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.

项目概要 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 有发展能力。哺乳动物的卵母细胞储存在卵巢中，停滞在减数分裂前期， 较长的时间（女性为数十年）。青春期后，卵母细胞进入生长期，其中 它们合成成熟过程所需的蛋白质。他们受到来自大脑的信号的刺激而成熟 垂体。卵母细胞在卵母细胞成熟过程中经历许多细胞质变化，即前期和成熟期之间的时期。 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+ 也会被内质网吸收，这是必要的 受精时卵子从内质网释放细胞内 Ca2+，这是早期胚胎发育所需的关键事件 发展。卵母细胞也会发生变化，使它们能够在 受精，一项重要的事件，使受精卵无法渗透到多个精子，因此 作为多精受精的预防机制。关于这些事件仍然存在许多疑问。目标1将 研究 ER 结构的变化和 ER-质膜 (ER-PM) 接触的形成是否与 发育能力所需的，以及调节 ER 重组、形成的机制 ER-PM 接触和成熟过程中 Ca2+ 的吸收。目标 2 将识别所需的蛋白质 组成型胞吐作用以及 Ca2+ 调节的皮质颗粒胞吐作用。这些实验将 利用蛋白质降解方法来研究特定蛋白质的作用，单独或与 其他候选蛋白质，使用分离的或卵泡封闭的卵母细胞。重要的是，我们将使用新的 开发了快速、急性降解内源蛋白质的方法。卵母细胞将被注射 荧光分子，可以使 ER 和 ER-PM 接触可视化，或者与 抗体/siRNA/吗啉代特异性地消耗蛋白质。这些研究得出的结论 将适用于了解女性卵母细胞的发育。目前，人类的能力已经成熟 体外卵母细胞是一个非常重要的领域，但卵母细胞成熟的方法并不完善。一个完整的 了解卵母细胞发育的各个方面可能会导致培养方法的改进，从而提高 辅助生殖的成功。