Korean Journal of Animal Reproduction (한국가축번식학회지)

The Korean Society of Animal Reproduction (한국동물번식학회)
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Effect of Cysteamine on In Vitro Maturation of Porcine Oocytes and Development of Porcine IVM/IVF Embryos

Cysteamine의 첨가배양이 돼지 난포란의 체외성숙과 배발달에 미치는 영향

  • 이경본 (충남대학교 농업생명과학대학 동물자원학부) ;
  • 한만희 (충남대학교 농업생명과학대학 동물자원학부)
  • Published : 2002.03.01
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Abstract

The present study was carried out to examine the effect of cysteamine in vitro maturation (IVM) of porcine oocytes and development of porcine IVM/IVF Embryos. The results were summarized as follows : 1. The rates of nuclear maturation, penetrated oocytes, pronuclear formation, polyspermic oocytes and mean numbers of the penetrated sperm were not different in NCSU23 maturation medium with 0, 25, 50 and 100 $\mu$M cysteamine (P〉0.05). 2. The rates of blastocyst formation at day 7 after in vitro fertilization in 0, 25, 50 and 100 $\mu$M cysteamine were 17.9$\pm$6.1, 17.4$\pm$6.3, 24.2$\pm$1.9 and 16.9$\pm$2.0%, respectively. And the total cells were 30.7$\pm$2.4, 34.9$\pm$2.8, 39.6$\pm$2.3 and 36.8$\pm$3.6, respectively. Fifty $\mu$M cystealnine group was significantly higher than those of any other treatment groups (P＜0.05). 3. The ratios of ICM/total cells in 20~40% category were 20.5, 41.6, 19.5 and 31.5%, respectively. Twenty five $\mu$M cysteamine group was higher than those of other groups. 4. The rates of blastocyst formation at day 7 in the NCSU-23 culture medium of porcine IVF-produced embryos with 0, 25, 50, and 100 $\mu$M cysteamine were 16.0$\pm$0.2, 13.6$\pm$1.7, 25.0$\pm$0.8 and 15.7$\pm$4.5%, respectively. And the total cells were 27.0$\pm$3.7, 36.1$\pm$4.8, 34.0$\pm$3.8 and 25.2$\pm$4.4, respectively. Fifty $\mu$M cysteamine group was significantly higher than those of any other treatment groups (P＜0.05). 5. The ratios of ICM/total cells in 20~40% category were 53.8, 30.0, 16.6 and 11.1%, respectively. The addition groups of cysteamine were lower than those of control group. In conclusion, these results suggested that the addition of 50 $\mu$M cysteamine in the IVM medium and 25~50 $\mu$M cysteamine in IVC medium were effective on the blastocyst formation and total cells of blastocysts.

본 연구는 돼지 난포란의 체외배양액과 배발달 배양액에 저분자량의 황화합물인 cysteamine을 각자 0, 25, 50 및 100$\mu$M 첨가배양함으로서 cysteamine이 돼지 난포란의 체외성숙과 체외배발달에 미치는 영향을 규명하고자 실시하였다. 본 연구에서 얻어진 결과를 요약하면 다음과 같다. 1. 돼지 난포란을 체외성숙배양액인 0.9mM CySH가 함유된 NCSU-23 배양액에 cysteamine을 각각 0, 25, 50 및 100$\mu$M 첨가배양하여 성숙을 유기한 다음, 체외수정을 실시한 결과, 모든 처리구에서 핵성숙률, 정자침투율, 웅성전핵형성률, 다정자침입률 및 평균침입정자수에서 유의적(P〉0.05)인 차이가 인정되지 않았다. 2. 체외수정을 실시한 후, 배발달배양액인 NCSU-23에 7일간 배양한 결과, 배반포형성률은 각각 17.9$\pm$6.1, 17.4$\pm$6.3, 24.2$\pm$l.9 및 16.9$\pm$2.0%로서 50$\mu$M의 cysteamine 처리구가 유의적(P＜0.05)으로 높은 결과를 나타냈고, 총세포수에 있어서도 각각 30.7$\pm$2.4, 34.9$\pm$2.8, 39.6$\pm$2.3 및 36.8$\pm$3.6개로서 50$\mu$M의 cysteamine 처리구가 유의적(P＜0.05)으로 높은 결과를 나타냈다. 3. 작성된 배반포기 수정란의 총세포수에 대한 내부세포괴세포(ICM/total cells)가 20~40%범주에 드는 비율은 각각 20.5, 41.6, 19.5 및 31.8로서 25$\mu$M 처리구가 가장 높은 성적을 나타냈다. 4. 체외에서 생산된 초기 수정란을 배발달배양액인 NCSU-23에 cysteamine을 각각 0, 25, 50 및 100$\mu$M 첨가하여 7일간 배양한 결과, 배반포기 도달률은 각각 16.0$\pm$0.2, 13.6$\pm$1.7, 25.0$\pm$0.8 및 15.7$\pm$4.5%로서 50$\mu$M의 cysteamine 처리구가 유의적(P＜0.05)으로 높은 결과를 나타냈고, 총세포수에 있어서는 각각 27.0$\pm$3.7, 36.1$\pm$4.8, 34.0$\pm$3.8 및 25.2$\pm$4.4개로서 25$\mu$M의 cysteamine 처리구가 유의적(P＜0.05)으로 높은 결과를 나타냈다. 5. 작성된 배반포기 수정란의 총세포수에 대한 내부세포괴세포(ICM/total cells)가 20~40% 범주에 드는 비율은 처리구가 대조구보다 낮은 결과를 나타냈다. 결론적으로 돼지난포란을 이용하여 체외성숙을 유기할 때 효과적인 cysteamine의 농도는 50$\mu$M이 적당하며, 초기배발달을 유기할 때의 효과적인 cysteamine의 농도는 25~50$\mu$M인 것으로 판단된다.

Keywords

References

  1. Abeydeera, L. R. and Day, B. N. 1997. In vitro penetration of pig oocytes in a modified tris-buffered medium: effect of BSA, caffeine and calcium. Theriogenology, 48:537-544 https://doi.org/10.1016/S0093-691X(97)00270-7
  2. Abeydeera, L. R., Wang, W. H., Canteley, T. C., Rieke, A., Murphy, C. N., Prather, R. S. and Day, B. N. 2000. Development and viability of pig oocytes matured in a protein-free medium containing eoidermal growth factor. Therioge-nology, 54:787-797 https://doi.org/10.1016/S0093-691X(00)00390-3
  3. Bing, Y. Z., Nagai, T. and Martinez, H. R. 2001. Effects of cysteamine, FSH and estra-dial-$17{\beta}$ on in vitro maturation of porcine oocytes. Theriogenology, 55:867-876 https://doi.org/10.1016/S0093-691X(01)00449-6
  4. Bing, Y. Z., Hirao, Y., Takenouchi, N., Che, L., Nakamura, H., Yodoi J. and Nagai, T. 2000. Effects of redox state on production of cattle embryos in vitro. Biol. Reprod., 62(Suppl.):319
  5. Byun, T. H., Lee, S. H. and Song, H. B. 1991. Development of a rapid staining method of the oocytes from domestics animal. Kor. J. Anim. Sci., 33;25-31
  6. Cheng, W. T. K., Polge C. and Moor, R. M. 1986. In vitro fertilization of pig and sheep oocytes. Theriogenology, 25: 146(abstr.)
  7. Dumoulin, J. C. M., Evers, J. L. H., Bras, M., Pieters, M. H. E. C. and Geraedts, 1. P. M. 1992. Positive effect of taurine on preimplanta-tion development of mouse embryos in vitro. J. Reprod. Fert., 94:373-380 https://doi.org/10.1530/jrf.0.0940373
  8. Edwards, R. G. 1965. Maturation of mouse, sheep, cow, pig, rhesus monkey, and human ovarian oocytes. Nature, 208:349-352 https://doi.org/10.1038/208349a0
  9. Franger, M. W., Hart, D. A. and Wells, J. V. 1970. Enhancement by reducing agent of the transformation of human and rabbit peripheral lymphocytes. J. Immunol., 105:1043-1045
  10. Gasparrini, B., Neglia, G., Di Palo, R., Cam-panile, G. and Zicarelli, L. 2000. Effect of cys-teamine during in vitro maturation on buffalo embryo development. Theriogenology, 54: 1537-1542
  11. Giles, J. R. and Foote, R. H. 1995. Rabbit blastocyst: allocation of cells to the inner cell mass and trophectoderm. Mol. Reprod. Dev., 41(2):204-211 https://doi.org/10.1002/mrd.1080410211
  12. Grupen, C. G., Nagashima, H. and Nattle, M. B. 1995. Cysteamine enhance in vitro develop-ment of IVM-IVF porcine oocytes. Theriogenology, 43:226(abstr.)
  13. Huxtable, R. J. 1992. Physiological actions of taurine. Physiol. Rev., 72(1):101-163
  14. Iritani, A., Niwa, K. and Imai, H. 1978. Sperm penetration in vitro of pig follicular oocytes matured in culture. J. Reprod. Fert., 54:379-383 https://doi.org/10.1530/jrf.0.0540379
  15. Johnson, M. H. and Nasr-Esfahani, M. H. 1993. Radical solutions and cultural problems: could free oxygen radicals be responsible for the impaired development of preimplantation mammalian-emryos in vitro? BioEssays, 16(1):31-38
  16. Kito, S. and Bavister, B. D. 1997. Male pronu-clear formation and early embryonic develop-ment of hamster oocytes matured in vitro with gonadotrophins, amino acids and cysteamine. J. Reprod. Fert., 110:35-46 https://doi.org/10.1530/jrf.0.1100035
  17. Machaty, Z., Day, B. N. and Prather, R. S. 1998. Development of early porcine embryos in vitro and in vivo. Biol. Reprod., 59:451-455 https://doi.org/10.1095/biolreprod59.2.451
  18. Matos, D. G. D., Furnus, C. C., Moses, D. F. and Baldassarre, H. 1995. Effect of cysteamine on glutathione level and developmental capacity of bovine oocytes matured in vitro. Mol. Re-prod. Dev., 42:432-436
  19. Matos, D. G. D., Fumus, C. C., Moses, D. F., Martinez, A. G. and Matkovic, M. 1996. Sti-mulation of glutathione synthesis of in vitro matured bovine oocytes and its effect on embryo development and freezability. Mol. Reprod. Dev., 45:451-457 https://doi.org/10.1002/(SICI)1098-2795(199612)45:4<451::AID-MRD7>3.0.CO;2-Q
  20. Mattioli, M., Bacci, M. L., Galeati, G. and Seren, E. 1989. Developmental competence of pig oocytes matured and fertilized in vitro. Theriogenology, 31:1201-1209
  21. Motlik, J. and Fulka, J. 1974. Fertilization of pig follicular oocytes cultivated in vitro. J. Reprod. Fertil., 36:235-237 https://doi.org/10.1530/jrf.0.0360235
  22. Oshima, R. 1978. Stimulation of the clonal growth and differentiation of feeder layer dependent mouse embryonal carcinoma cells by ${\beta}$-mercaptoethanol. Differentiation, 11: 149-155
  23. Petters, R. M. and Wells, K. D. 1993. Culture of pig embryos. J. Reprod. Fert.,(suppl.) 48:61-73
  24. Pincus, G. and Enzmann, E. V. 1935. The com-parative behaviour of mammalian eggs in vivo and in vitro. J. Exp. Med., 62:655-657
  25. Reed, M. L., Illera, M. J. and Petters, R. M. 1992. In vitro culture of pig embryos. The-riogenology, 37:95-109
  26. Rizzino, A. and Sato, G. 1978. Growth of em-bryonal carcinoma cells in serum-free medium. Proc. Natl. Acad. Sci., USA. 75(4):1844-1848
  27. SAS Insitute. 1996. SAS/$STAT{\circledR}$ user's guide, release 6.12 edition. SAS Institute Inc., Cary, NC. USA
  28. Takahashi, M., Nagai, T., Hamano, S., Kuwa-yama, M., Okamura, N. and Okano, A. 1993. Effect of thiol compounds on in vitro develpo-ment and intracellular glutathione content of bovine embryos. Biol. Reprod., 49:228-232
  29. Wang, W. H., Abeydeera, L. R., Cantley, T. C. and Day, B. N. 1997. Effects of oocyte matura-tion media on development of pig embryos or produced by in vitro fertilization. J. Reprod. Fert., 111:101-108 https://doi.org/10.1530/jrf.0.1110101
  30. Yamauchi, N. and Nagai, T. 1999. Male pronu-clear formation in denuded porcine oocytes after in vitro maturation in the presence of cystea-mine. Biol. Reprod., 61:828-833 https://doi.org/10.1095/biolreprod61.3.828
  31. Yoshida, M. 1993. Role of glutathione in the maturation and fertilization of pig oocytes in vitro. Mol. Reprod. Dev., 35:76-81
  32. 김하나. 2001 돼지 수정란 및 배반포발달에 영향을 미치는 체외수정 조건. 전남대학교 석사학위논문
  33. 양부근，박동헌，정희태，박춘근，김종복, 김정익. 1997. Thiol 화합물과 항산화제 첨가배양이 소 체외수정란의 체외발육과 세포내 Glutathi-one 농도 변화에 미치는 효과 : I. ${\beta}$-Mercap-toethanol과 Cysteamine 첨가가 소 체외수정란의 체외발육과 세포내 Glutathione농도 변화에 미치는 영향. 한국가축번식학회지，21(4):335-343
  34. 한만희. 2001. 항산화제와 산소조건이 돼지 난포란의 체외성숙，체외수정 및 체외배발달에 미치는 영향. 충남대학교 박사학위논문