Effects of superoxide dismutase on mouse in vitro fertilization and embryo culture system

  • Takafumi Nonogaki
  • Yoichi Noda
  • Katsuhiko Narimoto
  • Yoh Umaoka
  • Takahide Mori
Animal Investigations


We recently found that, for mouse embryos fertilized in vivo, the two-cell block could be attenuated by adding superoxide dismutase (SOD), a scavenger of superoxide radicals, to the culture medium. In this study, we evaluated the effects of SOD on the process of fertilization and on the further development of the embryos fertilized in vitro.


We performed incubation of mouse epididymal spermatozoa, in vitro fertilization, and further cultivation in Biggers—Whitten—Whittingham's medium supplemented with various concentrations of Cu · Zn—SOD.


High concentrations (2000 µg/ml or more) of SOD prevented loss of motility in mouse sperm over time. The addition of SOD (less than 2000 µg/ml) to the basic medium showed no significant difference in the fertilization rate. Also, no significant difference was observed in the rate of polyspermy or parthenogenesis between the basic and the SOD-supplemented media. However, 18% of the two-cell-stage embryos developed to the expanded blastocyst stage in the 500 µg/ml SOD-supplemented medium, while no blastocysts were found in the basic medium. Furthermore, the addition of SOD 7 hr after insemination increased the expanded blastocyst rate (28%).


These results indicate that the addition of SOD exerts a protecting effect from oxidative stress both on sperm viability and on the development of embryos fertilized in vitro as well as in vivo, while its addition showed no effect on the process of fertilization.

Key words

superoxide dismutase oxidative stress sperm motility mouse in vitro fertilization two-cell block 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Wright RW Jr, Bondioli KR: Aspects of in vitro fertilization and embryo culture in domestic animals. J Anim Sci 1981;53:702–729PubMedGoogle Scholar
  2. 2.
    Cole RJ, Paul J: Properties of cultured preimplantation mouse and rabbit embryos, and cell strains derived from them.In Preimplantation Stages of Pregnancy, GEW Wolstenholme, MW O'Connor (eds). London, Churchill, 1965, pp 82–122Google Scholar
  3. 3.
    Yanagimachi R, Chang MC: In vitro fertilization of golden hamster ova. J Exp Zool 1964;156:361–376PubMedGoogle Scholar
  4. 4.
    Whittingham DG: Fertilization, early development and storage of mammalian ova in vitro.In The Early Development of Mammals, B Balls, AE Wild (eds). Cambridge, Cambridge University Press, 1975, pp 1–24Google Scholar
  5. 5.
    Davis DL, Day BN: Cleavage and blastocyst formation by pig eggs in vitro. J Anim Sci 1978;46:1043–1053PubMedGoogle Scholar
  6. 6.
    Gandolfi F, Moor RM: Stimulation of early embryonic development in the sheep by co-culture with oviduct epithelial cells. J Reprod Fertil 1987;81:23–28PubMedGoogle Scholar
  7. 7.
    Bishop DW: Oxygen concentration in the rabbit genital tract.In Proceedings of the Third International Congress of Animal Reproduction, Section I, Cambridge, England, 1956, pp 53–58Google Scholar
  8. 8.
    Mastroianni L Jr, Jones R: Oxygen tension within the rabbit fallopian tube. J Reprod Fertil 1965;9:99–102Google Scholar
  9. 9.
    Maas DHA, Storey BT, Mastroianni L Jr: Oxygen tension in the oviduct of the rhesus monkey (Macaca mulatta). Fertil Steril 1976;27:1312–1317PubMedGoogle Scholar
  10. 10.
    Whitten WK: Nutrient requirements for the culture of preimplantation embryos in vitro.In Advances in the Biosciences 6, G Raspe (ed). Oxford, Pergamon Press, 1970, pp 129–141Google Scholar
  11. 11.
    Quinn P, Harlow GM: The effect of oxygen on the development of preimplantation mouse embryos in vitro. J Exp Zool 1978;206:73–80PubMedGoogle Scholar
  12. 12.
    Tervit HR, Whittingham DG, Rowson LEA: Successful culture in vitro of sheep and cattle ova. Nature 1972;179:1081–1082Google Scholar
  13. 13.
    McCord J, Fridovich I: Superoxide dismutase. J Biol Chem 1969;244:6049–6055PubMedGoogle Scholar
  14. 14.
    McCord JM, Keel BB, Fridovich I: An enzyme-based theory of obligate anoerabiosis: The physiological function of superoxide dismutase. Proc Natl Acad Sci USA 1971;68:1024–1027PubMedGoogle Scholar
  15. 15.
    Fridovich I: Superoxide radical and superoxide dismutase. Accts Chem Res 1972;5:321–326Google Scholar
  16. 16.
    Noda Y, Matsumoto H, Mori T: Superoxide dismutase overcomes 2-cell block in mouse embryos. Acta Obstet Gynaecol Jpn 1989;41:751–752Google Scholar
  17. 17.
    Noda Y, Matsumoto H, Umaoka Y, Tatsumi K, Kishi J, Mori T: Involvement of superoxide radicals in the mouse 2-cell block. Mol Reprod Dev 1991;28:356–360PubMedGoogle Scholar
  18. 18.
    Umaoka Y, Noda Y, Matsumoto H, Mori T: The effects of oxygen toxicity on early development of mouse embryo. Jpn J Fertil Steril 1990;35:285–292Google Scholar
  19. 19.
    Nasr-Esfahani MH, Aitken JR, Johnson MH: Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developedin vitro orin vivo. Development 1990;109:501–507PubMedGoogle Scholar
  20. 20.
    Nasr-Esfahani M, Johnson MH, Aitken JR: The effect of iron and iron chelators on the in-vitro block to development of the mouse preimplantation embryo: BAT6 a new medium for improved culture of mouse embryosin vitro. Hum Reprod 1990;5:997–1003PubMedGoogle Scholar
  21. 21.
    Nasr-Esfahani MH, Johnson MH: The origin of reactive oxygen species in mouse embryos culturedin vitro. Development 1991;113:551–560PubMedGoogle Scholar
  22. 22.
    Legge M, Sellens MH: Free radical scavengers ameliorate the 2-cell block in mouse embryo culture. Hum Reprod 1991;6:867–871PubMedGoogle Scholar
  23. 23.
    Biggers JD, Whitten WK, Whittingham DG: The culture of mouse embryos in vitro.In Methods in Mammalian Embryology, JC Daniel Jr (ed). San Francisco, Freeman, 1971, pp 86–116Google Scholar
  24. 24.
    Jones R, Mann T: Damage to ram spermatozoa by peroxidation of endogeneous phospholipids. J Reprod Fertil 1977;50:261–268PubMedGoogle Scholar
  25. 25.
    Jones R, Mann T: Toxicity of exogenous fatty acid peroxides towards spermatozoa. J Reprod Fertil 1977;50:255–260PubMedGoogle Scholar
  26. 26.
    Alvarez JG, Storey BT: Spontaneous lipid peroxidation in rabbit epididymal spermatozoa: Its effect on sperm motility. Biol Reprod 1982;27:1102–1108PubMedGoogle Scholar
  27. 27.
    Miyazaki T: Adverse effects of oxygen radicals on human spermatozoal motility—Lipid peroxides and superoxide dismutase in human spermatozoa and seminal plasma. Jpn J Fertil 1988;33:105–113Google Scholar
  28. 28.
    Mannella MR and Jones R: Properties of spermatozoal superoxide dismutase and lack of involvement of superoxides in metal-ion catalysed lipid-peroxidation reacions in semen. Biochem J 1980;191–297Google Scholar
  29. 29.
    Aitken RJ, Clarkson JS: Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil 1987;81:459–469PubMedGoogle Scholar
  30. 30.
    Wolf DP: The mammalian egg's block to polyspermy.In Fertilization and Embryonic Development in Vitro, L Mastroianni Jr, JD Biggers (eds). New York, Plenum Press, 1981, pp 183–197Google Scholar
  31. 31.
    Coburn M, Schudl H, Troll W: A hydrogen peroxide block to polyspermy in the sea urchin Arbacia punctulata. Dev Biol 1981;84:235PubMedGoogle Scholar
  32. 32.
    Foerder CA, Shapiro BM: Release of ovoperoxidase from sea urchin eggs hardens the fertilization membrane with tyrosine crosslinks. Proc Natl Acad Sci USA 1977;74(10):4214–4218PubMedGoogle Scholar
  33. 33.
    Nonogaki T, Noda Y, Narimoto K, Umaoka Y, Mori T: Protection from oxidative stress by thioredoxin and superoxide dismutase of mouse embryos fertilizedin vitro. Hum Reprod 1991;6:1305–1310PubMedGoogle Scholar
  34. 34.
    Umaoka Y, Noda Y, Mori T: The effects of SOD added low oxygen culture on mouse embryos after the first cell cycle. Jpn J Fertil Steril 1990;35:469–474Google Scholar

Copyright information

© Plenum Publishing Corporation 1992

Authors and Affiliations

  • Takafumi Nonogaki
    • 1
  • Yoichi Noda
    • 1
  • Katsuhiko Narimoto
    • 1
  • Yoh Umaoka
    • 1
  • Takahide Mori
    • 1
  1. 1.Department of Gynecology and Obstetrics, Faculty of MedicineKyoto UniversityKyotoJapan

Personalised recommendations