Cytoplasmic Maturation in Mouse Oocytes

项目总结 这个项目的目标是了解未成熟的卵细胞或卵母细胞成为 发展能力强的。哺乳动物的卵母细胞储存在卵巢中，在减数分裂前期停止，用于 延长的时间(女性为数十年)。在青春期之后，卵母细胞进入生长期 它们合成成熟过程所需的蛋白质。他们被来自于 脑下垂体。在卵母细胞成熟过程中，卵母细胞经历了许多细胞质的变化，在卵母细胞成熟的前期 其中包括内质网(ER)的戏剧性重组，其中 内质网集中在卵子皮质。钙离子也会被吸收到内质网中，这是 卵子在受精时从内质网释放细胞内钙离子，这是早期胚胎所需的关键事件 发展。卵母细胞也会发生变化，使它们在 受精，一个重要的事件，使受精卵不能被多个精子渗透，从而 作为一种多精受精的预防机制。关于这些事件，许多问题仍然存在。目标1将 研究内质网结构的改变和内质网-质膜(ER-PM)接触的形成是否 发育能力所需的，以及调节内质网重组的机制，形成 ER-PM接触，成熟过程中钙摄取。目标2将确定需要的蛋白质 皮质颗粒的结构性胞吐和钙离子调节的胞吐。这些实验将 利用蛋白质降解方法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，我们将使用一个新的 开发了快速和急性降解内源蛋白质的方法。卵母细胞将被注射入 允许显示ER和ER-PM接触的荧光分子，或与 抗体/siRNAs/Morolinos特异性地消耗蛋白质。从这些研究中得出的结论 将适用于了解女性的卵母细胞发育。目前，人类成熟的能力 卵母细胞的体外培养是一个非常重要的领域，但成熟卵母细胞的方法并不完善。一个完整的 对卵母细胞发育的各个方面的了解可能会导致改进培养方法，从而增加 辅助生殖的成功。

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.