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

Abstract

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 in vitro 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.

Fig 1. Effect of glucose on bovine cleavage and blastocyst development.

Fertilization and embryo culture was performed in culture medium without supplementary glucose (control) and in medium containing final glucose concentrations as indicated. A) Cleavage was evaluated on day 5 of post-fertilization culture. B) Blastocyst development was evaluated on day 8 of post-fertilization culture. Data are depicted as mean ± SD; columns with different letters differ significantly, p < 0.05.

Fig 2. Selected biochemicals detected in bovine blastocysts.

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.

Fig 3. Selected biochemicals detected in bovine embryo conditioned medium.

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 in vitro 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.

Fig 4. Schematic diagram of biochemicals in bovine blastocysts affected by glucose exposure.

Biochemical components of glycolysis, the tricarboxylic acid cycle, and the hexosamine pathway altered when bovine blastocysts were cultured in the presence of 3 mM (compared of 0 mM) glucose. Metabolites shown in green decreased and those in red increased in blastocysts exposed to 3 mM glucose, or in medium conditioned by these blastocysts, compared to control conditions (without glucose).