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脉冲受到严格调节，以维持生殖周期。脉冲GnRH释放是 GT1 GNRH细胞和内源性GNRH神经元的内在特性。根据GT1细胞中的发现，我们假设营地信号通路参与GNRH分泌的刺激和 GnRH脉冲的时间。我们的发现表明，营地的增加刺激了GNRH分泌营地门控阳离子（CNG）通道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增加的动作电位，CA2+振荡和GNRH中分泌。 camp水平的提高还激活了似乎启动负反馈途径的PKA。我们将研究这些信号分子在GT1 GNRH中体外的GNRH分泌调节的作用细胞系和转基因大鼠体内。我们将通过降低营地水平来降低神经元的兴奋性表达组成性活性磷酸二酯酶PDE4D1或通过抑制CNG通道活性表达CNG2通道亚基（DMCNG2）的显性/负（D/N）突变体。我们将增加神经元通过表达D/N突变体的表达来抑制PKA负反馈途径 PKA MRAB的调节亚基和通过表达组成型可溶性来提高营地水平腺苷酸环化酶（SAC）。在GT1细胞中，我们将使用腺病毒载体靶向遗传的表达探针。我们将研究GT1神经元兴奋性（CA2+振荡）的变化以及频率和 GnRH脉冲的振幅。我们现在表明，pDE4D1在GT1细胞中的表达抑制了Ca2+ 振荡和脉动GNRH释放。遗传探针证明在与GT1细胞实验中有效将使用大鼠GnRH基因专门针对转基因大鼠的GNRH神经元的细胞发起人/增强器。现在，我们已经表明，在转基因大鼠系中，PDE4D1的靶向表达降低了cast骨雄性和女性中LH脉冲的频率。女性不育，有钝的LH排卵或多囊卵巢。除了促进我们对信号的了解定时脉动gnRH分泌所涉及的途径这些动物将为研究GNRH脉动性改变对生殖功能的影响。这些发现有效可能与对人类疾病的理解有关。