Molecular Analysis of GnRH Action

描述(由申请人提供)：从全球角度来看，我们有义务通过制定有效控制怀孕率的社会和治疗战略来应对和应对世界人口不断增长的“可持续性”。或者，在美国和其他发达国家，相当多的夫妇面临不孕不育的问题。不孕不育和与意外怀孕有关的健康和社会问题的影响使美国卫生保健资源紧张，并在世界各地造成人类痛苦。我们对哺乳动物生育能力的理解需要广泛了解脑、脑垂体腺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而进行交流的机制。GnRH对细胞信号的整合是通过一个分隔的信号平台启动的，该平台专门与离散的膜室相关。GnRH信令网络中的中央信令“支柱”是ERK级联。我的实验室开发了一个关键的小鼠模型，检测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所绝对需要的垂体促性腺激素中的ERKs。这些令人兴奋的观察结果支持了我们的中心假设，即膜RAFT相关的GnRHR的区域化对于GnRHR与广泛的信号网络的耦合是必需的，例如ERK通路的激活。此外，GnRH在垂体促性腺激素中诱导ERK信号的性别特异性要求存在，并且对于哺乳动物的生育是必要的。提出了两个目标：目标1.确定存在于膜筏内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ERK2内的结构域对于膜筏定位的必要性和充分性，以及在雄性和雌性小鼠中，RAFT是否作为ERK激活的信号“枢纽”。我们将使用GnRHR免疫沉淀来分离膜筏蛋白；然后将使用蛋白质组学和质谱学方法来鉴定占据并促进来自该离散膜室的生产性信号的蛋白质队列。目的2.在体内检测ERK1和ERK2在垂体促性腺激素功能中的需求。目的2重点研究一种新的小鼠模型，以检测体内对促性腺激素释放激素信号网络中ERK的需求。研究调查潜在的发育异常与脑垂体器官发生过程中ERKs在细胞谱系中丢失有关。我们将在体内直接检测GnRH在没有ERKs的情况下促进基因转录的能力，包括评估可变的GnRH脉搏间隔；我们将确定改变CRE介导的脑垂体ERK基因切除的时机是否会影响性别特异性不孕症。最后，我们的小鼠模型提供了使用微阵列分析检测GnRH脉冲诱导的ERK依赖的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)通过调节脑下垂体的激素合成和分泌来控制生殖轴和生育能力。脑垂体源性促性腺激素反过来调节卵巢和睾丸的功能。操纵这一系统，使用特定的药物来影响促性腺激素释放激素的作用，可能会对生育产生很大影响。这些研究建议检查脑下垂体内重要信号分子的细胞内分区的作用，以及这种分区如何影响来自GnRH受体的信号。此外，这些研究将确定体内GnRH调节的关键信号分子的遗传缺失对生育能力的影响。

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.