Regulation of Gene Expression During Spermatogenesis

描述（由申请人提供）：雄激素是正常精子发生不可或缺的;然而，雄激素对这一复杂过程发挥作用的机制知之甚少。雄激素通过结合并激活支持细胞、管周细胞和间质细胞中的雄激素受体（AR）而作用于其靶细胞，但不作用于生殖细胞。据推测，雄激素对精子发生的作用是由支持细胞和管周细胞基因表达变化引起的旁分泌信号介导的。尽管几十年来进行了深入的研究，但传统方法未能确定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。 我们已经研究了一种过表达雄激素结合蛋白（ABP）的转基因小鼠模型，ABP是支持细胞的分泌产物。这些转基因动物的精子发生逐渐受损，并逐渐变得不育。我们对睾丸的形态学、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小管周围室和生精小管中生物可利用的雄激素浓度降低。 DNA微阵列技术提供了一种新的和强大的手段来分析基因表达在一个全面的，但有效的方式。该试验项目的具体目的是检验以下假设：ABP转基因小鼠曲细精管内生物可利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日龄野生型和转基因小鼠睾丸制备的cRNA进行探测。作为额外的对照，一组转基因动物将用人绒毛膜促性腺激素处理。已知这种治疗可增加睾丸内雄激素浓度，并应逆转雄激素耗竭引起的变化。 将根据表达水平、表达谱（树状图）和功能关系分析结果。将通过原位杂交和免疫细胞化学确定改变的基因表达的细胞定位。 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视图，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.