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. 1998 Oct 15;335(Pt 2):335–342. doi: 10.1042/bj3350335

The effects of a Ca2+ chelator and heavy-metal-ion chelators upon Ca2+ oscillations and activation at fertilization in mouse eggs suggest a role for repetitive Ca2+ increases.

Y Lawrence 1, J P Ozil 1, K Swann 1
PMCID: PMC1219787  PMID: 9761732

Abstract

During fertilization in mouse eggs, the sperm triggers a series of intracellular Ca2+ oscillations that lead to egg activation, as indicated by pronuclear formation. We show that Ca2+ oscillations in fertilized mouse eggs can be inhibited by addition of either the Ca2+ chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM) or the heavy-metal-ion chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) plus dithiothreitol (DTT). Both treatments inhibited Ca2+ oscillations, but they had different effects upon egg activation. Blocking Ca2+ oscillations with BAPTA-AM after the occurrence of just two Ca2+ spikes resulted in most eggs forming pronuclei. However, we found that BAPTA-AM-treated fertilizing eggs showed a decreased rate of protein synthesis, which by itself can promote egg activation. In contrast, blocking Ca2+ oscillations with TPEN plus DTT was accompanied by the inhibition of egg activation with no significant effect on protein synthesis. In eggs that were fertilized and then treated with TPEN plus DTT, there was a correlation between the number of Ca2+ spikes and the proportion of eggs that formed pronuclei, as well as between the number of Ca2+ spikes and the time taken for pronuclear formation and the first mitosis to occur. The addition of TPEN plus DTT did not block the generation of Ca2+ spikes or pronuclear formation when eggs were artificially stimulated by electroporation pulses. These data suggest that TPEN plus DTT inhibits pronuclear formation in fertilizing eggs via the inhibition of Ca2+ oscillations and that the number of Ca2+ spikes may regulate egg activation.

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Selected References

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  1. Arslan P., Di Virgilio F., Beltrame M., Tsien R. Y., Pozzan T. Cytosolic Ca2+ homeostasis in Ehrlich and Yoshida carcinomas. A new, membrane-permeant chelator of heavy metals reveals that these ascites tumor cell lines have normal cytosolic free Ca2+. J Biol Chem. 1985 Mar 10;260(5):2719–2727. [PubMed] [Google Scholar]
  2. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  3. Bos-Mikich A., Swann K., Whittingham D. G. Calcium oscillations and protein synthesis inhibition synergistically activate mouse oocytes. Mol Reprod Dev. 1995 May;41(1):84–90. doi: 10.1002/mrd.1080410113. [DOI] [PubMed] [Google Scholar]
  4. Cheek T. R., McGuinness O. M., Vincent C., Moreton R. B., Berridge M. J., Johnson M. H. Fertilisation and thimerosal stimulate similar calcium spiking patterns in mouse oocytes but by separate mechanisms. Development. 1993 Sep;119(1):179–189. doi: 10.1242/dev.119.1.179. [DOI] [PubMed] [Google Scholar]
  5. Cuthbertson K. S., Cobbold P. H. Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca2+. Nature. 1985 Aug 8;316(6028):541–542. doi: 10.1038/316541a0. [DOI] [PubMed] [Google Scholar]
  6. Cuthbertson K. S., Whittingham D. G., Cobbold P. H. Free Ca2+ increases in exponential phases during mouse oocyte activation. Nature. 1981 Dec 24;294(5843):754–757. doi: 10.1038/294754a0. [DOI] [PubMed] [Google Scholar]
  7. De Koninck P., Schulman H. Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations. Science. 1998 Jan 9;279(5348):227–230. doi: 10.1126/science.279.5348.227. [DOI] [PubMed] [Google Scholar]
  8. Di Virgilio F., Steinberg T. H., Silverstein S. C. Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers. Cell Calcium. 1990 Feb-Mar;11(2-3):57–62. doi: 10.1016/0143-4160(90)90059-4. [DOI] [PubMed] [Google Scholar]
  9. Fulton B. P., Whittingham D. G. Activation of mammalian oocytes by intracellular injection of calcium. Nature. 1978 May 11;273(5658):149–151. doi: 10.1038/273149a0. [DOI] [PubMed] [Google Scholar]
  10. Goldbeter A., Dupont G., Berridge M. J. Minimal model for signal-induced Ca2+ oscillations and for their frequency encoding through protein phosphorylation. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1461–1465. doi: 10.1073/pnas.87.4.1461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hofer A. M., Fasolato C., Pozzan T. Capacitative Ca2+ entry is closely linked to the filling state of internal Ca2+ stores: a study using simultaneous measurements of ICRAC and intraluminal [Ca2+]. J Cell Biol. 1998 Jan 26;140(2):325–334. doi: 10.1083/jcb.140.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jones K. T., Carroll J., Merriman J. A., Whittingham D. G., Kono T. Repetitive sperm-induced Ca2+ transients in mouse oocytes are cell cycle dependent. Development. 1995 Oct;121(10):3259–3266. doi: 10.1242/dev.121.10.3259. [DOI] [PubMed] [Google Scholar]
  13. Kline D., Kline J. T. Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg. Dev Biol. 1992 Jan;149(1):80–89. doi: 10.1016/0012-1606(92)90265-i. [DOI] [PubMed] [Google Scholar]
  14. Kurasawa S., Schultz R. M., Kopf G. S. Egg-induced modifications of the zona pellucida of mouse eggs: effects of microinjected inositol 1,4,5-trisphosphate. Dev Biol. 1989 May;133(1):295–304. doi: 10.1016/0012-1606(89)90320-5. [DOI] [PubMed] [Google Scholar]
  15. Kurebayashi N., Harkins A. B., Baylor S. M. Use of fura red as an intracellular calcium indicator in frog skeletal muscle fibers. Biophys J. 1993 Jun;64(6):1934–1960. doi: 10.1016/S0006-3495(93)81564-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lawrence Y., Whitaker M., Swann K. Sperm-egg fusion is the prelude to the initial Ca2+ increase at fertilization in the mouse. Development. 1997 Jan;124(1):233–241. doi: 10.1242/dev.124.1.233. [DOI] [PubMed] [Google Scholar]
  17. Meyer T., Stryer L. Calcium spiking. Annu Rev Biophys Biophys Chem. 1991;20:153–174. doi: 10.1146/annurev.bb.20.060191.001101. [DOI] [PubMed] [Google Scholar]
  18. Mezna M., Michelangeli F. Effects of thimerosal on the transient kinetics of inositol 1,4,5-trisphosphate-induced Ca2+ release from cerebellar microsomes. Biochem J. 1997 Jul 1;325(Pt 1):177–182. doi: 10.1042/bj3250177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ozil J. P., Swann K. Stimulation of repetitive calcium transients in mouse eggs. J Physiol. 1995 Mar 1;483(Pt 2):331–346. doi: 10.1113/jphysiol.1995.sp020589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ozil J. P. The parthenogenetic development of rabbit oocytes after repetitive pulsatile electrical stimulation. Development. 1990 May;109(1):117–127. doi: 10.1242/dev.109.1.117. [DOI] [PubMed] [Google Scholar]
  21. Preston S. F., Berlin R. D. An intracellular calcium store regulates protein synthesis in HeLa cells, but it is not the hormone-sensitive store. Cell Calcium. 1992 May;13(5):303–312. doi: 10.1016/0143-4160(92)90065-z. [DOI] [PubMed] [Google Scholar]
  22. Quinn P., Barros C., Whittingham D. G. Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa. J Reprod Fertil. 1982 Sep;66(1):161–168. doi: 10.1530/jrf.0.0660161. [DOI] [PubMed] [Google Scholar]
  23. Salama G., Abramson J. J., Pike G. K. Sulphydryl reagents trigger Ca2+ release from the sarcoplasmic reticulum of skinned rabbit psoas fibres. J Physiol. 1992 Aug;454:389–420. doi: 10.1113/jphysiol.1992.sp019270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schultz R. M., Kopf G. S. Molecular basis of mammalian egg activation. Curr Top Dev Biol. 1995;30:21–62. doi: 10.1016/s0070-2153(08)60563-3. [DOI] [PubMed] [Google Scholar]
  25. Shumaker D. K., Vann L. R., Goldberg M. W., Allen T. D., Wilson K. L. TPEN, a Zn2+/Fe2+ chelator with low affinity for Ca2+, inhibits lamin assembly, destabilizes nuclear architecture and may independently protect nuclei from apoptosis in vitro. Cell Calcium. 1998 Feb-Mar;23(2-3):151–164. doi: 10.1016/s0143-4160(98)90114-2. [DOI] [PubMed] [Google Scholar]
  26. Siracusa G., Whittingham D. G., Molinaro M., Vivarelli E. Parthenogenetic activation of mouse oocytes induced by inhibitors of protein synthesis. J Embryol Exp Morphol. 1978 Feb;43:157–166. [PubMed] [Google Scholar]
  27. Snitsarev V. A., McNulty T. J., Taylor C. W. Endogenous heavy metal ions perturb fura-2 measurements of basal and hormone-evoked Ca2+ signals. Biophys J. 1996 Aug;71(2):1048–1056. doi: 10.1016/S0006-3495(96)79305-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Speksnijder J. E., Corson D. W., Sardet C., Jaffe L. F. Free calcium pulses following fertilization in the ascidian egg. Dev Biol. 1989 Sep;135(1):182–190. doi: 10.1016/0012-1606(89)90168-1. [DOI] [PubMed] [Google Scholar]
  29. Steinhardt R. A., Epel D., Carroll E. J., Jr, Yanagimachi R. Is calcium ionophore a universal activator for unfertilised eggs? Nature. 1974 Nov 1;252(5478):41–43. doi: 10.1038/252041a0. [DOI] [PubMed] [Google Scholar]
  30. Stricker S. A. Repetitive calcium waves induced by fertilization in the nemertean worm Cerebratulus lacteus. Dev Biol. 1996 Jun 15;176(2):243–263. doi: 10.1006/dbio.1996.0131. [DOI] [PubMed] [Google Scholar]
  31. Swann K., Ozil J. P. Dynamics of the calcium signal that triggers mammalian egg activation. Int Rev Cytol. 1994;152:183–222. doi: 10.1016/s0074-7696(08)62557-7. [DOI] [PubMed] [Google Scholar]
  32. Vincent C., Cheek T. R., Johnson M. H. Cell cycle progression of parthenogenetically activated mouse oocytes to interphase is dependent on the level of internal calcium. J Cell Sci. 1992 Oct;103(Pt 2):389–396. doi: 10.1242/jcs.103.2.389. [DOI] [PubMed] [Google Scholar]
  33. Vitullo A. D., Ozil J. P. Repetitive calcium stimuli drive meiotic resumption and pronuclear development during mouse oocyte activation. Dev Biol. 1992 May;151(1):128–136. doi: 10.1016/0012-1606(92)90220-b. [DOI] [PubMed] [Google Scholar]
  34. Xu Z., Lefevre L., Ducibella T., Schultz R. M., Kopf G. S. Effects of calcium-BAPTA buffers and the calmodulin antagonist W-7 on mouse egg activation. Dev Biol. 1996 Dec 15;180(2):594–604. doi: 10.1006/dbio.1996.0331. [DOI] [PubMed] [Google Scholar]
  35. Yule D. I., Lawrie A. M., Gallacher D. V. Acetylcholine and cholecystokinin induce different patterns of oscillating calcium signals in pancreatic acinar cells. Cell Calcium. 1991 Feb-Mar;12(2-3):145–151. doi: 10.1016/0143-4160(91)90016-8. [DOI] [PubMed] [Google Scholar]

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