Effect of the Synchronization of Meiotic Resumption in Bovine Oocytes Matured In Vitro

감수분열 재개 동기화가 소 미성숙 난자의 체외 성숙에 미치는 영향

  • 박종임 (건국대학교 수의과대학 산과학교실) ;
  • 장유니 (건국대학교 수의과대학 산과학교실)
  • Published : 2003.12.01

Abstract

This study was carried out to test the efficacy of pharmacological inhibitors of the cell cycle transition in keeping bovine oocytes at the germinal vesicle(GV) stage and the reversibility of this inhibition. Bovine oocytes were incubated for 22∼24 hrs in the presence of various inhibitors : cycloheximide (2$\mu\textrm{g}$/$m\ell$), 6-DMAP (2 mM), and roscovitine (50$\mu$M). Bovine oocytes cultured with any of the inhibitors were significantly blocked at the GV stage. Reversibility of pharmacological inhibitors was assessed by culturing oocytes an additional 22∼24 hours in inhibitor-free medium. Examination of oocytes revealed that the inhibitory effect was fully reversible and effect of resuming meiotic progression on nuclear maturation varied according to the various inhibitors. This study suggests that cycloheximide, 6-DMAP and roscovitine can be applied to control meiotic arrest and resumption in maturation culture of bovine oocytes in vitro. More investigations are needed to better understand how the cell cycle of oocyte is blocked without problems to future developmental competence.

본 연구에서 소 미성숙 난자의 성숙 배양 시 세포주기 억제제인 cycloheximide, 6-DMAP, rosco-vitine를 첨가하였을 때 난자의 감수분열이 억제제비 첨가난자에 비해 높은 비율로 germinal vesicle 기에서 정지된 것이 관찰되었다. 또한 cyclohexi-mide, 6-DMAP, roscovitine을 배지에서 제거한 후 다시 추가적인 체외 성숙 배양을 실시하여 ger-minal vesicle기에 정지된 세포주기의 진행이 재개되어 난자의 감수분열 완료 및 성숙이 세포주기억제제 비 첨가난자와 거의 동등하게 나타난 것을 확인할 수 있었다. 본 연구를 통하여 cyclo-heximide, 6-DMAP, roscovitine의 소 미성숙 난자의 세포주기 조절작용이 가역적임을 알 수 있었으며 난자의 세포 주기 동기화에 이용할 수 있을 것으로 사료된다.

Keywords

References

  1. Alberio R, Zakhartchenko V, Motlik J and Wolf E. 2001. Mammalian oocyte activation: lessons from the sperm and implications for nuclear transfer. Int. J. Dev. BioI., 45:797-809
  2. Faerge I, Mayes M, Hyttel P and Sirard MA. 2001. Nuclear ultrastructure in bovine oocytes after inhibition of meiosis by chemical and biological inhibitors. Mol. Reprod. Dev., 59:459-467 https://doi.org/10.1002/mrd.1053
  3. Ie Beux G, Richard F and Sirard MA. 2003. Effect of cycloheximide, 6-DMAP, roscovitine and butyrolactone I on resumption of meiosis in porcine oocytes. Theriogenology, 60:1049-1058 https://doi.org/10.1016/S0093-691X(03)00124-9
  4. Kalous J, Kubelka M, Rimkevicova Z, Guerrier P and Motlik J. 1993. Okadaic acid accelerates germinal vesicle breakdown and overcomes cycloheximide- and 6-dimethylaminopurine block in cattle. Dev. BioI., 157:448-54 https://doi.org/10.1006/dbio.1993.1148
  5. Lonergan P, Dinnyes A, Fair T, Yang X and Boland M. 2000. Bovine oocyte and embryo development following meiotic inhibition with butyrolactone I. Mol. Reprod. Dev., 57:204-209 https://doi.org/10.1002/1098-2795(200010)57:2<204::AID-MRD12>3.0.CO;2-N
  6. Lonergan P, Khatir H, Carolan C and Mermillod P. 1997. Bovine blastocyst production in vitro after inhibition of oocyte meiotic resumption for 24 h. J. Reprod. Fertil., 109:355-365 https://doi.org/10.1530/jrf.0.1090355
  7. Lonergan P, Monaghan P, Rizos D, Boland MP and Gordon I. 1994. Effect of follicles size on bovine oocyte quality and developmental competence following maturation, fertilization, and culture in vitro. Mol. Reprod. Dev., 37:48-53 https://doi.org/10.1002/mrd.1080370107
  8. Mermillod P, Tomanek M, Marchal R, Meijer L. 2000. High developmental competence of cattle oocytes maintained at the germinal vesicle stage competence of cattle oocytes maintained at the germinal vesicle stage for 24 hours in culture specific inhibition MPF kinase activity. Mol. Reprod. Dev., 55:89-95 https://doi.org/10.1002/(SICI)1098-2795(200001)55:1<89::AID-MRD12>3.0.CO;2-M
  9. Motlik J and Kubelka M. 1990. Cell-cycle aspects of growth and maturation of mammalian oocytes. Mol. Reprod. Dev., 27:366-375 https://doi.org/10.1002/mrd.1080270411
  10. Ponderato N, Crotti G, Turini P, Duchi R, Galli C and Lazzari G. 2002. Embryonic and foetal development of bovine oocytes treated with a combination of butyrolactone I and roscovitine in an enriched medium prior to IVM and IVF. Mol. Reprod. Dev., 62:513-518 https://doi.org/10.1002/mrd.10134
  11. Ponderato N, Lagutina I, Crotti G, Turini P, Galli C and Lazzari G. 2002. Bovine oocytes treated prior to in vitro maturation with a combination of butyrolactone I and roscovitine at low doses maintain a normal developmental capacity. Mol. Reprod. Dev., 60:579-585 https://doi.org/10.1002/mrd.1123
  12. Richard FJ and Sirard MA. 1996. Effects of follicular cells on oocytes maturation II: thecal cell inhibition of bovine oocyte maturation in vitro. Biol. Reprod., 54:22-28 https://doi.org/10.1095/biolreprod54.1.22
  13. Saeki K, Nagao Y, Kishi M and Nagai M. 1997. Developmental capacity of bovine oocytes following inhibition of meiotic resumption by cycloheximide or 6-dimethylaminopurine. Theriogenology, 48:1161-1172 https://doi.org/10.1016/S0093-691X(97)00349-X
  14. Sirard MA. 2001. Resumption of meiosis: mechanism involved in meiotic progression and its relation with developmental competence. Theriogenology, 55:1241-1254 https://doi.org/10.1016/S0093-691X(01)00480-0
  15. Sirard MA, Richard F and Mayes M. 1998. Controling meiotic resumption in bovine oocytes: a review. Theriogenology, 49:483-497 https://doi.org/10.1016/S0093-691X(97)00420-2
  16. Vigneron C, Nuttinck F, Perreau C, Reinaud P, Charpigny G and Mermillod P. 2003. Effect of Roscovitine, a cdkl inhibitor, and of the presence of oocyte on bovine cumulus cell expansion and cyclooxygenase-2 expression. Mol. Reprod. Dev., 65:114-121 https://doi.org/10.1002/mrd.10265