Clinical and Experimental Reproductive Medicine

The Korean Society for Reproductive Medicine (대한생식의학회)
권호 표지

Effects of Media Volume on Blastocyst Formation, Cell Numbers and ICM Proportion in Mouse Two-cell Embryos

배양액 용량이 마우스 2-세포기 배의 배반포 형성, 세포수 및 내세포괴 비율에 미치는 영향

  • Kim, Sung-Yob (Department of OB/Gyn, School of Medicine, Cheju National University) ;
  • Park, Kee-Sang (Department of OB/Gyn, Kyungpook National University Hospital)
  • 김성엽 (제주대학교 의과대학 산부인과) ;
  • 박기상 (경북대학교 의과대학 산부인과)
  • Published : 2004.03.30
Download PDF
⟨ Previous Next ⟩

Abstract

연구 목적: 본 연구의 목적은 마우스에서 배양액의 용량이 배반포 배 형성과 세포수에 미치는 영향을 조사하기 위하여 실시하였다. 연구 재료 및 방법: $3{\sim}4$주령 ICR 암마우스에게 48시간 간격으로 5 IU PMSG와 hCG 주사 후 (hCG 주사 후 수컷과 동숙) $46{\sim}50$시간에 난관으로부터 총 138개의 2-세포기 배를 회수하여 2 ml (group I) 또는 $50{\mu}l$ (group II)의 배양액 (Dulbecco's Modified Eagle Medium + 20% human follicular fluid)에서 72시간 동안 배반포기까지 배양하였다. 배반포 배는 zona-intact (ZiB)와 zona-escape (ZeB)로 등급을 구분하고 나서, propidium iodide와 bisbenzimide를 이용한 differential staining 방법으로 염색하여 평균 세포수, 내세포괴(ICM) 세포수, 영양배엽(TE) 세포수, 총 세포수에 대한 ICM의 비율 (%ICM) 및 ICM:TE 비율을 조사하였다. 결과에 대한 유의성 검정은 $X^2$ test와 t-test를 이용하였으며, p<0.05일 때 통계적인 차이가 있는 것으로 하였다. 결 과: Group I과 II에서, 총 배반포 ($62.3{\pm}20.7%$ vs. $63.8{\pm}22.9%$), ZiB ($31.9{\pm}24.0%$ vs. $30.4{\pm}18.2%$)와 ZeB 형성율 ($30.4{\pm}20.8%$ vs. $33.3{\pm}22.3%$)은 차이가 없었다. 87개의 배반포 배를 염색 시도하였는데, 명확하게 differential staining된 41개의 배반포 배만을 대상으로 세포수를 조사하였다. 평균 세포수 ($61.6{\pm}19.5$ vs. $63.7{\pm}26.8$), ICM 세포수 ($13.0{\pm}10.6$ vs. $12.8{\pm}10.5$), TE 세포수 ($49.0{\pm}19.0$ vs. $47.8{\pm}18.7$), %ICM ($21.0{\pm}12.6%$ vs. $21.1{\pm}13.2%$) 및 ICM:TE 비율 (1:$3.77{\pm}4.9$ vs. 1:$3.72{\pm}4.8$)에서도 group I과 II에서 차이가 없었다. 결 론: 마우스에서 배 발생 능력의 척도로 쓰이는 배반포 배 형성율 배반포 배의 등급 세포수 및 %ICM 등이 20% 난포액을 첨가한 MEM 배양액의 용량에 따라 영향을 받지 않았다.

Keywords

References

  1. Park KS, Choi IK, Lee JS, Song HB. The effects of glutamine on blastulation of human embryos on vero cells in vitro. Kor J Fertil Steril 1998; 25: 65-70
  2. Park KS, Lee TH, Choi IK, Song HB, ChunSS. Comparison of blastulation and pregnancy rates of fertilized human oocytes obtained after conventional in vitro fertilization and intracyto-plasmic sperm injection. J Mamm Ova Res 2000; 17: 51-7 https://doi.org/10.1274/jmor.17.51
  3. Park KS, Song HB, Chun SS. Late fertilization of unfertilized human oocytes in vitro fertiliza-tion and intracytoplasmic sperm injection cycles: conventional insemination versus ICSI. J Assist Reprod Genet 2000; 17: 419-24 https://doi.org/10.1023/A:1009409100941
  4. Park KS, Song HB, Lee TH, Chun SS. Subse-quent embryo transfers (SET) on day 2 and day 5: it's safety and effectiveness. Kor J Fertil Ste-ril 2000; 27: 165-72
  5. Chun SS, Park KS. Birth of a healthy infant after in vitro fertilization and embryo transfer in patient of total uterine prolapse. J Assist Re-prod Genet 2001; 18: 346-8 https://doi.org/10.1023/A:1016676403705
  6. Lee HJ, Park KS, Song HB, Lee TH, Cho YL, Chun SS. Comparison of embryonic developmen-tal capacity by different co-culture time of oocy-tes in IVF-ET cycles. Kor J Fertil Steril 2002; 29: 21-2
  7. Kim DW, Park KS, Song HB, Lee TH, Chun SS. The clinical outcomes after embryo transfer (ET) on day 2 and day 5 or subsequent ET on day 2-5, 2-6, 2-7, 3-5 and 4-7 in in vitro fertilization-ET cycles. Kor J Fertil Steril 2003; 30: 31-7
  8. Wiley LM, Yanami S, Van Muyden D. Effect of potassium concentration, type of protein supplement and embryo density on mouse preimplantation development in vitro. Fertil Steril 1986; 45: 111-9 https://doi.org/10.1016/S0015-0282(16)49107-7
  9. Paria PC, Dey SK. Preimplantation embryo development in vitro: cooperative intractions among embryos and role of growth factors. Proc Natl Acad Sci USA 1990; 87: 4756-60 https://doi.org/10.1073/pnas.87.12.4756
  10. Canseco RS, Sparks AET, Pearson, RE, Gwazdauskas FC. Embryo density and medium volume effects on early murine embryo development. J. Assist Reprod Genet 1992; 9: 454-7 https://doi.org/10.1007/BF01204051
  11. Lane M, Gardner DK. Effect of incubation volume and embryo density on the development and viability of preimplantion mouse embryos in vitro. Hum Reprod 1992; 7: 558-62 https://doi.org/10.1093/oxfordjournals.humrep.a137690
  12. Salahuddin S, Ookutsu S, Goto K, NakanishiY, Nagata Y. Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in-vitro fertilized mouse embryos. Hum Reprod 1995; 10: 2382-5 https://doi.org/10.1093/oxfordjournals.humrep.a136303
  13. Park KS, Lee TH, Chun SS, Song HB. Effects of medium on blastocyst formation, cell number and percentage of ICM in mice. Kor J Fertil Steril 2002; 29: 237-43
  14. Bowman P, MaLaren A. Cleavage rate of mouse embryos in vivo and in vitro. J Embryol Exp Morphol 1970; 24: 203-7
  15. Harlow GM, Quinn P. Development of preimplantation mouse embryos in vitro and in vivo. Aus J Biol Sci 1982; 35: 187-93 https://doi.org/10.1071/BI9820187
  16. Streffer C, Van Beuningen D, Molls M, Zanboglou N, Schulz S. Kinetics of cell proliferation in the preimplantation mouse embryo in vivo and in vitro. Cell Tissue Kinet 1980; 13: 135-43
  17. Borland RM, Hazra S, Biggers JD, Lechene CP. The elemental composition of the environments of the gemetes and preimplantation embryo during the initiation of pregnancy. Biol Reprod 1977; 16: 147-57 https://doi.org/10.1095/biolreprod16.2.147
  18. Borland RM, Biggers JD, Lechene CP, Taymer ML. Elemental composition of fluid in the human fallopian tube. J Reprod Fertil 1980; 58: 479-82 https://doi.org/10.1530/jrf.0.0580479
  19. Gardner DK, Leese HJ. Concentrations of nutrients in mouse oviduct fluid and their effects on embryo development and metabolism in vitro. J Reprod Fertil 1990; 88: 361-8 https://doi.org/10.1530/jrf.0.0880361
  20. Larson MA, Kubisch HM. The effects of group size on development and interferon-$\tau$ secretion by in-vitro fertilized and cultured bovine blastocysts. Hum Reprod 1999; 14: 2075-9. https://doi.org/10.1093/humrep/14.8.2075
  21. Park KS, Park SB, Lee TH, Chun SS, Song HB. Effects of exposure of propidium iodide and bisbenzimide on differential staining of mouse blastocysts. Kor J Fertil Steril 2002; 29: 317-22
  22. Keefer CL, Stice SL, Paprocki AM, Golueke P. In vitro culture of bovine IVM-IVF embryos: coorperative interaction among embryos and the role of growth factors. Theriogenology 1994; 41: 1323-31 https://doi.org/10.1016/0093-691X(94)90491-Z