| Cadmium (Cd2+), one of the most toxic environmental and industrial pollutants, is known to exert gonadotoxic and spermiotoxic effects. In the present study, we examined the toxic effect of Cd2+ on the testis of freshwater crab, Sinopotamon henanense. Crabs were exposed to different Cd2+ concentrations (from 0 to 116.00 mg/L) for 1,3,5 and 7d respectively. In order to study the effect of Cd2+ on oxidative stress in the testis of freshwater crab, the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), as well as the content of malonyldialdehyde (MDA) in the testes were investigated. And in order to explore whether Cd2+ would further induce apoptosis after testicular oxidative damage, typical morphological characteristic and physiological changes of apoptosis were observed using a variety of methods (H.E. staining, AO/EB double fluorescent staining, TEM observation and DNA fragmentation analysis). In addition, the mechanisms of apoptosis in testis of the crab after Cd2+ administration were preliminarily explored. The activities of apoptosis-associated enzymes were investigated including Caspase-3 and Caspase-9, and hydrogen peroxide (H2O2) content and cytochrome c oxidase (CCO) activity were also detected. The results were showed as follows:(1) The activities of SOD,CAT and GPx increased initially and decreased subsequently with the increasing concentration of Cd2+and exposure periods. However, the content of MDA was continuatively increased. Low concentration of Cd2+ can significantly increase antioxidant enzymes, high concentrations of Cd2+ can lead to decrease enzyme activities, or even significantly lower than the control group. Inhibition of antioxidant enzymes activities and increasing of MDA content indicated that the crabs testes suffered severe oxidative damage.(2) The light microscopy examination of the testis of the control crabs had normal structure with evidence of well-organized distribution of cells in the seminiferous epithelium. With the increase of Cd2+ concentration and duration of treatment, a series of pathological changes were observed including disordered arrangement of germ cells, decreased number of sperms in the lumina of the seminiferous tubules, exhibited extensive necrosis in the germinal layer of the seminiferous tubules, etc. The fluorescence microscopic analysis results show that the number of apoptotic and necrotic cells were significantly increased with increasing Cd2+concentrations. The testis cells showed typical morphological characteristic of apoptosis in the TEM after exposure to cadmium, including nuclear chromatin condensation and marinating, nuclear membrane folding, etc. The Cd2+-induced apoptotic DNA fragmentation in testis was clearly indicated on the agarose gel as detected by ethidium bromide fluorescence. This series of results showed that a certain concentration and treatment time of cadmium induced apoptosis in the testis of freshwater crab.(3) At 7d, with the increasing concentration of Cd+, H2O2 content, Caspase-3 activity, Caspase-9 activity showed an increasing tendency, while CCO activity showed a declining trend. Cadmium induced a large number of reactive oxygen species in the testis of freshwater crab, destroyed the mitochondria electron transport chain, and led to germ cell apoptosis, possibly through Caspase-9 dependent mitochondrial pathway.Conclusions:(1) In order to clear the Cd2+-induced excessive reactive oxygen species, low concentrations of Cd2+could activate antioxidant enzyme activities in the testis of freshwater crab. However, high concentration of Cd2+was able to inhibit the activity of antioxidative enzymes, resulting in redox balance broken in vivo, reactive oxygen species and lipid peroxidation increased. Cd2+ could induce severe oxidative damage.(2) Acute exposure to Cd2+led to genotoxicity and apoptosis in the testis cells of freshwater crab, which may lend strong support to the conclusion that acute exposure to Cd2+resulted in a cumulative and/or progressive testis injury. The possible mechanism of apoptosis induced by Cd2+in the testis of freshwater crab was mitochondria-dependent apoptosis pathway involving the activation the activity of Caspase-9.(3)MDA content could be used as biomarkers of environmental pollution, which provided scientific basis for water quality monitoring and health of aquatic products breeding. On the other hand, these results were significant to provide a theoretical basis for the prevention of cadmium poisoning in crustaceans, and to maintain ecological balance. |
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