Damage Of Ovarian Granulosa Cells And Alteration Of DNA Methylation Patterns In Adult F1 Rats After Cadmium Exposure During Pregnancy

Objective:To study and elucidate the effects of cadmium exposure during pregnancy on the growth and hormonal synthesis of ovarian granulosa cells in adult F1 rats and the changes in DNA methylation patterns,providing further insights into the cytotoxicity and epigenetic mechanisms of cadmium-induced ovarian granulosa cells.Method:SPF adult SD rats were exposed to 0,0.5,2,and 8 mg/kg CdCl2 on the 1st to 20 th days of pregnancy,and were intragastrically infused once a day.F1 generation females took ovarian granulosa cells on the 56 th day after birth to stably culture for 24 h and adhered to collect cells and supernatant for use.Part I Effect of cadmium exposure during pregnancy on apoptosis of ovarian granulosa cells in adult F1 rats.1 Observe the granular cell substructure(TEM);2 Detect the apoptosis rate of ovarian granulosa cells(AnnexinV-FITC/PI double staining);3 Detect the mRNA(RT-PCR)and protein,s expression(Western-blot)of Bcl-2,Bax,Bcl-xl,Caspase3,Caspase8 in ovarian granulosa cells.Part II Effects of cadmium exposure during pregnancy on ovarian granulosa cytokine synthesis in adult F1 rats 1 Detect progesterone and estradiol levels in granule cell supernatants(ELISA);3 Detect the mRNA(RT-PCR)and protein,s expression(Western-blot)of StAR,SF-1,Cyp11 a1,Cyp17 a1,and Cyp19 a1 in ovarian granulosa cells.Part III whole Genomic DNA Promoter Methylation of Ovarian Granulosa Cells in adult F1 Rats 1 Detect differential gene expression profiles of the whole genomic DNA methylation promoter region in ovarian granulosa cells(n=3)(co-IP);2 GO Analysis of Differentially methylated genes;3 KEGG Analysis of differentially methylated genes.Part IV According to the results of microarray analysis and bioinformatics analysis to verify the DNA methylation status in Eif2s1 and Atf4 promoter regions.1.Methl Primer Express and SignalMap determined the promoter regions of Eif2s1 and Atf4 genes in CpG islands;2.verified methylation status of Eif2s1 and Atf4 gene promoter regions(BSP);3.Detect the expression of Eif2s1 and Atf4 mRNA(RT-PCR)Results Part I The Effect of cadmium exposure during pregnancy on apoptosis of granulosa cells in F1 generation rats 1.Compared with the control group,the number of apoptotic cells and apoptotic bodies increased in the cadmium-treated group,and mitochondria,endoplasmic reticulum,nucleus and chromatin of the granular cells were damaged in different degrees.2.Compared with the control group,the apoptosis rate of each cadmium group increased,the difference was statistically significant(P <0.01).3.Compared with the control group,the mRNA and protein expression of Bcl-2,Bcl-xl and Bcl-2/Bax were down-regulated in different degrees,and the mRNA and protein expression of Caspase3 were up-regulated in different degrees.The difference was statistically significant.(p<0.01 or 0.05),there was no significant difference in expression of Bax and Caspases8 mRNA and protein in each cadmium group.Part II The Effect of cadmium exposure during pregnancy on granulocyte cytokine synthesis in adult F1 rats 1.Compared with the control group,the level of progesterone in the granulosa cells of each cadmium group was significantly decreased(P<0.01),but there was no significant difference in the level of estradiol(P>0.05).2.The expression of StAR and Cyp11 a1 mRNA and protein in each cadmium group were significantly down-regulated,the difference was statistically significant(P<0.05),and there was no significant difference in mRNA and protein expression of Cyp17 a1 and Cyp19 a1(P>0.05),SF-1 gene mRNA expression changes in varying degrees,but the protein difference was not statistically significant(P> 0.05).Part III: Methylation of Granulocyte Whole Genome Promoter in F1 Generation Rats 1.Compared with the control group,the number of hypomethylated genes in the genomic DNA of the 8.0 mg/kg group was higher than that of hypermethylated genes,and it mainly occurred in the high-density promoter and the medium-density promoter region.2.GO analysis showed that cadmium exposure during pregnancy had an impact on the biological processes,molecular functions,and cellular components of ovarian granulosa cells in adult F1 rats.3.Differential methylation Pathway analysis suggested that compared with the control group,cadmium exposure had effects on 48 passages of F1 generation rat granulosa cells,among which 14 genes including Eif2 s1 and Atf4 in the apoptotic pathway had high methyl groups in the promoter region.Hypomethylation occurred in the promoter region of BCL-XL gene.No methylation status of the promoter regions of Bcl-2,Bax,Caspases3,and Caspases8 genes was changed;and the gene promoter genes Cyp1 a1 and Hsd17 b1 were found to be low.Methylation did not see changes in methylation of the promoter regions of StAR,SF-1,Cyp11 a1,Cyp17 a1,and Cyp19 a1 genes.Part IV Verify DNA methylation status in Eif2s1 and Atf4 promoter regions dependent on chip detection and bioinformatics analysis results 1.A high methylation peak appeared in a specific region of the promoter of Eif2s1 and Atf4 genes,and this peak fell on the corresponding gene CpG island.2.Compared with the control group,the expression of Eif2s1 and Atf4 mRNA in each cadmium group was down-regulated,and the difference was statistically significant(P<0.05).3.Compared with the control group,the average methylation rate of the Eif2s1 promoter region in the 8.0 mg/kg group was significantly higher(P<0.05),and the mean methylation rate of the Atf4 promoter was not statistically different.(P>0.05).Conclusion Dependent on the above results,following conclusions could be drawn: 1.Cadmium exposure during pregnancy could cause effects on growth and function of ovarian granulosa cells in adult F1 rats.2.The cadmium burst during pregnancy changes the whole genome DNA methylation status of granulosa cells in adult F1 generation rats,causing apoptosis and changes in methylation patterns of hormone synthesis-related genes.3.Under our experimental condition,DNA methylation may be involved in its inhibition of Eif2s1 mRNA expression regulation,but may not be involved in its inhibition of Atf4 gene mRNA expression regulation.