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Studies on production of pig embryos from growing oocytes

利用生长中的卵母细胞生产猪胚胎的研究

基本信息

批准号：
09556061
负责人：
KATO Seishiro
金额：
$ 9.09万
依托单位：
KOBE UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09556061/
关键词：
pig oogenesis folliculogenesis oocyte growth oocyte maturation in vitro culture in vitro fertilization embryo production ブタ体外発育卵母細胞体外成熟卵母細胞

项目摘要

Several hundred thausand primordial follicles are contained in the ovaries of domestic animals. The development of in vitro culture systems for the growth of small oocytes in primordial or small follicles is expected to provide a new source of a large population of oocytes for livestock production. This study was conducted to develop Culture systems for small oocytes from pig ovaries.Preantral follicles containing oocytes with various sizes were isolated from pig ovaries and cultured in collagen gel for up to 16 days in the presence of serum, FSH and oestradiol. Waymouth MB7 52/1 was used as a basic culture medium for oocyte growth. After culture, the oocytes enclosed by one or two layers of cumulus cells were cultured for meiotic maturation and inseminated with spermatozoa. The results obtained were as follows.1. Pig growing oocytes grew to their final size, acquired meiotic competence du ring in vitro culture, and were penetrated by spcermatozoa in vitro, although there was a very lo … More w level of success in fertilization. In contrast to the growing oocytes, no culture methods were available for much smaller oocytes. We tried to culture primordial follicles which contained small oocytes 30 μm in diameter, but the integrity of the follicles was soon broken and the follicles survived for only a few days.2. Using culture systems, we demonstrated that granulosa cells regulate oocyte growth, and that antrum formation of granulosa cells was induced only by meiotic-competent oocytes, indicating that the oocytes secrete a substance(s) that induces differentiation of granulosa cells.3. Of four glycosaminoglycans examined, hyalronic acids and chondroitin sulfate A supported the development of in vitro-matured and -fertilized pig oocytes to the blastocyst stage.4. In addition, degradation of cyclin B1 molecules and MAP kinase deph osphorylation during fertilization, localisation of phosphorylated MAP kinase during the transition from meiosis I to meiosis II, assembly and transformation of the spindle during the progression through meiotic cell cycle, and the level and localization of CENP-E and kinetochore numaber during maturation were examined. Less

家畜卵巢中含有数百个原始卵泡。在原始卵泡或小卵泡中培养小卵母细胞的体外培养系统的发展有望为畜牧业生产提供大量卵母细胞的新来源。本研究从猪卵巢中分离出含有不同大小卵母细胞的腔前卵泡，在胶原蛋白凝胶中加入血清、FSH和雌二醇培养16天。Waymouth MB 7 52/1用作卵母细胞生长的基础培养基。卵母细胞体外培养后，被一层或两层卵丘细胞包裹的卵母细胞进行减数分裂成熟培养，并与精子进行授精。主要研究结果如下：1.猪卵母细胞在体外培养中发育至最终大小，获得减数分裂能力，并在体外被孢子虫穿透，尽管有很低的穿透率， ...更多信息在受精成功的水平。与生长中的卵母细胞相反，对于小得多的卵母细胞没有可用的培养方法。我们尝试培养含有直径30 μm的小卵母细胞的原始卵泡，但卵泡的完整性很快被破坏，卵泡只能存活几天.利用培养系统，我们证明了颗粒细胞调节卵母细胞的生长，并且颗粒细胞的腔形成仅由减数分裂有能力的卵母细胞诱导，表明卵母细胞分泌一种诱导颗粒细胞分化的物质.在四种糖胺聚糖中，透明质酸和硫酸软骨素A支持体外成熟和受精的猪卵母细胞发育到囊胚阶段。此外，在受精过程中的细胞周期蛋白B1分子的降解和MAP激酶去磷酸化，定位磷酸化的MAP激酶在从减数分裂I到减数分裂II的过渡过程中，组装和转化的纺锤体在通过减数分裂细胞周期的进展，并在成熟过程中CENP-E和动粒数的水平和定位进行了检查。少