Apoptosis

, Volume 17, Issue 5, pp 439–448 | Cite as

High cytosolic free calcium level signals apoptosis through mitochondria-caspase mediated pathway in rat eggs cultured in vitro

Original Paper

Abstract

The present study was aimed to find out whether an increase of cytosolic free calcium level induces egg apoptosis through mitochondria-caspase mediated pathway. To increase cytosolic free calcium level and morphological apoptotic changes, ovulated eggs were cultured in Ca2+/Mg2+ free media-199 with or without various concentrations of calcium ionophore (0.5, 1, 2, 3, 4 μM) for 3 h in vitro. The morphological apoptotic changes, cytosolic free calcium level, hydrogen peroxide (H2O2) concentration, catalase activity, cytochrome c concentration, caspase-9 and caspase-3 activities and DNA fragmentation were analyzed. Calcium ionophore induced morphological apoptotic features in a concentration-dependent manner followed by degeneration at higher concentrations (3 and 4 μM). Calcium ionophore increased cytosolic free calcium level, induced generation of hydrogen peroxide (H2O2) and inhibited catalase activity in treated eggs. The increased H2O2 concentration was associated with increased cytochrome c concentration, caspase-9 and caspase-3 activities that resulted in the induction of morphological features characteristic of egg apoptosis. The increased caspase-3 activity finally induced DNA fragmentation as evidenced by TUNEL positive staining in calcium ionophore-treated eggs. These findings suggest that high cytosolic free calcium level induces generation of H2O2 that leads to egg apoptosis through mitochondria-caspase mediated pathway.

Keywords

Cytosolic free calcium Hydrogen peroxide Cytochrome c Caspases activity DNA fragmentation Egg apoptosis 

Supplementary material

10495_2012_702_MOESM1_ESM.tif (267 kb)
Supplementary material 1 (TIFF 266 kb)

References

  1. 1.
    Berridge MJ, Bootman MD, Lipp P (1998) Calcium: a life and death signal. Nature 395:645–648PubMedCrossRefGoogle Scholar
  2. 2.
    Tosti E (2006) Calcium ion currents mediating oocyte maturation events. Reprod Biol Endocrinol 4:26–34PubMedCrossRefGoogle Scholar
  3. 3.
    Miyazaki S, Shirakawa H, Nakada K (1993) Essential role of inositol 1,4,5-trisphosphate receptor/Ca2+ release channel in Ca2+ waves and Ca2+ oscillations at fertilization of mammalian eggs. Dev Biol 158:62–78PubMedCrossRefGoogle Scholar
  4. 4.
    Xu Z, Abbott A, Kopf GS, Schultz RM, Ducibella T (1997) Spontaneous activation of ovulated mouse eggs: time-dependent effects on M-phase exit, cortical granule exocytosis, maternal messenger ribonucleic acid recruitment, and Inositol 1,4,5-trisphosphate sensitivity. Biol Reprod 57:743–750PubMedCrossRefGoogle Scholar
  5. 5.
    Sergeev IN, Norman AV (2003) Calcium as a mediator of apoptosis in bovine oocytes and preimplantation embryos. Endocrine 22:169–176PubMedCrossRefGoogle Scholar
  6. 6.
    Chaube SK, Dubey PK, Mishra SK, Shrivastav TG (2007) Verapamil inhibits spontaneous parthenogenetic activation in aged rat eggs cultured in vitro. Cloning Stem Cells 9:15–624CrossRefGoogle Scholar
  7. 7.
    Vincent C, Cheek TR, Johnson MH (1992) Cell cycle progression of parthenogenetically activated mouse oocytes to interphase is dependent on the level of internal calcium. J Cell Sci 103:389–396PubMedGoogle Scholar
  8. 8.
    Lu Q, Chen ZJ, Gao X, Ma SY, Li M, Hu JM, Li Y (2006) Oocyte activation with calcium ionophore A23187 and puromycin on human oocytes that failed to fertilize after intracytoplasmic sperm injection. Zhonghua Fu Chan Ke Za Zhi 41:182–185PubMedGoogle Scholar
  9. 9.
    Chaube SK, Khatun S, Mishra SK, Shrivastav TG (2008) Calcium ionophore-induced egg activation or apoptosis is associated with the generation of intracellular hydrogen peroxide. Free Radic Res 42:212–220PubMedCrossRefGoogle Scholar
  10. 10.
    Ruddock NT, Machaty Z, Cabot RA, Prather RS (2001) Porcine oocyte activation: roles of calcium and pH. Mol Reprod Dev 59:227–234PubMedCrossRefGoogle Scholar
  11. 11.
    McConkey DJ, Orrenius S (1997) The role of calcium in the regulation of apoptosis. Biochem Biophys Res Commun 239:357–366PubMedCrossRefGoogle Scholar
  12. 12.
    Penzo D, Petronilli V, Angelin A, Cusan C, Colonna R, Scorrano L, Pagano F, Prato M, Di-Lisa F, Bernardi P (2004) Arachidonic acid released by phospholipase A(2) activation triggers Ca(2+)-dependent apoptosis through the mitochondrial pathway. J Biol Chem 279:25219–25225PubMedCrossRefGoogle Scholar
  13. 13.
    Tan AR, Cai AY, Deheshi S, Rintoul GL (2011) Elevated intracellular calcium causes distinct mitochondrial remodelling and calcineurin-dependent fission in astrocytes. Cell Calcium 49:108–114PubMedCrossRefGoogle Scholar
  14. 14.
    Cho SY, Lee JH, Bae HD, Jeong EM, Jang GY, Kim CW, Shin DM, Jeon JH, Kim IG (2010) Transglutaminase 2 inhibits apoptosis induced by calcium-overload through down-regulation of Bax. Exp Mol Med 42:639–650PubMedCrossRefGoogle Scholar
  15. 15.
    Wang ZG, Wang W, Yu SD, Xu ZR (2008) Effects of different activation protocols of preimplantation development, apoptosis and ploidy of bovine parthenogenetic embryos. Anim Reprod Sci 105:292–301PubMedCrossRefGoogle Scholar
  16. 16.
    Ma W, Zhang D, Hou Y, Li Y-H, Sun Q-Y, Sun X-F, Wang W-H (2005) Reduced expression of MAD2, BCL2 and MAP Kinase activity in pig oocytes after in vitro aging are associated with defects in sister chromatid segregation during meiosis II and embryo fragmentation after activation. Biol Reprod 72:373–383PubMedCrossRefGoogle Scholar
  17. 17.
    Takasu N, Yamada T, Shimizu Y (1987) Generation of H2O2 is regulated by cytoplasmic free calcium in cultured porcine thyroid cells. Biochem Biophys Res Commun 148:1527–1532PubMedCrossRefGoogle Scholar
  18. 18.
    Zoccarato F, Valente M, Alexandre A (1993) Identification of an NADH plus iron dependent, Ca2+ activated hydrogen peroxide production in synaptosomes. Biochem Biophys Acta 1176:208–214PubMedCrossRefGoogle Scholar
  19. 19.
    Przygodzki T, Sokal A, Bryszewska M (2005) Calcium ionophore A23187 action on cardiac myocytes is accompanied by enhanced production of reactive oxygen species. Biochem Biophys Acta 1740:481–488PubMedGoogle Scholar
  20. 20.
    Gordo AC, Rodrigues P, Kurokawa M, Jellerette T, Exley GE, Warner C, Fissore R (2002) Intracellular calcium oscillations signal apoptosis rather than activation in vitro aged mouse eggs. Biol Reprod 66:1828–1837PubMedCrossRefGoogle Scholar
  21. 21.
    Cetica PD, Pintos LN, Dalvit GC, Beconi MT (2001) Antioxidant enzyme activity and oxidative stress in bovine oocyte in vitro maturation. IUBMB Life 51:57–64PubMedCrossRefGoogle Scholar
  22. 22.
    Whitaker BD, Knight JW (2008) Mechanisms of oxidative stress in porcine oocytes and the role of anti-oxidants. Reprod Fertil Dev 20:694–702PubMedCrossRefGoogle Scholar
  23. 23.
    Tripathi A, Khatun S, Pandey AN, Mishra SK, Chaube R, Shrivastav TG, Chaube SK (2009) Intracellular levels of hydrogen peroxide and nitric oxide in oocytes at various stages of meiotic cell cycle and apoptosis. Free Radic Res 43:287–294PubMedCrossRefGoogle Scholar
  24. 24.
    Qian T, Herman B, Lemasters JJ (1999) The mitochondrial permeability transition mediates both necrotic and apoptotic death of hepatocytes exposed to Br-A23187. Toxicol Appl Pharmacol 154:117–125PubMedCrossRefGoogle Scholar
  25. 25.
    Petrocillo G, Ruggiero FM, Pistolese M, Paradies G (2004) Ca2+-induced reactive oxygen species production promotes cytochrome c release from rat liver mitochondria via mitochondrial permeability transition (MPT)-dependent and MPT-independent mechanisms. J Biol Chem 279:53103–53108CrossRefGoogle Scholar
  26. 26.
    Zhang X, Li XH, Ma X, Wang ZH, Lu S, Guo YL (2006) Redox-induced apoptosis of human oocytes in resting follicles in vitro. J Soc Gynecol Investig 13:451–458PubMedCrossRefGoogle Scholar
  27. 27.
    Ramalho-Santos J, Varum S, Amaral S, Mota PC, Sousa AP, Amaral A (2009) Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells. Hum Reprod Update 15:553–572PubMedCrossRefGoogle Scholar
  28. 28.
    Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD (1997) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275:1132–1136PubMedCrossRefGoogle Scholar
  29. 29.
    Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, Jones DP, Wang X (1997) Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275:1129–1132PubMedCrossRefGoogle Scholar
  30. 30.
    Braun T, Dar S, Vorobiov D, Lindenboim L, Dascal N, Stein R (2003) Expression of Bcl-x(S) in Xenopus oocytes induces BH3-dependent and caspase-dependent cytochrome c release and apoptosis. Mol Cancer Res 1:186–194PubMedGoogle Scholar
  31. 31.
    Wang Q, Frolova AI, Purcell S, Adastra K, Schoeller E (2010) Mitochondrial dysfunction and apoptosis in cumulus cells of type I diabetic mice. PLoS One 5:e15901–e15911PubMedCrossRefGoogle Scholar
  32. 32.
    Zou H, Li Y, Liu X, Wang X (1999) An APAF-1 cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 274:11549–11556PubMedCrossRefGoogle Scholar
  33. 33.
    Acehan D, Jiang X, Morgan DG, Heuser JE, Wang X, Akey CW (2002) Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation. Mol Cell 9:423–432PubMedCrossRefGoogle Scholar
  34. 34.
    Fuentes-Prior P, Salvesen GS (2004) The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem J 384:201–232PubMedCrossRefGoogle Scholar
  35. 35.
    Martin MC, Allan LA, Lickrish M, Sampson C, Morrice N, Clarke PR (2005) Protein kinase A regulates caspase-9 activation by Apaf-1 downstream of cytochrome c. J Biol Chem 280:15449–15455PubMedCrossRefGoogle Scholar
  36. 36.
    Carroll J, Swann K (1992) Spontaneous cytosolic calcium oscillations driven by inositol trisphosphate occur during in vitro maturation of mouse oocytes. J Biol Chem 267:11196–11201PubMedGoogle Scholar
  37. 37.
    Gavet O, Pines J (2010) Progressive activation of cyclinB1-cdk1 coordinates entry to mitosis. Dev Cell 18:533–543PubMedCrossRefGoogle Scholar
  38. 38.
    Tripathi A, Premkumar KV, Pandey AN, Khatun S, Mishra SK, Shrivastav TG, Chaube SK (2011) Melatonin protect against clomiphene citrate-induced generation of free radicals and egg apoptosis in rat. Eur J Pharmacol 667:419–424PubMedCrossRefGoogle Scholar
  39. 39.
    Chaube SK, Prasad PV, Thakur SC, Shrivastav TG (2005) Hydrogen peroxide modulates meiotic cell cycle and induces morphological features characteristic of apoptosis in rat oocytes cultured in vitro. Apoptosis 10:863–874PubMedCrossRefGoogle Scholar
  40. 40.
    Chaube SK, Prasad PV, Khillare B, Shrivastav TG (2006) Extract of Azadirachta indica (Neem) leaf induces apoptosis in rat oocytes cultured in vitro. Fertl Sterl 85(suppl 1):1223–1231CrossRefGoogle Scholar
  41. 41.
    Kajitani N, Kobuchi H, Fujita H, Yano H, Fujiwara T, Yasuda T, Utsumi K (2007) Mechanism of A23187-induced apoptosis in HL-60 cells: dependency on mitochondrial permeability transition but not on NADPH oxidase. Biosci Biotechnol Biochem 71:2701–2711PubMedCrossRefGoogle Scholar
  42. 42.
    Pepperell JR, Porterfield DM, Keefe DL, Behrman HR, Smith PJ (2003) Control of ascorbic acid efflux in rat luteal cells: role of intracellular calcium and oxygen radicals. Am J Physiol Cell Physiol 285:C642–C651PubMedGoogle Scholar
  43. 43.
    Priyadarsini RV, Murugan RS, Maitreyi S, Ramalingam K, Karunagaran D, Nagini S (2010) The flavonoid quercetin induces cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells through p53 induction and NF-κB inhibition. Eur J Pharmacol 649:84–91CrossRefGoogle Scholar
  44. 44.
    Jurisicova A, Acton BM (2004) Deadly decisions: the role of genes regulating programmed cell death in human preimplantation embryo development. Reproduction 128:281–291PubMedCrossRefGoogle Scholar
  45. 45.
    Ansari B, Coates PJ, Greenstein BD, Hall PA (1993) In situ end-labelling detects DNA strand breaks in apoptosis and other physiological and pathological states. J Pathol 170:1–8PubMedCrossRefGoogle Scholar
  46. 46.
    Roth Z, Hansen PJ (2004) Involvement of apoptosis in disruption of developmental competency of bovine oocytes by heat shock during maturation. Biol Reprod 71:1898–1906PubMedCrossRefGoogle Scholar
  47. 47.
    Henkel R, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, Menkveld R, Gips H, Schill WB, Kruger TF (2004) Influence of deoxyribonucleic acid damage on fertilization and pregnancy. Fertil Steril 81:965–972PubMedCrossRefGoogle Scholar
  48. 48.
    Hao Y, Lai L, Mao J, Im GS, Bonk A, Prather RS (2004) Apoptosis in parthenogenetic preimplantation porcine embryos. Biol Reprod 70:1644–1649PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Cell Physiology Laboratory, Biochemistry Unit, Department of ZoologyBanaras Hindu UniversityVaranasiIndia

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