Genome-Wide DNA Methylation Profiling in Cultured Eutopic and Ectopic Endometrial Stromal Cells

<div><p>The objective of this study was to characterize the genome-wide DNA methylation profiles of isolated endometrial stromal cells obtained from eutopic endometria with (euESCa) and without endometriosis (euESCb) and ovarian endometrial cysts (choESC). Three samples were analyzed in each group. The infinium methylation array identified more hypermethylated and hypomethylated CpGs in choESC than in euESCa, and only a few genes were methylated differently in euESCa and euESCb. A functional analysis revealed that signal transduction, developmental processes, immunity, etc. were different in choESC and euESCa. A clustering analysis and a principal component analysis performed based on the methylation levels segregated choESC from euESC, while euESCa and euESCb were identical. A transcriptome analysis was then conducted and the results were compared with those of the DNA methylation analysis. Interestingly, the hierarchical clustering and principal component analyses showed that choESC were segregated from euESCa and euESCb in the DNA methylation analysis, while no segregation was recognized in the transcriptome analysis. The mRNA expression levels of the epigenetic modification enzymes, including DNA methyltransferases, obtained from the specimens were not significantly different between the groups. Some of the differentially methylated and/or expressed genes (NR5A1, STAR, STRA6 and HSD17B2), which are related with steroidogenesis, were validated by independent methods in a larger number of samples. Our findings indicate that different DNA methylation profiles exist in ectopic ESC, highlighting the benefits of genome wide DNA methylation analyses over transcriptome analyses in clarifying the development and characterization of endometriosis.</p></div

The DNA methylation status as determined by the methylation-sensitive high resolution analyses (MS-HRMA) of NR5A1 (A), STAR (B), STRA6 (C) and HSD17B2 (D) in euESCa and choESC.

<p>Sample No.-wide DNA methylation and transpciptome as euESCa, and sample No. 8, 9, 10 were also analyzed as choESC. Sample No. 1 and 10 were analyzed for bisulfite sequencing as euESCa and choESC, respectively. The confidence value calculated by MS-HRMA indirectly indicates the DNA methylation level. The DNA methylation status of euESCa-1 was shown as 100% identical with regard to the DNA methylation. The confidence values of each sample were calculated in comparison with euESCa-1. In NR5A1 and STAR, the confidence values were lower in choESC than those in euESCa, indicating that the choESC are hypomethylated compared with the euESCa. In STRA6 and HSD17B2, the DNA methylation status of euESCa-1 was shown as −100% (arbitrary defined reverse axis value) identical regarding DNA methylation, indicating a DNA hypomethylation status. In STRA6, the confidence values were higher in choESC than those in euESCa, indicating that the choESC are hypermethylated compared with the euESCa. In HSD17B2, the DNA methylation status varied among individuals with euESCa and choESC. The samples of euESCa and choESC were isolated from seven and six patients, respectively.</p

Hypermethylation in choESC compred to euESCa.

<p>Lists of statistically significant GO terms (Biological process and molecular function) and KEGG pathway terms in hypomethylated genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083612#pone-0083612-t001" target="_blank">Table 1</a>) and in hypermethylated genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083612#pone-0083612-t002" target="_blank">Table 2</a>) in choESC compared to euESCa.</p

The mRNA levels of NR5A1 (A), STAR (B), STRA6 (C) and HSD17B2 (D) in ESCa, choESC, eutopic endometrium and chocolate cysts.

<p>ESCa (n = 7), choESC (n = 5), eutopic endometria (n = 17) and chocolate cysts (n = 6) were subjected to total RNA isolation followed by real-time RT-PCR. The relative mRNA expression normalized to that of TBP (an internal control) was calculated. The values are the means ±SD. *, <i>p</i><0.01. ND: not detected.</p

Volksrecht : Sozialdemokratisches Tagblatt für die politischen Bezirke Aussig und Leitmeritz.

The "Volksrecht" is a socialist newspaper for the political districts of Aussig (Ústí nad Labem) and Leitmeritz (Litoměřice).Electronic reproduction.Description based on: Jahrgang 25, Nr. 276 (1. Dez. 1920); caption title.Latest issue consulted: Jahrgang 25, Nr. 300 (31. Dez. 1920

Hypomethylation in choESC compred to euESCa.

<p>Hypomethylation in choESC compred to euESCa.</p

The results of the sodium bisulfite sequencing analyses of NR5A1 (A), STAR (B), STRA6 (C) and HSD17B2 (D) in the euESCa and choESC samples.

<p>The DNA methylation profile in the genomic regions of the NR5A1, STAR, STRA6 and HSD17B2 genes was analyzed by a sodium bisulfite sequencing method in a pair of euESCa and choESC from one individual, which had already been analyzed by the Infinium method. In NR5A1 and STRA6, the DNA methylation status of the proximal promoter and first exon was analyzed. The primer pairs BP-A and BP-B amplify region A and B, respectively, in STRA6. In STAR, the distal promoter region was analyzed. In HSD17B2, the first intron and second exon region were analyzed. The arrows indicate the positions of the bisulfite primers. Closed triangles represent the CpG sites analyzed by the Infinium method, and are accompanied by the identification names. •, methylated CpG sites; ◯, unmethylated CpG sites; BP, bisulfite primer.</p

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

<div><p>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.</p></div

Expression and Epigenetic Analysis of TESCs.

<p>(<b>A</b>) Relative expression of pluripotency markers by quantitative real-time RT-PCR analysis at passage 10. All data represent the mean and SEM (n = 3). *<i>P</i> < 0.05. (<b>B</b>) Analysis of DNA methylation profiles. DNA methylation levels in <i>Oct3/4</i> and <i>Nanog</i> genes were determined by Bio-COBRA in TESCs and ESCs. All data represent the mean and SEM (n = 3). *<i>P</i> < 0.05. (<b>C</b>) Immunostaining of pluripotency markers. DAPI was used to stain DNA. A representative figure is shown. Scale bar, 50 μm.</p

Production and Characterization of Tetraploid Embryonic Stem Cells (TESCs).

<p>(<b>A</b>) Diagram of production of TESCs with elecrofused diploid embryos. Two or three embryos were seeded onto a mouse embryonic fibroblast (MEF) feeder layer. (<b>B</b>) Flow cytometry analysis of DNA content after 10 passages. A representative figure is shown. (<b>C</b>) Karyotyping analysis of metaphase chromosome spreads. Tetraploid ESCs normally had 80 chromosomes, while control diploid ESCs had 40 chromosomes at passage 10. A representative figure is shown. (<b>D</b>) Proliferating TESCs formed typical round-shaped mouse ESC colonies with clear boundaries similar to control ESCs. TESC colonies also stained positive for the control ESC-positive marker alkaline phosphatase (ALP). A representative figure is shown. (<b>E</b>) Growth rates. The relative cell number was based on the cell number at day 0 (2 × 10⁴ cells). All data represent the mean and SEM (n = 3). *<i>P</i> < 0.001. Scale bar, 100 μm.</p