68 Flux analysis of aerobic glycolysis in bovine blastocysts and CT1 cells

Abstract

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 1.5 mM. 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 8 h 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.