Molecular Mechanism Of Toxic Effects On Primordial Germ Cells Of Zebrafish Embryo After Cadmium Exposure

The genesis of germ cells is the basis of organism development and heredity.During the embryonic development,the germ cells are called primordial germ cells(PGCs).It has been found that the formation of PGCs is a key step in germ cell development and played an important role in maintaining genome information of species.Conservative maternal factors including vasa,nanos,piwi and dazl determine the formation,migration,proliferation and differentiation of PGCs.Therefore,we studied on reproductive genes such as vasa,nanos,piwi and dazl,to explore the changes of primordial germ cells.Cadmium was one of the most toxic metal pollutants in water,which has a strong toxic effect on reproductive system of aquatic organisms.Cadmium caused serious damage to the reproductive system of fish,resulting in organ damage and functional defects.However,the study of cadmium toxicity on PGCs in fish is still unclear.Therefore,we used Danio rerio as the research object,four cadmium concentration groups(0.0 ?mol/L,8.9 ?mol/L,17.8?mol/L,35.6 ?mol/L)were set,to explore the molecular pathway of cadmium exposure on PGCs toxicity of zebrafish.By observing the changes of the number of PGCs in zebrafish embryos after cadmium exposure,it was confirmed that cadmium had serious effects on the migration and number of PGCs.Using qPCR the changes of expression of vasa,nanos,piwi and dazl in zebrafish at different developmental stages after cadmium exposure was detected.Furthermore,DIG labeled probes were used to track the effects of cadmium on the expression and localization of reproductive regulatory genes.The results showed that cadmium inhibited the expression of vasa gene and affected the expression and localization of nanos and piwi,which resulted in the decrease of the number of PGCs and the inhibition of the gather and migration of PGCs.These data suggested that cadmium exposure destroy key reproductive regulation gene function,which influence the number and migration of PGCs.This study provided a theoretical basis on the study on the mechanism of reproductive toxicity after cadmium exposure in fish and has potential application value in aquaculture.The results are as follows:1.The changes of PGCs migration in zebrafish embryos after cadmium exposureIn this study,four cadmium concentration groups were set.The changes of PGCs in zebrafish embryos developed to 24 hpf,72 hpf and 7dpf after cadmium exposure were observed.The results showed that compared with the control group,the number of PGCs at different stages of development gradually decreased,and the PGCs were scattered and migration lagged with the increase of cadmium concentration.2.Effects of cadmium exposure on the expression and distribution of vasa gene in zebrafish embryo at different developmental stagesqPCR was used to detect the changes of vasa gene expression in zebrafish at different developmental stages after cadmium exposure.The results showed that vasa expression in 72 hpf and 7 dpf larvae were significantly decreased compared with the control group after cadmium exposure at different concentrations(P<0.01).The expression of vasa in NAC+Cd group was significantly higher than that in single cadmium exposure.The migration and number of vasa-labeled PGCs after cadmium exposure were detected by the whole in situ hybridization.The migration and number of vasa-labeled PGCs after cadmium exposure were detected by whole in situ hybridization.The results showed that,compared with the control group,the vasa hybridization signal decreased gradually with the increase of cadmium concentration.When the cadmium concentration reached 35.6 ?mol/L,the vasa hybridization signal was the weakest.In addition,after cadmium exposure,the number of vasa-labeled PGCs decreased sharply,and the cells could not correctly aggregate and migrate.3.Effects of cadmium exposure on the expression and distribution of nanos and piwiThe expressions of nanos,piwi and dazl genes in zebrafish embryos exposed to cadmium were detected by qPCR.The results showed that the expressions of nanos,piwi and dazl genes in embryos developed to different stages after cadmium exposure decreased significantly compared with the control group.The expression levels of nanos,piwi and dazl genes were significantly decreased at cadmium concentrations of 17.8?mol/L and 35.6 ?mol/L(P<0.01).However,the gene expression level of co-treated with cadmium and NAC treatment group was higher than that of cadmium treatment group alone.The results of piwi and nanos gene at24 hpf and 7 dpf seperately showed that the hybridization signal of nanos and piwi decreased gradually with the increase of cadmium concentration.And nanos and piwi labeled PGCs migration were delayed and cell dispersion could not properly migrate to the gonad anlag.When the concentration of cadmium was higher than 35.6 ?mol/L,the influence of PGCs was most serious.Conclutions:1.After cadmium exposure,the number of PGCs in zebrafish was significantly reduced,germ cells accumulation were prevented and migration was delayedwhich affected gonad formation.Particularly,the migration and number of early germ cells of larva at 7 dpf were the most significantly affected by cadmium.2.Cadmium inhibited the expression of vasa,nanos,piwi and dazl,which played a critical role in the development of zebrafish embryos.3.Cadmium disrupted the expression and localization of the key reproductive regulatory genes and affected the function of reproductive regulatory genes.These effects leaded to a significant decrease in the number of PGCs and the inability of PGCs to migrate to the gonad anlag.