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育问题。不孕症和意外怀孕相关的健康和社会问题的影响使美国的卫生保健资源紧张，并导致世界各地的人类痛苦。我们对哺乳动物生育能力的理解需要广泛了解大脑、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。GnRH对细胞信号传导的整合是通过一个与离散膜隔室特异性相关的隔室化信号传导平台启动的。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力对垂体促性腺激素细胞外信号调节激酶的绝对需求。这些令人兴奋的观察结果支持了我们的中心假设，即GnRHR的膜筏相关区室化对于GnRHR偶联到广泛的信号网络（以ERK通路激活为例）是强制性的。此外，在垂体促性腺激素细胞内存在GnRH诱导的ERK信号传导的性别特异性要求，并且是哺乳动物生育力所必需的。提出了两个目标：目标1。确定膜筏内存在的信号复合物的组织和组成，这是通过GnRHR进行信号传导所必需的。目的1检查ERK 2内的结构域的必要性和足够的膜筏定位，如果筏作为一个信号传导的“枢纽”ERK激活在男性和女性小鼠。我们将使用促性腺激素释放激素受体免疫沉淀分离膜筏蛋白，然后将使用蛋白质组学和质谱方法来确定蛋白质的队列，占据和促进生产信号从这个离散的膜室。目标二。研究ERK 1和ERK 2在体内垂体促性腺激素功能中的需求。目的2着重于一种新的小鼠模型，以检查GnRH信号网络中ERK的体内需求。研究调查了垂体器官发生过程中与细胞谱系特异性ERK缺失相关的潜在发育异常。我们将直接检查GnRH促进基因转录的能力，在体内的ERK的情况下，包括评估变量GnRH脉冲间隔;我们将确定是否不同的时间Cre介导的ERK基因切除垂体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。使用特定药物操纵该系统以影响GnRH作用可对生育力产生很大影响。这些研究旨在研究脑垂体内重要信号分子的细胞内区室化的作用，以及这种区室化如何影响GnRH受体的信号传导。此外，这些研究将确定关键GnRH调节信号分子的遗传丢失对生育力的体内影响。

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.