MOLECULAR MECHANISMS OF HFSH/B GENE REGULATION

描述(摘自申请人的摘要) 最常见和最难管理的生殖障碍之一 促性腺激素调节系统的改变与过度活动有关 下丘脑-垂体-肾上腺(HPA)轴。这样做的第一个具体目标是 指导性项目旨在了解 生殖系统，特别是hFSH-β基因的调节 通过促性腺激素特异性因子、促性腺激素释放激素(GnRH)和 性腺类固醇。这将由L完成)对比 HFSH-β基因对已知的cAMP反应、促性腺激素特异性、雌激素、 以及雄激素反应元件和已知的GATA结合位点调节 其他促性腺激素基因，2)电迁移率变化和DNA酶保护 证明发现的任何结合位点的DNA结合和突变的分析 评估它们对表达的重要性以及3)3‘缺失基因的表达 在转基因小鼠中构建映射候选增强子。以特定的目标 第二，β-内啡肽在调节应激诱导中的重要性 无排卵将在生理学实验中使用 β-内啡肽基因敲除小鼠。糖皮质激素在这方面的重要性 将通过演示糖皮质激素的DNA结合来测试效果 受体与应激诱导无排卵的生理学实验 肾上腺切除β-内啡肽缺陷小鼠。这一领域的工作一直是 因缺乏方便的表达系统而受阻；因此，继续 利用小鼠促性腺激素细胞建立卵泡刺激素分泌细胞系的研究 与hFSH-β启动子相连的SV40tsTAg永生化 优化瞬时转基因是这项研究的第三个具体目标 项目。对生理学和分子生物学的更深入的理解 生殖系统的调节和HPA轴的相互作用 使用该系统将改善对生殖影响的治疗 下丘脑轴的紊乱。通过有指导的职业发展计划， 首席调查员将加强生物化学和 通过分子生物学课程工作，提高应用技能 基础科学研究以了解内分泌学，获得专业知识 研究设计和统计分析，并获得对 负责任的科学行为原则。到五年结束时 在辅导期，这将导致在 研究项目的设计和实施。

DESCRIPTION (Taken from the applicant's Abstract) One of the most common and difficult to manage disorders of the reproductive system is altered gonadotropin regulation related to overactivity of the hypothalamic-pituitary-adrenal (HPA) axis. The first Specific Aim of this mentored project is directed towards understanding the normal regulation of the reproductive system, in particular the regulation of the hFSH-beta gene by gonadotropin specific factors, gonadotropin releasing hormone (GnRH) and gonadal steroids. This will be accomplished by l) comparison of the hFSH-beta gene to known cAMP-responsive, gonadotropin-specific, estrogen, and androgen response elements and GATA-binding sites known to regulate other gonadotropin genes, 2) electromobility shift and DNase protection assays to demonstrate DNA binding and mutation of any binding sites found to assess their importance for expression and 3) expression of 3' deleted gene constructs in transgenic mice to map candidate enhancers. In Specific Aim Two, the importance of beta-endorphin in mediating stress-induced anovulation will be evaluated in physiologic experiments using beta-endorphin knockout mice. The importance of glucocorticoids for this effect will be tested by demonstrating DNA binding of glucocorticoid receptor and in physiologic experiments of stress-induced anovulation in adrenalectomized beta-endorphin deficient mice. Work in this field has been hampered by the lack of a convenient expression system; therefore, continued efforts to develop an FSH-producing cell line from mouse gonadotropin cells immortalized with the SV40tsTAg linked to the hFSH-beta promoter and optimizing transient transfections is the third Specific Aim of this project. An improved understanding of the physiologic and molecular regulation of the reproductive system and the interaction of the HPA axis with that system will lead to improved treatment of the reproductive effects of disorders of the HPA axis. Through a mentored career development plan, the Principal Investigator will enhance knowledge of biochemistry and molecular biology through course work, enhance skills in application of basic science research to understanding endocrinology, gain expertise in research design and statistical analysis, and gain appreciation of principles of responsible scientific conduct. By the end of the five year mentorship period, this will result in achievance of independence in the design and implementation of research projects.