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Fig. 3. Pyruvate causes the oxidation of cytosolic NADPH, but also a reduction
in mitochondrial NAD(P)+ and FAD++. (A)
Changes in NADH autofluorescence signal derived from measured global [large;
light-blue ROI indicated on FAD++ image (below, left); thick
light-blue trace], nuclear (small; dotted ROI in FAD++ image;
bottom light-blue trace) and mitochondrial areas (small; dark-blue ROI in
FAD++ image; dark-blue trace) in a fully grown GV oocyte. The cell
was incubated at time 0 in H-KSOM/AA alone; pyruvate and mitochondrial
reagents were added as indicated at the top of the graph (n=19
oocytes). The FAD++ image shows the oocyte analysed and the regions
of interest used to plot the graph, as this clearly shows the distribution of
mitochondria. (Below graph) NAD(P)H images of the oocyte collected at
different times. White line in the image at
t=0
indicates the line of
the scan in the graphs shown below each image. The line-scan analysis reveals
subcellular differences in the distribution of the changing NAD(P)H signal to
the manipulations. Time indicated in minutes. (B) Effect of pyruvate on
global FAD++ (large white ROI, green trace) and NAD(P)H (large
white ROI, light blue trace) and on mitochondrial NAD(P)H (small blue ROI,
dark blue trace) autofluorescence in an MII oocyte incubated in complete
H-KSOM/AA containing 5 mmol/l oxamate (n=11 oocytes). Pyruvate and
rotenone are added as indicated. The x-axes indicate time in
minutes.
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