15 research outputs found
Changes in Parthenogenetic Imprinting Patterns during Reprogramming by Cell Fusion
<div><p>Differentiated somatic cells can be reprogrammed into the pluripotent state by cell-cell fusion. In the pluripotent state, reprogrammed cells may then self-renew and differentiate into all three germ layers. Fusion-induced reprogramming also epigenetically modifies the somatic cell genome through DNA demethylation, X chromosome reactivation, and histone modification. In this study, we investigated whether fusion with embryonic stem cells (ESCs) also reprograms genomic imprinting patterns in somatic cells. In particular, we examined imprinting changes in parthenogenetic neural stem cells fused with biparental ESCs, as well as in biparental neural stem cells fused with parthenogenetic ESCs. The resulting hybrid cells expressed the pluripotency markers <i>Oct4</i> and <i>Nanog</i>. In addition, methylation of several imprinted genes except <i>Peg3</i> was comparable between hybrid cells and ESCs. This finding indicates that reprogramming by cell fusion does not necessarily reverse the status of all imprinted genes to the state of pluripotent fusion partner.</p></div
Bisulfite genome sequencing analysis of imprinted genes.
<p>DNA methylation patterns of paternally (<i>H19</i> and <i>Igf2</i>), and maternally imprinted genes (<i>Peg1</i> and <i>Peg3</i>) in ESCs, pESCs, NSCs, pNSCs, ES-pNSC, and pES-NSC hybrid cells. Black and white circles represent methylated and unmethylated CpGs, respectively.</p
Characterization of hybrid cells.
<p><b>(A)</b> Both ES-pNSC and pES-NSC hybrid cells are positive for alkaline phosphatase staining (100 ×). <b>(B)</b> RT-PCR analysis of <i>Oct4</i>, <i>Nanog</i>, <i>Sox2</i>, and <i>Nestin</i> expression in fusion partner and reprogrammed hybrid cells. Pluripotency markers, <i>Oct4</i> and <i>Nanog</i>, which were not expressed in NSCs and pNSCs were expressed in GFP<sup>+</sup> fusion hybrid cells. On the other hand, <i>Nestin</i>, which was expressed in NSCs and pNSCs was silenced after forming GFP<sup>+</sup> fusion hybrid cells. <b>(C)</b> Immunocytochemistry analysis of Oct4 and Nanog in ES-pNSC and pES-NSC hybrid cells (100 ×). <b>(D)</b> <i>In vitro</i> differentiation of ES-pNSC and pES-NSC hybrid cells into ectoderm (Tuj1), mesoderm (SMA), and endoderm (Sox17) lineages (200 ×). <b>(E)</b> In vivo differentiation potential of ES-pNSC and pES-NSC hybrid cells through teratoma assay. These hybrid cells were contributed to secretory epithelium (ectoderm), cartilage (mesoderm) and gut epithelium (endoderm), which were stained with PAS, Asian blue, and hematoxylin eosin, respectively. Each tissue was indicated by arrow head.</p
Quantitative RT-PCR analysis of imprinted gene expression.
<p>The expression profiles of paternal and maternal imprinted genes were analyzed by real-time RT-PCR. All data are normalized to <i>ACTB</i> expression and calibrated on the ESCs, whose gene expression was considered 1 for all genes. Error bars represent mean values ± SEM of three independent experiments. Student’s t-test: ***, p<0.001; **, p<0.01; *, p<0.05.</p
Generation of fusion hybrid cells between parthenogenetic and biparental cells.
<p><b>(A)</b> GFP fluorescence images of fusion between biparental ESCs and parthenogenetic neural stem cells (ES-pNSC), and between pESCs and biparental neural stem cells (pES-NSC) at day 3 after fusion (200 ×). <b>(B)</b> GFP fluorescence images of ES-pNSC and pES-NSC hybrids after FACS sorting (100 ×). <b>(C)</b> Representative tetraploid karyotype of the hybrid cells.</p
Generation of parthenogenetic ESCs (pESCs) from parthenogenetically activated embryos.
<p><b>(A)</b> Preimplantation development of parthenogenetically activated embryos from one-cells to blastocyst stage embryos (200 ×). <b>(B)</b> Efficiency of development of parthenogenetic embryos. About 83% of oocytes were successfully activated, of which about 62% progressed to blastocyst stage. <b>(C)</b> Embryonic stem cells derived from parthenogenetic blastocysts (pESCs) were positive for the alkaline phosphatase staining (100 ×). <b>(D)</b> Immunocytochemistry of pESCs using Oct4 and Nanog antibodies (200 ×). pESCs were stained positive for key pluripotency markers, Oct4 and Nanog.</p
xCT deficiency aggravates acetaminophen-induced hepatotoxicity under inhibition of the transsulfuration pathway
<p>Cystine, an oxidized form of cysteine (Cys), is imported into cells via the protein xCT, which is also associated with the export of glutamate as the counter amino acid. In the current study, we attempted to rationalize roles of xCT in the livers of male mice. While xCT was not expressed in the livers of ordinary mice, it was induced under conditions of glutathione depletion, caused by the administration of acetaminophen (AAP). To differentiate the role between xCT and the transsulfuration pathway on the supply of Cys, we employed an inhibitor of the enzyme cystathionine γ-lyase, propargylglycine (PPG). This inhibitor caused a marked aggravation in AAP-induced hepatic damage and the mortality of the xCT<sup>−/−</sup> mice was increased to a greater extent than that for the xCT<sup>+/+</sup> mice. While a PPG pretreatment had no effect on liver condition or Cys levels, the administration of AAP to the PPG-pretreated mice reduced the levels of Cys as well as glutathione to very low levels in both the xCT<sup>+/+</sup> and xCT<sup>−/−</sup> mice. These findings indicate that the transsulfuration pathway plays a major role in replenishing Cys when glutathione levels are low. Moreover, an ascorbic acid insufficiency, induced by Akr1a ablation, further aggravated the AAP-induced liver damage in the case of the xCT deficiency, indicating that glutathione and ascorbic acid function cooperatively in protecting the liver. In conclusion, while the transsulfuration pathway plays a primary role in supplying Cys to the redox system in the liver, xCT is induced in cases of emergencies, by compensating for Cys supply systems.</p
Male- and female-derived somatic and germ cell-specific toxicity of silver nanoparticles in mouse
<p>Silver nanoparticles (AgNPs) are widely used as an antibiotic agent in textiles, wound dressings, medical devices, and appliances such as refrigerators and washing machines. The increasing use of AgNPs has raised concerns about their potential risks to human health. Therefore, this study was aimed to determine the impact of AgNPs in germ cell specific complications in mice. The administration of AgNPs results in toxicity in mice; however, a more detailed understanding of the effects of AgNPs on germ cells remains poorly understood. Here, we demonstrate the effects of AgNPs (20 nm in diameter) in a mouse Sertoli and granulosa cells <i>in vitro</i>, and in male and female mice <i>in vivo</i>. Soluble silver ion (Ag<sup>+</sup>)-treated cells were used as a positive control. We found that excessive AgNP-treated cells exhibited cytotoxicity, the formation of autophagosomes and autolysosomes in Sertoli cells. Furthermore, an increase in mitochondrial-mediated apoptosis by cytochrome <i>c</i> release from mitochondria due to translocation of Bax to mitochondria was observed. In <i>in vivo</i> studies, the expression of pro-inflammatory cytokines, including tumor necrosis factor α, interferon-γ, −6, −1β, and monocyte chemoattractant protein-1 were significantly increased (<i>p</i> < 0.05). Histopathological analysis of AgNP-treated mice shows that a significant loss of male and female germ cells. Taken together, these data suggest that AgNPs with an average size of 20 nm have negative impact on the reproduction.</p
Effects of GW610742 on TGF-β2 expression.
<p><b>(A)</b> TGF-β2 (12.5 kDa) expression was assessed by immunoblot analyses in all groups on day 3, 7, and day 14 following surgery. Densitometric analysis shows the relative levels of TGF-β2 expression in each group. α-tubulin (42 kDa), used as a loading control, was not different between the groups. Representative blots are derived from three separate experiments. Values are represented as the mean ± SEM. *<i>P</i> < 0.05 and **<i>P</i> < 0.01. Sham (n = 3), MI (n = 10), MI + GW (n = 10). (<b>B)</b> Representative immunohistochemical images for TGF-β2 from the MI alone (MI) group and the MI treated with GW610742 (MI + GW) group on day 7 post-surgery. Scale bars = 100 μm. TGF-β2, transforming growth factor-beta 2.</p
Echocardiographic measurements of left ventricular remodeling.
<p>The consecutive measurements of the echocardiographic parameters were acquired at baseline (0, immediate post-operation), 7 days, 14 days, and 28 days after surgical procedure. *<i>P</i> <0.05 between the MI group vs. sham group. †<i>P</i> <0.05 between the MI + GW group vs. sham group. MI, myocardial infarction only; MI + GW, MI treated with GW610742, IVS, interventricular septal thickness; LVEDD, left ventricular dimension at end-diastole; LVESD, left ventricular dimension at end-systole; FS, fractional shortening; EF, ejection fraction; SV, stroke volume. Sham (n = 3/each day), MI (n = 28 of baseline, n = 18 of 7days, n = 17 of 14 days, n = 6 of 28days), MI + GW (n = 31 of baseline, n = 19 of 7days, n = 15 of 14 days, n = 6 of 28days).</p