Molecular Analysis of GnRH Action

描述（由申请人提供）：从全球的角度来看，我们有义务通过制定有效控制怀孕率的社会和治疗策略来面对和处理不断增长的世界人口的“可持续性”。另外，在美国和其他发达国家，有相当数量的夫妇面临不孕不育的问题。与不孕症和意外怀孕有关的健康和社会问题的影响使美国的卫生保健资源紧张，并加剧了全世界人类的痛苦。我们对哺乳动物生育能力的理解需要对大脑、脑垂体和性腺之间通过促性腺激素释放激素（GnRH）、促性腺激素和性腺激素的作用进行交流的机制有广泛的认识。GnRH的细胞信号整合是通过一个区室化的信号平台启动的，该信号平台与离散的膜区室特别相关。GnRH信号网络中的中心信号“支柱”是ERK级联。我的实验室开发了一种关键的小鼠模型，研究ERK信号在生殖轴中的作用。这些研究揭示了雌性小鼠正常生育对垂体促性腺激素中ERKs的绝对要求。这些令人兴奋的观察结果支持了我们的中心假设，即膜筏相关的GnRHR区隔化是GnRHR与ERK通路激活所体现的广泛信号网络耦合的必要条件。此外，垂体促性腺激素中存在gnrh诱导的ERK信号的性别特异性需求，并且是哺乳动物生育所必需的。提出了两个目标：目标1。确定膜筏中存在的信号复合体的组织和组成，这是通过GnRHR传递信号所必需的。目的1研究了ERK2中膜筏定位所必需和充分的结构域，以及膜筏是否作为ERK激活的信号“中枢”。我们将采用GnRHR免疫沉淀法分离膜筏蛋白；然后，蛋白质组学和质谱方法将用于鉴定占据并促进来自这种离散膜室的生产信号的蛋白质队列。目标2。体内垂体促性腺功能对ERK1和ERK2的需求。目的2侧重于一种新的小鼠模型，以检查GnRH信号网络中ERKs的体内需求。研究探讨垂体器官发生过程中与ERKs缺失相关的潜在发育异常。我们将直接检查GnRH在体内缺乏ERKs的情况下促进基因转录的能力，包括评估可变GnRH脉冲间隔；我们将确定在垂体中改变cre介导的ERK基因切除的时间是否会影响性别特异性不孕。最后，我们的小鼠模型提供了使用微阵列分析来检查由GnRH脉冲诱导的ERK依赖性垂体特异性基因网络的机会。公共卫生相关性：促性腺激素释放激素（GnRH）通过调节垂体的激素合成和分泌来控制生殖轴和生育能力。垂体衍生的促性腺激素反过来调节卵巢和睾丸的功能。操纵这个系统使用特定的药物来影响GnRH的作用可以对生育能力产生很大的影响。提出的研究检查垂体内重要信号分子的细胞内区隔化的作用，以及这种区隔化如何影响GnRH受体的信号传导。此外，这些研究将确定gnrh调节的关键信号分子基因缺失对生育能力的体内影响。

DESCRIPTION (provided by applicant): From a global perspective, we are obliged to confront and deal with the "sustainability" of a growing world population by developing social and therapeutic strategies that effectively control pregnancy rates. Alternatively, a significant number of couples in the US and other developed countries confront issues of infertility. The impact of infertility and unintended pregnancy-related health and social issues strains US health care resources and contributes to human suffering worldwide. Our understanding of fertility in mammals requires an extensive appreciation of the mechanisms of communication between the brain, pituitary gland and the gonad through the actions of gonadotropin releasing hormone (GnRH), gonadotropins and gonadal hormones. Integration of cell signaling by GnRH is initiated through a compartmentalized signaling platform specifically associated within discrete membrane compartments. The central signaling "pillar" within the GnRH signaling network is the ERK cascade. My lab has developed a critical mouse model examining the role of ERK signaling within the reproductive axis. These studies reveal an absolute requirement for ERKs within pituitary gonadotropes for normal fertility in female mice. These exciting observations support our central hypothesis that membrane raft-associated compartmentalization of the GnRHR is obligatory for GnRHR coupling to a broad signaling network exemplified by ERK pathway activation. Moreover, a gender- specific requirement for GnRH-induced ERK signaling within pituitary gonadotropes exists and is necessary for fertility in mammals. Two Aims are proposed: Aim 1. Determine the organization and composition of the signaling complex present within membrane rafts that is necessary for signaling through the GnRHR. Aim 1 examines the domains within ERK2 necessary and sufficient for membrane raft localization and if the raft serves as a signaling "hub" for ERK activation in male and female mice. We will use GnRHR immunoprecipitation to isolate membrane rafts proteins; proteomic and mass spectroscopy approaches will then be used to identify the cohort of proteins that occupy and facilitate productive signaling from this discrete membrane compartment. Aim 2. Examine the requirement for ERK1 and ERK2 in pituitary gonadotrope function in vivo. Aim 2 focuses on a novel mouse model to examine the in vivo requirements for ERKs within the GnRH signaling network. Studies investigate potential developmental abnormalities associated with the loss of ERKs on cell lineage specification during pituitary organogenesis. We will directly examine the ability of GnRH to promote gene transcription in the absence of ERKs in vivo, including assessment of variable GnRH interpulse intervals; and we will determine if varying the timing of Cre-mediated ERK gene excision in the pituitary affects gender-specific infertility. Finally, our mouse model provides the opportunity to examine the ERK- dependent pituitary-specific gene network that is induced by a pulse of GnRH using microarray analyses. PUBLIC HEALTH RELEVANCE: Gonadotropin-releasing hormone (GnRH) controls the reproductive axis and fertility by modulating hormone synthesis and secretion from the pituitary gland. Pituitary-derived gonadotropic hormones in turn regulate the function of the ovary and testis. Manipulation of this system using specific drugs to affect GnRH action can have a large impact on fertility. The studies proposed examine the role of intracellular compartmentalization of important signaling molecules within the pituitary gland and how this compartmentalization affects signaling from of the GnRH receptor. Moreover, these studies will define the in vivo impact of genetic loss of key GnRH-regulated signaling molecules on fertility.