Embryo quality and maternal recognition are crucial for successful initiation of bovine pregnancy. Previous studies have proposed that better quality embryos use aerobic glycolysis to meet a high demand for biomass components. While hexoses are the principal carbon sources that provide energy to glycolysis, little is known about partitioning of hexoses into metabolic pathways or alteration of partitioning when different hexoses are simultaneously available. Specific metabolic utilisation of 13C-labelled substrates can be quantified by gas chromatography-mass spectrometry, an excellent noninvasive approach for studying cellular metabolism. To assess hexose flux through central metabolism, bovine blastocysts and CT1 cells (a bovine trophectoderm cell line) were cultured in SOF-based media supplemented with combinations of 50% uniformly labelled (U) and 50% naturally abundant (NA) glucose (Glc) or fructose (Fru) (U−13C Glc+NA Glc, U−13C Fru+NA Fru, U−13C Glc+NA Fru, and U−13C Fru+NA Glc), such that total hexose concentration was 1.5mM. Metabolites in spent media from 24-h cultures of single or 5 blastocysts (40-μL drops; 5% CO2, 5% O2, 90% N2) and 1-, 2-, 3-, 6-, 8-, and 24-h incubations of CT1 cells (150 μL; ~3×104 cells per well; 5% CO2, 95% air) were extracted with a MeOH-CHCl3 reagent, derivatized, and analysed by gas chromatography-mass spectrometry. Measurement of mass isotopomer distributions of metabolites, chiefly pyruvate, lactate, and amino acids, followed by correction for natural abundances and metabolic modelling, revealed several insights. For instance, five Day 7 or Day 8 blastocysts (Day 0=fertilization) supplied with U−13C Glc+NA Fru displayed 13C enrichments of 80.3%±1.4% for pyruvate and 71.6%±2.8% for lactate, whereas when supplied with U−13C Fru+NA Glc, they displayed lower 13C enrichments of 5.7%±2.4% for pyruvate and 2.8%±0.4% lactate (mean±standard deviation, n=3 to 4). Metabolic modelling revealed that when Glc and Fru are simultaneously available, the blastocysts used 2.5±0.2 moles of Fru per 100 moles of Glc used. Furthermore, 13C enrichment of pyruvate was 42.0±0.6% when U−13C Glc+NA Glc was supplied and 37.8±2.7% when U−13C Fru+NA Fru was supplied. Lactate enrichments followed a similar trend. This indicates that, individually, Glc and Fru were utilised majorly through aerobic glycolysis with some involvement of the pentose phosphate pathway. Alanine was negligibly labelled in all of the experiments, suggesting either a low TCA flux or that alanine is diluted by extra- or intracellular amino or fatty acids. Single blastocysts and CT1 cells showed a similar labelling pattern when hexoses were available. Following Glc depletion at 8h in CT1 cultures, the 13C enrichments of alanine and citrate in the media increased, suggesting a sharp alteration of metabolic state. These findings demonstrate that metabolic flux can be comprehensively analysed for single bovine blastocysts and CT1 cell metabolism models that of the blastocyst.
This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2015-67015-23237 from the USDA National Institute of Food and Agriculture.
MIAMI––a tool for non-targeted detection of metabolic flux changes for mode of action identification
