Dynamic label-free imaging of lipid droplets and their link to fatty acid and pyruvate oxidation in mouse eggs

Mammalian eggs generate most of their ATP by mitochondrial oxidation of pyruvate from the surrounding medium, or from fatty acids which are stored as triacylglycerols within lipid droplets. The balance between pyruvate and fatty acid oxidation in generating ATP is not established. We have combined coherent anti-Stokes Raman scattering (CARS) imaging with deuterium labelling of palmitic acid to monitor turnover of fatty acids within lipid droplets of living eggs. We found that loss of labelled palmitic acid is promoted by pyruvate removal, but minimised with inhibited β-oxidation. Pyruvate removal also causes a significant dispersion of lipid droplets, while inhibiting β-oxidation causes droplet clustering. Live imaging of luciferase, or FAD autofluorecence from mitochondria, suggest that inhibiting β-oxidation in mouse eggs only leads to a transient decrease in ATP because there are compensatory uptake of pyruvate into mitochondria. Inhibiting pyruvate uptake and then β-oxidation caused similar and successive declines in ATP. Our data suggest that β-oxidation and pyruvate oxidation contribute nearly equally to resting ATP production in mouse eggs and that reorganisation of lipid droplets occurs in response to metabolic demand.