【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4～6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%)(P＜0.05)。2. 带下注射4～6精子后，有第1极体卵母细胞的存活率(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(P＞0.05)。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%)(P＜0.01)；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(P＞0.05)。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 MOUSE

Li Ziyi，Tan Jinghe^△， Sun Xingshen, Liu Zhonghuan, 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 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 (P＜0.05) than those (12.5% and 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% and 13.8%, respectively) in the oocytes without PB1, but the difference was not significant (P＞0.05). 3. The survival rate (21.4%) in the oocytes injected with a single spermatozoon into the cytoplasm was significantly lower (P＜0.01) than that (69.0%) in the oocytes injected with 4|6 spermatozoa into the 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 into the PVS, but they did not differ significantly(P＞0.05). 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 were 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 significantly 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; 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(micromanipulation-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，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6～8周龄、体重25～35g，雄鼠8～12周龄、体重35～45g，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1～2min(37℃)，去掉卵丘细胞，获取的卵母细胞置Hepes-CZB^［5］液中洗3次后备用。

3. 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 T^［6］₆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3～4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10μm(9～11μm)，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1.2mm×150mm玻璃毛细管，手工拉制成外径130～150μm的吸管，然后在微锻仪上加工成内径约20μm的固定吸管。将1.2mm×150mm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6μm，用于SUZI的注射针管内径8～10μm，用于球形精子细胞带下注射的注射针管内径约10μm。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φ90mm培养皿中，做9个50μl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(用于胞质内精子注射)或精子-获能液悬液(用于带下精子注射)或球形精子细胞悬液(用于带下注射)，周围8滴(Hepes-CZB液)各移入2～5个待注射卵(通常空出2个小滴，用于调试针管)。显微操作时，注射针管在含有精子或球形精子细胞的小滴中，将单个精子从尾部吸入注射针管尖端(胞质内精子注射)或吸入1～6个精子(带下注射)或吸入单个球形精子细胞(带下注射)。用固定吸管固定卵母细胞，使其极体(在有第1极体卵母细胞)朝向液滴内相当于时针“12”点或“6”点的位置，注射针管在相当于“3”点的位置穿过透明带刺入卵胞质，将单个精子连同微量的液体注入胞质；或注射针管穿过透明带后，将1个或多个精子连同极少量液体注入卵周隙或将单个球形精子细胞注入卵周隙(带下注射)。随后，慢慢撤出注射针管，并将卵母细胞从固定管释放下去。

6. 卵母细胞的电活化与电融合

在带下注射球形精子细胞之前，需对卵母细胞进行活化处理，所用仪器为KeFa-450型细胞融合仪(中国科学院发育生物学研究所