Fig. 5: Aberrant DNA methylations persisted from AE-F1 sperm to AE-F2 islets contribute to abnormal expression of β-cell functional genes.

a Venn diagram of the 727 DEGs in F2 islet and the 8571 genes with DMRs in F1 sperm, with 296 genes overlapping between the two conditions. b Top 10 pathways identified by KEGG enrichment analysis of the 296 overlapped genes in a. c Network analysis of the enriched pathways for 296 overlapped genes commonly altered between the WGBS and the RNA-seq datasets. d List of the genes in the insulin secretion pathway identified in b and c. It details the gene functions, differential methylation levels of insulin secretion genes in AE-F1 sperm, and gene expression changes in AE-F2 islet. e MeDIP-qPCR analyses for DNA methylation levels of Pdx1, Irs1, Ptprn2, and Cacna1c in Ctrl-F1 and AE-F1 sperms (n = 3). f MeDIP-qPCR analyses for DNA methylation levels of Pdx1, Irs1, Ptprn2, and Cacna1c in Ctrl-F2 and AE-F2 E18.5 pancreases (n = 3). g MeDIP-qPCR analyses for DNA methylation levels of Pdx1, Irs1, Ptprn2, and Cacna1c in Ctrl-F2 (n = 4) and AE-F2 adult islets (n = 5). h qRT-PCR analyses for mRNA expression levels of Pdx1, Irs1, Ptprn2, and Cacna1c in Ctrl-F2 (n = 4) and AE-F2 adult islets (n = 5). i Protein levels of PDX1, IRS1, PTPRN2, and CACNA1C were determined by western blot assay in islets from Ctrl-F2 and AE-F2 adult mice. j Methylation profile of the insulin secretion genes in F1‒F3 sperms. Vertical bars above the horizontal line indicate the methylation level (0‒1) at individual CpG site. The box indicates a DMR in the promoter or gene-body region. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01; significance is assessed by two-tailed unpaired Student’s t-test.