Signaling mechanisms that regulate meiosis in mammalian oocytes

描述（由申请人提供）：本研究的目的是研究调节哺乳动物卵母细胞减数分裂进程的信号事件。未成熟的卵母细胞被抑制在前期I，在黄体生成素LH（促黄体生成素）作用于卵泡细胞的作用下，它们重新进入减数分裂并成熟成卵。LH将其信号从卵泡细胞传递到卵母细胞的机制尚不清楚。本提案的目的1将研究黄体生成素启动小鼠卵母细胞减数分裂恢复的信号机制。在LH激增之前，减数分裂停止需要卵母细胞中高水平的cAMP；这种cAMP是由卵母细胞质膜上的g蛋白偶联受体GPRS产生的，它刺激g激活腺苷酸环化酶。作为对LH的反应，卵母细胞中的cAMP水平下降，导致减数分裂恢复。在卵母细胞中，LH可以作用的潜在靶点包括：1)GPR3，它可以通过G蛋白受体激酶（GRKs）和li-arrestins的激活而关闭，这是下调大多数gprrs的常见机制。2) Gs，其活性可被g蛋白信号（RGS）蛋白调控因子抑制。3) AKT/PKB信号通路，已被证明影响多种物种的减数分裂恢复。4)缝隙结点层面的调控。这些途径将使用最近开发的微注射卵泡封闭卵母细胞的方法进行研究。目的2将通过检查人类卵母细胞是否包含这些信号通路的相同成分，来检查人类卵母细胞的减数分裂停止和恢复是否依赖于与啮齿动物卵母细胞相同的途径。Aim 2还将描述人类卵母细胞成熟过程中通常发生的细胞质事件。这些研究将为啮齿动物模型的哪些方面可以应用于人类卵母细胞提供有价值的信息。减数分裂如何调控的问题与妇女健康问题高度相关。特别是，开发在体外成功使未成熟的人类卵母细胞成熟的方法具有很高的临床兴趣，这可能导致体外受精治疗的改进，并为妇女提供比目前可用的更多选择来保持其生育能力。体外成熟的改进将需要了解减数分裂停止的所有阶段和调节卵母细胞成熟起始的生理机制。拟议的研究将有助于奠定这些临床进展的基础科学背景。

DESCRIPTION (provided by applicant): The goal of this research is to examine the signaling events regulating meiotic progression in mammalian oocytes. Immature oocytes are arrested at prophase I, and in response to a surge of luteinizing hormone LH) that acts on the follicle cells, they re-enter meiosis and mature into eggs. The mechanism(s) by which LH transmits its signal from the follicle cells to the oocyte is not known. Aim 1 of this proposal will examine signaling mechanisms by which LH initiates meiotic resumption in mouse oocytes. Prior to the LH surge, meiotic arrest requires a high level of cAMP in the oocyte; this cAMP is produced by the G-protein coupled receptor GPRS, in the oocyte plasma membrane, that stimulates Gs to activate adenylate cyclase. In response to LH, cAMP levels in the oocyte fall, leading to meiotic resumption. Potential targets in the oocyte by which LH could act include the following: 1) GPR3, which could be turned off through the activation of G- protein receptor kinases (GRKs) and li-arrestins, a common mechanism for downregulating most GPCRs. 2) Gs, the activity of which could be inhibited by a regulator of G-protein signalling (RGS) protein. 3) The AKT/PKB signalling pathway, which has been shown to affect meiotic resumption in a variety of species. 4) Regulation at the level of gap junctions. These pathways will be investigated using recently developed methods for microinjecting follicle-enclosed oocytes. Aim 2 will examine if meiotic arrest and resumption in human oocytes depend on the same pathways as in rodent oocyte, by examining if human oocytes contain the same components of these signalling pathways. Aim 2 will also characterize cytoplasmic events that normally occur during human oocyte maturation. These studies will provide valuable information about which aspects of rodent models can be applied to human oocytes. The question of how meiosis is regulated is highly relevant to issues of women's health. In particular, it is of high interest clinically to develop methods for successfully maturing immature human oocytes in vitro, which could lead to improvements in in vitro fertilization treatments, as well as provide more options than are currently available for women to preserve their fertility. Improvements in in vitro maturation will require an understanding of all stages of meiotic arrest and the physiological mechanisms regulating the initiation of oocyte maturation. The proposed studies will contribute to the basic science background that underlies such clinical advances.