Studies on molecularbiological approach of male infertility-Isolation of androgen responsive genes by genomic binding-site method.

为了更好地了解雄激素对精子发生和精子形成的分子机制，我们采用基因组结合位点法分离雄激素反应基因。我们利用pET 1 5 b载体在大肠杆菌中表达了编码大鼠雄激素受体（AR-DBD）DNA结合域的组氨酸标签融合蛋白。用镍螯合树脂纯化了大量的融合蛋白。凝胶迁移实验表明AR-DBD蛋白能与真实的雄激素反应元件（ARE）特异性结合。将限制性内切酶消化的大鼠基因组DNA与AR-DBD蛋白混合，并通过硝酸纤维素过滤器选择与其结合的片段。将这些片段克隆到质粒载体中并扩增。将该选择扩增程序重复5至6次，最终分离出7个克隆。以这些片段为探针，经北方分析发现其中一个克隆在大鼠附睾中有强表达，但在LH-RH类似物处理后睾丸激素撤除后消失，表明我们分离到了在大鼠附睾中高表达的雄激素调节基因。进一步的研究应该是必要的，以评估该基因对男性生殖的功能。

To obtain a better understanding of molecular mechanism of androgen action on spermatogenesis and spermiogenesis, we performed genomic binding-site method to isolate androgen responsive genes. We have expressed histidine -tag fusion proteins encoding the DNA-binding domain of the rat androgen receptor (AR-DBD) in Escherichia col. using the pET1 5b vector. Large quantities of fusion proteins were produced and purified by nickelchelating resin. Gel shift assay revealed that AR-DBD protein could bind specifically with authentic androgen response elements(ARE). Rat genomic DNA digested by restriction enzymes was mixed with the AR-DBD protein and the fragments that bound to it were selected by nitrocellulose filter. These fragments were cloned into a plasmid vector and amplified. This sellection-amplification procedure were repeated five to six times and finally seven clones were isolated. Using these fragments as probes, northern analysis disclosed that one of the clones was strongly expressed in the rat epididymis and disappeared following withdrawal of testosterone by treatment of LH-RH analogue.It was concluded that we isolated the androgen regulated gene that was highly expressed in the rat epididymis. Further studies should be necessary to evaluate the function of this gene on male reproduction.