Inhibition of apoptosis by ascorbic and dehydroascorbic acids in Xenopus egg extracts
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- Saitoh, T., Tsuchiya, Y., Kinoshita, T. et al. Reprod Med Biol (2009) 8: 3. doi:10.1007/s12522-008-0001-x
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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.
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.
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.
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.
KeywordsAscorbic acidApoptosisCaspasesDehydroascorbic acidXenopus egg extracts
Aged eggs long after ovulation spontaneously degenerate and die mainly by apoptosis. It is poorly understood how the fate of ovulated eggs is switched from survival to death. Many reports suggest that the viability of isolated oocytes, eggs and early embryos from various animals is improved by the addition of antioxidants into culture medium. For example, ascorbic acid (AA) and/or α-tocopherol prevented the maternal aging and apoptosis of mouse oocytes [1–4]. Compromised oocyte function of cat oocytes was overridden by AA or cysteine . AA and α-tocopherol also enhanced the developmental competence of porcine oocytes [6, 7], but not of bovine oocytes . It is generally believed that antioxidants protect cells from reactive oxygen generated by intracellular metabolism and extracellular environments. However, many water-soluble antioxidants such as AA may not penetrate into eggs without specific transporters. Therefore, it is not clear whether antioxidants exert their pro-survival effects intracellularly, extracellularly, or both.
Xenopus egg cytoplasmic extracts are a suitable tool for biochemical analyses of apoptosis regulation [9–11]. The use of this cell-free system allows us to evaluate the direct intracellular effects of membrane-impermeable, water-soluble molecules. Incubation of egg extracts for more than 4 h induces the spontaneous release of cytochrome c from mitochondria, followed by the activation of caspases. Subsequently, caspase-mediated cleavages of substrate proteins including poly (ADP-ribose) polymerase (PARP) execute apoptosis. This phenomenon physiologically reflects the aging-induced apoptosis of oocytes and eggs in vivo.
The molecular mechanisms of apoptosis are evolutionarily conserved between mammals and Xenopus. We previously reported that xXIAP, a Xenopus homolog of mammalian X-linked inhibitor of apoptosis protein (XIAP), was a physiological apoptosis inhibitor in egg extracts [12, 13]. xEIAP/XLX, another structurally related protein, might be also involved in the inhibition of apoptosis. xXIAP was abruptly degraded at the onset of apoptosis, whereas xEIAP/XLX was highly unstable in egg extracts. Therefore, the proteolytic regulation of xXIAP and xEIAP/XLX may determine the timing for apoptotic execution, and apoptosis may be inhibited when the proteolysis of xXIAP and xEIAP/XLX is blocked. For both xXIAP and xEIAP/XLX, proteolysis was not inhibited by Z-VAD-FMK (a pan-caspase inhibitor) or MG-132 (a proteasome inhibitor), suggesting the involvement of hitherto unidentified proteolytic pathways.
We and others also reported that cytostatic factor (CSF)-arrested metaphase egg extracts required longer incubation time than interphase egg extracts to execute spontaneous apoptosis. This delay was dependent on the phosphorylation and activation of p42 mitogen-activated protein kinase (p42MAPK) in CSF-arrested egg extracts [12, 14]. p42MAPK is normally dephosphorylated and inactivated upon fertilization in eggs in vivo, and the addition of 0.4 mM CaCl2 into CSF-arrested egg extracts also dephosphorylates and inactivates p42MAPK in vitro. When p42MAPK dephosphorylation is blocked, sustained activity of p42MAPK may delay apoptosis in egg extracts.
In this study, we examined whether any of these events might be inhibited by the addition of AA into egg extracts. We also compared the cytoplasmic anti-apoptotic functions of AA with those of oxidized form of AA (dehydroascorbic acid, DHA) and unrelated acid (acetate, Ac).
Materials and methods
Preparation of Xenopus egg extracts and reagents
Animal care and use of female frogs (Xenopuslaevis) is approved by the Animal Research Committee for Animal Experimentation of Toho University. Preparations of fresh CSF-arrested, interphase, and apoptotic egg extracts were previously described [12, 13]. AA (Wako), DHA (Wako or Sigma), and Ac (Wako) were dissolved in water as 0.5 M stock solutions and added to egg extracts at 10 mM. The pH values of the extracts were routinely checked using pH indicator paper (Spezial Indikatorpapier pH 6.4–8.0, Macherey-Nagel). Where indicated, a pan-caspase inhibitor Z-VAD-FMK (Peptide Institute) or a proteasome inhibitor MG-132 (Peptide Institute) was added to the egg extracts at 100 μM.
SDS-PAGE, antibodies and western blot
SDS-PAGE was carried out using 12% separating gels for PARP, p42MAPK, and actin, or 15% separating gels for cytochrome c. Resolved samples were electrically transferred to Immobilon-P PVDF membranes (Millipore). Mouse monoclonal antibodies against PARP, MAPK, and phosphorylated MAPK (pT202/pY204) were purchased from BD Biosciences. Mouse monoclonal antibody against cytochrome c and rabbit polyclonal antibody against actin were from Lab Vision and Sigma, respectively. All antibodies were used at 1:1,000 dilutions for western blot as previously described [12, 13]. Alkaline phosphatase-linked anti-mouse and anti-rabbit secondary antibodies (Santa Cruz) were used, and signals were visualized using 5-bromo-4-chloro-3-indolyl phosphate (Sigma) and nitroblue tetrazolium (Wako).
Quantification of cytoplasmic cytochrome c
Egg extracts after incubation for indicated hours were filtered through 0.1 µm pore using Ultrafree-MC (Millipore) at 10,000×g for 30 min according to the method by Tashker et al. . The protein concentration of each filtrate was determined using Bio-Rad Protein Assay kit (Bio-Rad). Equal amounts of proteins were subjected to SDS-PAGE and western blot for cytochrome c and actin.
Quantification of caspase activities
To measure the caspase activity in Xenopus egg extracts, 1.5 µl of extracts was taken out at indicated times after incubation and rapidly frozen. Each sample was then thawed and mixed with 150 µl of reaction buffer (50 mM HEPES pH 7.4, 0.1 M NaCl, 1 mM EDTA, 10% glycerol, 0.1% CHAPS, 10 mM DTT) containing 200 µM Ac-DEVD-pNA (BIOMOL). The increase of absorbance at 405 nm during incubation for 1 h at room temperature was measured. For the assay of purified caspases in vitro, recombinant active human caspases and corresponding substrates (Ac-DEVD-pNA for caspases-3/7, Ac-LEHD-pNA for caspases-2/9, and Ac-VEID-pNA for caspase-6) were used (BIOMOL or Calbiochem). Caspase activities in the presence of various concentrations of AA, DHA, and Ac were determined according to manufacture’s instruction, except that optimal DTT concentration was experimentally determined for each caspase.
Protein stability assay using 35S-radiolabeled recombinant proteins
Open reading frames of Xenopus caspases (xCasp-2/3/6/7/9) were amplified by RT-PCR from Xenopus egg or stomach mRNA and cloned into either Nde I-Bam HI site or Xho I-Eco RI site of pET-15b (Novagen) [15, 16]. In vitro translations of 35S-radiolabeled, N-terminally 6XHis-tagged Xenopus caspases, xEIAP/XLX, and xXIAP in rabbit reticulocyte lysates (TnT Quick T7, Promega) were carried out as previously described [12, 13]. Rabbit reticulocyte lysates containing recombinant proteins were mixed with egg extracts at 1:9 and incubated at room temperature for indicated hours. After incubation, samples were resolved by SDS-PAGE using 12% separating gels for xXIAP and xEIAP/XLX, or 15% separating gels for Xenopus caspases, and detected by a BAS-5000 image analyzer (Fuji Film).
As previously reported, recombinant 35S-radiolabeled 6XHis-xXIAP produced in rabbit reticulocyte lysates was rather stable in healthy egg extracts, but was rapidly degraded at the onset of apoptosis after 4 h incubation [12, 13]. In the presence of AA, DHA, and Ac, degradation of 6XHis-xXIAP at 4 h was completely inhibited (Fig. 4, 6XHis-xXIAP). In addition, upward electrophoretic mobility shift of 6XHis-xXIAP was observed in these three samples, suggesting that 6XHis-xXIAP was modified in acid-treated egg extracts. In contrast, neither Z-VAD-FMK nor MG-132 was able to inhibit 6XHis-xXIAP degradation at 4 h, and upward electrophoretic mobility shift was not observed in the egg extracts containing these inhibitors. Further studies will be required to identify the molecular basis of this electrophoretic mobility shift. The degradation of recombinant 35S-radiolabeled 6XHis-xEIAP produced in rabbit reticulocyte lysates was also blocked in egg extracts containing AA, DHA, and Ac, but not in egg extracts treated with Z-VAD-FMK or MG-132 (Fig. 4, 6XHis-xEIAP). These results indicate that the stabilized xXIAP and xEIAP/XLX could also contribute to the inhibition of apoptosis in acid-treated egg extracts [18, 19]. Altogether, multiple apoptosis-regulating pathways were affected by the addition of acidic solutions to egg extracts.
We showed that the anti-apoptotic effects of AA, DHA, and Ac in egg extracts observed in this study were primarily due to their acidifying function. Other studies indicated that the artificial modifications of cytoplasmic pH changed the timing of maturation in Xenopus oocytes [20, 21]. Therefore, cytoplasmic pH is critical for various physiological events in Xenopus oocytes and eggs. In contrast, the antioxidant activity of AA did not show pro-survival roles in Xenopus egg extracts. Other reports using Xenopus egg extracts also failed to show the anti-apoptotic effects of reducing reagents such as reduced glutathione (GSH) and N-acetyl cysteine [9, 22]. Moreover, pretreatment of Xenopus oocytes with GSH-ethyl ester partially prevented sphingomyelinase-induced caspase-3 activation and oocyte death, but did not affect the spontaneous caspase-3 activation . These results indicate that increasing cytoplasmic antioxidant activity antagonizes artificial generation of reactive oxygen species, but does not prevent spontaneous apoptosis in this system. This may be because sufficient amounts of antioxidant activity are present and maintained in egg cytoplasm. Otherwise, antioxidant activity may be dispensable for the inhibition of spontaneous apoptosis in egg extracts. Rather, our data suggest that the oxidant activity of DHA can inhibit caspase activities directly in vitro. One possibility is that DHA generated by the oxidation of AA, rather than AA itself, may exert anti-apoptotic effects in cytoplasm. In the case of NF-κB signaling, AA quenches reactive oxygen whereas DHA inhibits IκBα kinases, and both reactions prevent NF-κB activation together . Further studies will be required to establish the unified mechanisms of cytoplasmic AA-DHA conversion and cellular survival.
In a number of reports suggesting the pro-survival roles of AA, the reagents are added to culture medium or administrated orally in cellular or animal experiments, respectively [1–8]. Intracellular uptake of AA requires the function of specific Na+-ascorbate co-transporter, and glucose transporters are necessary for the intracellular transport of DHA . Xenopus oocytes incorporate neither AA nor DHA significantly without exogenous expression of corresponding transporters . In our cellular assay, the presence of AA and DHA in the extracellular buffer did not improve the viability of Xenopus eggs (data not shown). It is not known whether mammalian oocytes and eggs express these transporters to transport AA and DHA into the cytoplasm. The pro-survival activity of AA in culture medium may be to prevent the oxidation of extracellular materials or medium components, and this extracellular effect may be specific to mammalian oocytes and eggs. In contrast, our study using cytoplasmic extracts is aimed to clarify the intracellular roles of AA and DHA. The metabolic and apoptotic pathways are evolutionarily conserved, and our study will complement the previous cellular studies to elucidate the physiological functions of AA for reproductive biology.
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.