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First published online 21 December 2006
doi: 10.1242/dev.02744

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Regulation of redox metabolism in the mouse oocyte and embryo

¹ Laboratoire de Biologie du Développement UMR 7009 CNRS/Paris VI, Observatoire, Station Zoologique, Villefranche sur Mer, 06230 France.
² Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK.

View larger version (29K): [in this window] [in a new window]   Fig. 1. NAD(P)H and FAD++ autofluorescence originate from both mitochondrial and cytosolic compartments in mouse oocytes. Schematic representation of the metabolic pathways producing NADH and NADPH in the cytosol and the mitochondria of a eukaryotic cell. The possible sources of FAD++ fluorescence in the mouse oocyte are highlighted in green and are the pyruvate dehydrogenase (PDH) complex and the complex I of the mitochondrial electron transport chain. See main text for details. -KG, alpha-ketoglutarate; Ac-CoA, acetyl coenzyme A; ANT, adenine nucleotide translocase; CiC, citrate carrier; LDH, lactate dehydrogenase; ME, malic enzyme; MDH, malate dehydrogenase; MCT, mono carboxylate transporter; NAD(P)+, oxidised nicotinamide adenine dinucleotide (phosphate); NADH, reduced nicotinamide adenine dinucleotide, NADP-ICDH, NADP-dependent isocitrate dehydrogenase; NK, NAD kinase; PentosePP, pentose phosphate pathway, TrH, transhydrogenase.

View larger version (23K): [in this window] [in a new window]   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.

View larger version (40K): [in this window] [in a new window]   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.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Effect on global autofluorescence of inhibition of mitochondrial respiration and pyruvate uptake. (A) Timecourse of changes in global NAD(P)H and FAD++ autofluorescence in a mature MII oocyte incubated in H-KSOM/AA alone (n=18 oocytes). Pyruvate is added after inhibition of mitochondrial respiration with rotenone and cyanide (CN-, as indicated). (B) Timecourse of changes in global NAD(P)H and FAD++ autofluorescence in a mature MII oocyte after cinnamate (CIN) perfusion (n=15 oocytes). The efficacy of CIN as an inhibitor of mitochondrial pyruvate uptake is confirmed by the lack of effect of the subsequent addition of pyruvate on FAD++ autofluorescence. (C) Effect of CIN perfusion on global autofluorescence of a mature MII oocyte after inhibition of LDH with oxamate (n=12 oocytes). Rotenone was added to a final concentration of 15 µmol/l. The x-axes indicate time in minutes.

View larger version (26K): [in this window] [in a new window]   Fig. 5. NADP-ICDH but not malate metabolism regulates NAD(P)H levels in the mouse oocyte. (A) Timecourse of changes in global NAD(P)H and FAD++ autofluorescence in a mature MII oocyte incubated in H-KSOM/AA after perfusion of dimethyl-malate (2-Me-malate, n=7 oocytes). Rotenone was then added to inhibit mitochondria. (B) Timecourse of changes in global (light-blue trace) and cytosolic (black trace) NAD(P)H and of global FAD++ autofluorescence (green trace) in a mature MII oocyte incubated in H-KSOM/A after sequential addition of oxalomalate and oxamate (n=25 oocytes). (C) Timecourse of changes in global NAD(P)H (blue trace) and in global FAD++ autofluorescence (green trace) after sequential addition of oxamate and oxalomalate on a mature MII oocyte incubated in H-KSOM/A (n=25 oocytes). The x-axes indicate time in minutes.

View larger version (13K): [in this window] [in a new window]   Fig. 6. Correlation between the level of GSH and the redox potential during development. (A) Relative amounts of NAD(P)H (grey bars), FAD++ (white bars) and GSH (black bars) in mature MII oocytes (M2 control), MII oocytes treated with 2 mmol/l BCNU for 1 hour (M2+ 2 mmol/l BCNU) and in MII oocytes treated with 2 mmol/l diamide for 15 minutes (M2+2 mmol/l diamide). The asterisks indicate that the values are significantly different from the control condition. (B) Relative amounts of NAD(P)H (grey bars), FAD++ (white bars) and GSH (black bars) in GV oocytes (fresh GV), in mature MII oocytes (fresh M2) and in two-cell embryos (2 cell). The asterisks indicate that the values are significantly different from the MII oocytes. P=0.019.

View larger version (28K): [in this window] [in a new window]   Fig. 7. Redox metabolism in the mouse zygote. Schematic representation of the metabolic pathways producing NADH, NADPH and GSH functioning in the cytosol and mitochondria of a mouse oocyte. See discussion for details. GSH, reduced glutathione, GSSG, oxidised glutathione, GPx, glutathione peroxidase, GR, glutathione reductase; H2O2, hydrogen peroxide; O2·-, superoxide.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Regulation of early development by the redox potential. (A) Effect of high pyruvate (1 mmol/l) on early development of mouse embryo. The bars indicate the percentage of embryos at each stage of development observed after 5 days of culture in control conditions (black bars), in H-KSOM/AA containing 1 mmol/l pyruvate (white bars) and in H-KSOM/AA containing 1 mmol/l pyruvate and 8 mmol/l TEMPO (stippled bars). Asterisk a (*a) indicates that this value is significantly different from the control value. Asterisk b (*b) indicates that this value is significantly different from the value of the white bar (high pyruvate treatment). (B) Effect of oxamate on early development of mouse embryos. Black bars: control condition. White bars: culture in the presence of 10 mmol/l oxamate. Asterisk indicates that this value is significantly different from the control value. (C) Effect of oxalomalate on early development of mouse embryos. Black bars: control condition. White bars: culture in the presence of 9 mmol/l oxalomalate. Asterisk indicates that this value is significantly different from the control value. P=0.05.