NEUROTRANSMITTER CONTROL OF REPRODUCTION AND OOCYTE MATURATION

神经递质控制生殖和卵母细胞成熟

• 批准号: 6240143
• 负责人: JEFFREY L RAM
• 金额: $ 2.69万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240143
• 关键词: Bivalvia; G protein; alternatives to animals in research; biological models; egg /ovum; immunocytochemistry; in situ hybridization; laboratory rabbit; molecular cloning; neurophysiology; neurotransmitters; polymerase chain reaction; reproduction; serotonin receptor; sperm

The goal of this project is to understand mechanisms of oocyte maturation and spawning in a model organism, the zebra mussel (Dreissena polymorpha). We have previously shown that serotonin can induce germinal vesicle breakdown (GVBD) and spawning in zebra mussels. Proposed experiments will investigate the hypothesis that GVBD and spawning are mediated by G- protein-coupled serotonin receptor(s) that activates a concerted response involving both inhibition of adenylyl cyclase and stimulation of phospholipase C. Prior supporting data for the hypothesis includes the pharmacology of spawning and GVBD in zebra mussels, which resembles ligand-binding data of 5HT1ym, a cloned molluscan receptor that has sequence similarities to fly and vertebrate serotonin receptors that are coupled to both adenylyl cyclase inhibition and phospholipase activation. Furthermore, in other organisms meiosis reinitiation is inhibited by cyclic AMP, correlated with increases in phospholipase C products, and affected by manipulation of G- proteins. Specific research aims will be to determine primary sequences of zebra mussel gonadal serotonin receptor(s) and G protein(s), to compare ligand binding data for the cloned receptor to spawning and GVBD pharmacology, to assess serotonin receptor location by sequence specific immunocytochemistry and in situ hybridization and by investigating serotonin responses of non- gonadal tissues, and to study roles of G proteins by measuring putative mediating enzymes and second messengers and effects of sequence specific inhibitors. Specific student participation aims; one graduate student and one undergraduate will learn molecular biological techniques, including RT-PCR, gel electrophoresis, cloning, sequence analysis, antibody production, immunohistology, and in situ hybridization; one undergraduate will investigate pharmacology of spawning and GVBD in vivo and in vitro; one undergraduate will investigate effects of serotonin and other chemicals on non-reproductive behaviors of zebra mussels. Students will become expert at one or more techniques, will discuss their experiments with the P.I. frequently, will report on their work at weekly group meetings, will write or participate in the writing of publications on completed work, and will present their results at regional and national meetings, including the annual MBRS Symposium. Thus, as has been successfully accomplished with MBRS and other students, students become actively involved in data acquisition, reduction, analysis, and presentation, and are co-authors of reviewed publications from the laboratory. Health relevance: This project will investigate, in a model system, the most poorly understood mechanisms in the reinitiation of meiosis, the coupling of membrane receptors to the process of GVBD and oocyte maturation. This is relevant to understanding fertility problems. In addition, the serotonin receptor we are investigating resembles to some degree the pharmacology of a receptor that may be involved in the mechanisms of migraine headache treatment.

该项目的目标是了解卵母细胞成熟的机制 并在模式生物斑马贻贝 (Dreissena polymorpha) 中产卵。 我们之前已经证明血清素可以诱导生发囊泡 斑马贻贝的分解（GVBD）和产卵。 拟议的实验将 研究 GVBD 和产卵是由 G-介导的假设 激活协同反应的蛋白质偶联血清素受体 涉及腺苷酸环化酶的抑制和腺苷酸环化酶的刺激 磷脂酶C. 该假设的先前支持数据包括 斑马贻贝的产卵和 GVBD，类似于 配体结合数据 5HT1ym，一种克隆的软体动物受体，与苍蝇具有序列相似性 和与腺苷酸偶联的脊椎动物血清素受体 环化酶抑制和磷脂酶激活。 此外，在其他 生物体减数分裂重新启动受到环 AMP 的抑制，与 磷脂酶 C 产物增加，并受 G- 操纵的影响 蛋白质。 具体研究目标是确定斑马的初级序列 贻贝性腺血清素受体和 G 蛋白，比较配体 克隆受体与产卵和 GVBD 药理学的结合数据， 通过序列特异性免疫细胞化学评估血清素受体位置 和原位杂交并通过研究非血清素反应 性腺组织，并通过测量假定的 G 蛋白来研究 G 蛋白的作用 介导酶和第二信使以及序列特异性的影响 抑制剂。 具体的学生参与目标； 一名研究生和一名 本科生将学习分子生物学技术，包括RT-PCR、 凝胶电泳、克隆、序列分析、抗体生产、 免疫组织学和原位杂交；一名本科生将 研究体内和体外产卵和 GVBD 的药理学；一 本科生将研究血清素和其他化学物质对 斑马贻贝的非繁殖行为。 学生将成为专家 在一项或多项技术上，将与 P.I. 讨论他们的实验。 经常，会在每周的小组会议上报告他们的工作，会写 或参与撰写已完成作品的出版物，并将 在区域和国家会议上展示其成果，包括 年度 MBRS 研讨会。 因此，正如已成功完成的那样 MBRS和其他学生，学生积极参与数据 获取、还原、分析和演示，并且是 审查了实验室的出版物。 健康相关性：该项目将在模型系统中调查 人们对减数分裂重新启动的机制知之甚少， 膜受体与 GVBD 和卵母细胞过程的耦合 成熟。 这与理解生育问题有关。 在 此外，我们正在研究的血清素受体类似于某些 程度可能参与受体的药理学 偏头痛治疗的机制。