H3K27me3 in bovine embryos.

<p>Bovine day 8pf embryos cultured in SOF, 5iLA or NHSM were stained for H3K27me3 (red; representative picture in A; a-specific staining indicated by arrow heads). Nuclei (blue) without foci (A; double headed arrows and B; upper left and lower left) were scored as active X-chromosome, whereas nuclei containing ≥1 foci (A; single headed arrows and B; upper right and lower right) were scored as an inactive X-chromosome (Xi). The average percentage of cells (± SD) with H3K27me3 foci per embryo from 5 SOF embryos (blue), 7 5iLA embryos (red) and 5 NHSM embryos (green) was calculated (C). Different letters indicate significant differences (p<0.05).</p

XCI in bovine embryos.

<p>Relative expression levels of <i>XIST</i> (A) and <i>HPRT1</i> (B) were determined by qRT-PCR in day 8pf embryos cultured in 3iL (blue), 5iLA (red) and NHSM (green) and compared with SOF day 8pf embryonic expression levels set at 1 (dashed line). Relative expression levels of <i>XIST</i> (C) and <i>HPRT1</i> (D) in ICMs dissected from day 9pf embryos cultured in NHSM were obtained by microarray (mA; blue) and qRT-PCR (red) and compared with their SOF counterparts set at 1 (dashed line). Relative copy numbers (mean ± SD) of genomic SRY (E) was determined in parthenogenetically activated oocytes (OA), a combination of OA and IVF produced blastocysts cultured in SOF (green/blue), IVF SOF-cultured blastocysts alone (purple; set at 1), and cultured in NHSM (green) by PCR.</p

Microarray data.

<p>Pie diagram (A) showing the numbers of genes examined by microarray analysis. Number of genes up regulated in NHSM medium is indicated in red and down regulated gene number in blue. Heatmap comparing four different comparisons between NHSM and SOF ICMs (B). These results were obtained from four separate microarrays showing a similar expression in NHSM-cultured embryos relative to SOF-cultured embryo expression. Scatter plot indicating differences in gene expression between Inner Cell Mass cells from NHSM-cultured and SOF-cultured embryos (C). Probes expressed at significantly higher levels in NHSM ICMs (red), in SOF ICMs (blue) or with no significantly different expression levels (grey) are depicted in this scatter plot. Diagonal lines indicate the 1.5-fold cut off used for analysis. Several genes discussed in the manuscript are indicated.</p

Expression differences of pluripotency related genes in the ICM and TE.

<p>Microarray data (GEO accession no. GSE63054; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172920#pone.0172920.ref014" target="_blank">14</a>]) were analyzed for the differential expression of a selection of pluripotency related genes comparing dissected ICM and TE from bovine day 9pf embryos cultured in SOF (A). 2Log TE expression was set at 0 and significant differences indicated by an asterisk (*). The same genes were assessed in the microarray performed for this study (B). 2Log average ICM expression levels (dashed lines) from SOF-cultured embryos was set at 0 with grey bars indicating SD. Relative 2Log expression levels in ICMs from NHSM-cultured embryos are depicted by dots and averages indicated by a horizontal bar. Significant average expression differences are indicated by an asterisk (*). Quantitative RT-PCR was performed to determine expression of 8 selected genes in the ICM (closed symbols) and TE (open symbols) of 5iLA- (squares) and SOF-cultured (triangles) day 9pf embryos (each 6 groups of 10 ICMs or TEs). 2Log average expression in the 5iLA-cultured ICM was set at 0 per gene. ICM or TE samples without detectable gene expression are indicated as nd. 2Log average gene expression is indicated by a horizontal bar and significant differences indicated by different letters.</p

Bovine embryos in 5iLA.

<p>Bovine embryos were cultured in 5iLA until day 23. Representative embryos at day 8 until day 23 are shown as indicated. Hypoblast detaching from trophectoderm is indicated (white arrow) as well as disorganized epiblast (black arrow). Scale bar indicates 100μm.</p

Lipids in bovine embryos.

<p>Representative pictures of embryos cultured in SOF, 3iL, 5iLA, NHSM, and SOF supplemented with KOSR stained by LD540 (pink) for lipid droplets and counterstained with DAPI (blue)are shown. Scale bars represent 100mm. Inserts show a higher magnification of trophectoderm cells with perinuclear lipid droplets.</p

Bovine ICM culture in 4 media and 2 coatings.

<p>Bovine ICM culture in 4 media and 2 coatings.</p

List of most differentially expressed genes.

<p>List of most differentially expressed genes.</p

Bovine in vitro embryo culture in human ‘naïve’ media.

<p>In embryos cultured in SOF (A), 3iL (B), 5iLA (C) and NHSM (D) cells were identified by nuclear DAPI staining (blue) and the average cell number (±SD) was determined in day 8pf (E) and day 9pf embryos (F). Scale bar represents 50μm. Significant differences (p<0.05) are indicated by different letters.</p

Fibrosis.

<p>Fibrosis scoring: α-SMA (hepatic stellate cell activation) and reticulin grading (collagen type III) in five COMMD1-deficient dogs in a time-dependent copper-induced hepatitis. Data are presented as median (range).</p><p>α-SMA grading; 0: no staining of HSCs; 1: a few positive HSCs; 2: diffuse staining of HSCs; 3: mild increased staining of HSCs; 4: marked increased staining of HSC and compared with age matches normal controls.</p><p>Reticulin grading; grade 0: normal, grade 1: local mild centrilobular increase, grade 2: multifocal mild to moderate centrilobular increase, grade 3: moderate increase with centro-central bridging or nodular transformation. The uncorrected p-values for comparison of each time point with 6 months of age are reported in the table. No significant differences are present when correcting these p-values for the number of tests.</p