| Cadmium is one of the major environmental pollutants.It can be used as a battery material to produce pigments,paints,fuels,tires and plastics which are closely related to our lives.Cadmium enters the body through food,drinking water,and air,and it can cause various degrees of damage to the liver,kidneys,lungs,brain,bones,blood,and reproductive tissues and systems.The mechanism of cadmium harming to the female reproductive system is still not fully understood.In this study,pregnant mice were used as an animal model to drink water containing cadmium chloride freely to study the effect of cadmium on fetal rat reproductive cells and its mechanism.On the basis of the establishment of whole tissue immunofluorescence staining 3D imaging method,the effect of cadmium on DNA methylation during the development of fetal mouse germ cells was analyzed.1.Establishment of whole tissue immunofluorescence 3D imaging methodIn order to analyze the development of fetal germ cells,germ cells were labeled with specific fluorescent antibodies on the basis of the complete morphology of the gonads.The confocal microscope was used to scan layer by layer after the different tissue-transparent methods treatment.Imaris software and statistical analysis method were used to generate a 3D model and the gonadal differentiation among number and distribution of germ cells.After comparing with the immunofluorescence staining of tissue sections,the whole tissue immunofluorescence 3D imaging method can excellently show the distribution and number of germ cells in the process of gonadal differentiation and scaling A2 sucrose is the optimal transparency method for gonads.The whole-tissue immunofluorescence 3D imaging method maintained the integrity of the tissue and stereoscopically displaying the distribution of cells in the tissue,and the statistical number of cells is more accurate.2.Effect of cadmium on the germ cells of female fetusesTo investigate the effect of cadmium on fetal rat germ cells,pregnant mice(0.5 days)were randomly divided into two groups.Pregnant mice in the experimental group were free to drink water containing cadmium in DDW water(64 mg/l).Pregnant mice or pups were sacrificed at 13.5 days of pregnancy(13.5 dpc),17.5 days(17.5 dpc),and 1 day of birth(1 dpp)respectively.Female gonads were collected and used for immunophenotyping.Germ cells were labeled and scanned by confocal laser scanning layer to determine the distribution of germ cells in the gonads.The 3D model was generated by Imaris software and the number of germ cells was counted.The results showed that immunofluorescence staining was widespread throughout the gonads at 13.5 dpc.there were many germ cells and there were no obvious boundaries between cells,and accurate cell counts could not be performed;However,the fluorescence intensity showed that difference was significant after the cadmium treatment and the number of germ cells in the gonads of 13.4 dpc female fetuses were decreased.Contrarily,the single germ cells were clearly visible in the gonads from 17.5 dpc and 1 d female.The germ cells were mainly located at the edge of the gonad and the middle distribution was less.The numbers of germ cells at 17.5 dpc and Id were 2564±65 and 1836±88 respectively(P<0.05).After cadmium treatment,there are more distributed from germ cells in female sex glands of 17.5 dpc was more toward the edge of the gonads and the number was 2105±67,which was significantly lower than that of the control group(P<0.05).After treatment with cadmium,the distribution of germ cells in the female gonads was not significantly different from that in the control group at 1 d,but the number was 1607±59 which have reached significant lever(P<0.05).The results showed that the number of germ cells showed a decreasing trend in the process of female gonad differentiation and development,and cadmium treatment could significantly accelerate this trend and lead to the lack of reproductive cell numbers in the offspring.3.The effect of cadmium on DNA methylation and related gene expression in gonadal of female fetusIn order to further study the mechanism of cadmium on female fetal rat germ cells,female gonads of 13.5 dpc,17.5 dpc and 1 dpp were collected from the model abovementioned Genomic DNA methylation(5-MC)levels were detected by ELISA,and BSP and COBRA methods was used to detect the DNA methylation levels of H19,Peg3,and LINE1 genes,then the expression levels of DNMTs and MTs genes were detected at the same time.The results showed that the level of 5-MC gradually decreased during the development of gonads,but the difference was not significant(P>0.05),and cadmium treatment could prevent the decline of 5-MC to a certain extent.DNA methylation analysis showed that the level of DNA methylation of the parental imprinted gene H19 decreased slowly during gonad development,but cadmium had no significant effect on the total(P>0.05);The DNA methylation of gene Peg3 showed a gradual upward trend.Cadmium interfered with this trend,making it increase first and then decrease,but overall there was no significant difference(P>0.05);The DNA methylation of the repeat sequence LINE1 was maintained in a relatively stable state,and cadmium had no significant effect on it(P>0.05);however,DNA methylation at some sites of these three genes was interfered by cadmium in different degrees.Analysis of gene expression showed that DNMT1o was expressed only in the gonads of female mice after birth.The expression level of DNMT1s gradually decreased during the development of gonads.Before birth,the expression level of DNMT3a was slightly lower and the level of DNMT3a was significantly increased after birth.The expression levels of DNMT1o,DNMT1s,and DNMT3a were decreased,but the difference was not significant.During the gonadal development,DNMT3b gene was not expressed.The expression of MTI gene was decreased and no longer expressed after birth.The expression of MTII gene remained relatively stable.The effect of cadmium on MTs gene expression was not significant.The results showed that the expression of DNMTs gene maintained the DNA methylation of genome and individual genes stably during gonad differentiation,and cadmium had no significant effect on them.At the same time,the expression of MTs gene was not significantly affected by cadmium.4.ConclusionThis study established a method for whole-tissue immunofluorescence 3D imaging and applied it to the study of the effects of cadmium on gonadal development.During the development of female gonadal,the number of germ cells showed a decreasing trend.Cadmium treatment can significantly accelerate this trend and lead to a decrease in the number of germ cells of the pups.During this process,The DNA methylation and the expression of related genes maintain a relatively constant,while the MTs gene is not affected by cadmium,indicating that the decrease in the number of cells by the cadmium treatment may not be accomplished through the interference of DNA methylation. |
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