| Objective:To investigate the growth, development of the first and second generation placenta exposure to intrauterine hyperglycemia, determine the risk of intrauterine hyperglycemia induced adult disease and intergenerational effect. Besides, we also detect the expression of imprinted genes Dlkl and Gtl2, and the methylation status of Dlkl/Gtl2differentially methylated region (DMR) of the first and second generation offspring exposure to intrauterine hyperglycemia, analyze the differential expression of imprinted genes associated with placental development, evaluate the risk of abnormal methylation, explore the relationship among placenta development, imprinted gene expression and methylation status.Materials and methods:We established a mouse model of gestational diabetes mellitus (GDM). The pups of intrauterine hyperglycemia (Fl-GDM) were fosted by normoglycemic female mice until they were weaned. We examined placenta weight, fetal mouse weight in day12.5, day15.5, day18.5of the first generation offspring of Control (C) and GDM (Fl-GDM). Further, we investigated placental morphology by HE staining. The female (♀) and male (?) F1adults of control (Control) and GDM mice were intercrossed. F2offspring were obtained from4groups including (1) C-(2) GDM(?)-C♀(3) C(?)-GDM♀(4) GDM(?)-GDM♀. The phenotypes of F2offspring were characterized in order to get an idea of placental development, we detected DLkl and Gtl2expression by real-time quantitative PCR. Trophoblast cells at embryonic day12-15were isolated and cultured for72h in media containing normal or high glucose, imprinted genes expression were determined by real-time quantitative PCR. The methylation statuses of DMRs on Dlkl/Gtl2region in mouse placenta were determined by bisulfite sequencing PCR (BSP).Results:We found placental weight and fetal mouse weight of F1-GDM offspring was significant lower than control in day12.5, day15.5, and day18.5. However, the ratio of fetal mouse weight/placental weight (%) was similar in both groups. HE staining showed that F1-GDM offspring placenta appeared severe dysplasia such as anomalously expanded spongiotrophoblastic layers and a malformed labyrinthine layer with an anomalous circulatory system. In F2offspring, compared with C(?)-C♀, placenta weight and fetal mouse weight, ratio of fetal mouse weight/placenta weight (%) of GDM(?)-C♀, C(?)-GDM♀, GDM(?)-GDM♀, there were no difference. Dlkl expression was reduced in F1-GDM placenta in three different stages, and we found Dlkl expression showed reverse U type. However, Gtl2expression was higher in F1-GDM placenta. Dlk1expression reduced more obvious in GDM(?)-C♀group, or Gtl2expression increased more significantly in C(?)-GDM♀group. Besides, by isolating trophoblast cells from placenta, after treating with high glucose concentration, we obtained similar results as these in vivo. In methylation region of Dlkl-Gtl2, compared with C(?)*-C♀, Dlkl DMR showed hypermethylation, however IG DMR and Gtl2DMR showed hypomethylation, which may help to explain the reason of abnormal expression of imprinted gene Dlk1and Gtl2.Conclusion:In the F1offspring from intrauterine hyperglycemia, placental weight and fetal mouse weight were abnormal. Histological Analysis showed F1-GDM placenta had the structural abnormality on day12.5, which indicated early-stage structural alteration may be fatal to placental development. Another potential candidate underlying placental abnormal development is altered expression of imprinted gene Dlkl and Gtl2of placenta.In vitro culture confirmed the direct effect of high glucose on imprinting genes expression of placenta. Intrauterine hyperglycemia causes hypermethylation at Dlkl DMR in both F1and F2offspring, and hypomethylation of IG DMR and Gtl2DMR in both two groups, which was the major mechanism for alteration expression of Dlkl and Gtl2. |
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