Signaling Pathways Regulating GnRH Secretion

描述（由申请人提供）：GnRH脉冲受到严格调节，以维持生殖周期。脉动性GnRH释放是GT1 GnRH细胞和内源性GnRH神经元的内在特性。基于在GT1细胞中的发现，我们假设cAMP信号通路参与了GnRH分泌的刺激和GnRH脉冲的定时。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。神经元兴奋性的增加反映在动作电位、Ca2+振荡和GnRH分泌的增加。cAMP水平的增加也激活了PKA，它似乎启动了负反馈通路。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对GnRH分泌的调节作用。我们将通过表达组成活性磷酸二酯酶PDE4D1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cAMP水平，从而降低神经元的兴奋性。我们将通过表达PKA mRAB调控亚基的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，从而增加神经元的兴奋性。在GT1细胞中，我们将使用腺病毒载体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PDE4D1在GT1细胞中的表达抑制Ca2+振荡和脉动性GnRH释放。在GT1细胞实验中证明有效的遗传探针将使用大鼠GnRH基因启动子/增强子特异性靶向转基因大鼠的GnRH神经元。我们现在已经证明，PDE4D1在转基因大鼠中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，并有钝化的黄体生成素排卵激增或多囊卵巢。除了提高我们对GnRH脉动性分泌的信号通路的认识外，这些动物将为研究GnRH脉动性改变对生殖功能的影响提供重要的模型。这些发现可能与理解人类疾病有关。

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.