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First published online 25 April 2006
doi: 10.1242/jcs.02927

Journal of Cell Science 119, 2048-2055 (2006)
Published by The Company of Biologists 2006
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Ca2+-linked upregulation and mitochondrial production of nitric oxide in the mouse preimplantation embryo

Rosemary C. Manser1 and Franchesca D. Houghton2,*

1 Department of Biology, University of York, PO Box 373, York, YO10 5YW, UK
2 Division of Human Genetics, University of Southampton, Duthie Building, Mailpoint 808, Southampton General Hospital, Tremona Road, Southampton, SO16 6YD, UK


Figure 1
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  		Fig. 1. Oxygen consumption of day 4 blastocysts cultured in KSOM or KSOMaa with or without 10 µM NONOate and/or 1 mM L-NAME. The number of determinations is shown in parentheses. Values are mean ± s.e.m. Significant differences in oxygen consumption in the various solutions were observed compared with levels in the relevant control (***P<0.001) and between the two control cultures in KSOM and KSOMaa (aP<0.001).

 

Figure 2
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  		Fig. 2. In situ production of NO throughout preimplantation development detected using DAF-FM. (a) zygote, (b) two-cell, (c) four-cell, (d) eight-cell, (e) morula, (f) blastocyst, (g) in vivo-derived mouse blastocyst. PB, polar body; ICM, inner cell mass; TE, trophectoderm, Bars, 20 µm.

 

Figure 3
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  		Fig. 3. A two-cell mouse embryo stained with DAF-FM and exposed to 10 µM digitonin to permeabilise cell and nuclear membranes. Images show DAF-FM staining (a) before, (b) after 1 minute, and (c) after 2 minutes of digitonin treatment. The punctate pattern revealed after release of cytoplasmic and nuclear DAF-FM into surrounding media appears to colocalise with mitochondria in the embryo stained with both DAF-FM and Mitotracker Deep Red (c,d,e). Bars, 20 µm.

 

Figure 4
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  		Fig. 4. Example of a FRET experiment. Two-cell mouse embryos were dual stained with DAF-FM and Mitotracker Deep Red. Images are those taken before (a,c) and after (b,d) photobleaching of the area indicated with the yellow circle. DAF-FM (a,b) and Mitotracker Deep Red (c,d) are separated for clarity. An increase in DAF-FM fluorescence is detected after photobleaching the selected area. Bars, 5 µm.

 

Figure 5
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  		Fig. 5. Relative fluorescence of DAF-FM in the area selected for bleaching. Two-cell embryos stained with DAF-FM with or without Mitotracker Deep Red were subjected to photobleaching using the 633nm laser line and DAF-FM fluorescence quantified using laser-scanning confocal microscopy. Values are means ± s.e.m. and are corrected for acquisitional bleach and normalised for comparison. The number of determinations is shown in parentheses.

 

Figure 6
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  		Fig. 6. Two-cell mouse embryos stained with DAF-FM or Fluo-3 AM in the presence (+) or absence (-) of 1 µM antimycin A (AMA) or 100 µM FCCP. Bars, 20 µm.

 




© The Company of Biologists Ltd 2006