Mechanisms of oxidative stress in porcine oocytes and the role of anti-oxidants
B. D. Whitaker A B and J. W. Knight AA Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA 24061, USA.
B Corresponding author. Email: bwhitaker@ferrum.edu
Reproduction, Fertility and Development 20(6) 694-702 https://doi.org/10.1071/RD08037
Submitted: 2 March 2008 Accepted: 26 May 2008 Published: 9 July 2008
Abstract
The mechanisms of oxidative stress in in vitro maturing porcine oocytes and the effects of anti-oxidant supplementation of the medium in ameliorating these effects were investigated in the present study. In addition to intracellular reduced glutathione (GSH) concentrations and DNA fragmentation, the present study focused on superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase activity. The anti-oxidants used were N-acetylcysteine (NAC) and its derivative NAC-amide (NACA). The results indicate that when SOD is inhibited, supplementation of the maturarion medium with 1.5 mm NAC or NACA compensates for the decrease in SOD activity by reducing the degree of DNA fragmentation (P < 0.05). When GPx is inhibited, supplementation of the maturarion medium with 1.5 mm NAC alleviates the effects of no GPx activity, as indicated by a decrease in the degree of DNA fragmentation (P < 0.05). When the maturarion medium was supplemented with 1.5 mm NACA, intracellular GSH concentrations decreased (P < 0.05) and SOD and catalase activities increased (P < 0.05) along with the degree of DNA fragmentation. These results indicate that the mechanisms of alleviating oxidative stress in porcine oocytes are very complex and supplementing maturing oocytes with anti-oxidants may enhance enzyme activities and eliminate free radicals.
Abeydeera, L. R. (2002). In vitro production of embryos in swine. Theriogenology 57, 257–273.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Abeydeera, L. R. , Wang, W. H. , Cantley, T. C. , Rieke, A. , and Day, B. N. (1998). Coculture with follicular shell pieces can enhance the developmental competence of pig oocytes after in vitro fertilization: relevance to intracellular glutathione. Biol. Reprod. 58, 213–218.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Aebi, H. (1984). Catalase in vitro. Methods Enzymol. 105, 121–126.
| PubMed |
![](../../media/share/OpenURL_Image.gif)
Ali, A. A. , Bilodeau, J. F. , and Sirard, M. A. (2003). Antioxidant requirements for bovine oocytes varies during in vitro maturation, fertilization and development. Theriogenology 59, 939–949.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Anderson, M. E. (1985). Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol. 113, 548–555.
| PubMed |
![](../../media/share/OpenURL_Image.gif)
Brash, D. E. , and Havre, P. A. (2002). New careers for antioxidants. Proc. Natl Acad. Sci. USA 99, 13 969–13 971.
| Crossref | GoogleScholarGoogle Scholar |
![](../../media/share/OpenURL_Image.gif)
Cetica, P. D. , Pintos, L. N. , Dalvit, G. C. , and Beconi, M. T. (2001). Antioxidant enzyme activity and oxidative stress in bovine oocyte in vitro maturation. IUBMB Life 51, 57–64.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
de Cavanagh, E. M. V. , Inserta, F. , Ferder, L. , Romano, L. , Ercole, L. , and Fraga, C. G. (1995). Superoxide dismutase and glutathione peroxidase activities are increased by enalapril and captopril in mouse liver. FEBS Lett. 361, 22–24.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Del Corso, A. , Cappiello, M. , and Mura, U. (1994). Thiol dependant oxidation of enzymes: the last chance against oxidative stress. Int. J. Biochem. 26, 745–750.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Ding, J. , and Foxcroft, G. R. (1994). Epidermal growth factor enhances oocyte maturation in pigs. Mol. Reprod. Dev. 39, 30–40.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Dumay, A. , Rincheval, V. , Trotot, P. , Mignotte, B. , and Vayssiere, J. (2006). The superoxide dismutase inhibitor diethyldithiocarbamate has antagonistic effects on apoptosis by triggering both cytochrome c release and caspase inhibition. Free Radic. Biol. Med. 40, 1377–1390.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Fiers, W. , Beyaert, R. , Declercq, W. , and Vandenabeele, P. (1999). More than one way to die: apoptosis, necrosis and reactive oxygen damage. Oncogene 18, 7719–7730.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Flohé, L. , and Ötting, F. (1984). Superoxide dismutase assays. Methods Enzymol. 105, 93–104.
| PubMed |
![](../../media/share/OpenURL_Image.gif)
Grinberg, L. , Fibach, E. , Am, J. , and Atlas, D. (2005). N-Acetylcysteine amide, a novel cell-permeating thiol, restores cellular glutathione and protects human red blood cells from oxidative stress. Free Radic. Biol. Med. 38, 136–145.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Grupen, C. G. , Nagashima, H. , and Nottle, M. B. (1995). Cysteamine enhances in vitro development of porcine oocytes matured and fertilized in vitro. Biol. Reprod. 53, 173–178.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Günzler, W. A. , Kremers, H. , and Flohé, L. (1974). An improved coupled test procedure for glutathione peroxidase in blood. Z. Klin. Chem. Klin. Biol. 12, 444–448.
![](../../media/share/OpenURL_Image.gif)
Halliwell, B. , and Gutteridge, J. M. (1988). Free radicals and antioxidant protection: mechanisms and significance in toxicology and disease. Hum. Toxicol. 7, 7–13.
| PubMed |
![](../../media/share/OpenURL_Image.gif)
Hayes, J. D. , and McLellan, L. I. (1999). Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defense against oxidative stress. Free Radic. Res. 31, 273–300.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Illera, M. J. , Lorenzo, P. L. , Illera, J. C. , and Petters, R. M. (1998). Developmental competence of immature pig oocytes under the influence of EGF, IGF-I, follicular fluid and gonadotropins during IVM-IVF processes. Int. J. Dev. Biol. 42, 1169–1172.
| PubMed |
![](../../media/share/OpenURL_Image.gif)
Issels, R. D. , Nagele, A. , Eckert, K. G. , and Wilmanns, W. (1988). Promotion of cystine uptake and its utilization for glutathione biosynthesis induced by cysteamine and N-acetylcysteine. Biochem. Pharmacol. 37, 881–888.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Jocelyn, P. C. (1959). Glutathione metabolism in animals. Biochem. Soc. Symp. 17, 43–65.
| PubMed |
![](../../media/share/OpenURL_Image.gif)
Laurincik, J. , Rath, D. , and Niemann, H. (1994). Differences in pronucleus formation and first cleavage following in vitro fertilization between pig oocytes matured in vivo and in vitro. J. Reprod. Fertil. 102, 277–284.
| PubMed |
![](../../media/share/OpenURL_Image.gif)
Liu, L. , Dai, Y. , and Moor, R. M. (1997). Role of secreted proteins and gonadotrophins in promoting full maturation of porcine oocytes in vitro. Mol. Reprod. Dev. 47, 191–199.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
McCord, J. M. , and Fridovich, I. (1969). Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049–6055.
| PubMed |
![](../../media/share/OpenURL_Image.gif)
Meister, A. , and Tate, S. S. (1976). Glutathione and the related γ-glutamyl compounds: biosynthesis and utilization. Annu. Rev. Biochem. 45, 559–604.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Misra, H. P. , and Fridovich, I. (1972). The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247, 3170–3175.
| PubMed |
![](../../media/share/OpenURL_Image.gif)
Nagai, T. (1996). In vitro maturation and fertilization of pig oocytes. Anim. Reprod. Sci. 42, 153–163.
| Crossref | GoogleScholarGoogle Scholar |
![](../../media/share/OpenURL_Image.gif)
Nagai, T. (2001). The improvement of in vitro maturation systems for bovine and porcine oocytes. Theriogenology 55, 1291–1301.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Nakata, K. , Kawase, M. , Ogino, S. , Kinoshita, C. , Murata, H. , Sakaue, T. , Ogata, K. , and Ohmori, S. (1996). Effects of age on levels of cysteine, glutathione and related enzyme activities in livers of mice and rats and an attempt to replenish hepatic glutathione levels of mouse with cysteine derivatives. Mech. Ageing Dev. 90, 195–207.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Nicholls, D. G. , and Budd, S. L. (2000). Mitochondria and neuronal survival. Physiol. Rev. 80, 315–360.
| PubMed |
![](../../media/share/OpenURL_Image.gif)
Nishikimi, A. , Mukai, J. , and Yamada, M. (1999). Nuclear translocation of nuclear factor kappa B in early 1-cell mouse embryos. Biol. Reprod. 60, 1536–1541.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Petters, R. M. , and Wells, C. D. (1993). Culture of pig embryos. J. Reprod. Fertil. Suppl. 48, 61–73.
| PubMed |
![](../../media/share/OpenURL_Image.gif)
Sawai, K. , Funahashi, H. , and Niwa, K. (1997). Stage-specific requirement of cysteine during in vitro maturation of porcine oocytes for glutathione synthesis associated with male pronuclear formation. Biol. Reprod. 57, 1–6.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Schulz, J. B. , Lindenau, J. , Seyfried, J. , and Dichgans, J. (2000). Glutathione, oxidative stress and neurodegneration. Eur. J. Biochem. 267, 4904–4911.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Shao, D. Z. , Lee, J. J. , Huang, W. T. , Liao, J. F. , and Lin, M. T. (2004). Inhibition of nuclear factor-kappa B prevents staphylococcal enterotoxin A-induced fever. Mol. Cell. Biochem. 262, 177–185.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Tatemoto, H. , Sakurai, N. , and Muto, N. (2000). Protection of porcine oocytes against apoptotic cell death caused by oxidative stress during in vitro maturation: role of cumulus cells. Biol. Reprod. 63, 805–810.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Tatemoto, H. , Ootaki, K. , Shigeta, K. , and Muto, N. (2001). 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. 65, 1800–1806.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Tatemoto, H. , Muto, N. , Sunagawa, I. , Shinjo, A. , and Nakada, T. (2004). Protection of porcine oocytes against cell damage caused by oxidative stress during in vitro maturation: role of superoxide dismutase activity in porcine follicular fluid. Biol. Reprod. 71, 1150–1157.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Werz, O. , Szellas, D. , Henseler, M. , and Steinhilber, D. (1998). Nonredox 5-lipoxygenase inhibitors require glutathione peroxidase for efficient inhibition of 5-lipoxygenase activity. Mol. Pharmacol. 54, 445–451.
| PubMed |
![](../../media/share/OpenURL_Image.gif)
Whitaker, B. D. , and Knight, J. W. (2004). Exogenous γ-glutamyl cycle compounds supplemented to in vitro maturation medium influence in vitro fertilization, culture, and viability of porcine oocytes and embryos. Theriogenology 62, 311–322.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Wu, W. , Goldstein, G. , Adams, C. , Matthews, R. H. , and Ercal, N. (2006). Separation and quantification of N-acetyl-l-cysteine and N-acetyl-cysteine-amide by HPLC with fluorescence detection. Biomed. Chromatogr. 20, 415–422.
| Crossref | l-cysteine and N-acetyl-cysteine-amide by HPLC with fluorescence detection.&journal=Biomed. Chromatogr.&volume=20&pages=415-422&publication_year=2006&author=W%2E%20Wu&hl=en&doi=10.1002/BMC.583" target="_blank" rel="nofollow noopener noreferrer" class="reftools">GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Xia, P. , Tekpetey, F. R. , and Armstrong, D. T. (1994). Effect of IGF-I on pig oocyte maturation, fertilization, and early embryonic development in vitro, and on granulosa and cumulus cell biosynthetic activity. Mol. Reprod. Dev. 38, 373–379.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Xu, D. X. , Chen, Y. H. , Wang, H. , Zhao, L. , Wang, J. P. , and Wei, W. (2005). Effect of N-acetylcysteine on lipopolysaccharide-induced intra-uterine fetal death and intra-uterine growth retardation in mice. Toxicol. Sci. 88, 525–533.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Yamauchi, N. , and Nagai, T. (1999). Male pronuclear formation in denuded porcine oocytes after in vitro maturation in the presence of cysteamine. Biol. Reprod. 61, 828–833.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Yang, J. G. , Chen, W. Y. , and Li, P. S. (1999). Effects of glucocorticoids on maturation of pig oocytes and their subsequent fertilizing capacity in vitro. Biol. Reprod. 60, 929–936.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)
Yoshida, M. , Ishigaki, K. , Nagai, T. , Chikyu, M. , and Pursel, V. G. (1993). Glutathione concentration during maturation and after fertilization in pig oocytes relevance to the ability of oocytes to form male pronucleus. Biol. Reprod. 49, 89–94.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
![](../../media/share/OpenURL_Image.gif)