昆明小鼠成熟卵母细胞体外受精及受精卵体外培养的研究

目的通过比较获能受精液成分及成熟卵母细胞的处理方法，观察对昆明小鼠体外受精(IVF)效果的影响和用于早期胚胎培养的三种发育培养液培养效果，探讨并建立起适合于昆明小鼠卵子体外受精与受精卵体外发育的实验体系。方法用T6或FM两种获能受精液对采自昆明雄鼠附睾尾的精子与成熟卵母细胞进行体外受精(IVF)，受精前对成熟卵母细胞选用透明质酸酶去除卵丘细胞和不去卵丘细胞(对照组)两种方法进行处理，受精卵分别用M16、改良的CZB液(mCZB)和改良的M16液(mM16)三种发育培养液对体外受精胚进行培养。结果选用FM获能受精液的受精率极显著高于T6液(92．3％对64．7％)，且体外受精前先用透明质酸酶处理的成熟卵母细胞比对照组成熟卵母细胞受精率要低(59．2％对64．7％)，但差异不显著；用mM16培养液进行培养时桑胚率及囊胚率(55．3％和35．6％)显著高于mCZB(13．3％和8．1％)和M16培养液(17．3％和14．7％)，后两者差异不显著。结论昆明小鼠卵子体外受精时宜选用FM受精获能液，不宜用透明质酸酶去除卵丘细胞，且宜选用mM16培养液对昆明小鼠体外受精卵进行早期培养，其中的亚硫磺酸钠可有效的克服2-细胞胚后的体外发育阻滞现象。

Mature Oocyte in vitro Fertilization and Embryo Culture of Kunming Mice

Objective The experiments were carried out to develop an improved in vitro fertilization (IVF) system for Kunmimg mice by comparing the effect of fertilization media and treatment methods on IVF of mature oocytes and the effect of three culture media for early embryo development. Methods Sperm was prepared by sqeezing cauda epididymidis from male mouse and mixed with mature oocyte from superovulated female mouse in T6 or FM media, and mature oocytes were treated with hyaluronidase for removing cumulus cells. The control was not treated before fertilization. The IVF embryos were cultured in M16, modified CZB media (mCZB) or modified M16 media (mM16) media. Results The fertilization rate in FM media was significantly higher than that in T6 media (92 3% vs. 64 7%). The fertilization rate of treatment method to mature oocyte without hyaluronidase before fertilization was higher than that of hyaluronidase treatment method (64 7% vs. 59 2%), but no significant difference was found. The developmental rate of morulae and blastocysts in M16 media (55 3% and 35 6%) were significantly higher than that in mCZB media (17 3% and 14 7%). Conclusion It is indicated that FM medium is more appropriate for in vitro fertilization of oocytes, and it is not necessary to remove the cumulus cells of mature oocyte by hyaluronidase. MM16 media is the most appro priate for in vitro culture of early embryos of Kunming mice, and hypotaurine can efficiently overcome embryo development retardation after 2 cell stage. reofearlyembryos