Effects On The Early Embryonic Development And The Expression Of Key Reproductive Genes After Cadmium Exposure In Zebrafish,Danio Rerio

To investigate the toxic effects of cadmium on early embryonic development and germ cells of zebrafish,Danio rerio,fertilized eggs were exposed to different cadmium concentrations.Firstly,the deformity rate and mortality rate of embryos in cleavage period(2 hour post-fertilization(hpf)),glastula period(6 hpf),segmentation period(10 hpf) and pharyngula period(24 hpf) were analyzed,which confirmed that cadmium exposure caused serious toxic effects on the early embryos.By detecting the changes of the level of reactive oxygen species(ROS),malondialdehyde(MDA) content and superoxide dismutase(SOD) activity in embryos treated with cadmium or co-treated with cadmium and N-acetylcysteine(NAC),the oxidative damage effect of cadmium and the protective effect of NAC on early embryos were demonstrated.Furthermore,the expression of vasa,nanos1,piwi and dazl of 2 hpf,6 hpf,10 hpf and 24 hpf embryos were detected after cadmium exposure by real-time PCR,which revealed the reproductive toxic effects of cadmium on early embryos.This study provides a theoretical basis for exploring the mechanism on the reproductive toxicity of early embryos after cadmium exposure.The study is mainly divided into three parts as follows:1.Toxic effects on early embryonic development of zebrafish after cadmium exposure.Cadmium exposure leaded to abnormal development of early embryos and even caused the embryo to die.The deformity rate of embryos at 2 hpf,6 hpf,10 hpf and 24 hpf were increased significantly in the high concentration group(17.80 ?mol/L,26.70 ?mol/L,35.60 ?mol/L),and the embryos were characterized by pericardial protuberance,abnormal division,delayed epiboly,yolk sac edema,tail bending.The mortality rate of 2 hpf embryos was no significant change,but the mortality rate of 6 hpf,10 hpf and 24 hpf embryos increased significantly when cadmium concentration was higher than 8.90 ?mol/L(P<0.01).2.Oxidative damage to early embryos of zebrafish after cadmium exposure.The ROS detection by DCFH-DA fluorescence probe showed that the content of ROS in early embryos increased after cadmium exposure,showing a concentration-effect relationship.Especially,the content of ROS increased significantly when cadmium concentration was higher than 8.90 ?mol/L(P<0.05).Compared with embryos treated with cadmium,the content of ROS in embryos decreased co-treated with cadmium and NAC,and even returned to the normal level.The results showed that cadmium induced a large number of ROS in early embryos.The results showed that the content of MDA in early embryos increased gradually with the increasing of cadmium concentration,and the content of MDA increased significantly after exposed to higher cadmium concentration(17.80 ?mol/L and 35.60 ?mol/L).The activity of SOD in embryos promoted when exposed to lower cadmium concentration,while inhibited by higher cadmium concentration.However,compared with the cadmium treatment group,the content of MDA decreased and the activity of SOD increased after co-treated with cadmium and NAC.These data confirmed that cadmium caused serious oxidative damage to the early embryos of zebrafish.3.Effects on the expression of key reproductive genes in zebrafish early embryos after cadmium exposure.The expression of reproductive genes in embryos after cadmium exposure was detected by real-time PCR.The results showed that the expression of vasa,nanos1,piwi and dazl did not change significantly when the embryos developed to 2 hpf after cadmium exposure.The expression of nanos1 decreased significantly in the high concentration group(17.80 ?mol/L,26.70 ?mol/L,35.60 ?mol/L),when the embryos developed to 6 hpf.The expression of vasa decreased significantly when cadmium concentration was higher than 8.90 ?mol/L,and the expression of nanos1 decreased obviously when the cadmium concentration was higher than 17.80 ?mol/L at 10 hpf embryos.The expression of vasa,nanos1,piwi and dazl decreased significantly followed 17.80 ?mol/L and 35.60 ?mol/L cadmium exposure when the embryos developed to 24 hpf(P<0.05).The genes expression of embryos co-treated with cadmium and NAC were retrieved to the levels of the control group.Conclutions:1.The deformity rate and mortality rate of early embryos significantly increased after cadmium exposure.Embryos showed a variety of abnormal phenotypes at different developmental stages,and even cause embryo to die.In particular,mortality rate of early embryos increased significantly when embryos developed to glastula and segmentation period.2.Cadmium exposure resulted in the level of ROS and the content of MDA increased,and the activity of SOD decreased,which caused serious oxidative damage to embryos.3.Cadmium exposure had different effects on the expression of reproductive regulatory genes.Early embryos of zebrafish after cadmium exposure resulted in a significant decrease in the expression of vasa,nanos1,piwi and dazl,especially in the expression of vasa and nanos1.Studies have shown that cadmium exposure down-regulated the expression of reproductive-related genes in early embryo.