Cellular Basis for Non-Parallel Gonadotropin Release

描述(申请人提供)：不孕不育问题在过去十年中加速增长。生育力是由下丘脑的促性腺激素释放激素(GnRH)脉冲调节的，GnRH对促性腺激素的释放有不同的调节作用。目前尚不清楚GnRH脉冲频率的变化如何调节黄体生成素和卵泡刺激素(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控的一个部位。在过去的十年中，检测到促性腺激素β亚基的mRNAs的研究表明，就在促黄体生成素激增之前，单激素促性腺激素更加丰富，这表明它们可能被选择性地增加，可能是由于促性腺激素释放激素的快速脉冲。这项研究旨在检验GnRH快速脉冲将选择性刺激单激素促性腺激素亚群的假设，这些单激素促性腺激素也表达更高密度的GnRH受体。较慢的GnRH脉冲可能有利于单激素FSH细胞。拟议的研究将使用新兴的双重原位杂交技术来了解LHb和FSHB基因的单激素或双激素表达调节背后的机制。目的1研究将确定在发情或发情期间，体内单激素促性腺激素是否被选择性地刺激增加。他们将比较短暂培养(24小时)后细胞的表达与周期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中，该阵列允许对数百个切片/载玻片进行细胞化学标记和分析。图像分析将提供关于每个基因产物的促性腺激素亚群的百分比及其标记的密度和面积的信息。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结合淋洗和生物磁铁技术收集和纯化这些亚群中的单独部分。来自这些组分的细胞将用于AIM 3分泌物的研究，并保存它们的mRNA样本用于未来的基因图谱。单一的单激素和双激素促性腺激素将通过激光捕获显微切割收集并存储在同质细胞组中，或单独用于未来的基因图谱。目的2研究将了解单激素促性腺激素是否在周期中期表达更多的GnRH受体。将开发检测3mRNAs或异核RNAs(HnRNA)和一个mRNAs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研究将使用灌流系统来了解单激素促黄体生成素细胞是否对促性腺激素释放激素快速脉冲的反应增加。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和Aim 1研究中收集的纯化种群中促性腺激素的反应，以了解反应的大小或模式是否取决于邻近垂体腺细胞的因素。这些研究的总体目标将是使用现有的和新兴的新技术的组合来了解更多关于促性腺激素种群的可塑性，因为我们开发了两种方法，可以收集纯化的、正常的促性腺激素用于未来的基因图谱。事实上，近一半的促黄体生成素或促卵泡激素促性腺激素是单激素的，就mRNA的表达而言，这表明了尚未认识到的巨大潜力。了解选择性调控这些细胞中基因表达的机制将为生育力如何调控提供重要信息。

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.