FSH - Responsive Genes in Mouse Sertoli Cells

描述(申请人提供)：在男性，卵泡刺激素(FSH)与支持细胞上的G蛋白偶联跨膜受体结合，控制其发育和功能。支持细胞是睾丸的体细胞，为增殖和分化的生殖细胞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，此后停止增殖并终末分化。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的异常可能导致男性不育。尽管如此，FSH调控Sertoli细胞增殖和分化的分子基础尚不清楚。研究FSH在男性体内的作用机制的一个主要挑战是快速分离出准确反映其体内功能的纯支持细胞(不受污染的睾丸细胞类型)。这个项目的长期目标是了解FSH如何通过协调各种基因/蛋白质网络来调节Sertoli细胞的不同发育程序。在特定的目标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，基于支持细胞独特的FSH受体的细胞表面表达，将使用一种快速的磁性细胞分离方法来选择性地丰富支持细胞。在第二种方法中，Sertoli细胞将从两个不同品系的转基因小鼠中浓缩，这些转基因小鼠在Sertoli细胞谱系中表达增强的绿色荧光蛋白(EGFP)标记(由Mullerian抑制物质或Pem同源框启动子驱动)。此外，通过杂交，GFP转基因将被引入FSH缺失的背景中。用流式细胞术从这两种品系的小鼠(有或没有FSH)中分离出纯的支持细胞群。在特定目标2中，为了开始鉴定FSH反应基因，我们将使用消减杂交策略来丰富Sertoli细胞特异性的cDNA，或使用cDNA微阵列来分析支持细胞纯群体(在特定目标1中分离)在增殖(第10天)和分化(第42天)阶段大规模基因表达的变化。总之，这些实验将使我们能够监测体内表达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，在这项试点拨款中产生的GFP转基因小鼠和Sertoli细胞特异性cDNA和大规模基因表达谱将是有价值的试剂，并将提供给一些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.