Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
권호 표지

Effects of Maturation Duration and Activation Treatments on Activation and Development of Porcine Follicular Oocytes

돼지 난모세포의 단위발생에 있어서 성숙시간과 활성화 처리가 활성화와 발달에 미치는 영향

  • Kim H. J. (National Livestock Research Institute) ;
  • Choi S. H. (National Livestock Research Institute) ;
  • Han M. H. (National Livestock Research Institute) ;
  • Son D. S. (National Livestock Research Institute) ;
  • Ryu I. S. (National Livestock Research Institute) ;
  • Kim I. C. (National Livestock Research Institute) ;
  • Lee J. H. (National Livestock Research Institute) ;
  • Kim I. H. (Chungbuk National University) ;
  • Im K. S. (Seoul National University) ;
  • Cho S. R. (National Livestock Research Institute)
  • Published : 2005.04.01
Download PDF
⟨ Previous Next ⟩

Abstract

This study is a part of research that development of effective genetic resources preservation system using the in vitro spermatogenesis, in vitro insemination and culture system. We aimed for establishment of in vitro culture system with in vitro activated porcine oocytes. The porcine oocytes were matured for 48 hours in $TCM199+10\%$ FCS and activated with $7\%$ ethanol. The activated oocytes were cultured for 7 days in $TCM199+10\%$ FCS or $NCSU23+0.4\%$ BSA medium. The activated oocytes were not developed to the blastocyst stage in $TCM199+10\%$ FCS medium. However in $NCSU23+0.4\%$ medium, those were developed to blastocyst with $3\%$ of treated oocytes. We extended maturation duration of porcine follicular oocytes fur 48, 52, 56, 60, 64, 68, and 72 hours and activated with $7\%$ ethanol and cultured using $NCSU23+0.4\%$ BSA medium. The six percents of activated oocytes were developed to blastocyst in 48 hours and $10\%$ in 52 hours with comparatively low rates suggested to be not fully activated by regenerated MPF. Maturation durations from 56 hours to 68 hours supported to develop upto $11.9\~18.3\%$ of blastocysts. However the developmental rate was declined to $7.2\%$ at 72 hours of maturation duration because of cytoplasmic deterioration. The assumed time window for activation will be $56\~68$ hours of maturation duration. When the matured oocytes were activated with electric pulse of 1, 1.2, 1.4, 1.6, 1.8 and 2.0kV/cm for $80{\mu}s$, although appling the electric current once was not enough for activation, appling twice with 1.6kV/cm for $80{\mu}s$ was shown the highest developmental rate with $11.3\%$. When those were compared with activating methods, $15.7%$ of blastocyst rate was obtained in the $7\%$ ethanol. That was higher than those in electric pulse with $9.5\%$ and calcium ionophore method with $5.8\%$. In this experimental condition, the $7\%$ ethanol treatment was the most effective method for activating porcine oocytes.

본 연구는 가축유전자원의 효율적 보존방법의 개발을 위해 수행되는 체외 정자 세포 생산 연구의 일부로, 생산된 정자 세포의 발생능을 검사하기 위한 체외배양 시스템을 확립 할 목적으로 수행되었다. 성숙 배양시간을 48시간으로 하여 $7\%$ ethanol로 활성화처리한 후 TCM199에 $10\%$의 소 태아 혈청으로 배양하였을 때 배반포까지 발달하지 못하였으나, NCSU23에 $0.4\%$ 소 혈청 알부민으로 배양하였을 때 $3\%$의 활성화된 난모세포가 배반포기까지 발달하였다. 성숙시간을 연장하여 48, 52, 56, 60, 64, 68, 그리고 72시간까지 성숙배양을 실시한 후 $7\%$ 에탄올로 활성화 처리하여 $NCSU23+0.4\%$ BSA로 배양하였을 때 56시간부터 68시간까지 배발달율이 증가하였으나 72시간 성숙배양할 때 배발달율이 다시 저하하여 활성화와 세포질 퇴행간의 윈도우가 56시간부터 68시간 사이인 것으로 추정되었다. 전기자극의 강도를 1, 1.2, 1.4, 1.6, 1.8 그리고 2.0kV/cm로 난모세포를 활성화 처리하였을때 1회 통전으로는 적절한 활성화가 일어나지 않았으며 1.6kV/cm, $80{\mu}s$, 2회 통전이 본 실험조건에서 가장 높은 배발달율을 보였다. 난모세포를 인위적으로 활성화하는데 주로 이용되는 $7\%$ 에탄올법, 전기자극법, 그리고 calcium ionophore법을 직접적으로 비교하였을 때 $7\%$ 에탄올법이 $15.7\%$, 전기 자극법이 $9.5\%$, calcium ionophore법이 $5.8\%$의 배반포 발달율을 보여, 본 실험조건에서는 $7\%$ 에탄올법이 배 발달을 활성화시키는데 가장 효율적인 것으로 나타났다.

Keywords

References

  1. Bing YZ, Che LM, Hirao Y, Takenouchi N, Kuwayama M and Nagai T. 2002. In vitro development of porcine oocytes activated by electric pulse: effect of maturation culture period. Cloning and Stem Cell, 3: 209 (Abstr 16-02)
  2. Bing YZ, Che L, Hirao Y, Takenouchi N, Rodriguez-Martinez H and Nagai T. 2003. Parthenogenetic activation and subsequent development of porcine oocytes activated by a combined electric pulse and butyrolactone I treatment. J. Reprod. Dev., 49:159-166 https://doi.org/10.1262/jrd.49.159
  3. Cui XS, Jeong YJ, Lee HY, Cheon SH and Kim NH. 2004. Fetal bovine serum influences apoptosis and apoptosis-related gene expression in porcine parthenotes developing in vitro. Reproduction, 127: 125-130 https://doi.org/10.1530/rep.1.00039
  4. Edwards LJ, Batt PA, Gandolfi F and Gardner DK. 1997. Modifications made to culture medium by bovine oviduct epithelial cells: changes to carbohydrates stimulate bovine embryo development. Mol. Reprod. Dev., 46:146-154 https://doi.org/10.1002/(SICI)1098-2795(199702)46:2<146::AID-MRD5>3.0.CO;2-Q
  5. Gasparrini B, Boocia L, Rosa AD, Palo RD, Campanile G and Zicarelli L. 2004. Chemical activation of buffalo (Bubalus bualis) oocytes by different methods: effects of aging on postparthenogenetic development. Theriogenology, 62:1627-1637 https://doi.org/10.1016/j.theriogenology.2004.03.005
  6. Goto Y, Kaneyama K, Kobayashi S, Imai K, Shin-noh M, Tsujino T, Nakano T, Matsuda S, Nakane S and Kojima T. 1999. Birth of cloned claves derived from cultured oviductal epithelial cells of a dairy cow. Jpn. Anim. Sci. J., 70:243-245
  7. Grupen CG, Mau JC, Mcllfatrick SM, Maddocks S and Nottle MB. 2002. Effect of 6-dimethyl-aminopurine on electrically activated in vitro matured porcine oocytes. Mol. Reprod. Dev., 62:387-396 https://doi.org/10.1002/mrd.10126
  8. Ikumi S, Asada M, Sawai K and Fukui Y. 2003. Effect of activation methods for bovine oocytes after intracytoplasmic injection. J. Reprod. Dev., 49:37-43 https://doi.org/10.1262/jrd.49.37
  9. Im KS, Kim HJ, Oh SJ, Yang BS and Jin DI. 1997. Effects of strength and duration of direct current (DC) pulse and age of donor embryo on fusion and development of bovine nuclear transfer embryos. Korean J. Anim. Sci., 39: 493-500
  10. Katoh M, Araki A, Ogura T and Valdivia RP. 2004. 6-dimethylaminopurine(6-DMAP), which is used to produce most cloned animals, is mutagenic in Salmonella typhimurium TA1535. Mutat. Res., 560:199-201 https://doi.org/10.1016/j.mrgentox.2004.03.003
  11. Kaufman MH. 1979. Mammalian parthenogenetic development. Bibliography of Reproduction, 33: 261-4
  12. Kaufman MH. 1983. Methodology: in vitro and in vivo activation techniques. In early mammalian development: parthenogenetic studies. ed. MH Kaufman, pp. 20-62. Cambridge University press, England
  13. Kim YS, Lee SL, Ock SA, Balasubramanian S, Choe SY and Rho GJ. 2004. Development of cloned pig embryos by nuclear transfer following different activation treatments. Mol. Reprod. Dev., 29:308-313
  14. Kim NH, Moon SJ, Prather RS and Day BN. 1996. Cytoskeletal alteration in aged porcine oocytes and parthenogenesis. Mol. Reprod. Dev., 43: 513-518 https://doi.org/10.1002/(SICI)1098-2795(199604)43:4<513::AID-MRD14>3.0.CO;2-#
  15. Kline D and Kline JT. 1992. Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg. Dev. Biol., 149:80-89 https://doi.org/10.1016/0012-1606(92)90265-I
  16. Leal CLV and Liu L. 1998. Differential effects of kinase inhibitor and electrical stimulus on activation and histone HI kinase activity in pig oocytes. Anim. Reprod. Sci., 52:51-61 https://doi.org/10.1016/S0378-4320(98)00084-0
  17. Lee JW, Tian XC and Yang X. 2004. Optimization of parthenogenetic activation protocol in porcine. Mol. Reprod. Dev., 68:51-57 https://doi.org/10.1002/mrd.20043
  18. Liu CT, Chen CH, Cheng SP and Ju JC. 2002. Parthenogenesis of rabbit oocytes activated by different stimuli. Anim. Reprod. Sci., 70:267-276 https://doi.org/10.1016/S0378-4320(01)00185-3
  19. Liu L and Yang X. 1999. Interplay of maturation-promoting factor and mitogen-activated protein kinase inactivation during metaphaseto-interphase transition of activated bovine oocytes. Biol. Reprod., 61:1-7 https://doi.org/10.1095/biolreprod61.1.1
  20. Martinez Diaz MA, Suzuki M, Kagawa M, Ikeda K and Takahashi Y. 2003. Effects of cycloheximide treatment on in vitro development of porcine parthenotes and somatic cell nuclear transfer embryos. Jpn, J. Vet. Res., 50:147-155
  21. Moses RM and Masui Y. 1994. Enhancement of mouse egg activation by the kinase inhibitor, 6-dimethylaminopurine (6-DMP). J. Exp. Zool., 270:211-218 https://doi.org/10.1002/jez.1402700210
  22. Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, Hanada H and Perry ACF. 2000. Pig cloning by microinjection of fetal fibroblast nuclei. Science, 289: 1188-1190 https://doi.org/10.1126/science.289.5482.1188
  23. Petters RM and Well KD. 1993. Culture of pig embryos. J. Reprod. Fertil., 48:61-73
  24. Roh SH and Hwang WS. 2002. Technical report: In vitro development of porcine parthenogenetic and cloned embryos: comparison of oocyte-activating techniques, various culture systems and nuclear transfer methods. Reprod. Fert. Dev., 14:93-99 https://doi.org/10.1071/RD01090
  25. Rosenkrans CF and First NL. 1991. Culture of bovine zygotes to the blastocyst stage : effects of amino acids and vitamins. Theriogenology, 35:266(Abstract) https://doi.org/10.1016/0093-691X(91)90242-6
  26. Ruddock NT, Machaty Z, Milanick M, and Prather RS. 2000. Mechanism of intracellular pH increase during parthenogenetic activation of in vitro matured porcine oocytes. Biol. Reprod., 63:488-492 https://doi.org/10.1095/biolreprod63.2.488
  27. Russo GL, Wilding M, Marino M and Dale B. 1998. Ins and outs of meiosis in ascidians. Semin. Cell Dev. Biol., 9:559-567 https://doi.org/10.1006/scdb.1998.0250
  28. Sagata N. 1998. Introduction: meiotic maturation and arrest in animal oocyte. Semin. Cell Dev. Biol., 9:535-53 https://doi.org/10.1006/scdb.1998.0247
  29. Wakayama T, Perry ACF, Zuccotti M, Johnson KR and Yanagimachi R. 1998. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature, 394:369-374 https://doi.org/10.1038/28615
  30. Wang WH, Abeydeera LR, Prather RS and Day BN. 1998. Functional analysis of activation of porcine oocytes by spermatozoa, calcium ionophore, and electrical pulse. Mol. Reprod. Dev., 51:346-353 https://doi.org/10.1002/(SICI)1098-2795(199811)51:3<346::AID-MRD15>3.0.CO;2-0
  31. Ware CB, Barnes FL, Maiki-Laurila M and First NL. 1989. Age dependence of bovine oocyte activation. Gamete Res., 22:265-275 https://doi.org/10.1002/mrd.1120220304
  32. Yamashita M. 1998. Molecular mechanisms of meiotic maturation and arrest in fish and amphibian oocytes. Semin. Cell Dev. Biol., 9:569-579 https://doi.org/10.1006/scdb.1998.0251
  33. Zhu J, Telfer EE, Fletcher J, Springbett A, Dobrinsky JR, De Sousa PA and Wilmut I. 2002. Improvement of an electrical activation protocol for porcine oocytes. Biol. Reprod., 66:635-641 https://doi.org/10.1095/biolreprod66.3.635