Signaling Pathways Regulating GnRH Secretion

调节 GnRH 分泌的信号通路

• 批准号: 6580675
• 负责人: RICHARD Ira WEINER
• 金额: $ 31.39万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6580675
• 关键词: Adenoviridae; Xenopus oocyte; adenylate cyclase; biological signal transduction; cyclic AMP; enzyme activity; fertility; gene expression; genetic enhancer element; genetic promoter element; genetically modified animals; gonadotropin releasing factor; laboratory rat; mutant; neurons; nucleic acid probes; phosphodiesterases; protein kinase A; secretion; tissue /cell culture; transfection /expression vector; voltage /patch clamp

DESCRIPTION (provided by applicant): GnRH pulses are tightly regulated for the maintenance of reproductive cycles. Pulsatile GnRH release is an intrinsic property of GT1 GnRH cells and endogenous GnRH neurons. Based on findings in GT1 cells, we hypothesize that the cAMP signaling pathway participates in the stimulation of GnRH secretion and the timing of GnRH pulses. Our findings show that increases in cAMP stimulate GnRH secretion by opening cAMP-gated cation (CNG) channels leading to increased excitability and depolarization of the neuron. Increased neuron excitability is reflected in increased action potentials, Ca2+ oscillations and GnRH secretion. Increased cAMP levels also activate PKA that appears to initiate negative feedback pathways. We will study the role of these signaling molecules on the regulation of GnRH secretion in vitro in the GT1 GnRH cell lines and in vivo in transgenic rats. We will decrease neuron excitability by lowering cAMP levels by expressing the constitutively active phosphodiesterase, PDE4D1, or inhibiting CNG channel activity by expressing a dominant/negative (D/N) mutant of the CNG2 channel subunit (DMCNG2). We will increase neuron excitability by inhibiting the PKA negative feedback pathway by expression of the D/N mutant of the regulatory subunit of PKA mRAB and by increasing cAMP levels by expressing a constitutively active soluble adenylate cyclase (sAC). In GT1 cells we will use adenovirus vectors to target expression of the genetic probes. We will study changes in GT1 neuron excitability (Ca2+ oscillations) and the frequency and amplitude of GnRH pulses. We have now shown that expression of PDE4D1 in GT1 cells inhibits Ca2+ oscillations and pulsatile GnRH release. Genetic probes shown to be effective in experiments with GT1 cells will be cell specifically targeted to GnRH neurons in transgenic rats using the rat GnRH gene promoter/enhancer. We have now shown that targeted expression of PDE4D1 in a line of transgenic rats decreased the frequency of LH pulses in castrated males and females. Females were infertile and had blunted LH ovulatory surges or polycystic ovaries. In addition to advancing our knowledge of the signaling pathways involved in timing pulsatile GnRH secretion these animals will provide important models for studying the effects of alterations in GnRH pulsatility on reproductive function. Potentailly these findings may be relevant to the understanding of human disorders.

描述（由申请人提供）：GnRH 脉冲受到严格调节以维持生殖周期。脉冲式 GnRH 释放是 GT1 GnRH 细胞和内源性 GnRH 神经元的固有特性。基于 GT1 细胞的研究结果，我们假设 cAMP 信号通路参与 GnRH 分泌的刺激和 GnRH 脉冲的计时。 我们的研究结果表明，cAMP 的增加通过打开 cAMP 门控阳离子 (CNG) 通道来刺激 GnRH 分泌，从而导致神经元的兴奋性和去极化增加。神经元兴奋性的增加反映在动作电位、Ca2+振荡和 GnRH 分泌的增加上。 cAMP 水平升高还会激活 PKA，从而启动负反馈通路。 我们将研究这些信号分子在 GT1 GnRH 细胞系体外和转基因大鼠体内 GnRH 分泌调节中的作用。 我们将通过表达组成型活性磷酸二酯酶 PDE4D1 来降低 cAMP 水平，或通过表达 CNG2 通道亚基 (DMCNG2) 的显性/阴性 (D/N) 突变体来抑制 CNG 通道活性，从而降低神经元兴奋性。 我们将通过表达 PKA mRAB 调节亚基的 D/N 突变体来抑制 PKA 负反馈通路，并通过表达组成型活性可溶性腺苷酸环化酶 (sAC) 来增加 cAMP 水平，从而增加神经元兴奋性。在 GT1 细胞中，我们将使用腺病毒载体来靶向基因探针的表达。 我们将研究 GT1 神经元兴奋性（Ca2+ 振荡）的变化以及 GnRH 脉冲的频率和幅度。我们现在已经证明 GT1 细胞中 PDE4D1 的表达抑制 Ca2+ 振荡和脉冲式 GnRH 释放。 在 GT1 细胞实验中显示有效的基因探针将是使用大鼠 GnRH 基因启动子/增强子专门针对转基因大鼠中 GnRH 神经元的细胞。 我们现在已经证明，在一系列转基因大鼠中 PDE4D1 的靶向表达降低了去势雄性和雌性的 LH 脉冲频率。女性不育，LH 排卵高峰或多囊卵巢减弱。 除了增进我们对脉动 GnRH 分泌定时所涉及的信号通路的了解之外，这些动物还将为研究 GnRH 脉动变化对生殖功能的影响提供重要模型。 这些发现可能与理解人类疾病有关。