FSH - Responsive Genes in Mouse Sertoli Cells

描述（由申请人提供）：在男性中，卵泡刺激素（FSH）与支持细胞上的g蛋白偶联跨膜受体结合并控制其发育和功能。支持细胞是睾丸的体细胞，为生殖细胞的增殖和分化提供物理支架和各种信号因子。在小鼠睾丸发育过程中，支持细胞快速分裂至出生后第14天，随后停止增殖并最终分化。支持细胞的数量决定了生殖细胞的能力，从而决定了男性的生殖潜力。支持细胞增殖和分化异常可能导致男性不育。尽管有这些知识，卵泡刺激素调节支持细胞增殖和分化的分子基础是未知的。研究雄性卵泡刺激素作用机制的一个主要挑战是快速分离出能够准确反映其体内功能的纯支持细胞（没有污染睾丸细胞类型）。该项目的长期目标是了解卵泡刺激素如何通过协调各种基因/蛋白质网络来调节支持细胞中不同的发育程序。在特异性目标1中，我们将通过两种方法分离支持细胞的纯群体。在第一种方法中，基于Sertoli细胞表面独特的fsh受体表达，将使用快速磁细胞分离方法选择性地富集Sertoli细胞。在第二种方法中，将从两种不同的转基因小鼠菌株中富集Sertoli细胞，这些转基因小鼠在Sertoli细胞谱系中特异性表达增强的绿色荧光蛋白（EGFP）标记（由缪勒管抑制物质或Pem同源盒启动子驱动）。此外，通过杂交，GFP转基因将被引入FSH-null背景。流式细胞术将从这两种小鼠（含和不含FSH）中分离出纯的支持细胞群。在Specific Aim 2中，为了开始鉴定fsh应答基因，我们将使用减法杂交策略来富集Sertoli细胞特异性cDNA，或使用cDNA微阵列来分析Sertoli细胞纯群体（在Specific Aim 1中分离）在增殖（第10天）和分化（第42天）阶段的大规模基因表达变化。总的来说，这些实验将使我们能够在体内监测表达gfp的Sertoli细胞的发育，开发快速纯化Sertoli细胞的方法，并鉴定和表征其中的fsh反应基因。最后，GFP转基因小鼠和Sertoli细胞特异性cdna和大规模基因表达谱将成为有价值的试剂，并将提供给许多从事男性生殖内分泌学和发育生物学研究的研究人员。

DESCRIPTION (provided by applicant): In the male, follicle stimulating hormone (FSH) binds to G-protein coupled transmembrane receptors on the Sertoli cells and controls their development and function. Sertoli cells are the somatic cells of the testis that provide physical scaffolding and various signaling factors to the proliferating and differentiating germ cells. During the mouse testis development, Sertoli cells rapidly divide up to postnatal day 14, thereafter cease to proliferate and terminally differentiate. The number of Sertoli cells determines the germ cell capacity and thus the male reproductive potential. Aberrations in Sertoli cell proliferation and differentiation may cause male infertility. Despite this knowledge, the molecular basis for FSH regulation of Sertoli cell proliferation and differentiation is unknown. A major challenge to study the mechanism of FSH action in the male has been to rapidly isolate pure Sertoli cells (free of contaminating testis cell types) that accurately reflect their in vivo function. The long-term goal of this project is to understand how FSH regulates distinct developmental programs in Sertoli cells by orchestrating various gene/protein networks. In Specific Aim 1, we will isolate pure populations of Sertoli cells by two approaches. In the first approach, a rapid magnetic cell separation method will be used to selectively enrich Sertoli cells based on their unique cell surface expression of FSH-receptors. In the second approach, Sertoli cells will be enriched from two different strains of transgenic mice expressing an enhanced green fluorescent protein (EGFP) marker (driven by either Mullerian inhibiting substance or Pem homoebox promoter) specifically in the Sertoli cell lineage. Additionally, by crossbreeding, the GFP transgenes will be introduced into the FSH-null background. Pure populations of Sertoli cells will be isolated from both these strains of mice (with and without FSH) by flow cytometry. In Specific Aim 2, to begin to identify FSH-responsive genes, we will use subtractive hybridization strategies to enrich Sertoli cell-specific cDNAs or use cDNA microarrays to analyze large-scale gene expression changes in pure populations of Sertoli cells (isolated in Specific Aim 1) during proliferation (day 10) and differentiation (day 42) phases. Collectively, these experiments will enable us to monitor the development of GFP-expressing Sertoli cells in vivo, develop rapid methods to purify Sertoli cells and identify and characterize the FSH-responsive genes in them. Finally, the GFP transgenic mice and the Sertoli cell - specific cDNAs and large-scale gene expression profiles generated in this pilot grant will be valuable reagents and will be made available to a number of investigators engaged in male reproductive endocrinology and developmental biology research.