Exposure to elevated glucose concentrations alters the metabolomic profile of bovine blastocysts

<div><p>Chronically high blood glucose concentrations are a characteristic of diabetes mellitus. Maternal diabetes affects the metabolism of early embryos and can cause a delay in development. To mimic maternal diabetes, bovine <i>in vitro</i> fertilization and embryo culture were performed in fertilization medium and culture medium containing 0.5, 2, 3, and 5 mM, glucose whereas under control conditions, the medium was glucose free (0 mM). Compared to control conditions (0 mM, 31%), blastocyst development was decreased to 23% with 0.5 and 2 mM glucose. Presence of 3 or 5 mM glucose in the medium resulted in decreased blastocyst rates (20% and 10% respectively). The metabolomic profile of resulting day 8 blastocysts was analysed by UPLC-MS/MS, and compared to that of blastocysts cultured in control conditions. Elevated glucose concentrations stimulated an increase in glycolysis and activity of the hexosamine pathway, which is involved in protein glycosylation. However, components of the tricarboxylic acid cycle, such as citrate and alpha-ketoglutarate, were reduced in glucose stimulated blastocysts, suggesting that energy production from pyruvate was inefficient. On the other hand, activity of the polyol pathway, an alternative route to energy generation, was increased. In short, cattle embryos exposed to elevated glucose concentrations during early development showed changes in their metabolomic profile consistent with the expectations of exposure to diabetic conditions.</p></div

Selected biochemicals detected in bovine blastocysts.

<p>Box and whisker plots for biochemical components detected in day 8 blastocysts produced in medium in the presence or absence (control) of 3 mM glucose. A) glucose (p = 0.0396), B) 3-phosphoglycerate (p = 0.0015), C) phosphoenolpyruvate (p = 0.0077), D) pyruvate (p = 0.6239), E) lactate (p = 0.6635) and F) oxidized glutathione (GSSG) (p = 0.0146). The upper whiskers represent the maximum, and the lower whiskers the minimum values. The plus-signs indicate the mean values, while the median values are represented by a black line within the boxes. Boxes with different letters differ significantly. Light blue—d8 blastocysts without supplementary glucose; blue—d8 blastocysts cultured in the presence of 3 mM glucose.</p

Selected biochemicals detected in bovine embryo conditioned medium.

<p>Box plots for biochemical components detected in SOF medium conditioned by bovine embryos with more than eight cells cultured for 3 days (day 5 –day 8 of <i>in vitro</i> culture). A) mannitol/sorbitol (p = 0.0131), B) fructose (p = 0.0002) and C) pyruvate (p = 0.0902). The upper whiskers represent the maximum, and the lower whiskers the minimum values. The plus-signs indicate the mean values, while the median values are represented by a black line within the boxes. Boxes with different letters differ significantly. Light blue—conditioned glucose-free medium; blue—conditioned 3 mM glucose supplemented medium.</p

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

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

Bovine ICM culture in 4 media and 2 coatings.

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

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

List of most differentially expressed genes.

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