| The toxicity of several waterborne inorganic contaminants to aquatic organisms is associated with oxidative damages due to the formation of reactive oxygen species (ROS). In the present work, the reproductive toxicity of the heavy metal cadmium (Cd2+) was tested with the sperm of the freshwater crab(Sinopotamon henanense). Crabs were exposed to 0,7.25, 14.5,29,58 and 116 mg/L of Cd2+ for 3,5 and 7 d. An induction of reactive oxygen species (ROS) and the total antioxidant capacity (TAC) were measured. Biomarkers of oxidative damage to lipids (malondialdehyde, MDA), proteins (protein carbonyl derivates, PCO) and DNA (DNA-protein crosslinks, DPC) were investigated to address Cd2+ effects on crucial macromolecules of S. henanense sperm. In order to determine whether the morphology of sperm was also affected by Cd2+ exposure, hematoxylin-eosin staining (H.E.) was used so that the sperm morphology changes were observed under an optical microscope. In addition, the transmission electron microscopy (TEM) was also used to observe sperm ultrastructural changes under moderate and high Cd2+ groups (Cd2+:29,116 mg/L) after exposure for 7 d. To further determine whether the sperm quality was affected, specific fluorescent dyes and flow cytometry (FCM) were used so that the sperm quality was investigated including sperm viability, plasma- and acrosomal-membrane integrity and chromatin integrity.(1) With the increase of Cd2+ concentration, the ROS levels were significantly elevated at days 3,5 and 7 and TAC declined at day 7 (p<0.05). This resulted in an increase of MDA content (5 d,7 d), PCO content (7 d), and of DPC levels (3 d,7 d) (p<0.05).(2) The microscope observation indicated that acute Cd2+ exposure could result in sperm morphological damages like acrosomal loss and nucleus injuries. Ultrastructural observations showed in addition that Cd2+ exposure after 7 d could result in sperm membrane wrinkling, chromatin irregularities and acrosomal integrities, which became worse with the increase of Cd2+ concentration. Our results showed that acute Cd2+ exposure caused morphological injury to sperm of crabs we investigated here, which might have an effect on the subsequent function like fertilization.(3) With the increase of Cd2+ concentration and exposure time, the viability was decreased, the intact rate of sperm plasma- and acrosomal-membrane were decreased and the ratio of abnormal chromatin was increased. When exposed by 58 and 116 mg/L Cd2+ for 5 d, sperm viability was significantly reduced; sperm membrane integrity and chromatin structure were also significantly damaged by 116 mg/L Cd2+; the acrosomal integrity were also damaged starting at 29 mg/L Cd2+. When exposed by 29, 58, and 116 mg/L Cd2+ for 7 d, the sperm viability and plasma- and acrosomal-membrane integrity were significantly lowered. The chromatin structure, however, were damaged at all Cd2+-exposed groups. Our results indicated that exposure to acute cadmium can exert obvious injury to sperm quality of the crabs investigated.The experimental results indicated that:(1) Under the acute Cd2+ exposure, sperm produce excessive ROS that exceeds the capacity of its TAC, leaving the sperm under the attack of ROS. Its major macromolecules such as lipids, proteins and DNA all underwent oxidative damages and ultimately put the sperm in a state of oxidative stress.(2) As a result of the acute Cd2+-induced oxidative stress, changes of the sperm structure appeared, the organelles with membranes were seriously damaged, the sperm plasma- and acrosomal-membrane were no longer complete, while the chromatin condensation appeared in the nucleus and the abnormal chromatin rate were increased.(3) Changes in sperm morphology caused by Cd2+-induced oxidative stress finally resulted in a decrease in sperm quality. Sperm viability was decreased, the ratio of intact sperm plasma- and acrosomal-membrane were lowered significantly, and the proportion of single-stranded DNA were obviously increased, indicating a severe damage in the DNA structure. All these results lend a credit to the fact that the sperm quality of the crabs were declined after being exposed to acute Cd2+. |
PDF Full Text Request