Transgenerational Effects Of Cadmium Exposure During Pregnancy On The Apoptosis Of Ovarian Granulosa Cells In The Offspring

ObjectiveWe investigate ovarian granulosa cell damage in offspring induced by cadmium exposure during pregnancy,and we also observe the transgenerational effects of epigenetic modifications of related genes.Through the above,we provide a scientific basis for further studies on the epigenetic mechanism of cadmium on ovarian cross-generation.MethodSD Rats of SPF are randomly divided into four groups after conception,exposed to cadmium(doses of 0,0.5,2.0,and 8.0(mg/kg),)from day 0 to day 20 of pregnancy.After delivered normally,obtaining the F1 generation.F1 females are mated with a new batch of healthy male rats after adulthood to produce the F2 generation.F3 generations are obtained similarly.We measure the apoptotic bodies(transmission electron microscopy),apoptosis rate and mitochondrial membrane potential levels(cell flow cytometry)in ovarian granulosa cells of offspring as adults.We also detecte the levels of apoptosis-related genes for Bax,Bcl2,caspase-3,caspase-8,caspase-9 in mRNA(PCR)and protein(Westernblot).We use DNA methylation sequencing to depict the methylation levels of gene promoter region,and use bioinformatics methed to analysis the differentially methylated genes in promoter regions.The mRNA levels of methylation-modifying enzymes(DNMT1,DNMT3 A,TET1)are determined by RT-PCR.We also use miRNA microarrays,literature,and databases to screen apoptosis-related miRNAs.We performe bioinformatics analysis(GO enrichment analysis,KEGG pathway analysis,protein interaction network construction,GO term similarity calculation)on the target genes of miRNAs.We also screene F1,F2,and F3 generations for apoptosis-related miRNAs.We measure their expression levels separately by using RT-PCR.We selecte the transcription related factors(the total of 16)that regulate apoptosis-related miRNAs in ovarian granulosa cells.We also measured the mRNA levels of miRNA maturation-related enzymes(DICER,DROSHA,DGCR-8).Results1.The apoptosis level of ovarian granulosa cells in F2 and F3We don’t observe the presence of apoptotic bodies in ovarian granulosa cells of each dose group in the F2 generation.We compare the apoptotic rate in each dose group and find no significant difference(p > 0.05).However,we find that the level of mitochondrial membrane potential in each dose group is significantly decreased compared with the control group,while we find that the difference was statistically significant(p < 0.05).In the F3 generation,we found no apoptotic bodies in any of the dose groups.At the same time,there was no significant difference in the apoptosis rate and mitochondrial membrane potential between each dose group and the control group(p > 0.05).2.The expression of apoptosis-related genes of ovarian granulosa cells in F2 and F3 generationsIn the F2 generation,we find that the mRNA expression of the anti-apoptotic gene Bcl-xl decreased at each dose,while we find that the difference is statistically significant(p < 0.05).However,at each dose of pro-apoptotic genes,we find no significant difference(p > 0.05).Except for caspase-3 mRNA,which decreased at low dose,the difference is statistically significant(p < 0.05).We find no significant differences in Bcl2,Bax protein,Bcl2/Bax protein ratio,and the protein of caspase-3to caspase-9 among the dose groups has no significant differences.In the ratio of caspase-3 to caspase-9 protein cleavosome\uncut in each dose group,we find a significant difference(p < 0.05).In the F3 generation,the exception for the mRNA levels of the anti-apoptotic gene Bcl-xl,and the pro-apoptotic gene caspase-9,which are significantly different compared with the control group(p < 0.05).There is no significant difference in mRNA,protein and their ratio of other genes between the control group and each dose group(p > 0.05).3.Expression of promoter methylation and the mRNA of methylation-modifying enzyme in ovarian granulosa cellsThe biological pathways of the differentially methylated genes on promoters enriched in both F2 and F3 generations involved the apoptotic process,but the GO terms are less similar in both generations,with 0.698 of DNA hypermethylation in promoter regions and 0.691 of DNA hypomethylation in promoter regions.In the F1 generation,the mRNA change of methylase DNMT3 A is significantly different between the medium and high dose groups compared with the control group(p < 0.05),and the other genes are not significantly changed(p > 0.05).There is a significant difference in the mRNA change of methylase DNMT1 at the medium dose group of F2 generation(p < 0.05)and the other genes do not find a significant change(p > 0.05).In the F3 generation,there is a significant difference in the mRNA change of DNMT1 under the medium and high dose groups(p < 0.05).At each dose group,there are significant differences in methylase DNMT3 A and demethylase TET1 mRNA changes(p < 0.05).4.miRNA microarray detection and RT-PCR validation in offspring ovarian granulosa cellsThere are 17 miRNAs are targeted to apoptosis-related genes and significantly up-regulated in the F2 generation.There are 8 miRNAs were targeted by F3 generation apoptosis-related genes and significantly up-regulated.The results show that the species composition of differentially expressed miRNAs is similar between multiple generations,and the microarray reveal that miRNAs have the same expression trend between F2 and F3 generations.However,the F2 and F3 generation miRNAs have high functional similarity(GO term similarity = 0.922).RT-PCR validation of the screening results showed that 20 apoptosis-related miRNAs are up-regulated in the F1 generation,there are 7 apoptosis-related miRNAs are up-regulated in the F2 generation,and there is only one miRNA is up-regulated in the F3 generation.Only two miRNAs(miR-3084 b,miR-532)are present with the same trend in F1 and F2 representatives.5.miRNA maturation-related gene expression and expression of transcription factors that regulate miRNAsThe expression of miRNA maturation-related genes(DICER,DROSHA,DGCR-8)in ovarian granulosa cells of offspring is detected,respectively,of which only DICER gene expression increase and is significantly different in the F1 generation,and there are no differentially expressed genes in the F2 generation.DICER,DROSHA,and DGCR-8 genes are all up-regulated in the F3 generation and the difference is statistically significant.There are 16 transcription factors regulating apoptosis-related miRNAs are detected in the three generations,respectively,and the results showed that they are up-regulated in the F1 generation: C-MYC,SP1,MAZ,RUNX1,STAT1,TRIM,HIF1,KDM5,LARP7,MAX,CTCF,E2F6,and EP300.F1 represents up to down-regulation and the difference is statistically significant for E2F1.F2 represents C-MYC,E2F1,SP1,RUNX1,CTCF,EP300 up to up-regulation.F2 represents MAZ and HIF1 up to down-regulation.F3 represents up to up-regulation of SP1,RUNX1,KDM5.F3 represents that CTCF and EP300 are down-regulated.Among them,SP1 and RUNX are up-regulated in F1,F2,and F3 generations.ConclusionUnder this exposure model:1.progeny apoptosis gradually disappeared in F2 and F3 generations,and the maternal transgenerational effect was not significant.2.miRNAs and DNA methylation that regulate ovarian granulosa cell apoptosis are altered and have intergenerational and transgenerational effects.Epigenetic modifications may be important in cadmium-induced apoptosis and repair of ovarian granulosa cells.3.In the regulatory mechanism of miRNAs,transcription factors such as SP1 may be more important in miRNA intergenerational and transgenerational effects..