Abstract
Purpose
The viability of mammalian eggs after ovulation is reported to be improved by the presence of ascorbic acid in the culture medium. However, the pro-survival mechanisms of ascorbic acid are poorly understood. The molecular pathways of apoptosis are evolutionarily conserved among animal species, and Xenopus eggs are technically and ethically more suitable for biochemical analyses than mammalian eggs. We used Xenopus egg cytoplasmic extracts to examine the direct intracellular effects of ascorbic acid.
Methods
Incubation of egg extracts for more than 4 h induces the spontaneous release of cytochrome c from mitochondria. This event triggers the activation of caspases, cleavage of substrate proteins, and execution of apoptosis. Multiple signal transduction pathways including proteolysis and protein phosphorylation are also involved in this process. We examined whether any of these events might be inhibited by the addition of ascorbic acid.
Results
Ascorbic acid showed no effect against cytochrome c release, but prevented caspase activation and substrate cleavage. Ascorbic acid also blocked the proteolysis of apoptosis inhibitor proteins and the dephosphorylation of p42 MAP kinase. However, dehydroascorbic acid (oxidized form of ascorbic acid) and acetate (unrelated acid) were equally effective, indicating that these effects were primarily due to their acidity. In addition, dehydroascorbic acid inhibited caspase activities directly in vitro.
Conclusions
The anti-apoptotic effect of ascorbic acid in Xenopus egg extracts is mainly due to cytoplasmic acidification rather than its intracellular antioxidant activity. Instead, oxidative conversion of ascorbic acid into dehydroascorbic acid may inhibit apoptosis through the inhibition of caspases.
References
Tarín JJ, Vendrell FJ, Ten J, Cano A. Antioxidant therapy counteracts the disturbing effects of diamide and maternal ageing on meiotic division and chromosomal segregation in mouse oocytes. Mol Hum Reprod. 1998;4:281–8.
Eppig JJ, Hosoe M, O’Brien MJ, Pendola FM, Requena A, Watanabe S. Conditions that affect acquisition of developmental competence by mouse oocytes in vitro: FSH, insulin, glucose and ascorbic acid. Mol Cell Endocrinol. 2000;163:109–16.
Guérin P, El Mouatassim S, Ménézo Y. Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum Reprod Update. 2001;7:175–89.
Tarín JJ, Pérez-Albalá S, Cano A. Oral antioxidants counteract the negative effects of female aging on oocyte quantity and quality in the mouse. Mol Reprod Dev. 2002;61:385–97.
Comizzoli P, Wildt DE, Pukazhenthi BS. Overcoming poor in vitro nuclear maturation and developmental competence of domestic cat oocytes during the non-breeding season. Reproduction. 2003;126:809–16.
Tatemoto H, Ootaki K, Shigeta K, Muto N. Enhancement of developmental competence after in vitro fertilization of porcine oocytes by treatment with ascorbic acid 2-O-α-glucoside during in vitro maturation. Biol Reprod. 2001;65:1800–6.
Tao Y, Zhou B, Xia G, Wang F, Wu Z, Fu M. Exposure to l-ascorbic acid or α-tocopherol facilitates the development of porcine denuded oocytes from metaphase I to metaphase II and prevents cumulus cells from fragmentation. Reprod Domest Anim. 2004;39:52–7.
Dalvit G, Llanes SP, Descalzo A, Insani M, Beconi M, Cetica P. Effect of α-tocopherol and ascorbic acid on bovine oocyte in vitro maturation. Reprod Domest Anim. 2005;40:93–7.
Newmeyer DD, Farschon DM, Reed JC. Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria. Cell. 1994;79:353–64.
von Ahsen O, Newmeyer DD. Cell-free apoptosis in Xenopus laevis egg extracts. Methods Enzymol. 2000;322:183–98.
Deming P, Kornbluth S. Study of apoptosis in vitro using the Xenopus egg extract reconstitution system. Methods Mol Biol. 2006;322:379–93.
Tsuchiya Y, Murai S, Yamashita S. Apoptosis-inhibiting activities of BIR family proteins in Xenopus egg extracts. FEBS J. 2005;272:2237–50.
Tsuchiya Y, Yamashita S. p42MAPK-mediated phosphorylation of xEIAP/XLX in Xenopus cytostatic factor-arrested egg extracts. BMC Biochem. 2007;8:5.
Tashker JS, Olson M, Kornbluth S. Post-cytochrome c protection from apoptosis conferred by a MAPK pathway in Xenopus egg extracts. Mol Biol Cell. 2002;13:393–401.
Yaoita Y, Nakajima K. Induction of apoptosis and CPP32 expression by thyroid hormone in a myoblastic cell line derived from tadpole tail. J Biol Chem. 1997;272:5122–7.
Nakajima K, Takahashi A, Yaoita Y. Structure, expression, and function of the Xenopus laevis caspase family. J Biol Chem. 2000;275:10484–91.
Bode AM, Cunningham L, Rose RC. Spontaneous decay of oxidized ascorbic acid (dehydro-l-ascorbic acid) evaluated by high-pressure liquid chromatography. Clin Chem. 1990;36:1807–9.
Holley CL, Olson MR, Colón-Ramos DA, Kornbluth S. Reaper eliminates IAP proteins through stimulated IAP degradation and generalized translational inhibition. Nat Cell Biol. 2002;4:439–44.
Greenwood J, Gautier J. XLX is an IAP family member regulated by phosphorylation during meiosis. Cell Death Differ. 2007;14:559–67.
Flament S, Browaeys E, Rodeau J-L, Bertout M, Vilain J-P. Xenopus oocyte maturation: cytoplasm alkalization is involved in germinal vesicle migration. Int J Dev Biol. 1996;40:471–6.
Sellier C, Bodart J-F, Flament S, Baert F, Gannon J, Vilain J-P. Intracellular acidification delays hormonal G2/M transition and inhibits G2/M transition triggered by thiophosphorylated MAPK in Xenopus oocytes. J Cell Biochem. 2006;98:287–300.
Nutt LK, Margolis SS, Jensen M, Herman CE, Dunphy WG, Rathmell JC, et al. Metabolic regulation of oocyte cell death through the CaMKII-mediated phosphorylation of caspase-2. Cell. 2005;123:89–103.
Coll O, Morales A, Fernández-Checa JC, Garcia-Ruiz C. Neutral sphingomyelinase-induced ceramide triggers germinal vesicle breakdown and oxidant-dependent apoptosis in Xenopus laevis oocytes. J Lipid Res. 2007;48:1924–35.
Cárcamo JM, Pedraza A, Bórquez-Ojeda O, Zhang B, Sanchez R, Golde DW. Vitamin C is a kinase inhibitor: dehydroascorbic acid inhibits IκBα kinase β. Mol Cell Biol. 2004;24:6645–52.
De Tullio MC, Arrigoni O. Hopes, disillusions and more hopes from vitamin C. Cell Mol Life Sci. 2004;61:209–19.
Daruwala R, Song J, Koh WS, Rumsey SC, Levine M. Cloning and functional characterization of the human sodium-dependent vitamin C transporters hSVCT1 and hSVCT2. FEBS Lett. 1999;460:480–4.
Acknowledgments
We thank the members of our laboratories for discussion. This study was supported by Project Research Grant 18-10 from Toho University School of Medicine and in part by Grants-in Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Saitoh, T., Tsuchiya, Y., Kinoshita, T. et al. Inhibition of apoptosis by ascorbic and dehydroascorbic acids in Xenopus egg extracts. Reprod Med Biol 8, 3–9 (2009). https://doi.org/10.1007/s12522-008-0001-x
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DOI: https://doi.org/10.1007/s12522-008-0001-x