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Production of Live Young form Mouse Oocytes and Fetal Germ Cells by a Novel Culture System

通过新型培养系统生产活的年轻小鼠卵母细胞和胎儿生殖细胞

基本信息

批准号：
17500294
负责人：
KADA Hidemi
金额：
$ 2.11万
依托单位：
Junior College of Tokyo University of Agriculture
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17500294/
关键词：
oocyte culture in vitro growth in vitro maturation in vitro oocyte maturation co-culture mouse oocyte-granulosa cell complexes 生殖細胞

项目摘要

The aim of this study was to establish the efficient culture method for mouse oocytes in vitro. A novel system was designed by co- culture with feeder cells derived from ovaries. B6CBF1 female mice (12 days old) were used in this study. Oocyte-granulosa cell complexes (OGCs) were isolated from ovaries by collagenase digestion in Leibovi's L-15 medium with 5% fetal bovine serum (FBS). The OGCs were group-cultured for 10 or 12 days on the feeder cells on collagen-coated insert membrane in a-minimum essential medium (MEM) supplemented with 100mlU/ml follicle-stimulating hormone, 10m1U/mIluteinizing hormone, 50ng/ml insulin-like growth factor type I, 50ng/ml stem cell factor at 37℃. Half amount of medium was exchanged on alternate days. The oocyte growth in vitro was achieved by this system. The oocyte maturation was induced by addition of final concentration of 1.5lU/ml human chorionic gonadotrophin and 5ng/ml epidermal growth factor on day 10 or 12 of culture. Release and mucification of the OCCs was observed at 17 hours after the maturation induction. The survival rate, the volume increase, the maturation competence and the developmental competence were improved by addition of gonadotropin, growth factor and co-culture, compared with old system. In culture in vitro from fetal ovaries (16-17dpc), the IVG-IVM oocytes showed no embryonic development, unfortunately. Thus, further improvement of culture conditions is required. However, the effectiveness of this culture system was shown in culture in vitro from the OGCs.

本研究旨在建立高效的小鼠卵母细胞体外培养方法。设计了一种与卵巢饲养细胞共培养的新体系.本研究使用B6CBF1雌性小鼠（12日龄）。采用胶原酶消化法从卵巢中分离出卵母细胞-颗粒细胞复合体（OGC）。将OGC置于含100mlU/ml促卵泡激素、10mlU/ml促黄体生成素、50ng/ml胰岛素样生长因子Ⅰ、50ng/ml干细胞因子的最低必需培养基（MEM）中，以胶原包被的插入膜为饲养层，于37 ℃培养10天或12天。隔日更换一半量的培养基。利用该系统实现了卵母细胞的体外培养。在培养的第10天或第12天加入终浓度为1.5IU/ml的人绒毛膜促性腺激素和5ng/ml的表皮生长因子诱导卵母细胞成熟。在成熟诱导后17小时观察到OCC的释放和粘液化。与旧系统相比，添加促性腺激素、生长因子和共培养可以提高细胞的存活率、体积增长、成熟能力和发育能力。在体外培养的胚胎卵巢（16 - 17 dpc），IVG-IVM卵母细胞显示没有胚胎发育，不幸的是。因此，需要进一步改善培养条件。然而，该培养系统的有效性在体外培养的OGC中显示。