Intrauterine hyperglycemia exposure results in intergenerational inheritance via DNA methylation reprogramming on F1 PGCs.

BACKGROUND: The 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. RESULTS: We 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. CONCLUSION: We 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.

Androgen-induced alterations in endometrial proteins crucial in recurrent miscarriages.

High androgen level impairs endometrial receptivity in women experiences the recurrent miscarriage. The mechanism of androgen actions on endometrium is still uncertain. We hypothesized that androgen has a direct effect on the endometrium in women with recurrent miscarriage. In the present study, we assess the impact of androgen (A2) at high concentration (10-7 M) on Ishikawa cells compared with the physiological concentration of androgen (10-9 M). To go into deeper analysis, we use global stable isotopes labeled profiling tactic using iTRAQ reagents, followed by 2D LC-MS/MS. We determine 175 non-redundant proteins, and 18 of these were quantified. The analysis of differentially expressed proteins (DEPs) identified 8 up-regulated proteins and 10 down-regulated in the high androgen group. These DEPs were examined by ingenuity pathway (IPA) analysis and established that these proteins might play vital roles in recurrent miscarriage and endometrium receptivity. In addition, proteins cyclin-dependent kinase inhibitor 2a (CDKN2a), endothelial protein C receptor (EPCR), armadillo repeat for velocardiofacial (ARVCF) were independently confirmed using western blot. Knockdown of CDKN2a significantly decreased the expression level of CDKN2a protein in ishikawa cells, and decreased migration (p < 0.01), invasion (p < 0.05), proliferation (p < 0.05), and the rate of Jar spheroid attachment (p < 0.05) to Ishikawa cell monolayer. The present results suggest that androgen at high concentration could alter the expression levels of proteins related to endometrium development and embryo implantation, which might be a cause of the impaired endometrial receptivity and miscarriage.

Hypertriglyceridemia in female rats during pregnancy induces obesity in male offspring via altering hypothalamic leptin signaling.

Maternal obesity influence the child's long-term development and health. Though, the mechanism concerned in this process is still uncertain. In the present study, we explored whether overfeeding of a high-fat diet during pregnancy in female rats altered metabolic phenotypes in an F1 generation and authenticated the contribution of hypothalamic leptin signaling. Leptin responsiveness and the number of immunopositive neurons for phosphorylated signal transducer and activator transcription 3 (pSTAT3) were analyzed. Neuropeptide Y in the arcuate nucleus of the hypothalamus and in nucleus tractus solitaries was examined. Triglycerides and leptin levels were increased in the high-fat diet mother. The number of neuropeptide Y positive cell bodies and neurons was significantly increased in the high-fat diet-F1 offspring (HDF-F1) as compared to Chow-F1. Leptin administration significantly decreased the food intake and increased the pSTAT3 expression levels in neurons in the arcuate nucleus of Chow-F1. However, leptin did not show any effect on food intake and had a reduced effect on pSTAT3 expression levels in neurons in the arcuate nucleus of HDF-F1. From the present domino effect, we conclude that mothers exposed to high-fat diet during pregnancy may pass the obese phenotype to the succeeding generation via altering hypothalamic leptin signaling.

Regulatory Effect of HBV Integrated Fragment on PCNA Promoter.

H7C is a HBV integrated fragment isolated from a human hepatocellular carcinoma, containing the promoter of preS2 and the C-terminal truncated preS/S open reading frame. We have studied the effect of the 3'-truncated preS/S on human proliferating cell nuclear antigen (PCNA) promoter by co-transfection of the expression plasmids. Result showed that the product, pKSH7C-Hpa I, which contained the intact H7C and the flanking cellular sequences, stimulated the expression from PCNA promoter dose-dependently, and its effect was 1-2 folds higher than that on SV40 promoter. However, two subclones, pKSH7C-XHX and pKSH7C-XbH, which would not express preS/S, showed no stimulatory effect. Furthermore, when if the -45 bp ATF-like site was mutated, the activation effect became diminished. This showed that the ATF-like site might be important in mediating the transactivating process. This is the first report of the effect of a HBV integrated fragment on the promoter of a replicating protein factor.