Signal Transduction at Fertilization

描述（由申请人提供）：本申请是关于卵母细胞成熟和受精过程中信号转导的研究项目的续期。它解决了长期悬而未决的黄体生成素（LH）如何导致哺乳动物卵泡封闭卵母细胞减数分裂恢复的问题。最近的研究发现，卵泡体细胞之间的间隙连接在LH的作用下迅速关闭，这为本项目的3个目标奠定了基础：1)研究LH诱导的卵泡间隙连接关闭是否由TACE蛋白酶介导的EGF受体激活引起，2)研究LH作用下卵泡细胞中Cx43间隙连接的磷酸化介导关闭是否需要恢复减数分裂，3)研究LH作用下卵泡细胞中间隙连接关闭在调节cAMP和cGMP中的作用。用荧光示踪剂显微注射活卵泡封闭卵母细胞，并通过双光子显微镜和光漂白后的荧光恢复分析间隙结通透性。这些研究将通过研究连接蛋白43在特定调控位点的磷酸化来补充。使用新开发的光学探针和共聚焦显微镜监测卵泡封闭卵母细胞中cAMP和cGMP的浓度。这些活组织显微镜的方法是一个重大的进步，因为它们避免了为了研究其功能而破坏卵泡调节环境的必要性。将使用转基因小鼠，结合这些显微镜方法，以验证EGF受体配体的蛋白水解加工和鉴定丝氨酸上连接蛋白43的磷酸化对于卵泡体细胞向卵母细胞的信号转导至关重要的假设。拟议的研究将促进对关键生物学过程的认识，并为未来的临床发展奠定基础，特别是在治疗不孕症方面。体外卵母细胞成熟是人类体外受精方法的一个新兴组成部分，了解控制卵母细胞减数分裂的信号机制将促进这一进展。公共卫生相关性：拟议的研究涉及激素信号导致储存在卵巢中的哺乳动物卵母细胞发育到可受精阶段的机制。这项研究将促进对关键生物学过程的认识，并为未来的临床发展奠定基础，特别是在治疗不孕症方面。

DESCRIPTION (provided by applicant): This application is for renewal of a project concerned with signal transduction during oocyte maturation and fertilization. It addresses the long-standing unanswered question of how luteinizing hormone (LH) causes meiotic resumption in mammalian follicle- enclosed oocytes. Recent findings, which establish that gap junctions between the somatic cells of the follicle close rapidly in response to LH, form the basis for the 3 aims of this project: 1) to investigate if LH-induced gap junction closure in the follicle is caused by TACE protease-mediated activation of the EGF receptor, 2) to investigate whether phosphorylation-mediated closure of Cx43 gap junctions in the follicle cells is required for resumption of meiosis in response to LH, and 3) to investigate the role of gap junction closure in regulating cAMP and cGMP in the oocyte in response to LH. Gap junction permeability will be investigated by microinjection of live follicle-enclosed oocytes with fluorescent tracers, and analysis by 2-photon microscopy and fluorescence recovery after photobleaching. These studies will be complemented by investigations of the phosphorylation of connexin 43 on specific regulatory sites. Concentrations of cAMP and cGMP will be monitored in follicle-enclosed oocytes using newly developed optical probes and confocal microscopy. These methods for live tissue microscopy are a major advance, because they avoid the necessity of disrupting the regulatory environment of the follicle in order to investigate its function. Genetically modified mice will be used, in combination with these microscopic approaches, in order to test the hypothesis that proteolytic processing of EGF receptor ligands and phosphorylation of connexin 43 on identified serines are essential for transduction of signals from the somatic cells of the follicle to the oocyte. The proposed research will advance knowledge of a crucial biological process, and establish a basis for future clinical developments, especially in the treatment of infertility. In vitro oocyte maturation is an emerging component of methods for human in vitro fertilization, and understanding of signaling mechanisms that control oocyte meiosis will facilitate such advances. PUBLIC HEALTH RELEVANCE: The proposed research concerns the mechanisms by which hormonal signals cause mammalian oocytes, stored in the ovary, to develop to the fertilizable stage. This research will advance knowledge of a crucial biological process, and establish a basis for future clinical developments, especially in the treatment of infertility.