Regulation of Gene Expression During Spermatogenesis

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；然而，雄激素在这一复杂过程中发挥作用的机制还知之甚少。雄激素通过结合和激活存在于支持细胞、肾小管周围细胞和间质细胞的雄激素受体(AR)作用于靶细胞，但不存在于生殖细胞。推测雄激素在精子发生中的作用是通过支持细胞和小管周细胞基因表达变化所产生的旁分泌信号来介导的。尽管几十年来进行了密集的研究工作，但传统方法未能准确定位关键的雄激素调节基因，这些基因的激活对精子发生至关重要。 我们研究了一种过度表达雄激素结合蛋白(ABP)的转基因小鼠模型，ABP是支持细胞的一种分泌产物。这些转基因动物会逐渐损害精子发生，并逐渐变得不育。我们对睾丸的形态、免疫细胞化学和流式细胞术分析的结果表明，这种损害最可能的原因是管周小管和生精小管中生物可用雄激素浓度的减少。 DNA微阵列技术为全面而高效地分析基因表达提供了一种新颖而有力的手段。这个试点项目的具体目的是检验这样一个假设，即ABP转基因小鼠生精小管中生物可用雄激素的慢性减少表现为DNA微阵列分析可检测到的基因表达的有序变化。小鼠基因组寡核苷酸阵列将通过从30天大的野生型和转基因小鼠的睾丸中制备的CRNAs来探测。作为额外的对照，一组转基因动物将被人绒毛膜促性腺激素治疗。众所周知，这种治疗可以增加睾丸内雄激素浓度，并应逆转雄激素耗竭所引起的变化。结果将根据表达水平、表达谱(树状图)和功能关系进行分析。改变基因表达的细胞定位将通过原位杂交和免疫细胞化学来确定。 这一策略可能首次提供哺乳动物睾丸中雄激素依赖基因的全面观察，为进一步研究提供丰富的信息来源，为理解和治疗某些人类男性不育病例提供新的线索，并为男性避孕提供新的潜在分子靶点。

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.