REGULATION AND FUNCTION OF HEAT SHOCK FACTORS IN TESTIS

睾丸热休克因子的调节及功能

• 批准号: 2673772
• 负责人: Kevin D Sarge
• 金额: $ 10.25万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2673772
• 关键词: DNA binding protein; DNA footprinting; cell type; developmental genetics; gene expression; genetic promoter element; genetic regulation; germ cells; hyperthermia; immunocytochemistry; in situ hybridization; laboratory mouse; male; messenger RNA; northern blottings; nuclear runoff assay; polymerase chain reaction; protein structure function; protein transport; spermatogenesis; stress proteins; testis; tissue /cell culture; transcription factor; western blottings

Spermatogenesis is the process by which immature male germ cells, through a complex series of events involving mitosis, meiosis, and differentiation of distinct spermatogenic cell types, are transformed into mature spermatozoa capable of fertilizing an ovum. Each spermatogenic cell type displays unique patterns of gene expression which ensure the production of proteins important for the specialized functions of these cells. The long- term goals of this project are to elucidate the mechanisms which regulate gene expression in spermatogenic cells and to understand the functions of regulated gene products in these cells. As a means to realize these goals, we propose to study the expression of Heat Shock Transcription Factor 2 (HSF2) in spermatogenic cells and its function in regulating hsp gene expression in these cells. Previous studies indicate that expression of several members of the hsp70 and hsp90 gene families (hsp70.2, hsc70t, hsp86) is regulated in spermatogenic cells, and that the promoters of these genes contain heat Shock Elements (HSEs), the HSF2 recognition sequence. Our previous studies demonstrate that HSF2 expression is regulated in germ cells, that the HSF2 protein exhibits constitutive DNA-binding activity in testis, and that HSF2 interacts with sequences in the hsp70.2 gene promoter. The hypothesis to be tested is that HSF2 expression and activity is regulated in germ cells to control hsp gene expression. We propose to precisely define the spatial and temporal patterns of HSF2 mRNA expression in testis and identify the mechanism which regulates HSF2 mRNA levels in testis cell types. HSF2 function in regulating gene expression in germ cells will be explored by determining the correlation between cellular localization of active HSF2 protein and hsp gene expression , by examining HSF2 interactions with hsp gene promoter sequences, both in vivo and in vitro, and by inhibiting HSF2 expression in germ cells and measuring resulting alterations in hsp gene expression. A second, related hypothesis to be tested is that the cellular stress response, which is mediated by Heat Shock Transcription Factor 1 (HSF1) and functions to protect cells from harmful effects of elevated temperature on cellular proteins, is involved in the well-known inhibitory effects of heat o spermatogenesis . This occurs either because spermatogenic cells lack the stress response and so are sensitive to heat-induced loss of protein function, or because stress response induction disrupts normal patterns of gene expression, thus interfering with essential cellular functions. To test this hypothesis, the stress-responsiveness of spermatogenic cells will be measured using probes for the three major parameters of the stress response; HSF1 DNA-binding activity, hsp70 mRNA, and hsp70 protein. The proposed studies will increase our understanding of the mechanisms of gene regulation in spermatogenic cells and of the importance of regulated gene expression for the specialized functions of these cells, and will also contribute to our understanding of how elevated temperature inhibits spermatogenesis. This information will provide a framework for exploring disease processes which affect spermatogenesis, and for development of new male contraceptives.

精子发生是不成熟的男性生殖细胞通过 涉及有丝分裂、减数分裂和分化的一系列复杂的事件 不同的生精细胞类型，转化为成熟的 精子能使卵子受精。 每种生精细胞类型 显示出独特的基因表达模式， 对这些细胞的特殊功能很重要的蛋白质。 很长的- 本项目的长期目标是阐明调节 基因在生精细胞中的表达，并了解 调节这些细胞中的基因产物。 作为实现这些目标的手段， 我们打算研究热休克转录因子2的表达， HSF 2在生精细胞中的表达及其对hsp基因的调控作用 在这些细胞中表达。 先前的研究表明， hsp 70和hsp 90基因家族的几个成员（hsp70.2，hsc 70 t， hsp 86）在生精细胞中受到调控，这些基因的启动子 基因包含热休克元件（HSE），即HSF 2识别序列。 我们以前的研究表明，HSF 2的表达在生殖细胞中受到调控， 细胞，HSF 2蛋白表现出组成型DNA结合活性， 睾丸，并且HSF 2与hsp70.2基因中的序列相互作用 启动子 待检验的假设是HSF 2的表达和活性 在生殖细胞中被调节以控制HSP基因表达。 我们建议 精确定义HSF 2 mRNA表达的空间和时间模式 并确定HSF 2 mRNA水平的调节机制， 睾丸细胞类型 热休克因子2在生殖细胞基因表达调控中的作用 将通过确定细胞之间的相关性来探索细胞 定位活性HSF 2蛋白和hsp基因表达，通过检查 HSF 2与hsp基因启动子序列的相互作用，无论是在体内还是在体外， 体外，并通过抑制生殖细胞中的HSF 2表达和测量 导致HSP基因表达的改变。 第二个有待检验的相关假设是， 反应，这是由热休克转录因子1（HSF 1）介导的， 功能，以保护细胞免受高温对细胞的有害影响。 细胞蛋白质，参与众所周知的热抑制作用 o精子发生。 这是因为生精细胞缺乏 应激反应等对热诱导蛋白质损失敏感 功能，或者因为应激反应诱导破坏了正常的 基因表达，从而干扰基本的细胞功能。 到 为了验证这一假设，生精细胞的应激反应将 使用探针测量应力的三个主要参数 反应; HSF 1 DNA结合活性，hsp 70 mRNA和hsp 70蛋白。 的 拟议的研究将增加我们对基因机制的理解， 生精细胞的调控及调控基因的重要性 表达这些细胞的特殊功能，并且还将 有助于我们理解高温是如何抑制 精子发生 这些信息将提供一个框架， 影响精子发生的疾病过程，以及新的 男性避孕药