Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
권호 표지

Optimization of Electrofusion Condition for the Production of Korean Cattle Somatic Cell Nuclear Transfer Embryos

  • Kim, Se-Woong (Cellular Reprogramming and Embryo Biotechnology Lab, Dental Research Institute and CLS21, Seoul National University School of Dentistry) ;
  • Kim, Dae-Hwan (Cellular Reprogramming and Embryo Biotechnology Lab, Dental Research Institute and CLS21, Seoul National University School of Dentistry) ;
  • Jung, Yeon-Gil (ET Biotech) ;
  • Roh, Sang-Ho (Cellular Reprogramming and Embryo Biotechnology Lab, Dental Research Institute and CLS21, Seoul National University School of Dentistry)
  • Received : 2011.02.16
  • Accepted : 2011.02.28
  • Published : 2011.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

This study was designed to determine the effect of electric field strength, duration and fusion buffer in fusion parameters on the rate of membrane fusion between the somatic cell and cytoplast for Korean cattle (HanWoo) somatic cell nuclear transfer (SCNT) procedure. Following electrofusion, effect of 5 or $10\;{\mu}M$ $Ca^{2+}$-ionophore of activation treatment on subsequent development was also evaluated. Cell fusion rates were significantly increased from 23.1% at 20 V/mm to 59.7% at 26 V/mm and 52.9% at 27 V/mm (p<0.05). Due to higher cytoplasmic membrane rupture or cellular lysis, overall efficiency was decreased when the strength was increased to 30 V/mm (18.5%) and 40 V/mm (6.3%) and the fusion rate was also decreased when the strength was at 25 V/mm or below. The optimal duration of electric stimulation was significantly higher in $25\;{\mu}s$ than 20 and $30\;{\mu}s$ (18.5% versus 9.3% and 6.3%, respectively, p<0.05). Two nonelectrolyte fusion buffers, Zimmermann's (0.28 M sucrose) and 0.28 M mannitol solution for cell fusion, were used for donor cell and ooplast fusion and the fusion rate was significantly higher in Zimmermann's cell fusion buffer than in 0.28 M mannitol (91.1% versus 48.4%, respectively, p<0.05). The cleavage and blastocyst formation rates of SCNT bovine embryos activated by $5\;{\mu}M$ $Ca^{2+}$-ionophore was significantly higher than the rates of the embryos activated with $10\;{\mu}M$ of $Ca^{2+}$-ionophore (70.0% versus 42.9% and 22.5% versus 14.3%, respectively; p<0.05). This result is the reverse to that of parthenotes which shows significantly higher cleavage and blastocyst rates in $10\;{\mu}M$ $Ca^{2+}$-ionophore than $5\;{\mu}M$ counterpart (65.6% versus 40.3% and 19.5% versus 9.7%, respectively; p<0.05). In conclusion, SCNT couplet fusion by single pulse of 26 V/mm for $25\;{\mu}s$ in Zimmermann's fusion buffer followed by artificial activation with $5\;{\mu}M$ $Ca^{2+}$-ionophore are suggested as optimal fusion and activation methods in Korean cattle SCNT protocol.

Keywords

References

  1. Choi JY, Kim CI, Park CK, Yang BK, Cheong HT (2004): Effect of activation time on the nuclear remodeling and in vitro development of nuclear transfer embryos derived from bovine somatic cells. Mol Reprod Dev 69:289-295. https://doi.org/10.1002/mrd.20131
  2. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA, Robl JM (1998): Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280:1256-1258. https://doi.org/10.1126/science.280.5367.1256
  3. Fissore RA, Robl JM (1992): Intercellular $Ca^{2+}$ response of rabbit oocytes to electrical stimulation. Mol Reprod Dev 32:9-16. https://doi.org/10.1002/mrd.1080320103
  4. Izant JG (1983): The role of calcium during mitosis. Calcium participates in the anaphase trigger. Chromosoma 88:1-10. https://doi.org/10.1007/BF00329497
  5. Karja NW, Otoi T, Wongsrikeao P, Shimizu R, Murakami M, Agung B, Fahrudin M, Nagai T (2006): Effects of electric field strengths on fusion and in vitro development of domestic cat embryos derived by somatic cell nuclear transfer. Theriogenology 66:1237-1242. https://doi.org/10.1016/j.theriogenology.2006.03.034
  6. Knott JG, Poothapillai K, Wu H, Li HeC, Fissore RA (2002): Porcine sperm factor support activation and development of bovine nuclear transfer embryos. BioI Reprod 66:1095-1103. https://doi.org/10.1095/biolreprod66.4.1095
  7. Kubiak JZ, Tarkowski AK (1985): Electrofusion of mouse blastomeres. Exp Cell Res 157:561-566. https://doi.org/10.1016/0014-4827(85)90143-0
  8. Ozil JP, Huneau D (2001): Activation of rabbit oo-cytes: the impact of the $Ca^{2+}$ signal regime on development. Development 128:917-928.
  9. Ozil JP, Modlinski JA (1986): Effects of electric field on fusion rate and survival of 2-cell rabbit embryos. J Embryol Exp Morphol 96:211-28.
  10. Park J, Oh H, Hong S, Kim M, Kim G, Koo O, Kang S, Jang G, Lee B (2011): Effective donor cell fusion conditions for production of cloned dogs by somatic cell nuclear transfer. Theriogenology 75:777-782. https://doi.org/10.1016/j.theriogenology.2010.10.016
  11. Rickords LF, White KL (1992): Effect of electrofusion pulse in either electrolyte or nonelectrolyte fusion medium on subsequent murine embryonic development. Mol Reprod Dev 32:259-264. https://doi.org/10.1002/mrd.1080320311
  12. Roh S (2005): Effect of the timing of oocyte activation on development of rat somatic cell nuclear transfer embryos. Reprod Dev BioI 29:229-234.
  13. Rosenkrans CFJ, First NL (1994): Effect of free amino acids and vitamins on cleavage and developmental rate of bovine zygotes in vitro. J Anim Sci 72:434-437. https://doi.org/10.2527/1994.722434x
  14. Shin SJ, Lee BC, Park JI, Lim JM, Hwang WS (2001): A separate procedure of fusion and activation in an ear fibroblast nuclear transfer program improves preimplantation development of bovine reconstituted oo-cytes. Theriogenology 55:1697-1704. https://doi.org/10.1016/S0093-691X(01)00513-1
  15. Sun FZ, Hoyland J, Haung X, Mason W, Moor RM (1992): A comparison of intracellular changes in porcine eggs after fertilization and electroactivation. Development 115:947-956.
  16. Tarkowski AK (1982) Embryonic development. part A. A.R. Liss New York Genetic aspects, pp 407-416.
  17. Trounson A, Lacham-Kaplan O, Diamente M, Gougoulidis T (1998): Reprogramming cattle somatic cells by isolated nuclear injection. Reprod Fertil Dev. 10:645-650. https://doi.org/10.1071/RD98095
  18. Tsunoda Y, Kato Y, Shioda Y (1987): Electrofusion for the pronuclear transplantation of mouse eggs. Gamete Res 17:15-20. https://doi.org/10.1002/mrd.1120170103
  19. Wakayama T, Perry AC, Zuccotti M, Johnson KR, 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
  20. Ware CB, Barnes FL, Miki-Laurila M, First NL (1989): Age dependence of bovine oocyte activation. Gamete Res 22:265-275. https://doi.org/10.1002/mrd.1120220304
  21. Wells DN, Misica PM, Tervit HR (1999): Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. BioI Reprod 60:996-1005. https://doi.org/10.1095/biolreprod60.4.996
  22. Wrenzycki C, Wells D, Herrmann D, Miller A, Oliver J, Tervit R, Niemann H (2001): Nuclear transfer protocol affects messenger RNA expression patterns. BioI Reprod 65:309-317. https://doi.org/10.1095/biolreprod65.1.309
  23. Zakhartchenko V, Durcova-Hills G, Stojkovic M, Schernthaner W, Prelle K, Steinborn R, Muller M, Brem G, Wolf E (1999): Effects of serum starvation and recloning on the efficiency of nuclear transfer using bovine fetal fibroblasts. J Reprod Fertil 115:325-331. https://doi.org/10.1530/jrf.0.1150325
  24. Zimmermann V, Vienken J (1982): Electric field-induced cell-to-cell fusion: Topical review. J Membrane BioI 67:158-174.
  25. Zimmermann U, Vienken J, Pilwat G (1984): Electrofusion of cells. In J Chayen, L Bitensky (eds): "Investigative Microtechniques in Medicine and Biology." New York: Marcel Dekker, Inc, 1:89-167.