Cytoplasmic Maturation in Mouse Oocytes

项目摘要 本项目的目的是了解未成熟的卵细胞或卵母细胞， 发育能力。哺乳动物卵母细胞储存在卵巢中，在减数分裂前期被阻止， 长期（女性数十年）。青春期后，卵母细胞进入生长期， 它们合成成熟过程所需的蛋白质。它们被来自大脑的信号刺激成熟。 脑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， I和中期II。这些包括内质网（ER）的戏剧性重组，其中 雌激素受体集中在卵皮质。Ca 2+摄取到ER中也发生，并且这是必需的， 卵子在受精时从ER释放细胞内Ca 2+，这是早期胚胎发育所需的关键事件。 发展卵母细胞也发生变化，允许它们在24小时内进行皮质颗粒胞吐作用。 受精，一个重要的事件，使受精卵不能渗透到一个以上的精子， 作为一种防止多精受精的机制。关于这些事件仍有许多问题。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 所需的发展能力，以及机制，调节ER重组，形成 ER-PM接触和成熟过程中的Ca 2+吸收。目标2将确定蛋白质所需的 组成性胞吐以及Ca 2+调节的皮质颗粒胞吐。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 开发了一种快速和急性降解内源性蛋白质的方法。卵母细胞将注射 荧光分子，其允许ER和ER-PM接触的可视化，或与 抗体/siRNA/吗啉代以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，成熟人类的能力 卵母细胞的体外培养是一个非常重要的领域，但是使卵母细胞成熟的方法还不完善。一个完整 了解卵母细胞发育的各个方面，可以改进培养方法， 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.