MOLECULAR MECHANISMS OF GNRH, LH & FSH GENE EXPRESSION

这项研究计划的总体目标是阐明 分子机制涉及细胞核和 细胞质，负责mRNA的改变 水平的三个重要的生殖激素，促性腺激素释放激素原，β- LH和β-FSH。 将采取跨学科方法 利用分子遗传学和组织学的工具 特征的啮齿动物生殖模型系统， 去势和雌激素/雄激素替代， 男性 这项建议可分为两方面 在解剖学上。 首先是促性腺激素的研究 视前区促性腺激素释放激素（GnRH）基因表达 老鼠 将使用原位杂交进行研究 技术、溶液杂交/核酸酶保护技术 和转录检测来确定促性腺激素释放激素原 在该模型系统中，基因在分子水平上受到调控。 在 此外，proGnRH基因启动子将融合到 氯霉素乙酰转移酶报告基因及其在 随后的基因转染研究进入各种类型的 培养细胞来确定哪些第二信使系统 和/或类固醇负责调节proGnRH基因 表情 促性腺激素释放激素原的原位杂交研究 大鼠视前区的表达区域将被 使用外显子和内含子特异性探针进行，以确定 细胞质mRNA和细胞核hnRNA基因表达水平， 分别 其次，研究也将在 分析β-FSH基因的分子机制 表达使用去势与类固醇替代模型， 雄性和雌性大鼠以及前额叶的原代培养物中 垂体细胞 研究将在原代培养物中进行 使用GnRH/GAP/GABIN/性类固醇来确定这些 这些因素是导致 β-FSH基因表达的调控。 分析将 通过在测定和溶液上运行转录进行 使用内含子/外显子的杂交/核酸酶保护测定 为大鼠β-FSH基因开发的连接探针。 原位 还将对β-FSH和 在去势中使用mRNA和内含子特异性探针检测β-LH 男性和女性的雌激素/雄激素替代方案 使用脑下垂体切片。 最后，对β-LH基因的研究 表达式将被扩展以分析分子机制 通过这种方式， 在β-LH基因表达中。 如果β-FSH基因的调节 表达也被发现是非转录的，类似的研究 将进行β-FSH检查。 这些研究将集中在 电子显微镜原位杂交研究分析了 β-LH hnRNA表达的亚核水平以及 β-LH hnRNA多聚腺苷酸化调控的研究。 这些研究应该提供更详细的了解， 参与这些调节的基本分子机制 非常重要的生殖肽/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.