Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
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Research Abstract |
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.
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