Maternal obesity and diabetes may cause DNA methylation alteration in the spermatozoa of offspring in mice

Abstract

Background: The adverse effects on offspring of diabetic and/or obese mothers can be passed to the next generation. However, the mechanisms behind this are still unclear. Epigenetics may play a key role during this process.

Methods: To confirm the hypothesis, we investigated the DNA methylation of several imprinted genes in spermatozoa of offspring from diabetic and/or obese mothers utilizing streptozotocin (STZ)- and high-fat-diet (HFD)-induced mouse models.

Results: We found that the DNA methylation of Peg3 was significantly increased in spermatozoa of offspring of obese mothers compared to that in spermatozoa of offspring of normal mothers. The DNA methylation of H19 was significantly higher in spermatozoa of offspring of diabetic mothers than that in spermatozoa of offspring of non-diabetic mothers.

Conclusions: These results indicate that pre-gestational diabetes and/or obesity can alter DNA methylation in offspring spermatozoa.

Figure 1

Average weight and blood glucose level in high-fat-diet (HFD)-induced mouse models. (A) At age of 21d (day), the female mice were randomly divided into two groups and fed with CD (control diet) and HFD (high-fat-diet), respectively. Twelve weeks later, mice fed with CD/HFD were mated with normal male mice, respectively. The pups of them were fed with diets as pre-pregnancy until weaning. Weaned male pups were fed with CD. At age of 7–8 weeks, spermatozoa of male pups were collected. After 12 weeks of treatment, the average weight of the HFD (n = 17) group was clearly heavier than that of the CD (n = 13) group, P < 0.001 (B), but the average blood glucose level of CD (n = 13) and HFD (n = 15) was similar between the two groups (C), P = 0.8209.

Figure 2

DNA methylation status in DMRs of imprinted genes in spermatozoa of OHFD analyzed by COBRA. DNA methylation patterns in DMRs of paternally imprinted genes H19 (423 bp), Gtl2 (425 bp), Rasgrf1 (284 bp) and maternally imprinted genes Peg3 (444 bp) and Snrpn (420 bp) in spermatozoa of OHFD (n = 9-10) and OCD mice (n = 10) were evaluated by COBRA. Oocytes were utilized as a control. Enzymes used are shown in the right column; Oo, oocyte. (A)H19; (B)Gtl2; (C)Rasgrf1; (D)Peg3, red arrowheads showed the digested bands; (E)Snrpn; (F) a shorter region located in DMR of H19 was amplified with Peg3 in the first-round PCR and the methylation pattern was analyzed by COBRA.

Figure 3

DNA methylation in spermatozoa of OHFD analyzed by BS. DNA methylation of paternally imprinted gene H19 and maternally imprinted gene Peg3 was further analyzed by BS. All the samples were pooled together for each group. (A) represents H19 methylation status; the number shows the methylation%; (B) shows the methylation patterns of Peg3. Black circle, methylated; white circle, unmethylated; blank loci, CpG lost.

Figure 4

Methylation patterns in DMRs of imprinted genes in sperm from offspring of diabetic (OD) and non-diabetic (ON) females. OD (n = 9)and ON (n = 9) (7–8 weeks old) mice were killed and spermatozoa were collected and treated with bisulfite and then amplified by nest-PCR. (A ~ C) The products of PCR were digested by enzymes and analyzed by 2.5% agarose gel electrophoresis. The red arrow heads point to the samples that were only partially digested by enzymes. (D, E) The methylation rates in DMRs of H19 and Gtl2 were evaluated by sequencing. Black circle, methylated; white circle, unmethylated; blank loci, CpG lost.