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Intrauterine hyperglycemia induces intergenerational Dlk1-Gtl2 methylation changes in mouse placenta.

ABSTRACT: An intrauterine hyperglycemic environment has long-lasting effects on the offspring. Recent studies focused on fetal tissues, whereas we studied the development and molecular alteration of the placenta. By intercrossing male and female adult control (C) and first-generation offspring mice with gestational diabetes mellitus (F1-GDM), we obtained four groups of second generation (F2) offspring: 1) C?-C?, 2) C?-GDM?, 3) GDM?-C?, 4) GDM?- GDM?. Placental weights in F1-GDM offspring were lower than in the control group. Placental weights in F2-offspring decreased through the paternal line. Placental RNA was extracted and analyzed using microarrays on day18.5 of pregnancy. This revealed 35 upregulated imprinted genes and 10 down-regulated imprinted genes. Dlk1and Gtl2 were especially down-regulated and up-regulated, respectively, due to their abnormal methylation status. These findings suggest that intrauterine hyperglycemia decreased placental weight in the first generation, and this was transmitted paternally to the second generation in mice. They also suggest intrauterine hyperglycemia leads to abnormal placental Dlk1-Gtl2 expression due to DNA methylation in first and second generation mice.

SUBMITTER: Jiang Y

PROVIDER: S-EPMC5976473 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Intrauterine hyperglycemia induces intergenerational Dlk1-Gtl2 methylation changes in mouse placenta.

Jiang Ying Y Yu Yi-Chen YC Ding Guo-Lian GL Gao Qian Q Chen Feng F Luo Qiong Q

Oncotarget 20180105 32

An intrauterine hyperglycemic environment has long-lasting effects on the offspring. Recent studies focused on fetal tissues, whereas we studied the development and molecular alteration of the placenta. By intercrossing male and female adult control (C) and first-generation offspring mice with gestational diabetes mellitus (F1-GDM), we obtained four groups of second generation (F2) offspring: 1) C♂-C♀, 2) C♂-GDM♀, 3) GDM♂-C♀, 4) GDM♂- GDM♀. Placental weights in F1-GDM offspring were lower than i ...[more]

PMID: 29854287

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Project description:BackgroundThe existing reports about intergenerational or transgenerational effects of intrauterine hyperglycemia have included both intrauterine and postnatal metabolic exposure factors, while the impact of intrauterine hyperglycemia per se has not been assessed alone. A number of studies suggest DNA methylation reprogramming of gametes plays a crucial role in the metabolic inheritance, but it is unclear when and how DNA methylation patterns are altered when exposed to intrauterine hyperglycemia. In this study, we selected nondiabetic F1- and F2-gestational diabetes mellitus (GDM) male mice as founders to examine metabolic changes in the next generation and performed methylome sequencing of day 13.5 primordial germ cells (PGCs) from F1-GDM to explore the underlying epigenetic mechanism.ResultsWe found that intrauterine hyperglycemia exposure resulted in obesity, insulin resistance, and/or glucose intolerance in F2 male mice, but no metabolic changes in F3 male mice at 8 weeks. Using reduced representation bisulfite sequencing, we found DNA methylome of day 13.5 PGCs from F1-GDM fetuses revealed differently methylated genes enriched in obesity and diabetes. Methylation validation of the insulin resistance and fat accumulation gene Fyn showed a consistent hypomethylation status in F1 PGCs, F1 fetal testes, sperm from F1/C-GDM mice, and somatic cells from F2-GDM male mice. In contrast, no methylation alteration was observed in F2-GDM male germ cells and F3-GDM somatic cells.ConclusionWe provide evidence that intrauterine hyperglycemia exposure per se contributes to intergenerational metabolic changes in the F2 but not F3 generation. And the aberrant DNA methylation reprogramming occurs as early as day 13.5 in PGCs of the F1 generation. Our findings suggest that intrauterine exposure alone is sufficient to cause the epigenetic inheritance in F2 offspring, and the epigenetic memory carried by DNA methylation pattern could be erased by the second wave of methylation reprogramming in F2 PGCs during fetal development.

Project description:BackgroundThe Dlk1 and Gtl2 genes define a region of mouse chromosome 12 that is subject to genomic imprinting, the parental allele-specific expression of a gene. Although imprinted genes play important roles in growth and development, the mechanisms by which imprinting is established and maintained are poorly understood. Differentially methylated regions (DMRs), which carry methylation on only one parental allele, are involved in imprinting control at many loci. The Dlk1-Gtl2 region contains three known DMRs, the Dlk1 DMR in the 3' region of Dlk1, the intergenic DMR 15 kb upstream of Gtl2, and the Gtl2 DMR at the Gtl2 promoter. Three mouse models are analyzed here that provide new information about the regulation of Dlk1-Gtl2 imprinting.ResultsA previously existing insertional mutation (Gtl2lacZ), and a targeted deletion in which the Gtl2 upstream region was replaced by a Neo cassette (Gtl2Delta5'Neo), display partial lethality and dwarfism upon paternal inheritance. Molecular characterization shows that both mutations cause loss of imprinting and changes in expression of the Dlk1, Gtl2 and Meg8/Rian genes. Dlk1 levels are decreased upon paternal inheritance of either mutation, suggesting Dlk1 may be causative for the lethality and dwarfism. Loss of imprinting on the paternal chromosome in both Gtl2lacZ and Gtl2Delta5'Neo mice is accompanied by the loss of paternal-specific Gtl2 DMR methylation, while maternal loss of imprinting suggests a previously unknown regulatory role for the maternal Gtl2 DMR. Unexpectedly, when the Neo gene is excised, Gtl2Delta5' animals are of normal size, imprinting is unchanged and the Gtl2 DMR is properly methylated. The exogenous DNA sequences integrated upstream of Gtl2 are therefore responsible for the growth and imprinting effects.ConclusionThese data provide further evidence for the coregulation of the imprinted Dlk1 and Gtl2 genes, and support a role for Dlk1 as an important neonatal growth factor. The ability of the Gtl2lacZ and Gtl2Delta5'Neo mutations to cause long-range changes in imprinting and gene expression suggest that regional imprinting regulatory elements may lie in proximity to the integration site.

Project description:Pregestational diabetes complicates one million pregnancies in the United States and is associated with placental dysfunction. Placental dysfunction can manifest as stillbirth, spontaneous abortions, fetal growth restriction, and preeclampsia in the mother. However, the underlying mechanisms of placental dysfunction are not well understood. We hypothesize that maternal hyperglycemia disrupts cellular processes important for normal vascular development and function. Hyperglycemia, defined as a non-fasting glucose concentration of >250 mg/dL was induced in eight-week-old female CD1 mice by injecting a one-time intraperitoneal dose of 150mg/kg streptozotocin. Control mice received an equal volume of normal saline. Hyperglycemic and control females were mated with CD-1 males. At Embryonic Day 17.5, the pregnant mice were euthanized. Sixty-eight placentas were harvested from the six euglycemic dams and twenty-six placentas were harvested from three hyperglycemic dams. RNA was extracted from homogenized placental tissue (N=12/group; 2-4 placentas per litter of each group). Total RNA was prepared and sequenced. Differentially expressed genes that were >2-fold change was considered significant. Placentas (9-20/group) were fixed in paraffin wax and sectioned at 6 μm. Cross-sectional areas of placental zones were evaluated using slides stained for hematoxylin and eosin, glycogen, collagen, proliferation and apoptosis. Quantification of staining intensity and percent positive nuclei was done using Leica Image Hub Data software. Data were compared between the control and experimental group using t-tests. Values of p < 0.05 were considered to be statistically significant. The average maternal blood glucose concentrations for control and diabetic dams were 112+/-24 and 473+/-47 respectively (p<0.0001). A higher rate of resorptions was noted in the hyperglycemia exposed placentas compared to euglycemic exposed placentas (24% vs 7%; p=0.04). A total of 24 RNA libraries (12/group) were prepared. Placentas from hyperglycemic pregnancies exhibited 1374 differentially expressed genes (DEGs). The 10 most significantly differentially expressed genes are Filip 1, Prom 2, Fam 78a, Pde4d, Pou3f1, Kcnk5, Dusp4, Cxcr4, Slc6a4 and D430019H16Rik. Their corresponding biologic functions are related to chemotaxis, ossification, cellular and vascular development. Histologically, we found that hyperglycemia exposed placentas demonstrated increased proliferation, apoptosis, and glycogen content and decreased collagen deposition. There was a higher rate of resorptions in the pregnancies of hyperglycemic dams. Pregestational diabetes resulted in significant changes in placental morphology, including increased glycogen content in the spongiotrophoblast, decreased collagen deposition, increased apoptosis and proliferation in the junction zone. Maternal diabetes causes widespread disruption in multiple cellular processes important for normal vascular development and sets the platform for placenta dysfunction.

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Intrauterine hyperglycemia induces intergenerational Dlk1-Gtl2 methylation changes in mouse placenta.

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Intrauterine hyperglycemia induces intergenerational Dlk1-Gtl2 methylation changes in mouse placenta.

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