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Confocal ratio-imaging of intercellular pH in unfertilised mouse oocytes

Published online by Cambridge University Press:  26 September 2008

C.R. House
C.R. House*
Affiliation:
Department Preclinical Veterinary Sciences, University of Edinburgh, Edinburgh, UK.
*
C.R. House, Department of Precilinical Veterinary Sciences, University of Edinburgh, Edinburgh EH9 1QH, UK. Telephone: 031 650 6105. Fax: 031 650 6576.
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Summary

Unfertilised mouse oocytes absorbed the pH-sensitive fluoroprobe SNARF-1-AM (carboxyseminaphthorhodafluor-1-acetoxymethylester), the ester being hydrolysed by an intracellular esterase. Ratioimaging of oocytes containing the resultant SNARF-1 excited by laser light (514nm) has been obtained by scanning confocal microscopy with appropriate barrier filters to monitor emission maxima about 590 and 640 nm recorded simultaneously in separate channels of the framestore. Images produced by pixel-by-pixel division of these channel images showed uniform distribution of SNARF-1 in equatorial regions in most cells. However, in some oocytes regions (about 4 μm diameter) with smaller ratios (i.e. lower pHi) were detected. The relation between the ratio of emitted maxima and the extracellular pH (pHo) in the presence of nigericin allowed a calibration procedure to determine the intracellular pH (pHi). With this mehod pHi was estimated to be 7.13±0.05 (mean ± SEM, n = 31). Whereas the application of a weak acid (butyric) caused a fall in the ratio and hence in pHi, exposure to weak bases (NH4Cl or trimethylamine) caused a rise. Large changes in pH0. did not evoke corresponding changes in the ratio and hence in pHi. Addition of 5% CO2 to the external solution buffered at the usual value of pH 7.4, however, did cause a fall in the ratio which was reversible only when HCO3− was present in the external solution.

Keywords

Confocal microscopyIntracellular pHMouse oocytesRatio-imagingSNARF-1
Type
Commentary
Information
Zygote , Volume 2 , Issue 1 , February 1994 , pp. 37 - 45
Copyright
Copyright © Cambridge University Press 1994

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References

Baltz, J.M., Biggers, J.D. & Lechene, C. (1990). Apparent absence of Na +/H+ antiport activity in the two-cell mouse embryo. Dev. Biol. 138, 421–9.CrossRefGoogle Scholar
Bassnett, S., Reinisch, L. & Beebe, D.C. (1990). Intracellular pH measurement using single excitation-dual emission fluorescence ratios. Am. J. Physiol. 258, C171–8.CrossRefGoogle ScholarPubMed
Blandau, R., Jensen, L., & Rummery, R. (1958). Determination of the pH values of the reproductive-tract fluids of the rat during heat. Fert. Steril. 9, 207–14.CrossRefGoogle Scholar
Boron, W.F. & Boulpaep, E.L. (1983). Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO3− transport. J. Gen. Physiol. 81, 53–94.CrossRefGoogle ScholarPubMed
Boron, W.F. & de Weer, P. (1976). Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors. J. Gen. Physiol. 67, 91–112.CrossRefGoogle ScholarPubMed
Boyarsky, G., Ganz, M.B., Sterzel, R.B. & Boron, W.F. (1988). pH regulation in single glomerular mesanglial cells. I. Acid extrusion in absence and presence of HCO3− Am. J. Physiol. 255, C844–56.CrossRefGoogle Scholar
Buckler, K.J. & Vaughan-Jones, R.D. (1990). Application of a new pH-sensitive fluoroprobe (carboxy-SNARF-1) for intracellular pH measurement in small, isolated cells. Pflugers Arch. 417, 234–9.CrossRefGoogle ScholarPubMed
Chaillet, J.R. & Boron, W.F. (1985). Intracellular calibration of a pH-sensitive dye in isolated, perfused salamander proximal tubules. J. Gen. Physiol. 86, 765–94.CrossRefGoogle ScholarPubMed
Dart, C. & Vaughan-Jones, R.D. (1992). Na+–HCO3− symport in the sheep cardiac Purkinje fibre. J. Physiol.(Lond). 451, 365–85.CrossRefGoogle ScholarPubMed
Dascalu, A., Nevo, Z. & Korenstein, R. (1992). Hyperosmotic activation of the Na+–H+ exchanger in rat bone cell line: temperature dependence adn activation pathways. J. Physiol.(Lond.) 456, 503–18.CrossRefGoogle Scholar
Dubé, F., Schmidt, T., Johnson, C.H. & Epel, D. (1985). The hierarchy of requirements for an elevated intracellular pH during early development of sea urchin embryos. Cell 40, 657–66.CrossRefGoogle ScholarPubMed
Eisner, D.A., Kenning, N.A., O'Neill, S.C., Pocock, G., Richards, C.D. & Valdeolmillos, M. (1989). A novel method for absolute calibration of intracellular pH indicators. Pflugers Arch. 413, 553–8.CrossRefGoogle ScholarPubMed
Epel, D. (1989). An ode to Edward Chambers: linkages of transport, calcium and pH to sea urchin egg arousal at fertilisation. In Mechanisms of Egg Activation, ed. Nuccitelli, R., Cherr, G.N. & Clark, W.H., pp. 271–284. New York: Plenum press.CrossRefGoogle Scholar
Erp, P.E.J. Van., Jansen, M.J.J.M., de Jongh, G.J., Boezeman, J.B.M. & Schalkwijk, J. (1991). Ratiometric measurement of intracellular pH in cultured human keratinocytes using carboxy-SNARF-1 and flow cytometry. Cytometry 12, 127–32.CrossRefGoogle ScholarPubMed
Georgiou, p.p. (1985). Calcium-activated potassium channels in mammalian eggs. phD thesis, Edinburgh University.Google Scholar
Georgiou, P., Bountra, C., McNiven, A. & House, C.R. (1987). The effect of lanthanum, quercetin and dinitrophenol on calcium-evoked electrical responses in hamster eggs. Q.J. Exp. Physiol. 72, 227–41.CrossRefGoogle ScholarPubMed
Georgiou, P., House, C.R., McNiven, A.I. & Yoshida, S. (1988). On the mechanism of a pH-induced rise in membrane potassium conductance in hamster eggs. J.Physiol.(Lond.) 402, 121–38.CrossRefGoogle ScholarPubMed
Grandin, N. & Charbonneau, M. (1989). Intracellular pH and the increase in protein synthesis accompanying activation of Xenopus eggs. Biol. Cell 63, 321–30.CrossRefGoogle Scholar
Grynkiewicz, G., Poenie, M. & Tsien, R.Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3340–450.CrossRefGoogle ScholarPubMed
Hall, B.V. (1936). Variation in acidity and oxidation-reduction potentials of rodent uterine fluids. Physiol. Zool. 9, 471–97.CrossRefGoogle Scholar
Igusa, Y., Miyazaki, S. & Yamashita, N. (1983). Periodic hyperpolarising responses in hamster and mouse eggs fertilised with mouse sperm. J. Physiol. (Lond.) 340, 633–47.CrossRefGoogle Scholar
McNiven, A.I. (1988). Electrophysiology of potassium channels in the hamster egg. phD thesis, Edinburgh University.Google Scholar
Nieminen, A.-L., Gores, G.J., Dawson, T.L., Herman, B. & Lemasters, J.J. (1990). Toxic injury from mercuric chloride in rat hepatocytes. J. Biol. Chem. 265, 2399–408.CrossRefGoogle ScholarPubMed
Roos, A. & Boron, W.F. (1981). Intracellular pH. Physiol. Rev. 61, 296–434.CrossRefGoogle ScholarPubMed
Seksek, O., Henry-Toulme, N., Sureau, F. & Bolard, J. (1991). SNARF-1 as an intracellular pH indicator in laser microspectrofluorimetry: a critical assessment. Anal. Biochem. 193, 49–54.CrossRefGoogle Scholar
Shen, S.S. & Steinhardt, R.A. (1978). Direct measurement of intracellular pH during metabolic derepression of the sea urchin egg. Nature 272, 253–4.CrossRefGoogle ScholarPubMed
shen, S.S. & Steinhardt, R.A. (1980). Intracellular pH controls the development of new potassium conductance after fertilisation of the sea urchin egg. Exp. Cell Res. 125,55–61.CrossRefGoogle ScholarPubMed
Szatkowski, M.S. & Thomas, R.C. (1985). Calculation of steady-state pHi from pHi changes by weak acids and bases in snail neurones. J. Physiol. (Lond.) 371, 153P.Google Scholar
Thomas, J.A., Buchsbaum, R.N., Zimniak, A. & Racker, E. (1979). Intracellular pH measurements in Ehrlich ascites tumor cells utilising spectroscopic probes generated in situ. Biochemistry 81, 2210–18.CrossRefGoogle Scholar
Thomas, R.C. (1976). Ionic mechanism of the H+ pump in a snail neurone. Nature 262, 54–5.CrossRefGoogle Scholar
Thomas, R.C. (1984). Experimential displacement of intracellular pH and the mechanism of its subsequent recovery. J. Physiol. (Lond.) 354, 3–22P.CrossRefGoogle Scholar
Tolkovsky, A.M. & Richards, C.D. (1987). Na+/H+ exchange is the major mechanism of pH regulation in cultured sympathetic neurones: measurements in single cell bodies and neurites using a fluorescent pH indicator. Neuroscience 22, 1093–102.CrossRefGoogle ScholarPubMed
Tsien, R.Y. & Waggoner, A. (1990). Flourophores for confocal microscopy: Photophysics and photochemistry. In Handbook of Biological Confocal Microscopy, ed. Pawley, J.B. pp. 169–78. New York: Plenum press.CrossRefGoogle Scholar
Vishwakarma, P. (1962). The pH and bicarbonated-ion content of the oviduct and uterine fluids. Fertil. Steril. 13, 481–5.CrossRefGoogle Scholar
Webb, D.J. & Nuccitelli, R. (1981). Direct measurement of the intracellular pH in Xenopus eggs at fertilisation and cleavage. J. Cell Biol. 91, 562–7.CrossRefGoogle ScholarPubMed
Winkler, M.M. & Steinhardt, R.A. (1981). Activation of protein synthesis in a sea urchin cell-free system. Dev. Biol. 84, 423–39.CrossRefGoogle Scholar