摘要
目的:建立小鼠腔前卵泡体外海藻酸盐包埋培养的三维培养(3-dimensions in vitro growth,3D-IVG)方法以及成熟卵母细胞体外受精体系。方法:采用机械法分离12日龄雌性昆明小鼠卵巢,获取结构完整的腔前卵泡,腔前卵泡经过包埋后体外立体化培养,激素超排,收集黏液化的卵丘-卵母细胞复合体(cumulus-oocyte-complex,COCs),分别计数发泡期(germinal vesicle,GV)卵母细胞、生发泡破裂期(germinal vesicle breakdown,GVBD)卵母细胞和含有第一极体(first polar body,PB)卵母细胞,制备卵母细胞染色体并进行C带染色分析。同时设小鼠体内超排卵母细胞(in vivo convention,IVC)作为对照。再将体外培养成熟的卵母细胞与获能后的小鼠精子受精,观察受精情况。结果:经过3D-IVG,卵泡存活率为82.5%,卵泡发育过程维持完整的三维结构,卵泡直径增长迅速,培养6d后,有91.7%成腔;在激素超排后,有82.6%COCs黏液化。GV、GVBD和含有PB的成熟卵母细胞分别为6.1%,45.4%和48.5%。3D-IVG获得的成熟卵母细胞百分率(48.5%)明显低于体内超排成熟卵母细胞百分率(82.9%),差异有统计学意义(P<0.05)。但3D-IVG所获得的卵母细胞染色体C带分析表明染色体数目为20条,结构未见异常,与体内超排卵母细胞一致。并观察到3D-IVG培养成熟的卵母细胞可以受精成功。结论:建立并完善了小鼠腔前卵泡的三维体外培养体系,为生殖毒性实验研究和胚胎工程研究提供了新的方法。
OBJECTIVE： To establish mouse preantral follicles 3-dimensions in vitro growth （3D-IVG） system and an efficient in vitro fertilization system. METHODS：Preantral follicles （PFs） of the 12 d old Kunming mice were isolated. 150 μm PFs were selected from each ovary. Then,PFs were cultured in 3D-IVG system for about 6 d. Ovulation of surviving follicles was induced by HCG and EGF. 36 h later,the mucificated cumulus-oocyte-complexes （COCs） and the matured oocytes were collected. Germinal vesicle breakdown （GVBD）,germinal vesicle （GV） oocytes and the matured oocytes were calculated under the stereomicroscope. Chromosomal abnormalities of matured oocytes including numerical and structural aberrations were examined and recorded by C-band strains compared with that of matured oocytes in vitro convention （IVC）.We put sperms and mature COCs together for 24-48 h,then, observed the zygote formation. RESULTS：In 3D-IVG system,the survival rate of the follicles was 82.5% and the mean diameter of follicles increased rapidly. After 6 d of culture ,the survival rate was 91.7%,the ovulation rate induced by hormone was 82.6%,6.1% oocytes arrested at GV stage,45.4% oocytes showed GVBD and 48.5% oocytes emitted the first polar body.After 6 d 3D-IVG culture,the development rate （48.5%） of PFs was strikingly lower than in vivo oocytes superovulation （IVC,82.9%） （P0.05）. However,the chromosomal abnormalities of 3D-IVG have no significant increase comparing with that of oocytes in vitro maturation （IVM） and oocytes superovulation in vivo. We observed that those matured oocytes cultured from 3D-IVG system could get fertilized,from which we can draw a conclusion that they obtained the ability of developing to embryos. CONLUSION：We established and optimized mouse preantral follicles 3D-IVG system,which may provide a new method for reproductive toxicology and embryo engineering.
出处
《癌变．畸变．突变》
CAS
CSCD
2013年第3期210-214,共5页
Carcinogenesis,Teratogenesis & Mutagenesis
基金
深圳市科技计划项目(201002080)
深圳大学自然科学基金(201147)
关键词
小鼠
腔前卵泡
三维培养
mice
preantral follicles
3-dimensions in vitro growth system
