| Objective To investigate the toxic effect of DEHP on the proliferative capacity of germ cells in mitotic zone and the differentiation ability of germ cells in meiotic zone and its toxic mechanism.Methods 1.Reproductive toxicity indictors such as the number of brood size,generation time,egg-laying rate and the number of fertilized eggs in the uterus were observed.The effects of DEHP on the fertility of C elegens were evaluated by Dianamidophenylindole(DAPI)staining to observe the changes of the number of germline cells in the mitotic zone and meiotic region of nematode.2.The number of germ cells in the mitotic zone and the size of the nucleus in the mitotic zone were observed by DAPI staining to evaluate the effect of DEHP on the mitotic region,the fluorescence intensity of DTC cells in JK2868[LAG-2::GFP] strain and the DNA damage of germ cells in WS1433[HUS-1::GFP] strain were observed under fluorescence microscope to study the effect of DEHP on DTC cells and the DNA damage of germ cells respectively.The mRNA expression levels of DNA damage genes and the genes of ATM-1/ATL-1 pathway were measured by RT-qPCR,then the knockout mutant WS2277/hus-1(op241),VC381/atm-1(gk186),VC728/atl-1(ok1063),WS3455/chk-2(gk212),JR1279/cep-1(w40),RB2075/phg-1(ok2741)were used for further verification of the ATM-1/ATL-1 pathway.3.The JH2623 [RAD-51::GFP] strain was used to observe the DNA damage repair in the meiosis region.The number of germ cells in the transition zone was observed by DAPI staining and the expression level of HIM-3 protein in JH2120[HIM-3::GFP] strain meiosis zone was observed under fluorescence microscope,the rate of males in the offspring was observed to determine whether the chromosome segregation were affected.DAPI staining was used to observe the number of germ cells in diakinesis and the size of the-2 egg cells under the differential interference differential microscope(DIC)and to explore the effect of DEHP in diakinesis,the mRNA expression levels of DNA damage and repair genes in meiosis were measured by RT-qPCR.Results 1.Effect of DEHP on the reproductive ability of C.elegans Compared with the control group,the number of brood size significantly reduced with each dose of DEHP.At the exposure doses of 1.0 and 10.0 mg/L,the number of fertilized eggs in the uterus decreased significantly and accompanied by the decrease of egg-laying rate,further confirms that DEHP has an effect on the oogenesis.The generation time was only prolonged in the 10.0 mg/L dose group.Compared to the control group,(DAPI)staining showed that the total number of germ cells and the number of germ cells in mitotic and meiotic zone were significantly decreased in 1.0 and 10.0 mg/L exposure groups.2.The mechanism of the decrease number in mitotic zone induced by DEHP After DEHP exposure,the number of germ cells in the mitotic region of C.elegans was significantly reduced in the 0.1,1.0,and 10.0 mg/L dose groups,and the difference was statistically significant.Exposured to high doses of DEHP could lead to an abnormal enlargement of germ cell nuclei in the mitotic zone.However,by further observing the fluorescence intensity of DTC in JK2868 strain,found that compared with the control group,the fluorescence intensity of DTC in JK2868 strain was not statistically different.With the increase of the exposure dose,the number of germ cells with DNA damage in WS1433[HUS-1::GFP] stain were gradually increased,and the difference was statistically significant.The mRNA expression levels of DNA damage-related genes hus-1,clk-2,and cep-1 were increased,indicating that DEHP induced DNA damage in germline,and the expression levels of atm-1,atl-1,chk-2 and phg-1 were also increased.After the gene knockout mutants WS2277/hus-1(op241),VC381/atm-1(gk186),VC728/atl-1(ok1063),WS3455/chk-2(gk212),JR1279/cep-1(w40),RB2075/phg-1(ok2741)were exposured to DEHP,we found that compared with N2 the number of germ cells in mitotic zone were increased.3.Mechanism for the decline of the number in meiotic zone caused by DEHP Compared to the control group,DEHP could significantly reduce the number of cells in meiotic zone at the exposure doses of 1.0 and 10.0 mg/L,and the difference were statistically significant.The DNA damage repair in the meiosis region were observed in JH2623 [RAD-51::GFP] strain,found that the expression of RAD-51 protein and DNA damage repair in meiosis region increased at 1.0 and 10.0 mg/L exposure doses and the difference was statistically significant.The number of germ cells in the transition zone showed a downward trend,but DEHP did not affect the pairing and synapsis of the chromosomes.Compared with the control group,the number of germ cells in the diakinesis were decreased in 1.0 and 10.0 mg/L exposure doses and the length of the horizontal axis of the-2 egg cells was significantly reduced at doses of 0.1,1.0,and 10.0 mg/L,the expression levels of rad-54,spo-11,pch-2,rad-51,mre-11,and msh-5 were increased at doses of 0.1,1.0,and 10.0 mg/L and the difference was statistically significant.It is indicated that DNA damage occurs in the meiosis region and DNA damage repair abnormality occurs.These results suggest that DEHP induced DNA damage in meiotic region and DNA damage repair abnormality.Conclusion DEHP could induces DNA damage in germ cells.In mitotic region,DEHP activates ATM-1/ATL-1 DNA damage pathway that lead to proliferation arrest in mitotic zone and reduction in the number of germ cells in mitotic zone.In meiosis zone,DEHP affects the repair of DNA damage in meiosis zone and activate rad-54,spo-11,pch-2,rad-51,mre-11,and msh-5 genes,induces DNA damage in meiosis region,which eventually led to the decrease in the number of germ cells in the meiosis region and finally led to a decrease in the reproductive capacity of Caenorhabditis elegans to perform its reproductive toxicity effect. |
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