小鼠腔前卵泡体外培养方法的建立  

Establishment of in-vitro mouse preantral follicle culture systems

作　　者：万旭英[1] 朱玉平[1] 马玺里[1] 朱江波[1] 侯娟[1] 刘真[1] 王飞[1] 张天宝[1] 

机构地区：[1]第二军医大学基础部卫生毒理学教研室,上海200433

出　　处：《卫生研究》2009年第2期153-158,共6页Journal of Hygiene Research

基　　金：国家自然科学基金资助项目(No.30571587)

摘　　要：目的建立可供毒理学研究的小鼠腔前卵泡体外培养方法。方法采用机械方法分离出生后12～14天(C57B1/6JxCBA/Ca)小鼠卵巢内直径为100～130μm的腔前卵泡,96孔板单个卵泡连续培养12天后诱导排卵16h,观察卵泡发育、激素分泌和卵子形成,并将体外卵母细胞的成熟情况(IVG)与体内卵母细胞的生长情况(IVM)进行比较,以判断体外培养方法是否成功。结果分离完整卵泡率为89.74%(376/419);培养12天卵泡存活率为96.81%(364/376),有腔形成率为48.94%(184/376);卵泡和卵母细胞直径显著增加。诱导排卵后卵丘细胞-卵母细胞复合体排出率为56.38%(212/376);培养卵泡经历从腔前卵泡到卵母细胞成熟和极体排出的形态学变化,也有部分卵泡不发生腔样结构改变。体外培养卵泡分泌E2和P激素与体内分泌特征一致。IVG组4.61%(10/217)卵母细胞不能恢复成熟[停止在生发泡期(GV期)],76.50%(166/217)停止减数分裂在生发泡破裂期(GVBD期),18.89%(41/217)卵母细胞停止在第2次减数分裂(MII期)(PB);而IVM组1.24%(3/241)的卵母细胞不能恢复成熟(停止在GV期),45.23%(109/241)停止减数分裂在GVBD,53.53%(41/241)卵母细胞停止在MII期(PB);卵母细胞染色体核型完整,无染色体数目和结构异常。结论本研究建立的小鼠腔前卵泡体外培养方法基本成功,可作为卵泡发育生理研究和毒理学研究的模型。Objective To develop mouse preantral follicle culture for toxicology study. Methods The ovaries were from prepubertal mice（C57B1/6JxCBA/Ca） （at the ages of 12- 14 days）. The ovaries were from prepubertal mice （C57B1/6JxCBA/Ca） （at the ages of 12 - 14 days）. The preantral follicles with a diameter around the range 100 - 130ptm were mechanically dissected and randomly allocated to 96 - well plates. After subsequently cultured individually for 12 days, the follicles were induced ovulation. The in-vitro developmental characteristics of the follicles were observed including hormonogenesis,follieulogenesis and oogenesis 16h later after ovulation. In-vitro growth and maturation of oocytes（IVG） were compared with in- vivo growth and in- vitro maturation of oocytes（IVM） to assess the in- vitro assay. Results 89.74 % （376/419） of follicles were visibly associated with theca cells and possessed a close follicle （granulosa） cell/oocyte apposition. At 12 days of culture, the average diameter of follicles were increased, the survival rate of follicles is 96.81% （364/376）, 48.94 % （184/376）of the cultured follicles reached the pre-ovulatory stage with a large antrum-like cavity, and 56.38% （212/376） COCs were released by maturation induction in this study. From preantral follicle to oocyte maturation and the formation of first polar body, the morphological observation showed that developmental characteristics of mouse preantral follicles in-vitro were similar to those in-vivo. The patterns of hormone secretion observed in preantral follicle culture were similar to the characteristics of in-vivo follicle hormone secretion. The numbers of oocytes with germinal vesicle （GV） did not differ between IVG and IVM groups, 18.89% oocytes form the first polar body in IVG groups and 53.53% oocytes form the first polar body in IVM groups. No abnormal chromosome segregation found in this study. Conclusion This study successfully developed in-vitro mouse pre-antral follicle culture system,which

关 键 词：腔前卵泡 体外培养 小鼠 

分 类 号：Q25[生物学—细胞生物学]