MOLECULAR MECHANISMS OF GNRH, LH & FSH GENE EXPRESSION

本研究计划的总体目标是阐明 核内和外周血中的分子机制 导致信使核糖核酸变化的细胞质 三种重要生殖激素：促性腺激素释放激素、β-激素释放激素水平 促黄体生成素和β-卵泡刺激素。将采取跨学科的方法 在井中使用分子遗传学和组织学的工具 具有特征的啮齿动物繁殖模型系统，即 女性和女性的去势和雌激素/雄激素替代 男性。这项提议有两个方面，可以分为两个方面 在解剖线条上。首先是促性腺激素的研究。 促性腺激素释放激素基因在青光眼视前区的表达 那只老鼠。研究将使用原位杂交进行 技术、溶液杂交/核酸酶保护技术 和转录运行检测以确定proGnRH如何 在这个模型系统中，基因在分子水平上受到调控。在……里面 此外，proGnRH基因启动子将与 氯苯二醇乙酰转移酶报告基因及其在 随后的基因转染法研究了各种类型的 培养的细胞以确定哪个第二信使系统 和/或类固醇负责调节proGnRH基因 表情。促性腺激素释放激素的原位杂交研究 大鼠视前区的表达区域将是 使用外显子和内含子特异的探针来确定 胞质mRNA和核hnRNA基因表达水平， 分别进行了分析。其次，研究也将在 垂体分析β-FSH基因的分子机制 用类固醇替代去势模型表达 雄性和雌性大鼠以及原代培养的大鼠 脑垂体细胞。研究将在原代培养中进行 使用GnRH/GAP/抑制素/性类固醇来确定以下哪种 这些因素是导致这些疾病的分子机制的原因。 β-FSH基因表达的调控。分析将是 通过在分析和溶液上运行转录来执行 利用内含子/外显子的杂交/核酸酶保护分析 为大鼠β-卵泡刺激素基因开发的连接性探针。就地 杂交研究也将在β-FSH和 用信使核糖核酸和内含子特异性探针检测去势后的β-黄体生成素 加上男性和女性的雌激素/雄激素替代范例 使用脑下垂体切片。最后，对β-黄体生成素基因的研究 表达将扩展到分析分子机制 通过GnRH的非转录效应引起的增加 β-黄体生成素基因的表达。β-FSH基因的IF调控 表达也被发现是非转录的，类似的研究 将对β-卵泡刺激素进行治疗。这些研究将集中在 电子显微镜原位杂交研究分析 β-LHhnRNA的亚核表达水平以及 β-黄体生成素核糖核酸多聚腺苷酸化调控的研究。 这些研究应该会让我们更详细地了解 参与调控的基本分子机制 极其重要的生殖多肽/蛋白质激素。

The overall objective of this research program is to elucidate the molecular mechanisms involved in both the nucleus and the cytoplasm which are responsible for the alterations in mRNA levels of three important reproductive hormones, proGnRH, beta- LH and beta-FSH. An interdisciplinary approach will be taken using the tools of molecular genetics and histology in a well characterized rodent reproductive model system, that of castration and estrogen/androgen replacement in female and male. There are two aspects to the proposal which can be divided upon anatomical lines. The first is the study of gonadotropin releasing hormone (GnRH) gene expression in the preoptic area of the rat. Studies will be performed using in situ hybridization techniques, solution hybridization/nuclease protection techniques and transcription run on assays to determine how the proGnRH gene is regulated at the molecular level in this model system. In addition, the proGnRH gene promoter will be fused to a chloramphenocol acetyl-transferase reporter gene and used in subsequent gene transfection studies into various types of cultured cells to determine which second messenger systems and/or steroids are responsible for regulation of proGnRH gene expression. In situ hybridization studies in the proGnRH expressing region of the preoptic area of the rat will be performed using exon and intron specific probes to determine levels of cytoplasmic mRNA and nuclear hnRNA gene expression, respectively. Secondly, studies will also be performed in the pituitary to analyze the molecular mechanisms of beta-FSH gene expression using the castration with steroid replacement model in male and female rats as well as in primary cultures of anterior pituitary cells. Studies will be performed in primary cultures using GnRH/GAP/inhibin/sex steroids to determine which of these factors are responsible for the molecular mechanisms of regulation of beta-FSH gene expression. Analyses will be performed by transcription run on assays as well as solution hybridization/nuclease protection assays using intron/exon junctional probes developed for the beta-FSH gene in rat. In situ hybridization studies will also be performed on beta-FSH and beta-LH using mRNA and intron specific probes in the castration plus estrogen/androgen replacement paradigm in male and female using pituitary gland sections. Finally, studies on beta-LH gene expression will be extended to analyze the molecular mechanism by which the non-transcriptional effects of GnRH elicits increases in beta-LH gene expression. If regulation of beta-FSH gene expression is also found to be non-transcriptional, similar studies will be performed for beta-FSH. These studies will focus upon electron microscopic in situ hybridization studies analyzing the subnuclear level of expression of beta-LH hnRNA as well as studies on regulation of polyadenylation of beta-LH hnRNA. These studies should provide a more detailed understanding the basic molecular mechanisms involved in the regulation of these extremely important reproductive peptide/protein hormones.