MOLECULAR BASIS OF HUMAN OOCYTE MATURATION

人类卵母细胞成熟的分子基础

• 批准号: 2403396
• 负责人: CHRISTOS COUTIFARIS
• 金额: $ 30.03万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2403396
• 关键词: antisense nucleic acid; biological signal transduction; chorionic gonadotropin; confocal scanning microscopy; cytoplasm; egg /ovum; exocytosis; female; fertility; fertilization; gonadotropin releasing factor; graafian follicles; granulosa cell; human subject; immunocytochemistry; integrins; light microscopy; luteinizing hormone; meiosis; microinjections; nucleic acid biosynthesis; plasminogen activator; polymerase chain reaction; tissue /cell culture; tubulin

The cellular and molecular mechanisms involved in the growth of human preantral follicles and the subsequent maturation of the oocyte to acquire competence to undergo meiosis remain largely unknown. Numerous studies have contributed to knowledge of these events in several animal species. The clinical application of this knowledge to the human is significant for the treatment of infertility, the improvement of the reproductive potential of women surviving childhood or early adulthood cancers as well as by its contribution to a basic understanding of reproductive events. In order to study the maturation of oocytes in vivo as well as in vitro, oocytes will be retrieved from volunteers (women undergoing tubal ligations) at different times following hCG administration. In addition, a unique aspect of our studies will involve excision of preantral follicles from ovarian tissue in order to construct culture systems that will permit maturation of a competent gamete. A major goal is to develop an informational database by characterizing the nuclear and cytoplasmic changes that occur during human oocyte maturation in vivo and in vitro. This will be evaluated by morphologic criteria including germinal vesicle breakdown as assessed by phase contrast light microscopy and indirect immunofluorescence. Tubulin immunohistochemistry and fluorescent DNA binding stain will determine spindle and nuclear morphology and thus assess the time to MI. Since integrin adhesion molecules have been detected on the plasma membrane of mammalian oocytes and our preliminary results indicate at least one integrin subunit (beta1) being present in human oocytes, we will investigate the expression of the repertoire of integrin subunits during the final stages of human oocyte maturation. This will be achieved through confocal microscopy using specific antibodies to the different integrins and an attempt will be made to assess their expression at the nucleotide level using RT-PCR. Their functional significance with respect to fertilizability of oocytes will then be tested with the use of functional antibodies or antisense mRNA microinjection. In parallel, the secretion of tissue type plasminogen activator by oocytes developing in vivo or in vitro will also be evaluated using substrate zymography. An additional goal is to evaluate culture systems for the in vitro development of preantral follicles that promote oocyte growth, acquisition of competence to resume meiosis, and capable of supporting normal fertilization and embryogenesis. The effects of various growth factors and hormones on follicular and oocyte development will be assessed. Finally, we propose to investigate the biochemical parameters of human egg activation following fertilization of in vivo and in vitro matured oocytes and to examine the potential signaling mechanisms involved in this process. This will be achieved by quantitatively evaluating calcium transients during fertilization, cortical granule exocytosis, recruitment of maternal mRNA, H1 kinase activity, pronuclear formation, and DNA synthesis and cleavage, using standard assay methods. These studies will define the events involved in normal human oocyte activation. Establishment of the optimal conditions for human oocyte culture from preantral follicles can have a tremendous impact in the enhancement of fertility,.

细胞和分子机制参与的增长， 人腔前卵泡和随后的成熟 获得减数分裂能力的卵母细胞 未知许多研究有助于了解这些 在几种动物中。临床应用 对人类的知识是重要的治疗 不孕症，提高妇女的生殖潜力 在儿童期或成年早期癌症中存活， 有助于对生殖事件的基本了解。在 为了研究卵母细胞在体内以及在体外的成熟， 在体外，将从志愿者（经历了 输卵管结扎）。 此外，我们研究的一个独特方面将涉及切除 从卵巢组织中提取腔前卵泡以构建 允许有能力的配子成熟的培养系统。 一个主要目标是通过以下方式建立信息数据库： 表征细胞核和细胞质的变化， 在人卵母细胞体内和体外成熟过程中。这将是 通过形态学标准进行评价，包括生殖囊泡 通过相差光学显微镜评估的击穿， 间接免疫荧光法微管蛋白免疫组化和 荧光DNA结合染色将确定纺锤体和核 形态学，从而评估至MI的时间。由于整合素粘附 在哺乳动物的质膜上检测到了 卵母细胞和我们的初步结果表明，至少有一个整合素 亚基（β 1）存在于人类卵母细胞，我们将研究 整合素亚单位库在细胞凋亡过程中的表达 人类卵母细胞成熟的最后阶段。完成这项工作的方法是 通过共聚焦显微镜使用特异性抗体 不同的整合素，并试图评估其 使用RT-PCR在核苷酸水平上表达。其功能 关于卵母细胞受精能力的重要性将 使用功能性抗体或反义mRNA进行测试 显微注射同时，组织类型的分泌 卵母细胞在体内或体外发育产生的纤溶酶原激活物 还将使用底物酶谱法进行评估。额外 目的是评估培养系统的体外开发， 促进卵母细胞生长的腔前卵泡， 恢复减数分裂的能力，并能够支持正常的 受精和胚胎发生。各种增长的影响 卵泡和卵母细胞发育的因素和激素将是 评估。最后，我们建议研究生物化学 受精后人类卵子激活的参数 体内和体外成熟的卵母细胞，并检查潜在的 这一过程中涉及的信号机制。这将是 通过定量评估钙瞬变来实现 受精，皮质颗粒胞吐，母体细胞募集 mRNA、H1激酶活性、原核形成和DNA合成 和切割。这些研究将 定义正常人类卵母细胞激活中涉及的事件。 人卵母细胞体外培养最佳条件的建立 从腔前卵泡中提取的蛋白质， 提高生育能力。