EXPRESSION OF HEAT-SHOCK GENES IN MOUSE SPERMATOGENIC CELLS

热休克基因在小鼠生精细胞中的表达

• 批准号: 3841150
• 负责人: E M EDDY
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3841150
• 关键词: affinity chromatography; antiserum; complementary DNA; developmental genetics; embryonic stem cell; environmental stressor; gene expression; genetic library; genetic recombination; genetic regulation; genetically modified animals; heat; laboratory mouse; male; meiosis; molecular cloning; nucleic acid sequence; protein structure function; regulatory gene; sperm; spermatogenesis; stress proteins; testis; western blottings

The synthesis of heat-shock proteins (hsp) following environmental stress is a highly conserved response and apparently protects cells against acute effects of detrimental conditions. One of the most abundant proteins (P70) in spermatogenic cells is related closely to hsp70, as shown by ATP affinity chromatography purification, immunoblots of two-dimensional protein gels, and peptide mapping. The P70 protein is synthesized during the meiotic phase of spermatogenesis. Although several members of the hsp70 gene family have been sequenced and their mRNA expression characterized, little is known about the proteins encoded by these genes. We developed antisera specific to P70 and hsp70 to investigate the expression and role of these proteins in male germ cells. The antiserum to P70 was also used to select clones from a mouse spermatocyte cDNA library. One of these clones hybridized to a testis-specific, developmentally regulated 3.0 kb mRNA whose expression was not affected by heat. Sequence analysis indicated that the clone has high homology to previously reported genes of the hsp70 family. However, it appears that spermatogenic cells do not produce hsp70 mRNA or protein in response to heat stress. This suggests that P70 gene expression is linked to suppression of the hsp70 gene in spermatogenic cells. However, the process of spermatogenesis is unusually sensitive to elevation of temperature and to many toxic agents and P70 may function in germ cell differentiation rather than to protect against the effects of environmental stress. Although P70 expression may be beneficial to the process of spermatogenesis, this process may also leave male germ cells particularly susceptible to environmental stress. Genomic clones have been isolated for the P70 gene and transgenic mice are being produced to examine the spermatogenic cell-specific regulation of expression of this gene. A construct is also being prepared to mutate the P70 gene by homologous recombination in embryonic stem cells. Mice will be produced without functional P70 genes to test the role of the P70 protein in spermatogenic cell function and differentiation.

环境应激后热休克蛋白（hsp）的合成 是一种高度保守的反应，显然可以保护细胞免受急性 不利条件的影响。 最丰富的蛋白质之一 (P70) ATP显示生精细胞中与hsp70密切相关 亲和层析纯化、二维免疫印迹 蛋白质凝胶和肽图谱。 P70蛋白的合成过程是 精子发生的减数分裂阶段。 尽管该组织的多名成员 hsp70基因家族已被测序及其mRNA表达 但对于这些基因编码的蛋白质知之甚少。 我们开发了针对 P70 和 hsp70 的抗血清来研究 这些蛋白质在雄性生殖细胞中的表达和作用。 抗血清为 P70 还用于从小鼠精母细胞 cDNA 文库中选择克隆。 这些克隆之一与睾丸特异性、发育性杂交 调节3.0 kb mRNA，其表达不受热影响。 顺序 分析表明该克隆与之前报道的具有较高的同源性 hsp70 家族的基因。 然而，生精细胞似乎确实 不产生 hsp70 mRNA 或蛋白质来应对热应激。 这 表明 P70 基因表达与 hsp70 的抑制有关 生精细胞中的基因。 然而，精子发生的过程是 对温度升高和许多有毒物质异常敏感 P70 可能在生殖细胞分化中发挥作用，而不是保护 对抗环境压力的影响。 尽管 P70 表达可能 有利于精子发生的过程，这个过程也可能 使雄性生殖细胞特别容易受到环境压力的影响。 已分离出 P70 基因的基因组克隆，并获得转基因小鼠 被生产来检查生精细胞的特异性调节 该基因的表达。 还正在准备一个构建体来突变 P70基因在胚胎干细胞中通过同源重组。 老鼠将会 生产不含功能性 P70 基因以测试 P70 蛋白的作用 生精细胞功能和分化。