Cytoplasmic Maturation in Mouse Oocytes

小鼠卵母细胞的细胞质成熟

• 批准号: 9885809
• 负责人: LISA M MEHLMANN
• 金额: $ 35.26万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-02 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9885809
• 关键词: Acute; Antibodies; Appearance; Area; Biology; Cell membrane; Cells; Competence; Cytoplasmic Granules; Cytoplasmic Protein; Development; Disease; Embryo; Embryonic Development; Endoplasmic Reticulum; Environmental Risk Factor; Event; Exocytosis; Family; Fertilization; Fluorescent Probes; Future; Growth; Human; In Vitro; Knowledge; Label; Lead; Meiosis; Meiotic Prophase I; Metaphase; Methods; Molecular; Movement; Mus; Nuclear; Oocytes; Ovary; Physiological Processes; Pituitary Gland; Pituitary Hormones; Prevention; Problem Solving; Process; Production; Prophase; Proteins; Puberty; RNA Interference; Regulation of Exocytosis; Reproductive Health; Research; Role; Secretory Cell; Signal Transduction; Site; Small Interfering RNA; Structure; Testing; Time; Visualization; Woman; assisted reproduction; clinical development; early pregnancy loss; egg; experimental study; extracellular; improved; infertility treatment; oocyte maturation; oocyte quality; ovulation time; poor egg quality; protein degradation; protein transport; rapid technique; response; sperm cell; success; syntaxin; uptake; zygote

PROJECT SUMMARY The objective of this project is to understand mechanisms by which immature egg cells, or oocytes, become developmentally competent. Mammalian oocytes are stored in the ovary, arrested at meiotic prophase, for extended periods of time (decades in women). Following puberty, oocytes enter a period of growth in which they synthesize proteins needed for the maturation process. They are stimulated to mature by signals from the pituitary. Oocytes undergo many cytoplasmic changes during oocyte maturation, the period between prophase I and metaphase II. These include a dramatic reorganization of the endoplasmic reticulum (ER) in which the ER becomes concentrated in the egg cortex. Ca2+ uptake into the ER also occurs, and that is necessary for the egg to release intracellular Ca2+ from the ER at fertilization, a critical event required for early embryonic development. Oocytes also undergo changes that permit them to undergo cortical granule exocytosis at fertilization, an important event that renders the fertilized egg impermeable to more than one sperm and thus serves as a polyspermy prevention mechanism. Many questions remain about these events. Aim 1 will investigate whether changes in ER structure and the formation of ER-plasma membrane (ER-PM) contacts are required for developmental competence, as well as mechanisms that regulate ER reorganization, formation of ER-PM contacts, and Ca2+ uptake during maturation. Aim 2 will identify proteins that are needed for constitutive exocytosis as well as the Ca2+-regulated exocytosis of cortical granules. These experiments will make use of protein degradation methods to study the role of specific proteins, alone or in combination with other candidate proteins, using isolated or follicle-enclosed oocytes. Importantly, we will use a newly developed method for the rapid and acute degradation of endogenous proteins. Oocytes will be injected with fluorescent molecules that allow the visualization of the ER and ER-PM contacts, or with antibodies/siRNAs/morpholinos to specifically deplete proteins. The conclusions reached from these studies will be applicable to understanding oocyte development in women. Currently, the ability to mature human oocytes in vitro is an area of high importance, but methods for maturing oocytes are imperfect. A complete knowledge of all aspects of oocyte development could lead to improved culturing methods that will increase the success of assisted reproduction.

项目摘要 该项目的目的是了解未成熟卵细胞或卵母细胞变成的机制 发育有能力。哺乳动物的卵母细胞存储在卵巢中，在减数分裂预言中被捕，因为 长时间（女性数十年）。青春期后，卵母细胞进入一个生长时期 它们合成成熟过程所需的蛋白质。它们通过来自 垂体。卵母细胞在卵母细胞成熟期间经历许多细胞质变化，预言之间的周期 I和中期II。其中包括内质网（ER）的戏剧性重组，其中 ER集中在卵皮质中。 Ca2+摄取急诊室也会发生，这对于 卵在受精时从ER中释放出细胞内Ca2+，这是早期胚胎的关键事件 发展。卵母细胞还会发生变化，使它们能够在 受精，这是一个重要的事件，它使受精的鸡蛋不渗透到一个以上的精子中，从而使 充当多植物预防机制。这些事件仍然存在许多问题。目标1意志 调查ER结构的变化和ER - 铂膜（ER-PM）接触的形成是否是 发展能力以及调节ER重组的机制所必需的 ER-PM触点和成熟过程中的Ca2+摄取。 AIM 2将确定所需的蛋白质 组成型胞吐作用以及Ca2+调节的皮质颗粒的胞吐作用。这些实验会 利用蛋白质降解方法来研究特定蛋白质的作用，或与之结合 使用分离或卵泡封闭的卵母细胞的其他候选蛋白质。重要的是，我们将使用新的 开发了内源性蛋白快速和急性降解的方法。卵母细胞会注射 允许可视化ER和ER-PM触点的荧光分子或与 抗体/siRNAS/形态的特异性耗尽蛋白质。这些研究得出的结论 将适用于了解女性的卵母细胞发展。目前，人类成熟的能力 体外卵母细胞是一个非常重要的领域，但是成熟的卵母细胞的方法不完善。完整 了解卵母细胞开发的各个方面可能会导致改进的培养方法，从而增加 辅助生殖的成功。