Maternal obesity alters DNA methylation of oocytes.

(A) Methylation level of 5mC and 5hmC in oocytes. 5mC, 5-methylcytosine; 5hmC, 5-hydroxymethylcytosine; DAPI, chromatin.

(B, C) Relative fluorescence intensity of 5mC and 5hmC in GV oocytes.

(D) Genomic methylation level of MII oocytes examined by single-cell whole genome bisulfite sequencing. Control group (CD) has two replicates, and obesity group (HFD) has three replicates.

(E) Average genomic CG methylation level in MII oocytes. CD, control group; HFD, obesity group; ** means p value < 0.01.

(F) CG methylation levels at different regions in MII oocytes. CGI, CpG Island; utr5, 5’ untranslated region; utr3, 3’ untranslated region; repeat, repeat sequence.

(G) Total differentially methylated regions (DMRs) in oocytes of control and obesity groups. hyper-DMRs, hypermethylated DMRs; hypo-DMRs, hypomethylated DMRs.

(H) Distribution of DMRs on chromosomes in MII oocytes. Outside-to-in: chromosome, hyper-DMRs, TE (transcription end region), and gene, hypo-DMRs.

(I) Enrichment of genes with DMRs at the promoter regions in KEGG pathways, and the top 20 pathways were presented.

(J) Schedule of breeding. Female C57BL/6 were fed with normal (CD) or high-fat diet (HFD) for 12 weeks marked as F0. F1 was produced by F0 mating with normal males, respectively, and marked as CF1 and HF1; F2 was produced by female F1 mating with normal males and marked as CF2 and HF2, respectively.

Transgenerational inheritance of metabolic disorders and altered DNA methylation.

(A-C) Glucose tolerance (GTT) and insulin tolerance ITT) were tested for female F0, F1, and F2, respectively. * p<0.05; ** p<0.01.

(D-F) DMR methylation at the promoter regions of Bhlha15, Mgat1, Taok3, Tkt, and Pid3cd in F0, F1, and F2 oocytes were respectively examined using bisulfite sequencing. At least 10 available clones from 80-100 oocytes were used to calculate the methylation level. White circle, unmethylated CG; black circle, methylated CG. * p<0.05; ** p<0.01.

(G) Inheritance of altered methylation in different generations was analyzed. * p<0.05; ** p<0.01.

Maternal obesity alters metabolomics of serum.

(A) Principal component analysis in CD and HFD mice.

(B) Differential metabolites in HFD serum compared to CD group. Red circle, up regulated metabolites; blue circle, down regulated metabolites.

(C) The enrichment of differential metabolites was analyzed using KEGG, and the top 10 enrichment term was presented.

(D) Heat map of the top 20 differential metabolites in HFD serum.

(E-G) Comparison of the concentrations of pyridoxine, methionine, and tyrosine between groups. * p<0.05; ** p<0.01; *** p<0.001.

(H-J) Concentrations of SAM, SAH and HCY in livers were examined by ELISA. Ns, there is no statistical significance between groups.

(K) Concentration of SAM in oocytes was analyzed using ELISA. ** p<0.01.

(L) Relative concentration of melatonin in serum. *** p<0.001.

(M) Genomic DNA methylation in oocytes was examined using immunofluorescence. CD, control group; HFD, obesity group; HFD+melatonin, obese mice were treated with exogenous melatonin for 14 days.

(N) Relative fluorescence intensity of 5mC was examined using Image J. * p<0.05; *** p<0.001.

Melatonin regulates DNA methylation in oocytes.

(A) Schedule of possible pathway of melatonin regulating DNA methylation in oocytes. According to previous studies, we predicted that melatonin might regulate DNA methylation in oocytes via cAMP/PKA/CREB pathway.

(B) Effects of melatonin and its inhibitor luzindole on oocyte methylation were examined using immunofluorescence.

(C, D) The relative fluorescence intensities of 5mC and 5hmC were analyzed using Image J. * p<0.05; *** p<0.001.

(E) The effects of melatonin and its inhibitor luzindole on the expression of adenylate cyclase (ADCYs) in oocytes were examined by qPCR. * p<0.05; ** p<0.01.

(F) Concentration of cAMP in oocytes was examined by ELISA. * p<0.05; ** p<0.01.

Role of cAMP in DNA methylation in oocytes.

(A) Female mice were respectively treated with ADCYs inhibitor SQ22536 or activator forskolin. Oocyte methylation was examined using immunofluorescence.

(B) The relative intensity of fluorescence in oocytes was analyzed using Image J. ** p<0.01; *** p<0.001.

(C) cAMP concentration in oocytes was examined using ELISA. * p<0.05; ** p<0.01.

(D) Female mice were treated with cAMP analogue 8-Bromo-cAMP, and oocyte methylation was examined using immunofluorescence. The relative fluorescence intensity of 5mC was analyzed using Image J (E). ** p<0.01. (F, G) Female mice were treated with PKA (protein kinase A) antagonist H 89 2HCL, and then oocyte methylation was examined using immunofluorescence. The relative fluorescence intensity of 5mC was analyzed using Image J (G). * p<0.05.

Effects of cAMP on CREB1.

(A) The mRNA expression of cAMP-response element binding (CREB) proteins in oocytes was examined by qPCR. * p<0.05.

(B and C) Phosphorylated CREB1 (pCREB1) in oocytes was examined using immunofluorescence, and the relative fluorescence intensity of pCREB1 was examined by Image J (C). * p<0.05; ** p<0.01; *** p<0.001.

(D and E) After treatment of cAMP analogue 8-Bromo-cAMP, pCREB1 in oocytes was examined using immunofluorescence. The relative fluorescence intensity was analyzed using Image J (E). *** p<0.001.

Role of melatonin/cAMP/PKA pathway in the expression of DNMTs.

(A) The expressions of DNMT1, DNMT3a and DNMT3l in oocytes were examined using qPCR after the treatment with SQ22536 and forskolin. * p<0.05.

(B) Relative expressions of DNMT1, DNMT3a and DNMT3l in oocytes were examined using qPCR after the treatment with luzindole and melatonin. * p<0.05.

(C) After 8-Bromo-cAMP treatment, the relative expression of DNMT3a in oocytes was examined using immunofluorescence and calculated by Image J (D). ** p<0.01.

(E and F) PKA antagonist H 89 2HCL treatment significantly reduced the level of DNMT3a in oocytes examined using immunofluorescence. ** p<0.01.

(G and H) DNMT1 localization in oocyte nucleus was examined using immunofluorescence after 8-Bromo-cAMP treatment. *** p<0.001.

(I and J) The localization of DNMT1 in oocyte nucleus was reduced by the treatment of PKA antagonist H 89 2HCL. ** p<0.01.

Melatonin regulates DNMTs expression via cAMP/PKA/CREB pathway in HFD oocytes.

(A) Relative expression of DNMT1, DNMT3a, and DNMT3l in HFD oocytes was examined using qPCR. * p<0.05; ** p<0.01.

(B) Concentration of cAMP in HFD oocytes was examined using ELISA. ** p<0.01.

(C) Relative expressions CREB1 and CREM in HFD oocytes were tested using qPCR. * p<0.05; ** p<0.01.

(D and E) The level of pCREB1 in oocytes was examined using immunofluorescence, and the relative fluorescence intensity was calculated by Image J (E). HFD, oocytes from obese mice; CD, oocytes from control mice; HFD + melatonin, oocytes from obese mice treated with exogenous melatonin. * p<0.05; *** p<0.001.

(F and G) PKA antagonist H89 2HCL treatment reduced the methylation level of HFD oocytes. ** p<0.01; *** p<0.001.

(H and I) The level of pCREB1 in HFD oocytes was also decreased by the treatment of PKA antagonist H89 2HCL. * p<0.05; ** p<0.01; ns, no statistical significance between groups.

(J and K) PKA antagonist H89 2HCL treatment reduced the localization of DNMT1 in HFD oocytes. ** p<0.01; *** p<0.001; ns, no statistical significance between groups.