EPIGENETIC CONTROL OF NORMAL EMBRYO DEVELOPMENT IN VITRO

体外正常胚胎发育的表观遗传控制

• 批准号: 2403146
• 负责人: BARRY DOUGLAS BAVISTER
• 金额: $ 39.78万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 2000-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2403146
• 关键词: Macaca mulatta; acidity /alkalinity; cellular respiration; confocal scanning microscopy; cooperative study; cytoplasm; early embryonic stage; embryo /fetus culture; embryo /fetus transplantation; embryo implantation; glycolysis; growth media; hamsters; homeostasis; in vitro fertilization; mammalian embryology; mitochondria; radiofluorescent probe; video microscopy

DESCRIPTION: The long-term goal of this proposal is to devise new culture media that support normal preimplantation embryo development. The developmental competence and viability of cultured embryos are compromised, showing that improvements in culture media are still needed. Improved media are urgently needed by human IVF clinics to increase the mean embryo viabili-ty, allowing fewer embryos, perhaps only one, to be transferred and support a successful pregnancy; this could eliminate the need for gonadotropin stimulation of patients. Other applications could benefit from improved embryo culture media, e.g., production of transgenic hamsters and rats for biomedical and toxicological research. Improvements are also needed to better support development of embryos stressed by cryopreservation or by micromanipulation (e.g., for preimplantation genetic diagnosis). Instead of using empirical methods for cultured medium design, the proposed research takes a mechanistic approach to find how hamster and rhesus monkey embryo development are regulated by key epigenetic factors, so these can be optimized and incorporated into new media formulations. The connecting theme of the re-search is "cytoplasmic homeostasis," targeting three critically important aspects if preimplantation embryo development the ionicregulation of intracellular protons (pHi) and calcium (pCai), oxidative metabolism and cytoplasmic organization, which are all perturbed in culture. The experimental strategy is to determine how culture conditions perturb these components, then modify the culture environment to compensate for these perturbations, and finally incorporate this new knowledge into improved culture media for preimplantation embryos. The proposed research is hypothesis-driven to provide answers to specific questions. Specific aims are to examine mechanisms of pHi homeostasis and relationships among pHi, calcium/magnesium and phosphate, and embryo development: to determine relationships among mitochondrial metabolism, glycolysis and development; to find how cytoplasmic components, including mitochondria and the cytoskeleton, are perturbed by culture conditions; and to test new media formulations on development. Techniques include fluorescent probe measurement of pHi and pCai; measuring substrate utilization by single embryos; static confocal and dynamic two-photon microscopic analysis of cell structural changes; objective analysis of embryo development by four-dimensional videomicroscopy; and embryo transfers.

描述：该提案的长期目标是设计新的文化 支持正常植入前胚胎发育的培养基。 的 培养胚胎的发育能力和生存力受到损害， 这表明培养基仍需要改进。 改进的媒体 人类试管婴儿诊所迫切需要增加平均胚胎 可行性，允许更少的胚胎，也许只有一个，被转移， 支持成功怀孕;这可以消除需要 促性腺激素刺激患者。 其他应用程序可以受益于 改进的胚胎培养基，例如，转基因仓鼠的生产， 用于生物医学和毒理学研究的大鼠。 改进也 需要更好地支持冷冻保存所强调的胚胎发育 或通过显微操作（例如，用于植入前遗传学诊断）。 而不是使用经验方法的培养基设计，建议 一项研究采用了一种机械的方法来发现仓鼠和恒河猴 胚胎发育受关键的表观遗传因素调节，因此这些因素可以 优化并纳入新的培养基配方中。 连接 研究的主题是“细胞质稳态”， 至关重要的方面，如果植入前胚胎发育， 细胞内质子（pHi）和钙（pCai）的离子调节，氧化 代谢和细胞质组织，这些都在培养中受到干扰。 实验策略是确定培养条件如何干扰 这些成分，然后修改培养环境，以补偿 这些扰动，并最终将这些新知识纳入 用于植入前胚胎的改良培养基。 拟议研究 是假设驱动的，以提供具体问题的答案。 具体 目的是研究pHi稳态的机制以及 pHi、钙/镁和磷酸盐以及胚胎发育：确定 线粒体代谢、糖酵解和发育之间的关系; 发现细胞质成分，包括线粒体和 细胞骨架，受培养条件的干扰;并测试新培养基 关于发展的提法。 技术包括荧光探针 pHi和pCai的测量;通过单次测量底物利用率 胚胎;细胞静态共聚焦和动态双光子显微分析 结构变化;胚胎发育的客观分析， 四维视频显微镜和胚胎移植。