Studies On Damage And Toxicity Mechanism Of Lead In Male Reproduction System Of The Freshwater Crab Sinopotamon Henanense

Lead?Pb?causes a series of toxic effects in aquatic organism.In order to understand the toxic mechanisms of Pb on the male reproduction system of freshwater crab Sinopotamon henanense,crabs were exposed to different Pb concentrations?0,3.675,7.35,14.7,29.4 and 58.8 mg/L?for 3,5 and 7 d,respectively.we firstly investigated Pb accumulation in the testes and the accessory glands,and the tissue damaging effects of Pb was also investigated by histopathological examination and analyses of antioxidant enzymes?including SOD,CAT and GPx?,lipid peroxidation?MDA?as well as the activity of acid phosphatase?ACP?.Then reactive oxygen species?ROS?production and apoptotic changes were detected in the testes,cell apoptosis was assessed by observing the ultrastructure of testes and employing in situ TUNEL staining,as well as the activity and mRNA expression of caspase-3,-8,-9.Furthermore,the effects of Pb on the accessory gland function in in sperm acrosome reaction induction and oxidative stress were investigated.In this work,effects of Pb on the sperm quality and oxidative stress were investigated too,sperm quality indices including survival rate of sperm measured by eosin Y stained,sperm plasma-membrane integrity and acrosomal-membrane integrity measured by flow cytometry,DNA integrity measured by fluorescence microscopy,the index of oxidative damage to lipids,proteins and DNA of sperm including malondialdehyde?MDA?,protein carbonylation?PCO?and DNA-protein crosslinks?DPC?.After we found that Pb reduced the acrosome reaction?AR?induction rate and acrosin activity,the role of calcium signaling,protein concentration of calmodulin?CaM?and VRP effect on Pb-induced stress were measured to evaluate whether Pb can enter the sperm through Ca²⁺ channels leading to sperm damage.The results showed that:1.Effects of lead on cell morphology and oxidative damage in testesand accessory glands of S.henanensePb levels in testes and accessory glands increased significantly following Pb exposure.Morphological changes identified histologically were discovered in testes,including a disordered arrangement of germ cells,a decreased number of sperm in the lumina of the seminiferous tubules,extensive necrosis in the germinal layer of the seminiferous tubules,etc.At the same time,histological abnormalities were discovered in accessory glands,the wall cells were separated from the basement membrane,and wall cells were missing partly.The activities of SOD,GPx and CAT in testes showed no statistically significant changes compared to the control for3 d,and initially increased and subsequently decreased with increasing Pb concentrations at 5 d and 7 d.The antioxidant enzyme activities in accessory glands initially increased and subsequently decreased with increasing Pb concentrations and Pb exposure.This was accompanied with an increase in malondialdehyde?MDA?content and a downward in activities of ACP in a concentration-dependent manner.2.Effect of lead on apoptosis of testes in S.henanenseROS content was increased in a concentration-dependent manner,evaluated by 2,7-Dichloro-uprescin-diacetate.Typical apoptotic characters such as shrunken and deformed nuclei and mitochondrial injury were observed by transmission electron microscopy.An increasing number of apoptotic cells were detected by the TUNEL test in Pb-treated groups.Furthermore,the activities and mRNA expression of caspase-3,-9 were increased in a concentration-dependent manner after Pb exposure,but no significant change in the activity and mRNA expression of caspase-8 were observed.3.Effect of Pb on accessory gland function in sperm acrosome reaction induction in S.henanenseThe acrosome reaction induction rate was high up to 78% under the combination of ionophore A23187 and accessory gland homogenate,itimplied that accessory gland could adjust the action of sperm acrosome reaction.The acrosome reaction induction rate was deceased significantly when the accessory gland taken from crabs which was treated by Pb.Meanwhile,the reactive oxygen species?ROS?levels and protein carbonylation?PCO?content were increased in a concentration-and time-dependent manner in accessory glands,however,DNA-protein crosslinks?DPC?lever was no significant change.4.Effects of lead on the sperm quality and oxidative stress in S.henanenseThe results showed that Pb levels in sperm were increased significantly upon Pb exposure in most treated groups,survival rate of sperm,sperm plasma-membrane integrity,acrosomal-membrane integrity,and DNA integrity were reduced at higher concentrations after 5 d and 7 d.Oxidiative stress of sperm induced by Pb was reflected in significant up-regulation of reactive oxygen species?ROS?levels.A significant reduction of the total antioxidant capacity levels occurred after exposure to 14.7 mg/L Pb and above at 7 d compared to the control.The results of oxidative damage to lipids,proteins and DNA of sperm showed that malondialdehyde,protein carbonylation and DNA-protein crosslinks were increased in a concentrationand time-dependent manner.5.Effect of lead on Ca²⁺ in the sperm of S.henanenseIt is showed that Pb reduced the acrosome reaction?AR?induction rate and acrosin activity in sperm.[Ca²⁺] was always lower under the combination of Pb+A23187 than A23187 alone.The protein concentration of calmodulin?CaM?was measured by ELISA method and it was showed that Pb elevated CaM content.The survival rate of sperm,DNA integrity,MDA and PCO content were always lower under the combination of verapamil?VRP?+Pb than Pb alone,VRP against Pb-induced stress in the sperm of S.henanense.Conclusions:1.Acute Pb exposure led to harmful effects at the cellular level of testes and accessory glands in S.henanense,which are most likely linked to Pb-induced oxidative stress.Meanwhile,it is speculated that Pb may be toxic to the testes and the accessory glands of the crab by decreasing activities of ACP.2.Pb could induce ROS as well as apoptosis of testes,and that the apoptotic process occurs through the mitochondrial caspase-dependent pathway.3.Pb destroyed the function of accessory gland in sperm acrosome reaction induction,which was associated with the increasing of ROS and PCO content.4.The results indicated Pb exposure lead to sperm quality decline,which is associated to the oxidative damage of sperm,and Ca²⁺ channels are the likely entry pathways of Pb into the sperm of the freshwater crab S.henanense.