Effect of β-Mercaptoethanol Supplement during In Vitro Maturation on IVM, IVF and Glutathione Level in Porcine Oocytes

돼지 미성숙 난포란의 체외성숙 시 β-mercaptoethanol의 첨가가 체외성숙, 체외수정 및 Glutathione 수준에 미치는 영향

  • Oh, Shin-Ae (Department of Animal Science & Technology, College of Industrial Sciences, Chung-Ang University) ;
  • Kim, Chang-Keun (Department of Animal Science & Technology, College of Industrial Sciences, Chung-Ang University) ;
  • Chung, Yung-Chai (Department of Animal Science & Technology, College of Industrial Sciences, Chung-Ang University) ;
  • Chang, Yoo-Min (Department of Animal Science & Technology, College of Industrial Sciences, Chung-Ang University) ;
  • Pang, Myung-Geol (Department of Animal Science & Technology, College of Industrial Sciences, Chung-Ang University)
  • 오신애 (중앙대학교 산업과학대학 동물자원과학과) ;
  • 김창근 (중앙대학교 산업과학대학 동물자원과학과) ;
  • 정영채 (중앙대학교 산업과학대학 동물자원과학과) ;
  • 장유민 (중앙대학교 산업과학대학 동물자원과학과) ;
  • 방명걸 (중앙대학교 산업과학대학 동물자원과학과)
  • Published : 2005.06.30


Experiments were conducted to determine the effects of beta-mercaptoethanol ($\beta$-ME) supplements to the in vitro maturation (IVM) medium on in vitro fertilization (IVF) and intracellular glutathione (GSH) concentration. Porcine cumulus-intact oocytes were matured in TCM-I99 medium containing porcine follicular fluid, sodium pyruvate, D-glucose, FBS, hormonal supplements, and $\beta$-ME (0, 25, 50 and 100 ${\mu}$M) for 36 to 46h. After culture, cumulus-free matured oocytes were co-incubated with epididymal spermatozoa for 18h. There were no significant differences in the maturation rate among treatment groups. However, increases (P < 0.05) in intracellular GSH concentration before and after. fertilization were observed in 50 ${\mu}$M $\beta$-ME supplements to the IVM medium. Also, increases (P < 0.05) in male pronuclear formation after IVF were observed in same treatment group. In conclusion, supplementing $\beta$-ME into the IVM medium increased intracellular GSH concentrations and increased fertilization in vitro.


$\beta$-Mercaptoethanol;Glutathione;IVM;IVF;Porcine oocyte


  1. Abeydeera, L. R. 2002. In vitro production of embryos in swine. Theriogenology 57:256-73
  2. Abeydeera, L. R., Wang, W. H., Cantley, T. C., Prather, R. S. and Day, B. N. 1998. Presence of ${\beta}-mercaptoethanol$ can increase the glutathione content of pig oocytes matured in vitro and the rate of blastocyst development after in vitro fertilization. Theriogenology 50:747-56
  3. Abeydeera, L. R., Wang, W. H., Cantley, T. C., Prather, R. S. and Day, B. N. 1999. Glutathione content and embryo development after in vitro fertilisation of pig oocytes matured in the presence of a thiol compound and various concentrations of cysteine. Zygote 7:203-10
  4. Caamano, J. N., Ryoo, Z. Y., Thomas, J. A. and Youngs, C. R. 1996. ${\beta}-mercaptoethanol$ enhances blastocyst formation rate of bovine in vitro maturated / in vitro fertilizaed embryos. Biol. Reprod. 55: 1179-84
  5. Corsby, I. M., Gandolfi, F. and Moor, R. M. 1998. Control of protein synthesis during cleavage of sheep embryos. J. Reprod. Fertil. 82:769-75
  6. de Matos, D. G., Fumus, C. C., Moses, D. F. and Baldassarre, H. 1995. Effect of cysteamine on glutathione level and developmental capacity of bovine oocyte matured in vitro. Mol. Reprod. Dev. 42:432-6
  7. de Matos, D. G., Furnus, C. C., Moses, D. F., Martinez, A. G. and Matkovic, M. 1996. Stimulation of glutathione synthesis of in vitro matured boovine oocytes and it's effect on embryo development and freezability. Mol. Reprod. Dev. 45:451-7<451::AID-MRD7>3.0.CO;2-Q
  8. de Matos, D. G., Furnus, C. C. and Moses, D. F. 1997. Gluta-taurine synthesis during in vitro maturation of bovine oocytes: Role of cumulus cells. Biol. Reprod. 57:1420-5
  9. Gardiner, C. S. and Donale, D. J. 1995. Synthesis of glutathione in the preimplantation mouse embryo. Arch. Biochem. Biophy. Vol. 318, 1:30-6
  10. 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: 173-8
  11. Halliwell, B. and Gutteridge, J. M. C. 1992. Free radicals in biology and medicine. Clarendin Press Oxford
  12. Kito, S. and Bavister, B. D. 1997. Male pronuclear formation and early embryonic development of hamster oocytes matured in vitro with gonadotropin, amino acid and cysteamine. J. Reprod. Fertil. 110:35-46
  13. Lafleur, M. V., Hoorweg, J. J., Joenje, H., Westmijze, E. J. and Retel, J. 1994. The ambivalent role of glutathione in the protection of DNA against singlet oxygen. Free Radic. Res. 21:9-17
  14. Li, J. and Foote, R. H. 1993. Culture of rabbit zygotes into blastocysts in protein-free medium with one to twenty percent oxygen. J. Reprod. Fertil. 98:163-7
  15. Nasr-Esfahani, M. H and Johnsin, M. H. 1991. Origin of reactive oxygen species in mouse embryos cultured in vitro. Development 113:551-60
  16. Perreault, S. D., Barbee, R. R., Elstein, K. H., Zucker, R. M. and Keefer, C. L. 1988. Interspecies differences in the stability of mammalian sperm nuclei assessed in vivo by sperm microinjection and in vitro by flow cytometry. Biol. Reprod. 39: 157-67
  17. 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
  18. Schoenbeck, R. A., Peters, M. S., Rickords, L. F., Stumpf, T. T. and Prather, R. S. 1992. Characterization of deoxyribonucleic acid synthesis and the transition from maternal to embryonic control in the 4-cell porcine embryo. Biol Reprod. 47:1118-25
  19. Takahashi, M., Nagai, T., Hamano, S., Kuwayama, M., Okamura, N. and Okano, A. 1993. Effect of thiol compounds on in vitro development and intracellular glutathione content of bovine embryos. Biol. Reprod. 49:228-32
  20. Wang, W. H. and Day, B. N. 2002. Development of porcine embryos produced by IVM/IVF in a medium with or without protein supplementation: effects of extracellular glutathione. Zygote 10:109-15
  21. Yoshida, M., Ishizaki Y., Kawagishi H., Bamba, K. and Kojima, Y. 1992. Effect of pig follicular fluid on maturaion of pig oocytes in vitro and on their subsequent fertilizing and developmental capacity in vitro. J. Biol. Reprod. 95:481-8
  22. Yoshida, M. 1993. Role of glutathione in the maturation and fertilization of pig oocytes in vitro. Mol. Reprod. Dev. 35:76-81
  23. 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
  24. Zuelke, K. A., Jeffay, S. C., Zucker, R. M. and Perreault, S. D. 2003. Glutathione (GSH) concentrations vary with the cell cycle in maturing hamster oocytes, zygotes, and pre-implantation stage embryos. Mol. Reprod. Dev. 64:106-12
  25. 양부근, 박동헌, 우문수, 정희태, 박춘근, 김종복, 김정익. 1997. Thiol 화합물과 항산화제 첨가배양이 소 체외수정란의 체외발육과 세포내 Glutathione 농도 변화에 미치는 효과; II. 항산화제 첨가와 체세포 공동배양이 소 체외수정란의 체외발육과 세포내 glutathione 농도변화에 미치는 영향. 한국가축번식학회지. 21:345-53
  26. 장현용, 오진영, 김종택, 박춘근, 정희태, 김정익, 이학교, 최강덕, 양부근. 2004. 돼지 체외수정란의 체외발육에 있어 항산화제의 효과. 한국동물번식학회지. 28:77-82
  27. 한만희, 박병권, 박창식, 서길웅, 이규승, 1998. ${\beta}-mercaptoethanol$ 및 cysteine이 돼지 미성숙난포란의 체외성숙에 미치는 영향. 한국가축번식학회지. 22:376-83
  28. Jarrell, V. L., Day, B. N. and Prather, R. S. 1991. The transition from maternal to zygotic control of development occurs during the 4-cell stage in the domestic pig, Sus scrofa: quantitative and qualitative aspects of protein synthesis. Biol. Reprod. 44:62-8
  29. Whitake, B. D. and Knight, J. W. 2004. Exgenous ${gamma}-glutamyl$ cycle compounds supplemented to in vitro maturation medium influence in vitro fertilization, culture and viability parameters of porcine oocytes and embryos. Theriogenology 62:311-22
  30. Mattioli, M., Bacci, M. L., Galeuti, G. and Seren, E. 1989. Developmental competence of pig oocytes matured and fertilized in vitro. Theriogenology. 31:1201-8