Effect of antigen masking of 5meC in three different mouse strains.

<p>PN4 and PN5 stage zygotes were collected from inbred (C57BL/6j), outbred (Quackenbush) and B6CBF1 (hybrid) females mated with the same strain male. Embryos were fixed and unmasked with acid alone (no trypsin, 1,2,5,6,9,10) or acid and trypsin (trypsin, 3,4,7,8,11,12) and stained with anti-5meC. The results are representative of three independent replicates.</p

Relative distribution of MBD1 and 5meC staining on metaphase chromosomes.

<p><b>A</b>) Triple staining of MBD1, 5meC and DNA in 1-cell and 2-cell metaphase chromosomes after antigenic unmasking by acid or acid plus trypsin. Zygotes (<b>A1</b> and <b>A3</b>) and 2-cell (<b>A2</b> and <b>A4</b>) embryos were collected at 31 and 53h after hCG, respectively, as they were entering metaphase. They were fixed and treated with acid alone (<b>A1</b> and <b>A2</b>) or acid followed by acid plus trypsin (HCl + trypsin) (<b>A3</b> and <b>A4</b>). DNA was counter-stained with DAPI (purple), anti-5meC (FITC, green) and MBD1 (red). Images shown are the merged result of these three channels. Regions of DNA in which anti-5meC and anti-MBD1 are co-localised appear white-yellow, regions of anti-MBD1 alone are pink, and those with anti-5meC alone stain blue-green. Images are z-stacks of multiple confocal sections through the chromosomes. Representative of three independent replicates. The scale bars are 10 µm. <b>B</b>) High resolution image of triple-stained metaphase chromosomes from zygotes and 2-cell embryos. Condensed chromosomes from zygotes (<b>B1</b>) were fixed by the air-dried method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030687#pone.0030687-Tarkowski1" target="_blank">[16]</a> and 2-cell condensed chromosomes (<b>B2</b>) by formaldehyde. Images were captured using a 100× oil objective and multiple confocal sections through the chromosomes were collected and the Z-stack images compiled into a two-dimensional representation. Regions of the genome are illustrated as follows: ↑, undecorated DNA; ∧, DNA stained only with anti-MBD1; *, DNA stained only with anti-5MeC; +, DNA dual stained with anti MBD1 and anti-5meC. Representative of three independent replicates. The scale bars are 10 um. <b>C</b>)The role of DNA methyltransferase in the maintenance of 5meC over the first cell cycles. Zygotes (25h post-hCG) were cultured in standard media <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030687#pone.0030687-ONeill1" target="_blank">[35]</a> (- RG108) or media supplemented with RG108 (5 µM) for 16 h. This incubation period covered the time of DNA synthesis in the 2-cell embryo <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030687#pone.0030687-Mu1" target="_blank">[37]</a>. The embryos were then fixed and stained with anti-5meC (control – <b>C1</b>, RG108 – <b>C2</b>) or the embryos were extensively washed and then cultured for another 24 h (control – <b>C3</b>, RG108 – <b>C4</b>) or 32 h (control – <b>C5</b>, RG108 – <b>C6</b>) prior to staining with anti-5meC. Non-immune controls (control – <b>C7</b>, RG108 – <b>C8</b>). Representative of five independent replicates with at least 10 embryos per treatment dose. The scale bars are 10 µm.</p

The effect of embryo manipulation on 5meC staining in zygotes.

<p><b>A</b>)Zygotes were created by routine mouse IVF <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030687#pone.0030687-ONeill2" target="_blank">[40]</a> and culture in vitro (8 h) to the PN5 stage (IVF) (<b>A1</b> and <b>A2</b>) ; collected after fertilization in the reproductive tract 17 h after hCG and then culture in vitro for 8 h to the PN5 stage (Zygote culture) (<b>A3</b> and <b>A4</b>), or they were collected directly from the reproductive tract at the PN5 stage, and fixed without further treatment (Fresh zygotes) (<b>A5</b> and <b>A6</b>). The zygotes were fixed, subjected to acid unmasking and then either buffer (no trypsin) (<b>A1</b>, <b>A3</b> and <b>A5</b>) or tryptic digestion (trypsin) (<b>A2</b>, <b>A4</b> and <b>A6</b>) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030687#pone-0030687-g002" target="_blank">Fig 2</a>. Zygotes were stained with anti-5meC (green) or PI (red) and both channels merged. Representative of three independent replicates. The scale bars are 10 µm. Zygotes were prepared as in (A) but were fixed with methanol by the air-dried method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030687#pone.0030687-Tarkowski1" target="_blank">[16]</a> and stained during chromosome condensation. In cultured zygotes a proportion showed variable segments of chromosome that stained for 5meC (<b>B1-3</b>). In Fresh zygotes no 5meC staining was observed (<b>B4</b>-<b>5</b>). The proportion of the total number of metaphase zygotes from each treatment that showed the illustrated pattern of staining is shown above the figures. No 5meC staining was observed in no-immune control (<b>B6</b>). The scale bars are 10 µm.</p

Pattern of anti-5meC staining in early preimplantation stage embryos.

<p>Zygotes were collected directly from the oviduct 16 h – 25 h after the ovulatory injection of hCG. Images show zygotes at PN stage 1-5 (<b>A1-5</b>) and condensing chromosomes (<b>B1</b>), 2-cell (<b>B2</b>), 4-cell (<b>B3</b>) and 8-cell (<b>B4</b>). Mitotic zygotes stained with non-immune IgG control is also shown (<b>B5).</b> Antigenic unmasking of 5meC (green) was by brief acid exposure. DNA was counter stained with propidium iodide (PI, Red). Images from these two channels were merged to show co-localization (merge). Images of zygotes were single-equatorial confocal sections (0.77 µm), images of nuclei from 2-cell to 8-cell stage were Z-stacks of multiple sections through each nuclei of the embryo. The images shown here are representative of at least seven independent replicate experiments with at least 10 embryos per observation group per replicated. The scale bars are 10 µm.</p

Effect of different methods of fixation on 5meC staining.

<p>Condensed chromosomes from zygotes (<b>A-C)</b> were fixed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030687#pone-0030687-g001" target="_blank">Figure 1</a> or were subjected to the air-dried and methanol fixation method (D-F). The resultant chromosomes from both methods failed to display any significant decoration by anti-5meC. The images show the anti-5meC (5meC), propidium (PI) staining and the merged imaged of these two channels (merge). The images shown here are representative of at least three independent replicate experiments with at least 10 embryos per observation group per replicated. The scale bars are 10 µm.</p

Specificity of 5meC staining.

<p>Zygotes at the PN5 stage were antigenically unmasked by combined acid and trypsin pretreatment and stained with anti-5meC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030687#pone-0030687-g001" target="_blank">Fig 1</a>, (<b>A-C</b>) or stained with anti-5meC in the presence of excess (0.6 µM ) free 5meC (Sigma) (<b>D-F</b>). To further asses specificity staining with an anti-5meC from an alternative source was performed (<b>G-I</b>). (mouse monoclonal to 5meC, used as 1∶100 dilution and incubated at 4°C for 18 h; Abcam ab73938). This showed the same pattern of staining as was the antibody used in the rest of the study. Representative of three independent replicates.</p

Localization of 5meC in the early embryo by staining for MBD1 and the effect of tryptic digestion on antigenic unmasking of 5meC.

<p>Embryos were freshly collected from the reproductive tract and fixed and antigen unmasked (HCl) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030687#pone-0030687-g001" target="_blank">Fig 1</a>. They were then stained with either anti-MBD1 (MBD1) (A and C ), anti-5meC (5meC) (B and D), non-immune IgG (E) (control). Some embryos were subjected to further antigenic unmasking by tryptic digestion (HCl + trypsin) (C, D, E). Embryos were assessed at the (1) zygotic PN2, (2) PN5, (3) zygotic metaphase, (4) interphase 2-cell, (5) 2-cell metaphase, (6) 4-cell, and (7) 8-cell stages. Each image is a single (0.77 µm) confocal section through embryos except metaphase 2-cell chromosomes and 8-cell embryos which are complied z-stacks of multiple sections. The images shown here are representative for at least seven independent replicate experiments with at least 10 embryos per observation group per replicated. The scale bars are 10 µm.</p

Staining pattern of 5meC and MBD1 antigens after epitope retrieval acid or acid plus trypsin treatment of confluent MEFs.

<p>Fixed and permeabilized MEFs were treated with HCl for 10 or 20<b>A</b> The proportion of nuclei (%) with focal 5meC staining (n, number of nuclei analysed). HCl (p<0.01) and trypsin (p<0.01) treatments revealed an increase in the focal staining of nuclear 5meC. 20 min HCl caused no further increase (p>0.05) after trypsin. <b>B</b> Typical examples of focal and diffuse staining of 5meC staining within the nuclei, yellow and red arrows indicate predominantly diffuse and focal staining, respectively. Non-immune IgG control staining is also shown. <b>C</b> The total level of 5meC staining of treatments shown in A. The results are the mean ± SEM of arbitrary units of optical density increased (**** p<0.0001). <b>D</b> The average number of 5meC foci per cell in A. <b>E</b> The average size (cross-sectional area) of 5meC foci in cells in A. <b>F</b> Representative images of MBD1 staining after 10 min HCl and trypsin 0, 1 or 2 min. Blue and red arrows indicate large and small foci of MBD1, respectively, and yellow arrow shows diffuse staining of MBD1. <b>G</b> The proportion of nuclei with focal MBD1 staining increased after 1 min trypsin (p<0.01). <b>H</b> The total level of MBD1 staining (arbitrary units of optical density) increased after 10 min HCl and 1 min trypsin (p<0.0001) Mean +/− standard mean of error. Results are from three independent replicates. ** p<0.01 and **** p<0.0001. Scale bar 10 µm. If not shown on individual graphs, the relevant statistical outcomes are stated within the results text.</p

The effect of cell growth on the pattern of co-staining for 5meC and bisBenzimide (Hoechst).

<p><b>A</b> co-staining of 5meC and DNA by Hoechst in cells during proliferation after acid alone (−trypsin) or acid followed by trypsin (+trypsin). 5meC foci are co-localized with Hoechst-dense regions in each cell. Representative non-immune IgG staining in proliferative cells is also shown. Scale bar 10 µm. <b>B</b> The number of Hoechst-dense foci per cell co-stained with 5meC or failed to co-stain with trypsin (+) or without (−). Tryptic digestion significantly (p<0.0001) affected the detection of co-localization of 5meC and DNA-dense areas. <b>C</b> The level of Hoechst staining (arbitrary units of optical density) of cells with trypsin (+) or without (−) during proliferation. Trypsin revealed a decrease in the detection of amount of Hoechst staining in each cell (p<0.0001) and cell growth affected Hoechst staining (p<0.0001). <b>D</b> Average size of Hoechst-rich foci per cell (arbitrary units of optical density). <b>E</b> The level of Hoechst foci staining per cell. <b>F</b> The level of 5meC foci staining per cell. Trypsin decreased the detectable amount of Hoechst and 5meC foci (p<0.0001). Cell growth affected the 5meC (p<0.0001) but not Hoechst (p>0.05). <b>G</b> The relationship between Hoechst and 5meC foci staining for each nucleus in (i) proliferative, (ii) confluent and (iii) quiescent cells (p<0.0001). Hoechst and 5meC are positively correlated in each growth. R-squared for each growth is given. The results are of three independent replicates.</p

The staining pattern of 5meC within nuclei in MEFs.

<p><b>A</b> Phase contrast images of the growth pattern of MEFs in (i) sparse (proliferative), (ii) confluent, and (iii) serum deprived confluent (quiescent) cells. Bar = 20 µm. <b>B</b> Typical examples of the 5meC staining patterns of MEFs in (i) proliferative, (ii) confluent, and (iii) quiescent growth phases. Arrows show cells with a predominantly focal pattern of staining and the other cells show a predominantly diffuse pattern of staining. Scale bar 5 µm. <b>C</b> The proportion (%) of nuclei with predominantly focal 5meC staining in each cell (n, number of nuclei analysed). The focal pattern of 5meC increased with cell proliferation (p<0.0001). The results are representative of three independent replicates. Epitope retrieval was by treatment with of cell with HCl 4N for 10 min at 22°C.</p