Ikeda M, Kodama H, Fukuda J, Shimizu Y, Murata M, Kumagai J, Tanaka T
Department of Obstetrics and Gynecology, Akita University School of Medicine, and Akita University College of Allied Medical Science, Akita city, 010-0041 Japan.
Biol Reprod. 1999 Aug;61(2):393-9. doi: 10.1095/biolreprod61.2.393.
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.
本研究旨在阐明活性氧在热应激诱导的睾丸生殖细胞凋亡中的作用。从未成熟大鼠分离的睾丸细胞在有或无温度升高的情况下进行培养，这些细胞凋亡的发生通过琼脂糖凝胶电泳后DNA片段化的出现以及通过流式细胞术对凋亡细胞进行定量来确定。在32.5℃时，整个培养期间<1%的细胞显示出凋亡迹象，而在热应激下，培养24小时后，在37℃时凋亡细胞比例增加到5%，或在43℃暴露1小时后，接着在32.5℃培养23小时后，凋亡细胞比例增加到14%。与热应激的作用类似，外源性供应的氧自由基也诱导凋亡。相反，用过氧化氢酶处理可显著减轻热应激诱导的凋亡。此外，通过荧光探针2',7'-二氯荧光素二乙酸酯测量，睾丸细胞的热应激与细胞内过氧化物水平升高有关。总之，我们的数据表明活性氧参与了热应激诱导的睾丸生殖细胞凋亡。本研究为睾丸生殖细胞凋亡的研究提供了一个有用的体外模型。
