Functional analysis of differentially expressed genes involved in testicular dysfunctions induced by endocrine disrupting chemicals

用温度敏感型猴病毒40大T(TsSV40LT)抗原感染原代培养的小鼠附睾头细胞，建立了条件永生化的附睾头细胞系MEPC5。在允许的温度33℃下，表达了大T抗原，细胞继续生长。而T抗原在39℃时的下调和33℃时细胞密度的上调与细胞生长停滞和p21^&lt；waf1&gt；蛋白表达增加有关。细胞表达ME1、SGP-2和雄激素受体等附睾头表达基因。有趣的是，在细胞生长受限的条件下，ME1和SGp-2的表达水平显著升高。从携带tsSV40LT抗原的转基因小鼠建立的睾丸Sertoli TTE3细胞在33℃持续增殖，而大T抗原失活39℃导致分化。为了阐明分化的详细机制，我们利用基因芯片研究了基因表达变化的时间进程。在分析的865个基因中，有14个基因的表达水平有所上升。实时定量聚合酶链式反应显示p21^&lt；waf1&gt；、乳脂球膜蛋白E8、热反应蛋白12和硒蛋白P的mRNA水平显著升高，小鼠附睾头上皮细胞MEPC5和小鼠睾丸支持细胞TTE3有望成为研究雄性生殖系统基因表达和内分泌干扰物的良好模型。在我们之前的实验中，31个基因，如fra-2、c-myc和鸟氨酸脱羧酶(ODC)等被鉴定为Sertoli TTE3细胞中对双酚A反应上调的基因。目前，已经利用这些已建立的细胞系研究了fra-2、c-myc和ODC在内分泌干扰物引起的男性生殖功能障碍中的作用。

A conditionally immortalized epididymis caput cell line, MEPC5,was established by infecting primary cultured mouse epididymis caput cells with a temperature-sensitive simian virus 40 large T (tsSV40LT)-antigen. At a permissive temperature 33℃, the large T-antigen was expressed and the cells grew continuously. However, the down regulation of T-antigen at a nonpermissive temperature of 39℃ and the upregulation of cell density at 33℃ were associated with growth arrest and the increased protein expression of p21^<waf1>. The cells expressed epididymal caput-expressed genes such as ME1,sulfated glycoprotein-2 (SGP-2) and androgen receptor. Interestingly, the expression levels of ME1 and SGP-2 were significantly elevated under the cell growth-restricted conditions.Testicular Sertoli TTE3 cells, established from transgenic mice bearing tsSV40LT-antigen, proliferated continuously at 33℃ whereas inactivation of the large T-antigen by 39℃ led to differentiation. To clarify the detailed mechanisms of differentiation, we investigated the time course of changes in gene expression using cDNA microarrays. Of the 865 genes analyzed, 14 genes showed increased levels of expression. Real-time quantitative PCR revealed that the mRNA levels of p21^<waf1>, milk fat globule membrane protein E8,heat-responsive protein 12,and selenoprotein P were markedly elevated.Taken together, mouse epididymis caput epithelial MEPC5 and mouse testicular Sertoli TTE3 cells should prove an excellent model for studies of gene expression and endocrine disrupting chemicals in the male reproductive systems. In previous our experiments, 31 genes such as fra-2,c-myc, and ornithine decarboxylase (ODC) were identified as up-regulated genes responsive to bisphenol A in Sertoli TTE3 cells. At present, roles of fra-2,c-myc, and ODC in male reproductive dysfunctions caused by endocrine disrupting chemicals have been investigated using these established cell lines.