Molecular analysis of reproductive fanction on the genes implicated to sex differentiation and gametogenesis

采用多柔比星(DXR)致小鼠睾丸生殖细胞毒性模型，用RT-PCR和免疫组织化学方法检测c-kit在睾丸生殖细胞基因表达和c-kit产生方面的意义。并观察中药(TJ41、TJ7)和GTCS对端粒酶活性和细胞凋亡的促进作用。用TdT介导的dUDP缺口末端标记(TUNEL)法标记凋亡细胞。DXR染毒后，睾丸重量显著下降至55-%，而GTCS与DXR联合染毒后，睾丸重量与对照组相近。在DXR染毒小鼠的睾丸中，组织学分析显示，对照组睾丸小管中的生殖细胞急剧减少约80%。然而，TJ41和GTCS联合应用可显著减轻生殖细胞损伤。DXR暴露组大鼠睾丸c-Kit及其基因表达降低，但未加促进剂的睾丸c-Kit及其基因表达降低。TJ41和GTCS联合应用时，TJ41和GTCS联合作用后，TJ41和GTCS诱导的细胞端粒酶活性明显升高，而未加TJ41和GTCS的DXR组端粒酶活性明显降低。使用DXR的促进剂显示，与不使用这些试剂的DXR暴露相比，凋亡细胞的数量显著减少。这些结果表明，暴露于有毒化学物质后的睾丸功能障碍可能是由c-kit及其基因表达引起的，而TJ41和GTCS可能通过抑制睾丸细胞凋亡而促进端粒酶活性，从而对DXR所致的睾丸毒性具有保护作用。

The implication of c-kit was examined on the aspect of gene expression and c-kit production at testicular germ cell by RT-PCR and immunohistochemistry in the mice testes damaged by doxorubicin (DXR) as a germ cell toxic model. And also the promoting effect of herbal medicine (TJ41, TJ7) and GTCs on telomerase activity and apoptosis were investigated. Apoptotic cells were labelled by the TdT-mediated dUDP nick-end labelling (TUNEL) assay. The weight of testes was significantly decreased to 55-64% by DXR exposure, while the combined treatment of GTCs with DXR resulted in parameters similar to the control. In the testes of DXR-exposed mice, germ cells in the testicular tubules of control were drastically reduced by about 80% by histological analysis. However, germ cell damage was significantly attenuated by TJ41 and GTCs coadministration. c-Kit and its gene expression were reduced in the DXR exposured testis, but not in the group with promoting agents. Although the telomerase activity evaluated by fluorescence-based TRAP assay was increased with coadministration of TJ41 and GTCs compared with control, that was decreased by DXR expoosure without these promoting agents. The promoting agents with DXR show a marked reduction in the number of apoptotic cells compared with DXR exposure without these agents. These results suggest that testicular dysfunction following exposure of toxic chemicals are determining at the mechanisms provoked by c-kit and its gene expression and TJ41 and GTCs may have eventful evidence of a protective effect against DXR-induced testicular toxicity via promoting telomerase activity by inhibition of testicular apoptosis.