Gene expression of Dnmt1 isoforms in porcine oocytes, embryos, and somatic cells.

In the mouse, the dynamics of genomic methylation and the initial events of gametic imprinting are controlled by the activity of an oocyte isoform of the DNA methyltransferase-1 (Dnmt1o) enzyme. The objectives of this study were to identify the alternative splicing variants of Dnmt1 in porcine oocytes and determine the gene expression pattern of the different Dnmt1 isoforms during embryo development. A rapid amplification of cDNA ends (RACE ) system was used to amplify the 5' cDNA end of Dnmt1 isoforms in porcine oocytes. RNA levels of the Dnmt1 isoforms were analyzed in porcine oocytes and embryos. DNMT1 protein expression of oocytes and somatic cells were analyzed by western blot and immunostaining. Two new Dnmt1o RNA isoforms were identified--Dnmt1o1 and Dnmt1o2. The previously reported somatic Dnmt1 isoform (Dnmt1s) was expressed at low but constant levels in oocytes and embryos from the two-cell to the blastocyst stage. Abundant RNA levels of Dnmt1o1 and Dnmt1o2 were detected in oocytes and embryos from the two- to the eight- to 16-cell stage. Levels of these Dnmt1o transcripts were low at the morula and blastocyst stages. Although Dnmt1s was present in all the somatic cell types analyzed, Dnmt1o1 and Dnmt1o2 were not detected in any somatic tissues. As predicted by the RNA sequence and verified by western blot analysis, Dnmt1o1 and Dnmt1o2 RNAs translate one DNMT1o enzyme. Western blot analysis confirmed that both the oocyte and the somatic forms of DNMT1 protein are present in porcine oocytes and early embryos, whereas somatic cells produce only DNMT1s protein. DNMT1o is localized mainly in the nuclei of oocytes and early embryos, whereas DNMT1s is expressed in the ooplasm cortex of oocytes and cytoplasm of early embryos.

High-saturated-fat diet induces gestational diabetes and placental vasculopathy in C57BL/6 mice.

Gestational diabetes mellitus (GDM) is a commonly encountered disorder of mid to late pregnancy that is characterized by hyperglycemia, hyperinsulinemia, and impaired glucose tolerance. Gestational diabetes mellitus is thought to be multifactorial in origin and derives from both genetic and environmental factors. However, the mechanisms underlying GDM are incompletely understood; and current GDM animal models do not appear to closely mimic the clinical situation in humans. The present study used environmental exposure to high-saturated-fat diet (HFD) in an effort to develop a GDM mouse model that closely simulates metabolic abnormalities seen in human GDM. This model was then used to determine the contributions of HFD-induced placental oxidative stress (OS) and vascular dysregulation, which are observed in GDM patients and are believed to contribute to the pathogenesis of the disease. Pathologic manifestations of the disease were quantified by evaluating the extent of placental lipid peroxidation and by determining protective effects of dietary antioxidant quercetin supplementation to reduce HFD-associated placental OS. In this study, female C57BL/6 mice were fed HFD for 1 month before conception and throughout gestation to mimic chronic maternal fast food consumption. Maternal body weight increased from gestation day (GD) 0 to GD19 by 41% with HFD, as compared with 23% in control dams; HFD dams also developed insulin resistance (66% increase in plasma insulin and 27% increase in plasma glucose levels by GD10) as compared with control dams. Placentas from HFD GD19 dams demonstrated loss of trophoblasts and OS-mediated labyrinthine endothelial cellular damage, the latter of which was prevented with quercetin supplementation. Our findings suggest that prenatal HFD alters glucose metabolism and elevates placental OS, which are believed to collectively relate to improper formation of the conceptus and impaired birth outcome.