Objective To investigate whether apoptosis induced by low-dose radiation （LDR） is regulated by mitochondrial pathways in testicular cells. Methods Male mice were exposed to whole-body LDR, and changes in mitochondrial function and in expression of apoptotic factors were analyzed in the testicular cells as follows. Total nitric-oxide synthase （T-NOS） and Na＋/K＋ ATPase activities were biochemically assayed. Reactive oxygen species （ROS） and mitochondrial membrane potential （Adjm） were determined by flow cytometry using fluorescent probes. Levels of mRNAs encoding cytochrome c （Cyt c） and apoptosis-inducing factor （AIF） were quantified by real-time reverse-transcription PCR （RT-PCR）. Expression of Cyt c, AIF, caspase-9, and caspase-3 at the protein level was assessed by western blotting and immunohistochemistry. Results LDR induced an increase in T-NOS activity and ROS levels, and a decrease in Na＋/K~ ATPase activity and mitochondrial A^m, in the testicular cells. The intensity of these effects increased with time after irradiation and with dose. The cells showed remarkable swelling and vacuolization of mitochondria, and displayed a time- and dose-dependent increase in the expression of Cyt c, AIF, procaspase-9, and procaspase-3. Activation of the two procaspases was confirmed by detection of the cleaved caspases. The changes in expression of the four apoptotic factors were mostly limited to spermatogonia and spermatocytes. Conclusion LDR can induce testicular cell apoptosis through mitochondrial signaling pathways
Summary： The study examined the role of endoplasmic reticulum stress （ERS） and signaling pathways of inositol-requiring enzyme-1 （IRE1）, RNA-activated protein kinase-like ER kinase （PERK） and activating transcription factor-6 （ATF6） in apoptosis of mouse testicular cells treated with low-dose radiation （LDR）. In the dose-dependent experiment, the mice were treated with whole-body X-ray irradiation at different doses （25, 50, 75, 100 or 200 mGy） and sacrificed 12 h later. In the time-dependent experiment, the mice were exposed to 75 mGy X-ray irradiation and killed at different time points （3, 6, 12, 18 or 24 h）. Testicular cells were harvested for experiments. H202 and NO concentrations, and Ca2＋-ATPase activity were detected by biochemical assays, the calcium ion concentration （[Ca2＋]i） by flow cytometry using fluo-3 probe, and GRP78 mRNA and protein expressions by quantitative real-time RT-PCR （qRT-PCR） and Western blotting, respectively. The mRNA expressions of S-XBP1, JNK, caspase-12 and CHOP were measured by qRT-PCR, and the protein expressions of IREla, S-XBP1, p-PERK, p-elF2a, ATF6 p50, p-JNK, pro-caspase-12, cleaved caspase-12 and CHOP by Western blot- ting. The results showed that the concentrations of H202 and NO, the mR_NA expressions of GRP78, S-XBP1, JNK, caspase-12 and CHOP, and the protein expressions of GRP78, S-XBP1, IREla, p-PERK, p-elF2a, ATF6 p50, p-JNK, pro-caspase-12, cleaved caspase-12 and CHOP were significantly increased in a time- and dose-dependent manner after LDR. But the [Ca2]i and Ca2-ATPase activities were sig nificantly decreased in a time and dose-dependent manner. It was concluded that the ERS, regulated by IRE 1, PERK and ATF6 pathways, is involved in the apoptosis of testicular cells in LDR mice, which is associated with ERS-apoptotic signaling molecules of JNK, caspase-12 and CHOP.
