GNRH MODULATION OF GONADOTROPIN GENE TRANSCRIPTION

促性腺激素释放激素以脉冲的形式释放，作用于脑下垂体以刺激 促黄体生成素和卵泡刺激素的合成和释放。这种脉动性对于 保持正常生育能力。我们之前展示了频率依赖的， 促性腺激素释放激素脉冲对大鼠促性腺激素基因的亚基特异性影响 体内和体外转录，现在有GnRH反应 荧光素酶(Luc)报告基因构建的大鼠α和LHbeta 基因。对转录的频率依赖效应可能产生于 钙离子和蛋白C(PCK)/有丝分裂原激活的差异激活 蛋白激酶(MAPK)级联和/或发散型GnRH反应基因 区域和转录因子。不同细胞内的作用 促性腺激素释放激素刺激的内源性促性腺激素基因转录信号 用促性腺激素治疗后的核径流分析进行评估 钙离子内流抑制物、PKC和MAPK的特异性激活物 没有或存在促性腺激素释放激素。转基因小鼠的脑垂体细胞 表达LHbetaLUC将被类似对待和Lhbeta启动子 测量到的活动。最后，alphaLUC和LHbetaLUC构造将是 转染克隆促性腺激素细胞系，并受GnRH刺激 在没有或存在钙抑制剂的情况下测量的活性 内流、MAPK和与共转染的显性阴性或结构性 活性形式的MAPK和钙-钙调蛋白激酶(CAMK)。MAPK和CAMK 酶活性将在处理过的细胞中直接测量。GnRH- 阿尔法和LHbeta基因的敏感元件将通过 利用报告基因缺失/突变构建物进行转基因分析 基因。分离出的DNA元件的活性将在 异源启动子和凝胶执行的DNA-核蛋白结合 移位和足迹分析以及西南印迹分析。一种能力 类固醇直接影响对GnRH的反应，并结合 核蛋白对GnRH敏感的基因区域将被确定。这些 研究将有助于确定频率依赖的潜在机制 调节促性腺激素的合成，并开始识别特异性 促性腺激素释放激素反应的细胞靶点。

GnRH is released in pulses and acts on the pituitary to stimulate the synthesis and release of LH and FSH. This pulsatility is crucial to maintain normal fertility. We previously demonstrated frequency-dependent, subunit-specific effects of GnRH pulses on rat gonadotropin gene transcription in vivo and in vitro, and now have GnRH-responsive luciferase (LUC) reporter gene constructs for the rat alpha and LHbeta genes. Frequency-dependent effects on transcription could arise from differential activation of Ca+2 and protein C(PCK)/mitogen-activated protein kinase (MAPK) cascade and/or divergent GnRH-responsive gene regions and transcription factors. The role of different intracellular signals in GnRH-stimulated endogenous gonadotropin gene transcription will be assessed by nuclear run-off assays in gonadotropes after treatment with specific activators of inhibitors of Ca+2 influx, PKC, and MAPK, in the absence or presence of GnRH. Pituitary cells from transgenic mice expressing LHbetaLUC will be similarly treated and Lhbeta promotor activity measured. Finally, alphaLUC and LHbetaLUC constructs will be transfected into clonal gonadotrope cell lines, and GnRH-stimulated activity measured in the absence or presence of inhibitors of calcium influx, MAPK, and with co-transfected dominant negative or constitutively active forms of MAPK and calcium-calmodulin kinase (CAMK). MAPK and CAMK enzyme activity will be measured directly in treated cells. GnRH- sensitive elements of the alpha and LHbeta genes will be identified by transfection analysis using deletion/mutation constructs of the reporter genes. Activity of the isolated DNA elements will be demonstrated on heterologous promoters, and DNA-nuclear protein binding performed by gels shift and footprinting assays and southwestern blot analysis. The ability of steroids to directly influence the response to GnRH, and binding of nuclear proteins to GnRH-sensitive gene regions will be determined. These studies will help define the underling mechanisms of frequency-dependent regulation of gonadotropin synthesis, and begin to identify specific cellular targets which confer the GnRH response.