Advertisement

Histochemistry

, Volume 92, Issue 6, pp 523–529 | Cite as

Quantitative X-ray microanalysis of calcium in sea urchin eggs after quick-freezing and freeze-substitution

Validity of the method
  • I. Gillot
  • B. Ciapa
  • P. Payan
  • G. De Renzis
  • G. Nicaise
  • C. Sardet
Article

Summary

Freeze-substitution was used to study the distribution of calcium in sea urchin eggs, and the validity of the technique was assessed. We followed the fate of both total and exchangeable calcium of sea urchin eggs in two species (Paracentrotus lividus and Arbacia lixula) after the various treatments needed for freeze-substitution and embedding. We compared the calcium content either by X-ray microanalysis of Epon-embedded sections of freeze-substituted eggs (6.2±0.71 mmoles/kg of Epon-embedded tissue) or by flame spectrometry analysis of living eggs (32.3±1.30 nmoles/mg protein). After standardization of units, both values lead to similar total calcium content. We also measured the movements of 45Ca from prelabelled eggs. Exchangeable 45Ca as well as total calcium appeared unaffected by the preparative treatment for X-ray microanalysis. In conclusion, our preparative technique for X-ray microanalysis can be considered appropriate for our material and allows us to undertake a subcellular quantification of calcium in various organelles.

Keywords

Public Health Calcium Spectrometry Analysis Calcium Content Total Calcium 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Arzania R, Chambers EL (1976) The role of divalent cations in activation of the sea urchin egg. 1. Effect of fertilization on divalent cation content. J Exp Zool 198:65–78Google Scholar
  2. Blaineau S, Julliard AK, Amsellem J, Nicaise G (1987) Quantitative X-ray microanalysis of calcium with the Camebax-TEM system in frozen, freeze-substituted and resin-embedded tissue sections. Histochemistry 87:545–555Google Scholar
  3. Carafoli E (1987) Intracellular calcium homeostasis. Annu Rev Biochem 56:395–433Google Scholar
  4. Cardasis CA, Schuel H, Herman L (1978) Ultrastructural localization of calcium in unfertilized sea-urchin eggs. J Cell Sci 77:101–115Google Scholar
  5. Giapa B, Crosseley I, De Renzis G (1988) Structural modifications induced by TPA (12-o-tetradecanoyl phorbol-13-acetate) in sea urchin eggs. Dev Biol 128:142–149Google Scholar
  6. Epel D (1975) The program of and mechanism of fertilization in the echinoderm eggs. Mar Zool 15:505–522Google Scholar
  7. Geyer G, Halbhuber KJ, Benser A (1974) Ultrahistochemical demonstration of calcium ions by a freeze-substitution method. Acta Histochem 48:257–261Google Scholar
  8. Hall TA (1979) Biological X-ray microanalysis. J Microsc 117:145–163Google Scholar
  9. Jaffe LF (1983) Sources of calcium in egg activation: A review and hypothesis. Dev Biol 99:265–276Google Scholar
  10. Jaffe LF (1985) The role of calcium explosions, waves and pulses in activating eggs. In: Metz CB, Monroy A (eds) Biology of fertilization, vol 3. Academic Press, New York, pp 128–165Google Scholar
  11. Monroy-Oddo A (1946) Variations in ca and Mg contents in Arbacia eggs as a result of fertilization. Experientia 2:371–372Google Scholar
  12. Neumann D, Janossy AGS (1981) Action of giberellic acid in a dwarf maize mutant: an X-ray microanalysis study. J Microsc 120:73–84Google Scholar
  13. Nicaise G, Gillot I, Julliard AK, Keicher E, Blaineau S, Amsellem J, Meyran JC, Hernandez-Nicaise ML, Ciapa B, Gleyzal C (1989) X-ray microanalysis of calcium containing organelles in resin embedded tissue. Scanning Microsc 3:199–220Google Scholar
  14. Ornberg RL, Reese TS (1980) Freeze-substitution method for localizing divalent cations: examples from secretory systems. Fed Proc 39:2802–2808Google Scholar
  15. Ornberg RL, Reese TS (1981) Quick freezing and freeze substitution for X-ray microanalysis of calcium. In: Hutchinson T, Somlyo AP (eds) Microprobe analysis of biological systems. Academic Press, New York, pp 213–228Google Scholar
  16. Orström A, Orström M (1942) Über die Bindung von Kalzium in Ei und Larve von Paracentrotus lividus. Protoplasma 36:475–490Google Scholar
  17. Payan P, Girard JP, Christen R, Sardet C (1981) Na+ movements and their oscillations during fertilization and the cell cycle in sea urchin eggs. Exp Cell Res 134:339–344Google Scholar
  18. Payan P, Girard JP, Sardet C, Whitaker M, Zimmerberg J (1986) Uptake and release of calcium by isolated cortices of sea urchin Paracentrotus lividus. Biol Cell 58:87–90Google Scholar
  19. Poenie M, Epel D (1987) Ultrastructural localization of intracellular calcium stores by a new cytochemical method. J Histochem Cytochem 35:939–956Google Scholar
  20. Roomans GM (1980) Quantitative X-ray microanalysis of thin sections. In: Hayat MA (ed) X-ray microanalysis in biology. Mac Millan, London, pp 401–454Google Scholar
  21. Rothschild Lord, Barnes H (1953) The inorganic constituents of the sea-urchin egg. J Exp Biol 30:534–544Google Scholar
  22. Sardet C, Chang P (1985) A marker of animal-vegetal polarity in the egg of the sea urchin Paracentrotus lividus. The pigment band. Exp Cell Res 160:73–82Google Scholar
  23. Spurr AR (1972) Freeze-substitution additives for sodium and calcium retention in cells studies by X-ray analytical electron microscopy. Bot Gaz 133:263–270Google Scholar
  24. Wilson JA, Philips CE, Steinbrecht RA, Müller B (1983) The effects of salines and fixative upon the size of an identified neuron. J Neurobiol 14:377–384Google Scholar
  25. Whitaker MJ, Steinhardt RA (1985) Ionic signaling in the sea urchin eggs at fertilization. In: Metz CB, Monroy A (eds) Biology of fertilization, vol 3. Academic Press, New York, pp 167–221Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • I. Gillot
    • 1
  • B. Ciapa
    • 2
  • P. Payan
    • 2
  • G. De Renzis
    • 2
  • G. Nicaise
    • 3
  • C. Sardet
    • 1
  1. 1.Station ZoologiqueUnité de Biologie Cellulaire Marine URA 671 CNRS/Paris VIVillefranche-sur-merFrance
  2. 2.Laboratoire de Physiologie Cellulaire et Comparée, CNRS URA 651Université de NiceNice CedexFrance
  3. 3.Laboratoire de Cytologie Expérimentale, CNRS URA 651Université de NiceNice CedexFrance

Personalised recommendations