Somatic cell nuclear transfer (SCNT) enables cloning of differentiated cells by reprogramming their nuclei to a totipotent state. However, successful full-term development of SCNT embryos is a low-efficiency process and arrested embryos frequently exhibit epigenetic abnormalities. Here, we generated genome-wide DNA methylation maps from mouse pre-implantation SCNT embryos. We identified widespread regions that were aberrantly re-methylated, leading to mis-expression of genes and retrotransposons important for zygotic genome activation. Inhibition of DNA methyltransferases (Dnmts) specifically rescued these re-methylation defects and improved the developmental capacity of cloned embryos. Moreover, combining inhibition of Dnmts with overexpression of histone demethylases led to stronger reductions in inappropriate DNA methylation and synergistic enhancement of full-term SCNT embryo development. These findings show that excessive DNA re-methylation is a potent barrier that limits full-term development of SCNT embryos and that removing multiple epigenetic barriers is a promising approach to achieve higher cloning efficiency.

Serotonin (5-HT) is an important mediator of allergic airway narrowing in several animal species. The present study was designed to characterize the receptor subtypes that mediate 5-HT-induced airway responses in the rat. To do this, we measured Ca2+ transients and adenosine 3',5'-cyclic monophosphate (cAMP) production evoked by 5-HT in cultured rat tracheal smooth muscle cells as well as 5-HT-induced contractions of isolated tracheal rings. 5-HT (10(-6) to 10(-4) M) triggered a rapid increase in intracellular Ca2+ concentration ([Ca2+]i) followed by a second phase of sustained, elevated, and sometimes oscillating levels of [Ca2+]i. Sustained but not peak [Ca2+]i levels were dependent on Ca2+ influx but were not attenuated by nifedipine (10(-5) M). Oscillations were observed in cells in Ca2+ -free medium, suggesting Ca2+ -induced Ca2+ release independent of Ca2+ influx. The effects of 5-HT were inhibited by thapsigargin (10(-6) M) and ketanserin (10(-7) M). In cells incubated with LY-278,584 (5-HT3 antagonist) and (-)pindolol (5-HT1 antagonist), 5-HT-evoked responses were not significantly different from the control values. 5-methyltryptamine (5-MT), a ligand with higher affinity for 5-HT2C receptors than "classical" 5-HT2 receptors, elicited higher responses than dipropyl-5-carboxamidotryptamine (DP-5-CT), which possesses lower affinity for 5-HT2C receptors than 5-MT, but an affinity for the classical 5-HT2 receptor similar to 5-MT, but an affinity for the classical 5-HT2 receptor similar to 5-MT.(ABSTRACT TRUNCATED AT 250 WORDS)

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Increasing evidence suggests that in vitro fertilization (IVF) may be associated with an increased risk of developing obesity and metabolic diseases later in life in the offspring. Notably, the addition of melatonin to culture medium may improve embryo development and prevent cardiovascular dysfunction in IVF adult mice. This study aimed to determine if melatonin supplementation in the culture medium can reverse impaired glucose metabolism in IVF mice offspring and the underlying mechanisms. Blastocysts used for transfer were generated by natural mating (control group) or IVF with or without melatonin (10 −6 M) supplementation (mIVF and IVF group, respectively) in clinical-grade culture media.Here, we first report that IVF decreased hepatic expression of Fbxl7, which was associated with impaired glucose metabolism in mice offspring. Melatonin addition reversed the phenotype by up-regulating the expression of hepatic Fbxl7. In vitro experiments showed that Fbxl7 enhanced the insulin signaling pathway by degrading RhoA through ubiquitination and was up-regulated by transcription factor Foxa2. Specific knockout of Fbxl7 in the liver of adult mice, through tail intravenous injection of recombinant adeno-associated virus, impaired glucose tolerance, while overexpression of hepatic Fbxl7 significantly improved glucose tolerance in adult IVF mice. Thus, the data suggest that Fbxl7 plays an important role in maintaining glucose metabolism of mice, and melatonin supplementation in the culture medium may rescue the long-term risk of metabolic diseases in IVF offspring.

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