小鼠极体重建卵母细胞的体外受精与发育

本实验分别以昆明系、ICR系和C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb系小鼠（即转绿色荧光蛋白基因小鼠，简称荧光标记小鼠）为主要试验动物，探索其卵母细胞第一极体核重组卵母细胞的生殖功能。超数排卵后采集小鼠卵丘卵母细胞复合体（COCs），利用链霉蛋白酶酶解法分离出第一极体（PbI）；以形态学和台盼蓝染色相结合的方法对不同温度保存条件下COCs中PbI进行活力鉴定和分级；采用显微操作术将PbI核供体注射到去核卵母细胞中，卵母细胞，进一步观察重建卵母细胞体外受精和体外培养发育能力。结果表明：注射hCG后12～14h回收的PbI活力最佳， 4℃条件下保存48h 后PbI仍保持活性；117枚昆明系和ICR系小鼠重建卵母细胞经体外受精获得13枚2-细胞胚胎， 38枚荧光标记小鼠的卵母细胞中有3枚卵裂成2-细胞胚胎。本研究初步证明小鼠第一极体重建卵母细胞可持续随后受精及其早期发育过程，为发掘和利用其它哺乳动物母本基因资源提供了参考依据。

In Vitro Fertilization and Development of the Mouse oocytes reconstructed by First Polar Bodies

The reproductive function of three strains of mice (Kunming, ICR and C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb) oocytes reconstructed by the nuclei of first polar body (abbreviated to PbI) was explored in these experiments, respectively. Cumulus oocyte complexes (COCs) from the mouses were collected after superovulation, then PbIs were obtained from the COCs by 2% pronase treatment. The survival PbIs under different temperature was identified and stapled by the methods of morphology and trypanblau staining, later the contents including nuclei and a little cytoplasm of PbI was injected into the oocyte that its nuclei had been enucleated by micromanipulation before, the reconstructed oocytes by PbI was fertilized and cultured in vitro in order to observe its ability of further development. The results showed that vigor of PbIs was in the best status within 12 to 14 hours after superovulation, and still maintained during 48 hours under 4℃. 13 out of 117 reconstructed oocytes from Kunming and ICR mice developed into 2-cell embryos, and 3 out of 38 reconstructed oocytes from C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb mice was cleavage as 2-cell embryos. The experiments confirmed that the mouse oocytes reconstructed by the nuclei of PbI could be normally maintained its fertilization and further development, this has offered valuable reference for further utilization of gene resources for other mammalian animals.