小鼠腔前卵泡三维培养成熟后受精体系的建立及优化

doi:10.3969/j.issn.1004-616x.2013.03.011

癌变·畸变·突变

小鼠腔前卵泡三维培养成熟后受精体系的建立及优化

曾荔苹¹,王晓梅²，*,侯震晖¹,林桂淼²,李慧²,林苏霞^2,林晓潭²,朱玥荃²,蔡志明³

( 1. 北京大学深圳医院，广东深圳 518036；2. 深圳大学泌尿生殖研究所，深圳市合成生物学工程实验室，广东深圳 518060；3. 深圳大学第一附属医院，广东深圳 518035 )

收稿日期:2013-02-04 修回日期:2013-03-29 出版日期:2013-05-30 发布日期:2013-05-30
通讯作者: 王晓梅，E-mail：xmwang@szu.edu.cn
作者简介:曾荔苹 (1966- )，女，广东省梅州市人，主任医师，硕士，研究方向：生殖医学。
基金资助:
深圳市科技计划项目 (201002080)；深圳大学自然科学基金 (201147)

Establishment and optimization of mouse preantral follicles 3-dimensions in vitro growth and fertilization system

ZENG Li-ping¹，WANG Xiao-mei²，*，HOU Zhen-hui¹，LIN Gui-miao²，LI Hui²，LIN Su-xia²，LIN Xiao-tan²，ZHU Yue-quan²，CAI Zhi-ming³

(1. Shenzhen Hospital of Beijing University，Shenzhen 518036；2. The Research Institute of Urinary and Reproduction，The Engineering Lab of Synthetic Biology，Shenzhen University，Shenzhen 518060；3. The First Affiliated Hospital of Shenzhen University，Shenzhen 518035, China)

Received:2013-02-04 Revised:2013-03-29 Online:2013-05-30 Published:2013-05-30
Contact: WANG Xiao-mei，E-mail：xmwang@szu.edu.cn

摘要/Abstract

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6 d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。

关键词: 小鼠, 腔前卵泡, 三维培养

Abstract:

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％) (P<0.05). 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．

Key words: mice, preantral follicles, 3-dimensions in vitro growth system

曾荔苹,王晓梅,侯震晖,林桂淼,李慧,林苏霞,林晓潭,朱玥荃,蔡志明. 小鼠腔前卵泡三维培养成熟后受精体系的建立及优化[J]. 癌变·畸变·突变, doi: 10.3969/j.issn.1004-616x.2013.03.011.

ZENG Li-ping，WANG Xiao-mei，HOU Zhen-hui，LIN Gui-miao，LI Hui，LIN Su-xia，LIN Xiao-tan，ZHU Yue-quan，CAI Zhi-ming. Establishment and optimization of mouse preantral follicles 3-dimensions in vitro growth and fertilization system[J]. Carcinogenesis, Teratogenesis & Mutagenesis, doi: 10.3969/j.issn.1004-616x.2013.03.011.

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小鼠腔前卵泡三维培养成熟后受精体系的建立及优化

Establishment and optimization of mouse preantral follicles 3-dimensions in vitro growth and fertilization system

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