Role of radical oxygen species in rat testicular germ cell apoptosis induced by heat stress

The present study was designed to clarify the role of radical oxygen species in testicular germ cell apoptosis induced by heat stress. Testicular cells isolated from immature rats were cultured with or without elevated temperature, and occurrence of apoptosis in these cells was defined by the appearance of DNA fragmentation following agarose gel electrophoresis and by flow cytometric quantification of apoptotic cells. At 32.5 degrees C, < 1% of cells showed signs of apoptosis throughout the culture period, whereas under heat stress, the proportion of apoptotic cells increased to 5% at 37 degrees C after 24 h of culture, or to 14% after 1-h exposure at 43 degrees C followed by 23-h culture at 32.5 degrees C. Similar to the effect of heat stress, exogenously supplied oxygen free radicals also induced apoptosis. In contrast, treatment with catalase significantly attenuated heat stress-induced apoptosis. Furthermore, heat stress of testicular cells was associated with an increased intracellular peroxide level as measured by a fluorescent probe, 2',7'-dichlorofluorescin diacetate. In conclusion, our data indicate the involvement of radical oxygen species during testicular germ cell apoptosis induced by heat stress. This study provides a useful in vitro model for the study of testicular germ cell apoptosis.