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Signaling Pathways Regulating GnRH Secretion

调节 GnRH 分泌的信号通路

基本信息

批准号：
7149186
负责人：
RICHARD Ira WEINER
金额：
$ 29.09万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-02-01 至 2008-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7149186
关键词：
Action Potentials Adenovirus Vector Adenylate Cyclase Animals Castration Cations Cell Line Cells Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cyclic Nucleotides Disease Dominant-Negative Mutation Dopamine Enhancers Estradiol Estrous Cycle Excision Feedback Female Fertility Follicle Stimulating Hormone Frequencies Genes Genetic Human Hydroxyprogesterone In Situ Hybridization In Vitro Infection Infertility Knowledge Maintenance Measures Neurons Numbers Pathway interactions Periodicity Physiologic pulse Polycystic Ovary Syndrome Polymerase Chain Reaction Progesterone Property Protein Overexpression Pulse taking RNA Splicing Rattus Regulation Research Personnel Role Signal Pathway Signal Transduction Signaling Molecule Study models Technology Testing Testosterone Time Transgenic Organisms Vagina Variant Waxes Xenopus oocyte base cyclic nucleotide-gated cation channel in vivo male mutant patch clamp phosphoric diester hydrolase programs promoter reproductive reproductive function research study transgene expression

项目摘要

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.

GnRH脉冲被严格调节以维持生殖周期。脉冲式GnRH释放是一种 GT 1 GnRH细胞和内源性GnRH神经元内在特性。基于GT 1细胞的发现，我们假设cAMP信号通路参与刺激GnRH分泌，促性腺激素释放激素脉冲的时机。我们的研究结果表明，cAMP的增加刺激GnRH分泌，通过开放 cAMP门控阳离子（CNG）通道导致神经元的兴奋性和去极化增加。增加的神经元兴奋性反映在增加的动作电位，Ca 2+振荡和GnRH 分泌物增加的cAMP水平也激活PKA，PKA似乎启动负反馈途径。我们将研究这些信号分子在体外GT 1 GnRH分泌调节中的作用。细胞系和转基因大鼠体内。我们将通过降低cAMP水平来降低神经元的兴奋性，表达组成型活性磷酸二酯酶PDE 4D 1，或通过抑制CNG通道活性表达CNG 2通道亚基（DMCNG 2）的显性/阴性（D/N）突变体。加大通过表达D/N突变体抑制PKA负反馈途径来抑制神经元兴奋性 PKA mRAB的调节亚基，并通过表达组成型活性可溶性腺苷酸环化酶（sAC）。在GT 1细胞中，我们将使用腺病毒载体来靶向表达基因。 probes.我们将研究GT 1神经元兴奋性（Ca 2+振荡）和频率的变化，促性腺激素释放激素脉冲的幅度。我们现在已经表明，在GT 1细胞中表达PDE 4D 1抑制Ca 2 + 振荡和脉冲式GnRH释放。基因探针在GT 1细胞实验中显示有效将使用大鼠GnRH基因在转基因大鼠中特异性靶向GnRH神经元启动子/增强子。我们现在已经证明，PDE 4D 1在转基因大鼠系中的靶向表达降低去势雄性和雌性LH脉冲频率。雌性不育，黄体生成素排卵高峰减弱或多囊卵巢。除了提高我们对信号的认识，这些动物将提供重要的模型，研究GnRH脉动性改变对生殖功能的影响。这些发现可能可能与人类疾病的理解有关。