Korean Journal of Animal Reproduction (한국가축번식학회지)

The Korean Society of Animal Reproduction (한국동물번식학회)
권호 표지

Effects of $\beta$-Mercaptoethanol on lipid Peroxidation and Fertilization Ability In Vitro by Xanthine-Xanthine Oxidase System in Pig

Xanthine-Xanthine Oxidase System,하에서 돼지 동결-융해정자의 Lipid Peroxidation과 체외수정능력에 대한 $\beta$-Mercaptoethanol의 영향

  • 사수진 (강원대학교 동물자원과학대학) ;
  • 정희태 (강원대학교 동물자원과학대학) ;
  • 이장희 (축산기술연구소) ;
  • 유일선 (축산기술연구소) ;
  • 양부근 (강원대학교 동물자원과학대학) ;
  • 김정익 (강원대학교 동물자원과학대학) ;
  • 박춘근 (강원대학교 동물자원과학대학)
  • Published : 2002.09.01
Download PDF
⟨ Previous Next ⟩

Abstract

This study was undertaken to evaluate the effects of $\beta$-mercaptoethanol ($\beta$-ME) on lipid peroxidation and fertilization ability in vitro by xanthine (X) - xanthine oxidase (XO) system in boar spermatozoa frozen-thawed. The boar spermatozoa were treated with X and/or XO, and the spermatozoa viability were measured by the eosin-nigrosin stain method. In control group, level of vitality in boar spermatozoa were higher than in medium with X, XO and X+XO groups. No significant differences, however, were observed under the all conditions. The percentage of spermatozoa that reached acrosome reaction were significantly (P<0.05) higher in sperm treated without that than with $\beta$-ME under the all conditions. On the other hand, when spermatozoa were inseminated in medium with X and/or XO, the penetration rates in all conditions were higher in medium with that than without $\beta$-ME. However, significant differences were not observed between medium with and without $\beta$-ME. The lipid peroxidation of sperm was evaluated on the basis of malondialdehyde (MDA) production. The MDA were higher in sperm treated without that than with $\beta$-ME under the above all conditions. However, significant differences were not observed between medium with and without $\beta$-ME. Sperm-SH group were higher detected in medium with that than without $\beta$-ME under the all conditions. The activity of sperm binding to Bona pellucida was also evaluated through binding to salt-stored porcine oocytes. In control group, sperm binding to zona pellucida were significantly (P<0.05) higher than in medium with X+XO groups. The sperm binding in all conditions were higher in medium with that than without $\beta$-ME. However, significant differences were not observed between medium with and without $\beta$-ME. These results suggest that addition of $\beta$-ME in X-XO system may play a positive role in improving of fertilization ability in vitro.

본 연구는 xanthine-xanthine oxidase system하에서 돼지 동결-융해 정자의 lipid peroxidation과 체외수정능력에 대한 $\beta$-mercaptoethanol ($\beta$-ME)의 영향을 검토하였다. 그 결과 돼지 동결-응해 정자가 X-XO system하에서 처리되었을 때, control구에서 높은 정자생존율이 관찰되었으나 처리구간의 유의차는 인정되지 않았다. 또한 첨체반응이 유기된 정자의 비율은 모든 처리구에서 $\beta$-ME 첨가시 보다 무첨가시가 유의적(P<0.05)으로 더 높았다. 한편, X-XO system하에서 체외수정시 난자에 대한 정자의 침입율은 모든 조건하에서 $\beta$-ME 첨가시가 무첨가시 보다 높은 경향을 나타냈지만, 유의적인 차이는 인정되지 않았다. 정자의 lipid peroxidation은 malondialdehyde (MDA)의 생성에 기초를 두고 평가하였는데, 모든 조건하에서 $\beta$-ME 첨가시 보다 무첨가시에 MDA의 생성이 높게 나타났지만, 유의적인 차이는 인정되지 않았다. 또한 동결-융해된 정자의 sulfhydryl (-SH) group의 함량을 측정한 결과 모든 처리구에서 $\beta$-ME 무첨가시 보다 첨가시에 높은 함량이 측정되었지만, 유의적인 차이는 인정되지 않았다. 한편, 체외에서 성숙시킨 난자의 투명대에 대한 동결-융해 정자의 접착 정도를 평가한 결과 모든 처리구에서 $\beta$-ME 첨가시 무 첨가시에 비해 다소 높은 경향을 보였으며, Control group의 경우 X+XO group에 비해 유의적(P<0.05)으로 높은 정자접착율이 관찰되었다 그렇지만, $\beta$-ME 첨가 유무에 따를 유의적인 차이는 인정되지 않았다. 된 연구의 결과는 X-XO system하에서 $\beta$-ME 첨가가 돼지에서의 체외수정능력 향상에 영향을 미치는 것으로 생각된다.

Keywords

References

  1. Aitken, R. J. 1994. A free radical theory of male infertility. Reprod. Fertil. Dev., 6:19-24 https://doi.org/10.1071/RD9940019
  2. Aitken, R. J. and Clarkson, J. S. 1987. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J. Reprod. Fertil., 81:459-469 https://doi.org/10.1530/jrf.0.0810459
  3. Aitken, R. J., Clarkson, J. S. and Fishel, S. 1989. Generation of reactive oxygen species, lipid peroxidation and human sperm function. Biol. Reprod., 40:183-197
  4. Aitken, R. J. and Fisher, H. 1994. Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bioassays, 16:259-267 https://doi.org/10.1002/bies.950160409
  5. Aitken, R. J., Harkiss, D. and Buckingham, D. W. 1993. Analysis of lipid peroxidation mechanism in human spermatozoa. Mol. Reprod. Dev., 35: 302-315 https://doi.org/10.1002/mrd.1080350313
  6. Aitken, R. J., Irvine, D. S. and Wu, F. C. 1991. Prospective analysis of sperm-oocyete fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. Am. J. Obstet. Genecol., 164:542-551 https://doi.org/10.1016/S0002-9378(11)80017-7
  7. Alvarez, R. J. and Storey, B. T. 1989. Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Gamete Res. 23:77-90 https://doi.org/10.1002/mrd.1120230108
  8. Baumber, J., Ball, B. A., Gravance, C. G., Medina, V. and Davies-Morel, M. C. G. 2000. The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. J. Andro., 21:895-902
  9. Bilodeau, J. F., Blanchette, S., Gagnon, C. and Sirard, M. A. 2001. Thiols prevent $H_{2}0_{2}$-mediated loss of sperm motility in cryopreserved bull semen. Theriogenology, 56:275-286 https://doi.org/10.1016/S0093-691X(01)00562-3
  10. Block, E. R. 1991. Hydrogen peroxide alters the physical state and function of the plasma membrane of pulmonary artery endotherial cells. J. Cell. Physiol., 146:362-369 https://doi.org/10.1002/jcp.1041460305
  11. de Lamirande, E. and Gagnon, C. 1992a. Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes. J. Androl., 13:368-378
  12. de Lamirande, E. and Gagnon, C. 1992b. Reactive oxygen species and human spermatozoa. II. Depletion of adenosine triphosphate plays an important role in the inhibition of sperm motility. J. Androl., 13:379-386
  13. de Lamirande, E. and Gagnon, C. 1995. Capacitation associated production of superoxide anion by human spermatozoa. Free Radic. Biol., 18:487-495 https://doi.org/10.1016/0891-5849(94)00169-K
  14. de Lamirande, E., Jiang, H., Zini, A., Kodama, H. and Gagnon, C. 1997. Reactive oxygen species and sperm physiology. Rev. Reprod., 2:48-54
  15. DasGupta, S., Mills, C. L. and Fraser, L. R. 1993. $Ca^{2+}$-related changes in the capacitation state of human spermatozoa assessed by a chlortetracycline fluorescence assay. J. Reprod. Fertil., 99:135-143 https://doi.org/10.1530/jrf.0.0990135
  16. Di-Simplicio, P., Cheeseman, K. H. and Slater, T. F. 1991. The reactivity of the SH group of bovine serum albumin with free radicals. Free Radical Res. Commun., 14:253-262 https://doi.org/10.3109/10715769109088954
  17. Green, C. M., Cockle, S. M., Watson, P. F. and Fraser, L. R. 1996. Fertilization promoting peptide, a tripeptide similar to thyrotrophin-releasing hormone, stimulates the capacitation and fertilizing ability of human spermatozoa in vitro. Human Reprod., 11:830-836 https://doi.org/10.1093/oxfordjournals.humrep.a019262
  18. Hecht, D. and Zick, Y. 1992. Selective inhibition of protein tyrosine phosphatase activities by $H_{2}0_{2}$ and vanadate in vitro. Biochim. Biophys. Res. Comm., 189:773-779
  19. Hsu, P. C., Hsu, C. C. and Guo, Y. L. 1999. Hydrogen peroxide induces premature acrosome reaction in rat sperm and reduces their penetration of the zona pellucida. 139:93-101
  20. Hsu, P. C., Liu, M. Y., Hsu, C. C., Chen, L. Y. and Guo, Y. L. 1997. Lead exposure causes generation of reactive oxygen species and functional impairment in rat sperm. Toxicology, 122:133-143 https://doi.org/10.1016/S0300-483X(97)00090-5
  21. Hsu, P. C., Liu, M. Y., Hsu, C. C., Chen, L. Y. and Guo, Y. L. 1998. Effects of vitamin E and/or C on reactive oxygen species-related lead toxicity in the rat sperm. Toxicology, 128: 169-179 https://doi.org/10.1016/S0300-483X(98)00068-7
  22. Ikeda, M., Kodama, H., Fukuda, J., Shimizu, V,. Murata, M., Kumagai, J. and Tanaka, T. 1999. Role of radical oxygen species in rat testicular germ cell apoptosis induced by heat stress. Biol. Reprod., 61:393-399 https://doi.org/10.1095/biolreprod61.2.393
  23. Iwasaki, A. and Gagnon, C. 1992. Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil. Steril., 57:409-416 https://doi.org/10.1016/S0015-0282(16)54855-9
  24. Kaul, G., Singh, S., Gandhi, K. K. and Anand, S. R. 1997. Calcium requirement and time course of capacitation of goat spermatozoa assessed by chlortetracycline assay. Andrologia, 29:243-251 https://doi.org/10.1111/j.1439-0272.1997.tb00478.x
  25. Kodama, H., Kuribayashi, Y. and Gagnon, C. 1996. Effect of spenn lipid peroxidation on fertilization. J. Androl., 17:151-157
  26. Lim, J. G., Kim, N. H., Lee, H. T. and Chung, K. S. 1997. Effects of extracellular potassium concentration on acrosome reaction and polyspermy during in vitro fertilization and subsequent development in vitro in the pig. Theriogenology, 48:843-851 https://doi.org/10.1016/S0093-691X(97)00306-3
  27. Marnmoto, A., Masumoto, N., Tahara, M., Yoneda, M., Nishizaki, T., Tasaka. K. and Miyake, A. 1996. Involvement of a sperm protein sensitive to sulfhydryl-depleting reagents in mouse sperm-egg fusion. Mol. Reprod. Dev. (in press)
  28. Myles, D. G. 1993. Molecular mechanisms of sperm-egg membrane binding and fusion in mammalians. Dev. Biol., 158:35-45 https://doi.org/10.1006/dbio.1993.1166
  29. O'Flaherty, C., Beconi, M. and Beorlegui, N. 1997. Effect of natural antioxidants, superoxide dismutase and hydrogen peroxide on capacitation of frozen-thawed bull spermatozoa. 29: 269-275. https://doi.org/10.1111/j.1439-0272.1997.tb00481.x
  30. O'Flaherty, C., Beorlegui, N. and Beconi, M. 1999. Reactive oxigen species requirements for bovine sperm capacitation and acrosome reaction. Theriogenology, 52:289-301 https://doi.org/10.1016/S0093-691X(99)00129-6
  31. Perez, L. J., Valcarcel, A., de las Heras, M. A., Moses, D. F. and Baldassarre, H. 1997. In vitro capacitation and induction of acrosomal exocytosis in ram spermatozoa as assessed by chlortetracycline assay. Theriogenology, 45:1037-1046
  32. Perry, R. L., Naeeni, M., Barratt C. L. R., Warren, M. A. and Cooke, I. D. 1995. A time course study of capacitation and the acrosome reaction in human spermatozoa using a revised chlortetracycline pattern classification. Fertil. Steril., 64:150-159
  33. Snyder, L. M., Fortier, N. L., Leb, L., McKenney, J., Trainor, J., Sheerin, H. and Mohandas, N. 1988. The role of membrane protein sulfhydryl groups in hydrogen peroxide-membrane damage in human erythrocytes. Biochim. Biophys. Acta., 937:229-240 https://doi.org/10.1016/0005-2736(88)90245-3
  34. Takahashi, M., Nagai, T., Okamura, N., Takahashi, H. and Okano, A. 2002. Promoting effect of ${\beta}$-mercaptoethanol on in vitro development under oxidative stress and cystine uptake of bovine embryos. Biol. Reprod., 6:562-567
  35. Ward, C. R. and Storey, B. T. 1984. Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay. Dev. Biol., 104:287-296 https://doi.org/10.1016/0012-1606(84)90084-8