Signaling Pathways Regulating GnRH Secretion

调节 GnRH 分泌的信号通路

• 批准号: 6986831
• 负责人: RICHARD Ira WEINER
• 金额: $ 27.83万
• 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/6986831
• 关键词: Signaling; Pathways; Regulating; GnRH; Secretion

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。GnRH脉冲性释放是GT1 GnRH细胞和内源性GnRH神经元的固有特性。根据对GT1细胞的研究结果，我们假设cAMP信号通路参与了GnRH分泌的刺激和GnRH脉冲的计时。我们的发现表明，cAMP的增加通过开放cAMP门控阳离子通道来刺激GnRH的分泌，从而增加神经元的兴奋性和去极化。神经元兴奋性的增强反映在动作电位的增加、钙振荡和GnRH的分泌上。CAMP水平的升高也激活了似乎启动负反馈通路的PKA。我们将研究这些信号分子在GT1 GnRH细胞株体外和转基因大鼠体内GnRH分泌调节中的作用。我们将通过表达固有活性的磷酸二酯酶PDE4D1来降低cAMP水平，或者通过表达CNG2通道亚单位的显性/负性(D/N)突变体(DMCNG2)来抑制CNG通道活性，从而降低神经元的兴奋性。我们将通过表达PKA MRAb调节亚基的D/N突变体来抑制PKA负反馈途径，并通过表达具有结构性活性的可溶腺苷环化酶(SAC)来提高cAMP水平，从而提高神经元的兴奋性。在GT1细胞中，我们将使用腺病毒载体来靶向表达基因探针。我们将研究GT1神经元兴奋性(钙振荡)以及GnRH脉冲的频率和幅度的变化。我们现在已经证明，PDE4D1在GT1细胞中的表达抑制了钙振荡和GnRH的脉动性释放。在GT1细胞实验中被证明有效的遗传探针将是使用大鼠GnRH基因启动子/增强子的转基因大鼠中GnRH神经元的细胞特异性靶点。我们现在已经证明，在转基因大鼠品系中靶向表达PDE4D1降低了去势雄性和雌性大鼠的促黄体生成素脉冲频率。女性不育，有钝的黄体生成素排卵峰或多囊卵巢。此外，这些动物还将为研究GnRH脉动性改变对生殖功能的影响提供重要的模型。这些发现可能与对人类疾病的理解有关。