Wang S H, Chen J H, Lin L Y
Institute of Radiation Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
J Cell Biochem. 1994 Aug;55(4):486-95. doi: 10.1002/jcb.240550408.
The presence and inducibility of the major cadmium (Cd) chelating protein metallothionein (MT) in testicular cells has been controversial. In this study, the induction and production of MT in testicular cells were studied using mouse Leydig and Sertoli cell lines. Metal accumulation was studied by subjecting the cells to increasing levels of Cd. The presence of transcription factors for MT synthesis was analyzed by transfecting the cells with a reporter gene under the control of the MT promoter. The dose- and time-dependent induction of MT were conducted by Northern analyses. Expression of MT genes occurred in both Leydig and Sertoli cells. To avoid cross hybridization of the MT probe with mRNAs encoding testicular metal binding proteins and to investigate the integrity of MT mRNA, isoMT mRNA identification and primer extension experiments were performed. Those studies show that the induced mRNA indeed encodes MT. The biosynthesis of MT was confirmed by following 35S-cysteine incorporation into the protein. Finally, cadmium tolerance of testicular cells is compared with that of fibroblast cells. By these studies, we conclude that the MT genes are functional and inducible in testicular cells.
睾丸细胞中主要的镉（Cd）螯合蛋白金属硫蛋白（MT）的存在及其可诱导性一直存在争议。在本研究中，使用小鼠睾丸间质细胞和支持细胞系研究了睾丸细胞中MT的诱导和产生。通过使细胞接触不断增加水平的镉来研究金属积累。通过用MT启动子控制下的报告基因转染细胞，分析MT合成转录因子的存在情况。通过Northern分析进行MT的剂量和时间依赖性诱导。MT基因在睾丸间质细胞和支持细胞中均有表达。为避免MT探针与编码睾丸金属结合蛋白的mRNA发生交叉杂交，并研究MT mRNA的完整性，进行了同工型MT mRNA鉴定和引物延伸实验。这些研究表明，诱导的mRNA确实编码MT。通过追踪35S-半胱氨酸掺入蛋白质中证实了MT的生物合成。最后，将睾丸细胞的镉耐受性与成纤维细胞的镉耐受性进行了比较。通过这些研究，我们得出结论，MT基因在睾丸细胞中具有功能且可诱导。
