Fig. 2. Glucose, pyruvate and lactate have distinct effects on NAD(P)H and FAD⁺⁺ autofluorescence, indicating distinct metabolic pathways. (A) Timecourse of changes in NAD(P)H (blue) and FAD⁺⁺ (green) autofluorescence in a MII oocyte incubated at time 0 in H-KSOM/AA that was devoid of glucose, lactate and pyruvate (n=35 oocytes). These substrates were added at the times indicated at the bottom of the graph. (B) Inhibition of the pentose phosphate pathway: timecourse of changes in autofluorescence in a fully grown GV oocyte incubated in complete H-KSOM/AA (n=12 oocytes). The PPP inhibitor DHEA was added to the chamber at the time indicated at the top of the graph. Continuous traces indicate global signals (large, dark-blue ROI in the FAD⁺⁺ image). Dashed traces indicate nuclear signals (small, light-blue ROI in the FAD⁺⁺ image, left). (C) Inhibition of LDH: timecourse of changes in autofluorescence in a mature MII oocyte incubated in H-KSOM/AA without lactate or pyruvate (n=18 oocytes). Lactate (2 mmol/l) was added, as indicated, followed by the addition of the LDH inhibitor, oxamate (10 mmol/l). The concentration of lactate in the medium was then increased to 8 mmol/l, as indicated. The x-axes indicate time in minutes.