Cellular Basis for Non-Parallel Gonadotropin Release

描述（由申请人提供）：在过去的十年中，不孕问题加速发展。生殖能力受来自下丘脑的促性腺激素释放激素（GnRH）脉冲的调节，这些脉冲对促性腺激素的释放有不同的调节。目前尚不清楚GnRH脉冲频率的变化如何调节LH和促卵泡激素（FSH）基因的差异表达。在过去的30年里，细胞化学家们推测单激素促性腺激素群体可能是差异调节的一个位点。过去十年中检测促性腺激素β亚基mrna的研究表明，单激素LH促性腺激素在LH激增之前更为丰富，这表明它们可能是有选择性地增加的，可能是由于GnRH的快速脉冲。本研究旨在验证以下假设：GnRH的快速脉冲将选择性地刺激单激素LH促性腺激素亚群，这些亚群也表达更高密度的GnRH受体。较慢的GnRH脉冲可能有利于单激素FSH细胞。本研究将利用新出现的双原位杂交技术来了解LHb和FSHb基因单激素或双激素表达调控背后的机制。目的1研究将确定是否有选择性地刺激单激素促性腺激素增加，在体内，在发情或发情期间。他们将比较短暂培养（24小时）后的细胞表达与循环雌性大鼠组织切片中促性腺激素的表达。切片将被收集在一个新的组织微阵列，允许细胞化学标记和分析数百个切片/幻灯片。图像分析将提供有关促性腺激素亚群的百分比以及每个基因产物的标记密度和面积的信息。这些研究将确定周期中单激素促性腺激素丰富的时间，并利用这些信息通过联合洗脱和生物磁体技术收集和纯化这些亚群的分离部分。来自这些部分的细胞将用于Aim 3的分泌研究，并保存其mRNA样本以备将来的基因分析。单激素和双激素促性腺激素将通过激光捕获显微解剖收集，并存储在均匀细胞组中，或单独存储，用于将来的基因分析。目的2研究将了解单激素促性腺激素是否在月经中期表达更多的GnRH受体。将开发检测3个mRNA或异核rna （hnRNA）和一个mRNA的三重标记技术，以优化单激素或双激素促性腺激素中CmRH受体的检测。目的3研究将使用灌注系统来了解单激素LH细胞是否会随着GnRH的快速脉冲而增加。这些研究还将测试较慢的脉冲是否会导致单激素FSH细胞的增加。Aim 3研究还将比较混合培养中促性腺激素的反应与Aim 1研究中收集的纯化人群的反应，以了解反应的大小或模式是否取决于邻近垂体细胞的因素。这些研究的总体目标将是结合现有的和新兴的新技术，更多地了解促性腺激素群体的可塑性，因为我们开发了两种方法，我们可以收集纯化的，正常的促性腺激素，用于未来的基因分析。事实上，近一半的LH或FSH促性腺激素是单激素的，就mRNA表达而言，这表明一个尚未被认识到的巨大潜力。对这些细胞中选择性调节基因表达的机制的理解将提供有关如何调节生育的重要信息。

DESCRIPTION (provided by applicant): Infertility problems have accelerated over the past decade. Fertility is regulated by pulses of gonadotropin releasing hormone (GnRH) from the hypothalamus that differentially regulate gonadotropin release. It is not known how changing frequencies of GnRH pulses regulate differential expression of LH and follicle stimulating hormone (FSH) genes. Cytochemists over the past 30 years have speculated that one site for differential regulation might be the monohormonal gonadotrope population. Studies during the past decade that detected mRNAs for the gonadotropin beta subunits showed that monohormonal LH gonadotropes were more abundant just before the LH surge, suggesting they may be selectively increased, perhaps by the rapid pulses of GnRH. This study is proposed to test the hypothesis that rapid pulses of GnRH will selectively stimulate a subset of monohormonal LH gonadotropes that also express a higher density of GnRH receptors. Slower pulses of GnRH may favor monohormonal FSH cells. The proposed studies will use emerging new dual in situ hybridization technology to learn mechanisms behind the regulation of monohormonal or bihormonal expression of LHb and FSHb genes. Aim 1 studies will determine if monohormonal gonadotropes are selectively stimulated to increase, in vivo during proestrous or estrous. They will compare expression of cells after brief culture (24h) with expression of gonadotropins in tissue sections from cycling female rats. The sections will be collected in a novel Tissue Microarray, which allows cytochemical labeling and analysis of hundreds of sections/slide. The image analysis will provide information about the percentages of gonadotrope subsets and the density and area of their label for each gene product. These studies will identify times during the cycle when monohormonal gonadotropes are abundant, and use this information to collect and purify separated fractions of these subsets by combined elutriation and bio-magnet techniques. The cells from these fractions will be used in Aim 3 studies of secretion, and samples of their mRNA saved for future gene profiling. Single monohormonal and bihormonal gonadotropes will be collected by Laser Capture Microdissection and stored in homogeneous cellular groups, or, singly, for future gene profiling. Aim 2 studies will learn if the monohormonal LH gonadotropes express more GnRH receptors at midcycle. Triple labeling techniques that detect 3 mRNAs or heteronuclear RNAs (hnRNA) and an mRNA will be developed to optimize the detection of CmRH receptors in monohormonal or bihormonal gonadotropes. Aim 3 studies will use perifusion systems to learn if monohormonal LH cells increase in response to rapid pulses of GnRH. These studies will also test if slower pulses cause an increase in monohormonal FSH cells. Aim 3 studies will also compare responses of gonadotropes in mixed cultures with those in the purified populations collected in Aim 1 studies to learn if the magnitude or pattern of the responses depends on factors from neighboring pituitary cells. The overall objective of these studies will be to use a combination of established and emerging new technologies learn more about the plasticity of the gonadotrope population, as we develop two methods whereby we can collect purified, normal gonadotropes for future gene profiling. The fact that nearly half of LH or FSH gonadotropes are monohormonal, with respect to mRNA expression, suggests a great potential that has not been recognized. An understanding of mechanisms that selectively regulate gene expression in these cells will provide important information about how fertility is regulated.