Kamber DEMİR, Serhat PABUCCUOĞLU, Süleyman BACINOĞLU, Sema BİRLER, Ümüt CİRİT, Elif KARAMAN

Parthenogenetic activation of sheep oocytes

Koyunlarda somatik ve embriyonik hücre nüklear transfer yöntemlerini laboratuvarımızda başlatabilmek için, iki farklı partenogenetik aktivasyon protokolü denendi. Oositler mezbahada kesilen koyunların ovaryumlarından elde edildi ve piruvat, hormonlar (FSH ve LH) ve %10 FCS katkılı medyum 199 içerisinde 24 saat olgunlaştırıldı. Olgunlaştırılan oositlere 80 usec süreyle 1.25 kV/cm elektrik akımı verildi ve oositlerin tamamı önce 5 dk ionomisin içerisinde, daha sonra ise 30 dk sitokalasin B içerisinde bırakıldı. İki gruba rastgele ayrılan oositler, 1. grupta 6-DMAP’lı medyumda 2 saat, 2. grupta ise sikloheksimid’li medyumda 3 saat inkübe edildikten sonra kültür medyumuna aktarıldı. Grup I ve II’de elde edilen embriyolarda yarıklanma oranları sırasıyla %82.7 ve %81.3 olurken blastosist oranları %2.3 ve %0 olarak bulundu (P>0.05). Sunulan çalışma koyunların klonlanması konusunda önemli aşamalardan birini oluşturan partenogenetik aktivasyon konusunda ülkemizde gerçekleştirilmiş ilk çalışmadır.

Koyun oositlerinin partenogenetik aktivasyonu

In order to establish somatic- and embryonic-cell nuclear transfer procedures in sheep in our laboratory, two activation protocols for parthenogenetic activation of sheep oocytes were tested. Oocytes obtained from ovaries of slaughtered ewes were matured for 24 h in medium 199 supplemented with pyruvate, hormones (FSH and LH) and 10% FCS. After maturation, all oocytes were exposed to single electrical stimulation of 1.25 kV/cm for 80 usec, then placed into ionomycine and cytochalasin B for 5 min and 30 min respectively, and then divided into 2 groups randomly. While oocytes in Group I were incubated in 6-dimethylaminopurine (6-DMAP) for 2 h, oocytes in Group II were kept in cycloheximide (CHX) for 3 h before transferring into culture medium. Cleavage and blastocyst rates in groups I and II were 82.7% and 81.3%, and 2.3% and 0% respectively (P>0.05). These are our preliminary results for the parthenogenetic development of sheep oocytes in Turkey and represent promising results in the direction to achieve sheep cloning.

PDF

___

1. Alberio, R., Zakhartchenko, V., Motlik, J., Wolf, E.: Mammalian oocyte activation: lessons from the sperm and implications for nuclear transfer. Int. J. Dev. Biol., 2001; 45: 797-809.
2. Alexander, B., Coppola, G., Di Berardino, D., Rho, G.J., St John, E., Betts, D.H., King, W.A.: 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., 2006; 73: 20-30.
3. Birler, S., Pabuççuoğlu, S., Alkan, S., Özdas, Ö.B., Atalla, H., İleri, İ.K.: Development of in vitro derived sheep embryos to the blastocyst stage. Turk J. Vet. Anim. Sci., 2002; 26: 891-894.
4. Birler, S., Pabuççuoğlu, S., Atalla, H., Alkan, S., Özdaş, Ö.B., Bacınoğlu, S., Cirit, Ü., Zavar, İ., Sönmez, M.E.C., İleri, İ.K.: Transfer of in vitro produced sheep embryos. Turk. J. Vet. Anim. Sci., 2002; 26: 1421-1426.
5. Chesne, P., Adenot, P.G., Viglietta, C., Baratte, M., Boulanger, L., Renard, J.P.: Cloned rabbits produced by nuclear transfer from adult somatic cells. Nat. Biotechnol., 2002; 20: 366–369.
6. Gómez, M.C., Jenkins, J.A., Giraldo, A., Harris, R.F., King, A., Dresser, B.L., Pope, C.E.: Nuclear transfer of synchronized African wild cat somatic cells into enucleated domestic cat oocytes. Biol. Reprod., 2003; 69:1032-1041.
7. Kubota, C., Yamakuchi, H., Todoroki, J., Mizoshita, K., Tabara, N., Barber, M., Yang, X.: Six cloned calves produced from adult fibroblast cells after long-term culture. Proc. Natl. Acad. Sci., 2000; 97: 956-957.
8. Lagutina, I., Lazzari, G., Duchi, R., Colleoni, S., Ponderato, N., Turini, P., Crotti, G., Galli, C.: Somatic cell nuclear transfer in horses: Effect of oocyte morphology, embryo reconstruction method and donor cell type. Reproduction, 2005; 130: 559-567.
9. Lee, B.C., Kim, M.K., Jang, G., Oh, H.J., Yuda, F., Kim, H.J., Shamim, M.H., Kim, J.J., Kang, S.K., Schatten, G., Hwang, W.S.: Dogs cloned from adult somatic cells. Nature, 2005; 436: 641.
10. Loi, P., Ledda, S., Fulka Jr, J., Cappai, P., Moor, R.M.: Development of parthenogenetic and cloned ovine embryos: Effect of activation protocols. Biol. Reprod., 1998; 58: 1177-1187.
11. Peura, T.T., Vajda, G.: A comparison of established and new approaches in ovine and bovine nuclear transfer. Cloning and Stem Cells, 2003; 5: 257-277.
12. Polejaeva, I.A., Chen, S.H., Vaught, T.D., Page, R.L., Mullins, J., Ball, S., Dai, Y., Boone, J., Walker, S., Ayares, D.L., Colman, A., Campbell, K.H.: Cloned pigs produced by nuclear transfer from adult somatic cells. Nature, 2000; 407: 86-90.
13. Wakayama, T., Perry, A.C., Zuccotti, M., Johnson, K.R., Yanagimachi, R.: Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature, 1998; 394: 369-374.
14. Wells, D.N., Misica, P.M., Tervit, H.R.: Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. Biol. Reprod., 1999; 60: 996-1005.
15. Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J., Campbell, K. H. S.: Viable offspring derived from fetal and adult mammalian cells. Nature, 1997; 385: 810-813.