REGULATION AND FUNCTION OF HEAT SHOCK FACTORS IN TESTIS

精子发生是未成熟的雄性生殖细胞通过 涉及有丝分裂、减数分裂和分化的一系列复杂事件 不同的生精细胞类型，转化为成熟的 能够使卵子受精的精子。 每种生精细胞类型 显示出独特的基因表达模式，确保生产 对这些细胞的特殊功能很重要的蛋白质。 长- 该项目的长期目标是阐明调节机制 生精细胞中的基因表达并了解其功能 这些细胞中受调节的基因产物。 作为实现这些目标的手段， 我们建议研究热休克转录因子2的表达 生精细胞中的HSF2及其对hsp基因的调节作用 这些细胞中的表达。 先前的研究表明，表达 hsp70 和 hsp90 基因家族的几个成员（hsp70.2、hsc70t、 hsp86）在生精细胞中受到调节，并且这些的启动子 基因含有热休克元件 (HSE)，即 HSF2 识别序列。 我们之前的研究表明HSF2的表达在细菌中受到调节 细胞中，HSF2 蛋白表现出组成型 DNA 结合活性 睾丸，并且 HSF2 与 hsp70.2 基因中的序列相互作用 发起人。 要测试的假设是 HSF2 表达和活性 在生殖细胞中受到调节以控制 hsp 基因表达。 我们建议 精确定义 HSF2 mRNA 表达的空间和时间模式 并确定睾丸中 HSF2 mRNA 水平的调节机制 睾丸细胞类型。 HSF2在调节细菌基因表达中的功能 将通过确定细胞之间的相关性来探索细胞 活性HSF2蛋白和hsp基因表达的定位，通过检查 HSF2 与 hsp 基因启动子序列在体内和体内的相互作用 体外，并通过抑制生殖细胞中的 HSF2 表达并测量 从而导致 hsp 基因表达的改变。 第二个需要检验的相关假设是细胞应激 反应，由热休克转录因子 1 (HSF1) 介导 具有保护细胞免受高温有害影响的功能 细胞蛋白，参与众所周知的热抑制作用 Ø 精子发生。 发生这种情况的原因要么是生精细胞缺乏 应激反应，因此对热引起的蛋白质损失敏感 功能，或者因为应激反应诱导破坏了正常的模式 基因表达，从而干扰基本的细胞功能。 至 检验这一假设，生精细胞的应激反应性将 使用探针测量应力的三个主要参数 回应； HSF1 DNA 结合活性、hsp70 mRNA 和 hsp70 蛋白。 的 拟议的研究将增加我们对基因机制的理解 生精细胞的调节以及调节基因的重要性 这些细胞的特殊功能的表达，并且还将 有助于我们了解高温如何抑制 精子发生。 这些信息将为探索提供一个框架 影响精子发生的疾病过程以及新的开发 男性避孕药。

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.