Shoichi Wakitani

Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers

Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.

Effects of leukemia inhibitory factor on lectin-binding patterns in the uterine stromal vessels of mice.

Lectin histochemistry was performed on mouse uteri to determine what effects leukemia inhibitory factor (LIF) has on carbohydrate epitope expressions at the time of implantation. Twenty-two biotinylated lectins were used in this study. Following injection of LIF, specific binding to the apical surface of the uterine glandular epithelium (GE) was recognized by six lectins. Particularly, binding of the lectin from Griffonia (Bandeiraea) simplicifolia was specific to the glandular epithelium close to the luminal epithelium. Succinylated wheat germ agglutinin (WGA), which has specificity for oligosaccharides recognized by WGA without sialic acid residues, showed weaker binding to the uterine luminal epithelium (LE) and the stroma than WGA, suggesting that terminal residues of glyco-conjugates on these tissues may be modified by sialic acids. Lectin binding to the glandular and luminal epithelium was not influenced by LIF. However, three lectins including a lectin from Dolichos biflorus showed specificity for stromal vessels 6h after LIF injection. Since the lectin from D. biflorus binds to neo-vascular vessels, LIF may play a role in regulating maternal angiogenesis directly and/or indirectly during implantation.

The differentially DNA-methylated region responsible for expression of runt-related transcription factor 2

Runt-related transcription factor 2 (Runx2) is essential for osteogenesis. This study aimes at identification of the genomic region differentially methylated in DNA for regulation of Runx2 expression. In the proximal promoter of mouse Runx2, DNA methylation was frequent at the region further than 3 kb relative to the transcription start site, in contrast to lower methylation status of the closer locus within 2 kb from the transcription start site. At the intermediate part, we identified a novel differentially methylated region in the Runx2 promoter region (Runx2-DMR): from −2.7 to −2.2 kb relative to the start site of Runx2 transcription in mice. In this region, the DNA methylation rate correlated negatively with Runx2 expression among mouse organs as well as among primary cultures of bone marrow from different dogs. Induction of mouse and dog mesenchymal-like cells into osteoblastic differentiation decreased the methylation rate of Runx2-DMR. Thus, in this study, we identified a novel genomic region in which DNA methylation status is related to Runx2 expression and detected demethylation of Runx2-DMR during osteoblastic differentiation in mouse and dog.

Influence of Atopic Dermatitis on Reproduction and Uterine Natural Killer Cells

ABSTRACT The causal relationship between severe allergic conditions and successful pregnancy remains unclear. We aimed to evaluate reproductive performance in an experimental mouse model of atopic disease (AD), and the appearance of uterine natural killer (uNK) cells that have crucial roles in placental formation was examined. In the NC/Nga pregnant mice with moderate skin allergic lesions and an 8.6-fold elevation of plasma IgE, significant differences were not detected in the reproductive indices of the number of normal fetuses, abortion rate and placental size. There were few uNK cells in the placenta of AD mice, and they showed a significant decrease regarding the immature subtype as compared with controls. These findings revealed that AD disturbs uNK cell differentiation and provides disadvantageous effects on placental formation, although it does not arrest the pregnancy process. It may be possible that specific immunological conditions behind AD operate favorably to recover the reproductive performance.

Multiple Acyl-CoA Dehydrogenation Deficiency (Glutaric Aciduria Type II) with a Novel Mutation of Electron Transfer Flavoprotein-Dehydrogenase in a Cat

Multiple acyl-CoA dehydrogenation deficiency (MADD; also known as glutaric aciduria type II) is a human autosomal recessive disease classified as one of the mitochondrial fatty-acid oxidation disorders. MADD is caused by a defect in the electron transfer flavoprotein (ETF) or ETF dehydrogenase (ETFDH) molecule, but as yet, inherited MADD has not been reported in animals. Here we present the first report of MADD in a cat. The affected animal presented with symptoms characteristic of MADD including hypoglycemia, hyperammonemia, vomiting, diagnostic organic aciduria, and accumulation of medium- and long-chain fatty acids in plasma. Treatment with riboflavin and L-carnitine ameliorated the symptoms. To detect the gene mutation responsible for MADD in this case, we determined the complete cDNA sequences of feline ETFα, ETFβ, and ETFDH. Finally, we identified the feline patient-specific mutation, c.692T>G (p.F231C) in ETFDH. The affected animal only carries mutant alleles of ETFDH. p.F231 in feline ETFDH is completely conserved in eukaryotes, and is located on the apical surface of ETFDH, receiving electrons from ETF. This study thus identified the mutation strongly suspected to have been the cause of MADD in this cat.