Effect On The Parental Gene Imprinting In Offspring Exposure To Intrauterine Hyperglycemia And Mechanism Involved

Part?Establishment of GDM mouse model and the phenotype of offspringObjective:To investigate the growth, development and glycometabolism of the first and second generation offspring exposure to intrauterine hyperglycemia, determine the risk of intrauterine hyperglycemia induced adult disease and transgenerational effect.Materials and methods:We established a mouse model of gestational diabetes mellitus (GDM). The pups of intrauterine hyperglycemia (F1-GDM) were fostered by normoglycemic female mice until they were weaned. We examined birth weight, body weight and pancreas mass of 3-week and 8-week of the first generation offspring of control (C) and GDM (F1-GDM). Further, we investigated fasting and random fed blood glucose level and glucose tolerance test (GTT) using blood glucose meter, fasting and random fed serum insulin level using ELISA kit, islet morphology by HE staining, islet ultrastructure by electronic microscopy. The female (?) and male(?) F1 adults of control (C) and GDM mice were intercrossed. F2 offspring were obtained from 4 groups including (1) C?-C?(2) C?-GDM?(3) GDM?-C?and (4) GDM?-GDM?. The phenotypes of F2 offspring were characterized.Result(s):We found birth weight of Fl-GDM offspring was significant lower than control. After fostered by normoglycemic mice, there were no differences in body weight (BW) of either male or female at 3-week age or 8-week age. Although the GTT was normal at early development (3-week-old), the ratio of pancreas/BW (%) in Fl-GDM offspring at 3-week age was significantly higher than control. Analyzed by transmission electron microscopy, the representative images of ultrastructure showed that compared with control, the structure of endoplasmic reticulum (ER) in islet cells were obviously swelling and disordered. At 8-week age, impaired glucose tolerance (IGT) was found in both male and female mice of Fl-GDM group. In F2 offspring, compared with C(?)-C?, the birth weight of GDM(?)-C?group and GDM(?)-GDM?group significantly increased, while there was no difference in C(?)-GDM?group, which showed obvious parental hereditary effect. However, there were no differences in body weight among groups of either male or female at 3-week old or 8-week old. With the same tendency of F1-GDM, the ratios of pancreas/BW (%) in all F2 offspring of intrauterine hyperglycemia at 3-week age were significantly higher than C(?)-C?. In F2 offspring, compared to C(?)-C?, either male or female, IGT appeared as early as 3-week-old through both parental lineages. Analyzed by transmission electron microscopy, the representative images of ultrastructure showed that compared with control, the structure of endoplasmic reticulum (ER) in islet cells were obviously swelling and disordered in F2 offspring at 3-week age, while the structure of ER in islet cells was repaired and almost become normal at 8-week age. However, IGT still existed at in F2 offspring 8-week age. Further analysis found that male F1 and F2 offspring from intrauterine hyperglycemia showed more obvious IGT than female.Conclusion(s):In the F1 and F2 offspring from intrauterine hyperglycemia, birth weight and glycometabolism were abnormal with parental hereditary characteristics. Male F1 and F2 offspring from intrauterine hyperglycemia showed more obvious IGT than female. The paternal factor of F1 offspring played more important role in growth and glycometabolism of F2 offspring, suggesting that epigenetic mechanism may play important role in the intergenerational and transgenerational transmission. Part?Effect of intrauterine hyperglycemia on islet function, imprinting genes expression and methylation status of mouse offspringObjective:To investigate the islet function, the expression of imprinting genes Igf2?H19 and Plagll, and the methylation status of Igf2/H19 differentially methylated region (DMR) of the first and second generation offspring exposure to intrauterine hyperglycemia, analyze the differential expression of imprinting genes associated with islet development and diabetes onset risk, evaluate the risk of abnormal methylation, explore the relationship among islet function, imprinting genes expression and methylation status.Materials and methods:Islets were isolated from 8-week-old mice following intraductal collagenase infusion. Islet function was examined by in vitro glucose-stimulated insulin section test. The expression of Igf2, H19 and Plagll was determined by real-time quantitative PCR. Islets of fetal mice at embryonic day 17 were isolated and cultured for 24 h in media containing normal or high glucose, imprinting genes expression was determined by real-time quantitative PCR. The methylation status of DMRs in Igf2/Hl9 in mouse islets was determined by bisulfite sequencing PCR (BSP).Result(s):Insulin secretary dysfunction was found in both F1 and F2 offspring from intrauterine hyperglycemia. Igf2 and H19 expression were reduced in isolated islet, while Plagll expression was normal. Differential imprinting genes expression was more obvious in male F1 and F2 offspring from intrauterine hyperglycemia than female. Igf2 and H19 expression of fetal islets were also reduced after cultured in media containing high glucose. The 12 CpG sites of Igf2 DMRO and 4 CpG sites of Igf2 DMR1 were all hypermethylated in control, F1-GDM and GDM?-GDM?groups. The 13 CpG sites of Igf2 DMR2 and 12 CpG sites of H19 DMR were differentially methylated in control group. However, the methylated status of Igf2 DMR2 and H19 DMR of F1-GDM and GDM?-GDM?groups were significant higher than control.Conclusion(s):Reduced glucose-stimulated insulin secretion may contribute to impaired glucose tolerance in both F1 and F2 offspring from intrauterine hyperglycemia. One potential candidate underlying insulin secretary dysfunction is altered expression of imprinting genes Igf2 and H19 of islets. In vitro culture confirmed the direct effect of high glucose on imprinting genes expression of islet. Intrauterine hyperglycemia causes hypermethylation at Igf2 DMR2 and H19 DMR in both F1 and F2 offspring, which was the major mechanism for reduced Igf2 and H19 expression.Part?Genomics of placenta and imprinting genes expression of gonad of mouse offspring conceived through intrauterine hyperglycemiaObjective:To investigate genomic expression profile in control, F1 and F2 offspring of intrauterine hyperglycemia, elucidate the significance of difference, analyze the differential expression of imprinting genes, and screen the critical differential genes. To examine the imprinting genes expression of gonad of F1 offspring exposure to intrauterine hyperglycemia, analyze the differential expression of imprinting genes which homologous sequence is associated with human imprinting diseases.Materials and methods:The gene chip array (Mouse Genome 430 2.0, Affymetrix company) was used for the analysis of the gene expression in the placenta of control?F1-GDM and GDM?-GDM?groups. The genes expression was analyzed by significance analysis of microarray (SAM). The differentially expressed genes were analyzed by hierarchical cluster, gene ontology (GO) classification and pathway analysis. The differentially expressed non-imprinting and imprinting genes were examined by real-time quantitative PCR. The testis and ovary of F1 offspring exposure to intrauterine hyperglycemia were collected to analyze the differential expression of imprinting genes which homologous sequence is associated with human imprinting diseases.Result(s):Gene chip microarray analysis showed that there were 124 differentially expressed genes between control and F1-GDM groups, there were 66 differentially expressed genes between control and GDM?-GDM?groups. These differentially expressed genes could be totally classified by hierarchical cluster. GO analysis according to molecular function, biological process and cell component showed that differentially expressed genes associated with growth, development and metabolism and so on. Pathway analysis found differentially expressed genes were involved in many signal pathways, including cell adhesion, nutriment metabolism and antigen presentation and so on. Twelve non-imprinting genes and 12 imprinting genes examined by real-time quantitative PCR confirmed the results of gene chip microarray analysis. Differentially expressed imprinting genes were found in the testis and ovary of F1 offspring born from intrauterine hyperglycemia.Conclusion(s):Some non-imprinting and imprinting genes, which homologous sequence is associated with human imprinting diseases, were altered in placenta of Fl and F2 offspring of intrauterine hyperglycemia. The differentially expressed genes may affect placenta function and thereby influence intrauterine development. Intrauterine hyperglycemia contributed to the abnormal imprinting genes expression of gonad of Fl offspring, maybe one of the mechanisms of transgenerational transmission.