Effects of serum starvation and ionomycin activation on the development of somatic cell nuclear transfer embryos in sheep

Abstract

Synchronization of donor cells and activation of the reconstructed oocytes are important factors affecting the success
rate in somatic cell cloning. In this study, it was aimed to investigate the effects of serum starvation in donor cell synchronization and
ionomycin treatment in the activation of reconstructed oocytes after somatic cell nuclear transfer in Kıvırcık sheep. Cumulus cells were
obtained from a slaughtered sheep ovaries and used as donor cells after serum starvation for 4 days (0.5% FCS; SS) or without serum
starvation (10% FCS; S). After reconstruction, oocytes were activated by ionomycin for 5 min plus 6-dimethylaminopurine for 3 h (I+)
or only with 6-dimethylaminopurine for 3 h (I-). All cleaved embryos (n= 44) at the second day of in vitro culture were transferred into
synchronized recipient ewes (n= 10). Cleavage rates of the embryos were 37.3, 44.1, 34.6 and 44.7% in SS/I+, S/I+, SS/I- and S/Igroups, respectively. Recipient ewes had serum progesterone levels >1 ng/ml at 18th day were 33.3, 50.0, 50.0 and 100.0%, respectively.
Only one pregnancy in the S/I- group continued after 40 days however the cloned lamb (7.1%, regarding to embryos transferred) died
10 days before term due to a maternal problem (uterine torsion). The results of this study reveal that somatic cell synchronization by
serum starvation and ionomycin treatment for the activation of oocytes can be omitted for the success of somatic cell nuclear transfer
in sheep  

Keywords

• Ionomycin,serum starvation,sheep,somatic cell nuclear transfer

References

1. 1. Akagi S, Geshi M, Nagai T (2013): Recent Progress in bovine somatic cell nuclear transfer. Animal Sci J, 84, 191- 199.
2. 2. Alberio R, Zakhartchenko V, Motlık J, et al. (2001): Mammalian oocyte activation: lessons from the sperm and implications for nuclear transfer. Int J Dev Biol 45, 797- 809.
3. 3. Alexander B, Coppola G, Berardıno DD, et al. (2006): The effect of 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and nuclear transfer embryos. Mol Reprod Dev, 73, 20-30.
4. 4. Arat S, Caputcu AT, Akkoc T, et al. (2011): Using cell banks as a tool in conservation programmes of native domestic breeds: the production of the first cloned Anatolian Grey cattle. Reprod Fertil Dev, 23, 1012-1023.
5. 5. Asgari V, Hosseini SM, Ostadhosseını S, et al. (2012): Specific Activation Requirements of In Vitro-Matured Sheep Oocytes Following Vitrification-Warming. Mol Reprod Dev, 79, 434-444.
6. 6. Ashtiani SK, Nasr-Esfahani MH, Hosseini SM, et al. (2008): Royana: Successful Experience in Cloning the Sheep.The Cell J , 10, 193-200.
7. 7. Baguisi A, Behboodi E, Melican DT, et al. (1999): Production of goats by somatic cell nuclear transfer. Nat Biotechnol , 17, 456-461, 1999.
8. 8. Birler S, Pabuccuoğlu S, Demir K, et al. (2010): Production of cloned lambs: transfer of early clevage stage embryos to final recipients. J Fac Vet Med Istanbul Univ, 36, 1-8.

9. 9. Campbell KHS, Alberio R, Choi I, et al. (2005): Cloning: eight years after dolly. Reprod Dom Anim, 40, 256-268.
10. 10. Cho JK, Lee BC, Park JI, et al. (2002): Development of bovine oocytes reconstructed with different donor somatic cells with or without serum starvation. Theriogenology, 57, 1819-1828.
11. 11. Choi I, Joon-Hee L, Fısher P, et al. (2010): Caffeine treatment of ovine cytoplasts regulates gene expression and foetal development of embryos produced by somatic cell nuclear transfer. Mol Reprod Dev, 76, 876-887.
12. 12. Cibelli JB, Stice SL, Golueke PJ, et al. (1998): Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science, 280, 1256-1258.
13. 13. Dalman A, Eftekhari-Yazdi P, Valojerdi MR, et al. (2010): Synchronizing Cell Cycle of Goat Fibroblasts by Serum Starvation Causes Apoptosis. Reprod Dom Anim, 45, 46–53.
14. 14. Demir K, Can A, Ertürk E, et al. (2014): Effects of Different Activation Techniques on Immature and In Vitro Maturated Cat Oocytes. Kafkas Univ Vet Fak Derg, 20, 565-570.
15. 15. Edwards JL, Dorado CM, Wilson TJ, et al. (2001): Development of cloned embryos reconstructed with serum fed or serum starved adult granulosa cells. Theriogenology, 55, 265.
16. 16. Galli C, Lagutina I, Crotti G, et al. (2003): Pregnancy: a cloned horse born to its dam twin. Nature, 424, 635.
17. 17. Gibbons J, Arat S, Rzucidlo J, Miyoshi K, et al. (2002): Enhanced survivability of cloned calves derived from roscovitine-treated adult somatic cells. Biol Reprod, 66, 895-900. 18. Hosseini M, Moulavi F, Foruzanfar M, et al. (2008): Effect of donor cell type and gender on the efficiency of in vitro sheep somatic cell cloning. Small Rum Res, 78, 162- 168.
18. 19. Kato T, Tsunoda Y (2010): Role of the donor nuclei in cloning efficiency: can the ooplasm reprogram any nucleus. Int J Dev Biol, 54, 1623-1629.
19. 20. Kikyo N, Wolffe AP (2000): Reprogramming nuclei: insights from cloning, nuclear transfer and heterokaryons. J Cell Sci, 113, 11-20.
20. 21. Lagutina I, Lazzari G, Duchi R, et al. (2007): Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep. Theriogenology, 67, 90-98.
21. 22. Lee BC, Kim MK, Jang G, et al. (2005): Dogs cloned from adult somatic cells. Nature, 436, 641.
22. 23. Loi P, Clinton M, Barboni B, et al. (2002): Nuclei of Nonviable Ovine Somatic Cells Develop İnto Lambs After Nuclear Transplantation. Biol Reprod, 67, 126-132.
23. 24. Loi P, Clinton M, Vackova I, et al. (2006): Placental abnormalities associated with post-natal mortality in sheep somatic cell clones. Theriogenology, 65, 1110-1121.
24. 25. Loi P, Ledda S, Fulka JJ, et al. (1998): Development of parthenogenetic and cloned embryos: effect of activation protocols. Biol Reprod, 58, 1177–1187.
25. 26. Niemann H, Lucas-Hahn A (2012): Somatic cell nuclear transfer cloning: practical applications and current legislation. Reprod Dom Anim, 47, 2-10.
26. 27. Peura TT (2001): Serum starvation can cause excessive DNA damage in sheep fetal fibroblasts. Theriogenology, 55, 285.
27. 28. Polejaeva IA, Chen SH, Vaught TD, et al. (2000): Cloned pigs produced by nuclear transfer from adult somatic cells. Nature, 407, 486-490.
28. 29. Ptak G, Clinton M, Tischner M, et al. (2002): Improving delivery and offspring viability of in vitro-produced and cloned sheep embryos. Biol Reprod, 67, 1719-1725.
29. 30. Renard JP, Zhou QI, LeBourhis D, et al. (2001): Nuclear transfer technologies: between successes and doubts. Theriogenology, 57, 203-222.
30. 31. Shin T, Kraemer D, Pryor J, et al. (2002): Cell biology: a cat cloned by nuclear transplantation. Nature, 415, 859.
31. 32. Wakayama T, Perry ACF, Zuccotti M, et al. (2003): Fullterm development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature, 394, 369-374.
32. 33. Wang C, Swanson WF, Herrick JR, et al. (2009): Analysis of cat oocyte activation methods for the generation of feline disease models by nuclear transfer. Reprod Biol Endocrinol, 7, 1-12.
33. 34. Watanabe S, Nagai T (2011): Survival of embryos and calves derived from somatic cell nuclear transfer in cattle: a nationwide survey in Japan. Animal Sci J 82, 360-365.
34. 35. Wilmut I, Schnieke AE, McWhir J, et al. (1997): Viable offspring derived from fetal and adult mammalian cells. Nature, 385, 810-813