Regulation of Gene Expression During Spermatogenesis

精子发生过程中基因表达的调控

• 批准号: 6723750
• 负责人: PETER PETRUSZ
• 金额: $ 7.28万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6723750
• 关键词: Sertoli cells; androgen receptor; androgens; fertility; gene expression; genetic regulation; genetically modified animals; immunocytochemistry; in situ hybridization; laboratory mouse; microarray technology; polymerase chain reaction; spermatogenesis; testis

DESCRIPTION (provided by applicant): Androgens are indispensable for normal spermatogenesis; however, the mechanism by which androgens exert their effect on this complex process is poorly understood. Androgens act on their target cells by binding to and activating the androgen receptor (AR) present in Sertoli cells, peritubular cells, and Leydig cells but not in germ cells. It is assumed that androgens' actions on spermatogenesis are mediated by paracrine signals resulting from changes in gene expression in Sertoli and peritubular cells. In spite of an intensive research effort for several decades, traditional methods have failed to pinpoint key androgen-regulated genes whose activation is critical for spermatogenesis. We have studied a transgenic mouse model that overexpresses androgen-binding protein (ABP), a secretory product of Sertoli cells. These transgenic animals develop a progressive impairment of spermatogenesis and become gradually infertile. The results of our morphological, immunocytochemical, and flow cytometric analyses of the testes indicate that the most likely cause of this impairment is the reduction in the concentration of bioavailable androgens in the peritubular compartment and the seminiferous tubules. DNA microarray technology offers a novel and powerful means to analyze gene expression in a comprehensive, yet efficient, way. The Specific Aim of this pilot project is to test the hypothesis that the chronic reduction of bioavailable androgens within the seminiferous tubules of the ABP-transgenic mice is manifested in ordered changes in gene expression detectable by DNA microarray analysis. Murine genome oligonucleotide arrays will be probed by cRNAs prepared from testes of wild type and transgenic mice of 30 days of age. As additional controls, a set of the transgenic animals will be treated with human chorionic gonadotropin. This treatment is known to increase intratesticular androgen concentration and should reverse the changes caused specifically by androgen depletion. Results will be analyzed in terms of expression levels, expression profiles (dendrograms) and functional relationships. Cellular localization of altered gene expression will be determined by in situ hybridization and immunocytochemistry. This strategy is likely to provide, for the first time, a comprehensive view of androgen-dependent genes in the mammalian testis, offering a rich source of information for further studies, new clues for understanding and treating certain cases of human male infertility, and new potential molecular targets for male contraception.

描述（由申请人提供）：雄激素对于正常的精子发生是必不可少的；但是，雄激素对这一复杂过程的影响的机制知之甚少。雄激素通过与Sertoli细胞，周期细胞和Leydig细胞中的雄激素受体（AR）结合并激活其靶细胞作用，但不在生殖细胞中。假定雄激素对精子发生的作用是由旁分泌信号介导的，这是由于Sertoli和周围细胞中基因表达的变化而导致的。尽管几十年来进行了深入的研究工作，但传统方法仍未确定雄激素调节的基因，其激活对于精子发生至关重要。 我们研究了一种过表达雄激素结合蛋白（ABP）的转基因小鼠模型，这是Sertoli细胞的分泌产物。这些转基因动物会产生精子发生的逐渐损害，并逐渐不育。我们的形态学，免疫细胞化学和流式细胞术的结果表明，这种损害的最可能原因是腹侧隔室中可生物可利用雄激素浓度的降低，并降低了细胞精中的小管。 DNA微阵列技术提供了一种新颖而强大的方法，可以以综合而有效的方式分析基因表达。该试验项目的具体目的是检验以下假设：ABP-转基因小鼠的生物可利用雄激素的长期降低在DNA微阵列分析中可检测到的基因表达的有序变化，以有序的变化表现出了ABP-转基因小鼠的生物可利用雄激素。鼠类基因组寡核苷酸阵列将由野生型和30天的转基因小鼠睾丸制备的CRNA探测。作为其他对照，将用人绒毛膜促性腺激素治疗一组转基因动物。已知这种治疗方法会增加内部雄激素浓度，并应扭转特异性雄激素消耗引起的变化。 结果将以表达水平，表达谱（树状图）和功能关系来分析。基因表达改变的细胞定位将通过原位杂交和免疫细胞化学来确定。 该策略可能首次提供了哺乳动物睾丸中雄激素依赖基因的全面观点，为进一步的研究提供了丰富的信息来源，了解和治疗人类男性不育症的某些病例的新线索以及男性避孕药的新潜在分子靶标。