Signaling Pathways Regulating GnRH Secretion

促性腺激素释放激素脉冲受到严格调控，以维持生殖周期。脉冲式GnRH释放是一种 GT1 GnRH细胞和内源性GnRH神经元的固有特性。基于在GT1细胞中的发现，我们 假设cAMP信号通路参与促性腺激素释放激素的分泌 GnRH脉冲的定时。我们的发现表明cAMP的增加通过开放刺激GnRH的分泌 CAMP门控阳离子(CNG)通道导致神经元兴奋性和去极化增加。 神经元兴奋性的增强反映在动作电位、钙振荡和促性腺激素释放激素的增加 分泌物。CAMP水平的升高也激活了似乎启动负反馈通路的PKA。我们 将在体外研究这些信号分子在GT1 GnRH分泌调节中的作用 转基因大鼠体内和细胞系的研究。我们将通过降低cAMP水平来降低神经元的兴奋性 表达具有结构性活性的磷酸二酯酶PDE4D1，或通过以下方式抑制CNG通道活性 表达CNG2通道亚单位的显性/负性(D/N)突变体(DMCNG2)。我们将增加 通过表达D/N突变体抑制PKA负反馈通路的神经元兴奋性 PKA MRAb调节亚基和通过表达一种结构性活性可溶性蛋白来增加cAMP水平 腺苷环化酶(SAC)。在GT1细胞中，我们将使用腺病毒载体来靶向表达基因 探测器。我们将研究GT1神经元兴奋性(钙振荡)的变化以及频率和 GnRH脉冲的幅度。我们现在已经证明PDE4D1在GT1细胞中的表达抑制钙离子 振荡和脉动性GnRH释放。基因探针在GT1细胞实验中被证明是有效的 将针对使用大鼠促性腺激素释放激素基因的转基因大鼠中的促性腺激素释放激素神经元 启动子/增强子。我们现在已经证明了PDE4D1在转基因大鼠系中的靶向表达 降低去势雄鼠和雌鼠的黄体生成素脉冲频率。女性是不育的，有 迟钝的黄体生成素排卵高峰或多囊卵巢。除了提高我们对信号的认识之外 这些动物参与调节促性腺激素释放激素脉动分泌的途径将为 研究促性腺激素释放激素搏动性改变对生殖功能的影响。潜在的这些发现 可能与对人类疾病的理解有关。

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.