Effects of Nitric Oxide Scavenger and Inhibitor on the Development of Bovine IVM/IVF Embryos

Nitric Oxide 화합물 첨가가 소 체외수정란의 체외발육에 미치는 효과

  • Jang H. Y. (College of Animal Resources Science, Kangwon National University) ;
  • Kim J. T. (College of Animal Resources Science, Kangwon National University) ;
  • Park C. K. (College of Animal Resources Science, Kangwon National University) ;
  • Cheong H. T. (College of Animal Resources Science, Kangwon National University) ;
  • Kim C. I. (College of Animal Resources Science, Kangwon National University) ;
  • Yang B. K. (College of Animal Resources Science, Kangwon National University)
  • 장현용 (강원대학교 동물자원과학대학) ;
  • 김종택 (강원대학교 동물자원과학대학) ;
  • 박춘근 (강원대학교 동물자원과학대학) ;
  • 정희태 (강원대학교 동물자원과학대학) ;
  • 김정익 (강원대학교 동물자원과학대학) ;
  • 양부근 (강원대학교 동물자원과학대학)
  • Published : 2004.09.01

Abstract

This study was designed to evaluate the effects of nitric oxide scavenger (hemoglobin) and inhibitor (L-nitro-L-arginine methyl ester; L-NAME) with or without cumulus cell on the development of bovine IVM/IVF embryos. When CR/sub 1aa/ medium were supplemented with different dosage (lug/m, 5ug/m and 10ug/ml) of hemoglobin at 48hrs for in vitro culture, the proportion of embryos developing beyond morulae stage in 0, 1ug/ml and 5ug/ml with or without cumulus cell were 23.8%, 33.3 % and 26.8%, and 39.5%, 54.8% and 48.8%, respectively. There was a significantly difference the developmental rate of 1ug/ml hemoglobin intact cumulus cells to any other groups (P<0.05). On the other hand, when added to hemoglobin at 96 hrs, 1 ug/ml hemoglobin with cumulus cell group was significantly increased the percentage of developing into morulae and blastocysts to any other groups (P<0.05), and similar trend that of added at 48hrs. The overall means of the percentage of developing into morulae and blstocysts in 1ug/ml hemoglobin group was significantly increased than those of any other groups (P<0.05) and cumulus co-culture with hemoglobin was increased the in vitro developing rate of IVM/IVF embryos. In CR/sub 1aa/ medium treated with L-NAME 0, 10, 50 and 100mM, the developmental rate of morula plus blastocysts were 55.6%, 64.9%, 58.8% and 66.7%, respectively. The L-NAME did not affect the developmental rate and the cell numbers of blastocysts in all treated groups. These results indicate that hemoglobin and cumulus co-culture can increase the proportion of embryos that developed into morulae and blastocysts, but cell numbers of blastocysts were not affect in all groups.

체외수정 후 체외배양48시간에 난구세포의 제거 유ㆍ무에 따라 CR/sub 1aa/ 배양액에 homoglobin을 0, 1, 5 ㎍/㎖를 첨가한 구의 상실배기 이상 체외발육성 적은 난구세포를 제거한 구에서 23.8%, 33.3% 및 26.8% 였으며, 난구세포를 제거하지 않은 구에서는 각각 39.5%, 54.8% 및 48.8%로서 난구세포를 제거하지 않은 구에 Hb를 1 ㎍/㎖를 첨가한 구가 여타구보다 통계적으로 높은 결과를 얻었다(P<0.05). 체외수정 후 체외배양 96시간 후 난구세포를 제거 유ㆍ무에 따라 Hb를 0, 1, 5 ㎍/㎖를 첨가하였을 때, 상실배기 이상 체외발육성적은 난구세포를 제거한 구에서 각각 28.6%, 46.2% 및 39.1%였으며, 난구세포를 제거하지 않은 구에서는 각각 33.9%, 67.2% 및 46.0%로서, 난구세포를 제거하지 않은 구에 Hb를 1 ㎍/㎖를 첨가구가 여타구에 비해 통계적으로 유의하게 높게 나타났다(P<0.05). CR/sub 1aa/ 배양액에 L-NAME를 0, 10, 50 및 100 mM을 첨가한 구에서 상실배 이상 발육된 체외발육성적은 각각 55.6%, 64.9%, 58.8% 및 66.7%로써 L-NAME 첨가구가 무첨가구에 비해 커다란 차이가 나타나지 않았다(P>0.05). 모든 처리구에 배반포까지 발육된 체외수정란의 세포수에는 커다란 차이가 인정되지 않았다.

Keywords

References

  1. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA (1990): Apprent hydrosyl radical production by peroxynitrite : implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci 87:1620-1624 https://doi.org/10.1073/pnas.87.4.1620
  2. Brackett BG, Oliphant G (1982): Capacitation of rabbit spermatozoa in vitro. Biol Reprod 12:260-274 https://doi.org/10.1095/biolreprod12.2.260
  3. Bredt OS, Hwang PM, Snyder SH (1990): Localization of nitric oxide synthase indicating a neural role of nitric oxide. Nature 347:768-770 https://doi.org/10.1038/347768a0
  4. Chatterjee S, Gangula PRR, Dong YL, Yallampalli C (1996): Mmunocytochemicallocalization of nitric oxide synthase-III in reproductive organs of female rats during the estrus cycle. J Histochem 28:715-723 https://doi.org/10.1007/BF02409009
  5. Fostermann V, Schmith HH, Pllock JS (1991): Isoforms of nitric oxide synthase : characterization and purification from different cell types. Biochem Pharmacol 42:1849-1857 https://doi.org/10.1016/0006-2952(91)90581-O
  6. Fukuda A, Hubbard TE, Breuel KF (1996): Production of nitric oxide from mouse embryo and effect of nitrite on mouse embryoic development in vitro. Biol Reprod 54:173(abstr) https://doi.org/10.1095/biolreprod54.1.173
  7. Gouge RCP, Marshbum Gordon BE, Nunly W, Huet-Hudson YM (1998): Nitric oxide as a regulator of embryonic development. Bio Reprod 58:875-879 https://doi.org/10.1095/biolreprod58.4.875
  8. Haddad EK, Ducios AJ, Baines MG (1997): Early embryo loss is associated with local production of nitric oxide by decidual mononuclear cells. J Exp Med 58:875-879
  9. Hibbs JB Jr, Taintor RR, Vavrin Z (1987): Macrophage cytotoxicity : Role for L-arginine deiminase and imino oxidation to nitrite. Science 235:473-476 https://doi.org/10.1126/science.2432665
  10. Hibbs JB, Jr, Taintor RR, Vivrin Z (1989): L- arginine is required for the expression of the activated macrophage effect or mechanism causing selective metabolic inhibition in target cells. J Immunol 138: 550-565
  11. Ischiropolous, Zhu HL, Chen J, Tsai M, Martin JCM, Smith CD, Beckman JS (1992): Peroxynitrite-mediated tyrosin nitration catalyzed by superoxide dismutase. Arch Biochem Biophys 298:431-437 https://doi.org/10.1016/0003-9861(92)90431-U
  12. Lim JM, Hansel W (1998): Improved development of in vitro-derived bovine embryos by use of a nitric oxide scanvener in a cumulus-granulosa cell coculture system. Mol Reprod Dev 50:45-53 https://doi.org/10.1002/(SICI)1098-2795(199805)50:1<45::AID-MRD6>3.0.CO;2-X
  13. Lim JM, Mei Y, Chen B, Gododke RA, Hansel W (1999): Development of bovine IVF oocytes cultured in medium supplemented with a nitric oxide scavenger or inhibitor in a co-coculture system. Theriogenology 51:941-949 https://doi.org/10.1016/S0093-691X(99)00040-0
  14. Moncada S, Palmer RMG, Higgs EA (1991): Nitric oxide; physiology, pathophysiology and pharmacology. Pharmacol Rev 43 : 109-142
  15. Norman JE, Cameron IT (1996): Nitric oxide in the human uterus. Rev Reprod 1:61-68 https://doi.org/10.1530/ror.0.0010061
  16. Novaro V, Gonzales E, Jawerbaurn A, Rettori V, Canteros G, Gimeno MF (1997): Nitric oxide synthase regulation during embryonic implantation. Reprod Fertil Dev 9:557-564 https://doi.org/10.1071/R97005
  17. Papaioannou DE, Ebert KM (1988): The preimplantation pig embryo; cell number ad allocation to trophetoderm adn inner cell mass of the blastocyst in vivo and in vitro. Development 102:793-803
  18. Shukovski L, Tsafriri A (1994): The involvement of NO in the ovulatory process in rat. Endocrinology 135:2287-2290 https://doi.org/10.1210/en.135.5.2287
  19. Sladex SM, Magness RR, Conrad KP (1997): Nitric oxide and pregnancy. Am J Physiol 272:R441-463
  20. Yallampalli C, Izumi H, Byam-Smith M, Garfield RE (1993): An L-arginine-nitric oxide cyclic guanosine monophosphate system exists in the uterus and inhibits contractility during pegnancy. Am J Obst Gynec 170:175-185
  21. Yallampalli C, Izumi H, Byam-Smith M, Garfield RE(1994): Steroid hormones modulate the production of nitric oxide and cGMP in the rat uterus. Endocrinology 134:1971-1974
  22. Yallampalli C, Dong YL, Gangula PR, Fang (1998): Role and regulation of nitric oxide in the uterus during pregnancy and parturition. J Soc Gynecol Invest 5:58-67 https://doi.org/10.1016/S1071-5576(97)00106-8