华周琪贺桂馨单位：李子义谭景和孙兴参刘忠华周琪贺桂馨东北农业大学生物工程系，哈尔滨150030关键词：精子；球形精子细胞；显微注射；电活化；电融合；小鼠【摘要】目的研究动物受精机理。方法以昆明白鼠为实验动物，利用显微注射技术，对精子和球形精子细胞的显微注射受精进行了研究。结果1. 带下注射受精，注射46个精子的卵的受精率(32.5%)和卵裂率(25.0%)显著高于注射单个精子的卵的受精率和卵裂率(分别为12.5%和6.3%)(P0.05)。2. 带下注射46精子后，有第1极体卵母细胞的存活率
2、(69.6%)、受精率(32.5%)和卵裂率(25.0%)分别高于无第1极体卵母细胞的存活率、受精率和卵裂率(分别为64.4%、20.7%和13.8%)，但两者无显著差异(P0.05)。3.胞质内精子注射卵的存活率(21.4%)极显著低于带下注射卵的存活率(69.6%)(P0.01)；而胞质内精子注射卵的受精率(35.5%)、卵裂率(32.3)%和囊胚率(20.0%)则高于带下注射卵的受精率(33.3%)、卵裂率(25.0%)和囊胚率(10.0%)，但无显著差异(P0.05)。4.将球形精子细胞注入经电活化处理的卵母细胞的透明带下，注射卵的存活率为90.5%，电融合率为34.6%，2-细胞分裂
3、率为28.3%。结论1.注射精子的数量对小鼠带下注射的受精率和卵裂率有显著影响。2.卵母细胞中第1极体存在与否对小鼠带下注射的受精率和卵裂率无显著影响。3. 不同注射方法对小鼠卵的存活率有极显著的影响，而对受精率、卵裂率和囊胚率无显著影响。4. 本实验所采用的电融合条件，尚未满足多数球形精子细胞与卵母细胞融合之需要。FERTILIZATION BY MICROINJECTION OF SPERMATOZOON AND ROUND SPERMATID INTO OOCYTE IN THE MOUSELi Ziyi，Tan Jinghe， Sun Xingshen, Liu Zhonghuan,
4、Zhou Qi, He Guixin(Department of Biotechnology, Northeast Agricultural University)【Abstract】 Objective To study fertilization mechanism in mammals and provide essential data for treating human male infertility.MethodsUsing microinjection technique, the fertilization by microinjection of spermatozoa
5、and round spermatids into oocytes in the mouse was systematically studied.Results1. Following subzonal injection of spermatozoa into occytes, the fertilization rate (32.5%) and cleavage rate(25.0%) in the oocytes injected with 4-6 spermatozoa were significantly higher (P0.05) than those (12.5% and 6
6、.3%, respectively) in the oocytes injected with a single spermatozoon. 2. Following subzonal injection of 4-6 spermatozoa into the oocytes, the survival rate (69.6%), fertilization rate (32.5%) and cleavage rate (25.0%) in those oocytes with polar body 1 (PB1) were higher than those (64.4%, 20.7% an
7、d 13.8%, respectively) in the oocytes without PB1, but the difference was not significant (P0.05). 3. The survival rate (21.4%) in the oocytes injected with a single spermatozoon into the cytoplasm was significantly lower (P0.01) than that (69.0%) in the oocytes injected with 4|6 spermatozoa into th
8、e perivitelline space (PVS). The fertilization rate (35.5%), cleavage rate (32.2%) and blastocyst rate (20.0%) in those oocytes injected with a single spermatozoon into the cytoplasm of oocytes were higher than those (33.3%, 25.0% and 10.0%, respectively) in the oocytes injected with 4-6 spermatozoa
9、 into the PVS, but they did not differ significantly(P0.05). 4. Following subzonal injection of a round spermatid into the oocyte, the survival rate, electrofusion rate and 2-cell rate were 90.5%, 34.6% and 28.3%, respectively.Conclusions1. The fertilization rate and cleavage rate in the oocytes wer
10、e significantly affected by the number of subzonal injection of spermatozoa. 2. The survival rate, fertilization rate and cleavage rate in the oocytes with PB1 or without PB1 were not significantly affected when subzonal injection of spermatozoa. 3. The survival rate in the oocytes were significantl
11、y affected by injection methods, and the fertilization rate, cleavage rate and blastocyst rate in the oocytes were not significantly affected by injection methods. 4. Most round spermatids and oocytes were not fused on the present condition of electrofusion.【Key words】 Spermatozoon; Round spermatid;
12、 Microinjection; Electrical activation; Electrofusion; Mouse正常的受精过程，精子必须穿过透明带并与卵质膜融合而进入卵中。随着显微操作技术和设备的日趋完善，使得由透明带和卵质膜所形成的人工授精障碍能够得以排除。胞质内精子注射(intracytoplasmic sperm injection, ICSI) 或带下注射(subzonal injection, SUZI),是利用显微操作仪将精子(或精子细胞)直接注射到卵胞质内或透明带下的受精过程，称为显微注射受精(microfertilization)或显微操作协助受精(micromanip
13、ulation-assisted fertilization)1。它是体外受精与显微操作技术相结合而形成的一项新技术，该技术在动物受精机理研究、治疗男性不育、动物育种和野生动物遗传资源保存方面，有着极为广阔的应用前景。小鼠带下注射受精已于1998年有子代出生2，而小鼠胞质内精子注射的研究一直进展缓慢。到1995年，Kimura等3改进了显微操作系统，Ahmadi等4先向胞质内注入Ca2+、再注入精子，先后获得小鼠胞质内精子注射受精后代。尽管已有后代出生，但有关精子(或精子细胞)显微注射受精的基础理论研究还很少。本实验以小鼠为实验动物，利用显微注射技术，对精子和球形精子细胞显微注射受精进行了研究
14、，旨在为深入研究动物受精机理，以及为治疗男性不育等提供基础数据。材料和方法1. 动物选择实验用昆明白鼠，雌鼠68周龄、体重2535g，雄鼠812周龄、体重3545g，购自哈尔滨市制药厂实验动物室，按常规方法饲养管理。2. 卵子准备雌鼠腹腔注射10IU孕马血清促性腺激素(PMSG)，间隔48h腹腔注射10IU人绒毛膜促性腺激素(hCG)，进行超排卵处理。注射hCG后16h，以颈椎脱臼法处死雌鼠，剪下输卵管，在实体解剖镜下，用眼科镊子划破输卵管壶腹部收集卵丘团。卵丘团经0.1%透明质酸酶(Sigma公司)处理12min(37)，去掉卵丘细胞，获取的卵母细胞置Hepes-CZB5液中洗3次后备用。3
15、. 精子与球形精子细胞准备以颈椎脱臼法处死雄鼠，取双侧附睾尾及部分输精管中的精子，移入含有1 ml T66液的小管中，用封口膜封住管口后，放入37温箱中待用。采用带下注射时，精子在T6液中获能2h(37温箱中)；胞质内注射时，可免去精子获能步骤，直接将1份精子悬液与34份用T6液配制的含10%聚乙烯吡咯烷酮(polyvinylpyrolidone, PVP)滞动液混合待用。球形精子细胞的制备参照Ogura等7方法进行。即取小鼠睾丸，剥离睾丸被膜，用眼科剪刀剪碎曲细精管，置Hepes-CZB中离心2次(1 000r/min， 5min次)，取沉淀物的表层置操作液滴中，球形精子细胞体积很小，直径约
16、10m(911m)，细胞中央有染色质团构成的轮廓明显的球形区。4. 显微操作工具制备将1.2mm150mm玻璃毛细管，手工拉制成外径130150m的吸管，然后在微锻仪上加工成内径约20m的固定吸管。将1.2mm150mm玻璃毛细管，经拉针仪、磨针仪和微锻仪制成注射针管。用于ICSI的注射针管内径为6m，用于SUZI的注射针管内径810m，用于球形精子细胞带下注射的注射针管内径约10m。玻璃毛细管在制备针管之前经超声波洗涤、洗液浸泡、高热灭菌处理。制备好的针管使用前再经紫外线照射20 min灭菌。5. 显微操作将注射针管和固定吸管安装至显微操作仪上，在90mm培养皿中，做9个50l的小滴(正中央1滴，周围成环形排列8滴)。正中央1滴为精子-PVP悬液(用于胞质内精子注
