Effects of Serum and Gonadotropins in In-Vitro Maturation Medium on Nuclear Maturation, Development and Cell Numbers of Korean Native Cow Embryos

체외성숙용 배지에 혈청과 호르몬의 첨가가 한우 난포란의 핵성숙과 배발달 및 배반포의 세포수에 미치는 영향

  • 박용수 (경상북도축산기술연구소) ;
  • 김재명 (포천중문의과대학 생리학교실) ;
  • 박흠대 (대구대학교 식품생명공학부)
  • Published : 2004.12.01

Abstract

The main objective of this study was to examine the effects of serum and gonadotropins supplement during in vitro maturation(IVM) of bovine oocytes on nuclear maturation and embryo development, and we also examine the cell number. 1 . The first polar body(PB) extrusion rates of Korean native cow(KNC) oocytes matured in medium with FBS or gonadotropins were similar among treatment groups. The development rate to the blastocyst stage was significantly higher in the group of both supplement FBS and gonadotropins(26.0%) than in the group of non-supplement(9.9%) and gonadotropins (12.0%). The numbers of inner cell mass (ICM) and trophectoderm (TE) cells and total cell numbers of blastocysts were highest in the group of both supplement FBS and gonadotropins, and the number of ICM cells was increased by FBS supplementation (p<0.05). 2. The PB extrusion rates of KNC oocytes matured in medium with FBS in the different duration of IVM was significantly higher in the 0-18hr(63.1%) and in the 9-18hr(63.4%) group than in the 0-9hr.(37.4%) group (p<0.05). The embryo development rates did not differ among treatment groups. The numbers of TE cells and total cell numbers of blastocysts were similar among treatment groups, but the number of ICM cells of the 0-18h. group were significantly higher than the other treatment groups (p<0.05). The results indicate that although TCM199 alone can support bovine oocyte maturation and development to the blastocyst stage, a high quality of blastocysts can be produced from oocytes matured in medium containing serum and gonadotropins.

본 연구에서는 소 체외수정란 생산에 있어서 체외성숙용 배지에 첨가하는 혈청과 호르몬의 효과를 검토하기 위하여 제1극체 출현율과 배발달율을 조사하였고, 생산된 배반포의 품질을 평가하기 위하여 세포수를 검토하였다. 1. 체외성숙용 배지에 혈청 및 성선자극호르몬의 첨가에 따른 한우 난포란의 제1극체 출현율은 비슷한 경향이었다. 배반포까지의 발달율은 혈청 및 성선자극호르몬 공동 첨가군(26.0%)이 대조군과 성선자극호르몬 단독 첨가군보다 유의하게 높았다(P<0.05). 혈청 및 성선자극호르몬 공동 처리군에서 생산된 배반포의 ICM, TE, 총 세포수가 가장 많았으며, ICM 세포는 혈청 첨가로 유의하게 증가하였다(p<0.05). 2. 체외성숙용 배지에 혈청의 첨가시기에 따른 한우 난포란의 제1극체 출현율은 체외성숙 18시간 동안의 처리군과 체외성숙 9시간째부터 첨가한 처리군이 미첨가군보다 높았으며, 체 외 성숙 후 9시간까지의 처리군보다는 유의하게 높았다(p<0.05). 체외성숙용 배지에 혈청의 첨가시기에 따른 한우 난포란의 배발달율은 전군에서 비슷한 수준이었다. 배반포의 TE와 총세포수는 비슷한 경 향이었으나, ICM 세포수는 체외성숙 18시간 동안 처리군이 혈청 미첨가군보다 유의하게 높았다(p<0.05).

Keywords

References

  1. Abeydeera LR, Wang WH, Cantley TC, Rieke A and Day BN. 1998. Coculture with follicular shell pieces can enhance the developmental competence of pig oocytes after in vitro fertilization: relevance to intracellular glutathione. Biol. Reprod., 58:213-218 https://doi.org/10.1095/biolreprod58.1.213
  2. Ali A and Sirard MA. 2002. Effect of the absence or presence of various protein supplements on further development of bovine oocytes during in vitro maturation. Biol. Reprod., 66:901-905 https://doi.org/10.1095/biolreprod66.4.901
  3. Behbooi E, Anderson GB, Bondurant RH, Cargill SL, Kreusher BR, Medrano JF and Murlay JD. 1995. Birth of large calves that developed from in vitro-derived bovine embryos. Theriogenology, 44:227-232 https://doi.org/10.1016/0093-691X(95)00172-5
  4. Blondin P and Sirard MA. 1995. Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes. Mol. Reprod. Dev., 41:54-62 https://doi.org/10.1002/mrd.1080410109
  5. Choi SH, Ryu IS, Kim IH, Park SB, Yeon SH, Jin HJ, Suh CS and Son DS. 1999. Effects of in vitro maturation condition on bovine IVF embryos development. Korean J. Emb. Trans., 14: 113-119
  6. Choi YH, Carnevale EM, Seidel GE and Squires EL. 2001. Effects of gonadotropins on bovine oocytes matured in TCM-199. Theriogenology, 56:661-670 https://doi.org/10.1016/S0093-691X(01)00597-0
  7. Edwards RG. 1965. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature, 208:349-51 https://doi.org/10.1038/208349a0
  8. Eppig JJ, Schroeder AC and O'Brien MJ. 1992. Developmental capacity of mouse oocytes matured in vitro: effects of gonadotrophic stimulation, follicular origin and oocyte size. J. Reprod. Fertil., 95:119-127 https://doi.org/10.1530/jrf.0.0950119
  9. Fukui Y and Ono H. 1989. Effects sera, hormonse and granulosa cells added to culture medium for in vitro maturation, fertilization, cleavage and development of bovine oocytes. J. Reprod. Fertil., 86:501-506 https://doi.org/10.1530/jrf.0.0860501
  10. Gardner DK. 1994. Mammalian embryo culture in the absence of serum or somatic cell support. Cell Biol Int., 18:1163-1179 https://doi.org/10.1006/cbir.1994.1043
  11. Gardner DK and Lane M. 1993. Embryo culture systems. In: Trounson A, Gardner DK (eds.), Handbook of In vitro Fertilization. Boca Raton, FL : CEC Press; 85-114
  12. Geshi M, Yonai M, Sakaguchi M and Nagai T. 1999. Improvement of in vitro co-culture systems for bovine embryos using a low concentration of carbon dioxide and medium supplemented with $\beta$ -mercaptoethanol. Theriogenology, 51:551-558 https://doi.org/10.1016/S0093-691X(99)00009-6
  13. Hunter MG. 2000. Oocyte maturation and ovum quality in pigs. Rev. Reprod., 5:122-130 https://doi.org/10.1530/ror.0.0050122
  14. Iwasaki S, Yoshida N, Ushijima H, Watanabe S and Nakahara T. 1990. Morphology and proportion of inner cell mass of bovine blastocysts fertilized in vitro and in vivo. J. Reprod. Fertil., 90:279-84 https://doi.org/10.1530/jrf.0.0900279
  15. Khatir H, Lonergan P and Mermillod P. 1998. Kinetics of nuclear maturation and protein profiles of oocytes from prepubertal and adult cattle during in vitro maturation. Theriogenology, 50:917-929 https://doi.org/10.1016/S0093-691X(98)00196-4
  16. Kim DJ, Chung HJ, Kim DH, Uhm SJ, Lee HT and Chung KS. 2001. Effects of FSH and LH on maturation of bovine preantral follicle. Korean J. Animal Reprod., 25:101-111
  17. Kito S and Bavister BD. 1991. Male pronuclear formation and early embryonic development of hamster oocytes matured in vitro with gonadotropins, amino acid and cysteamine. J. Reprod. Fertil., 110:35-46
  18. Lee ES and Fukui Y. 1996. Synergistic effect of alanine and glycine on bovine embryos cultured in a chemically defined medium and amino acid uptake by vitro-produced bovine morulae and blastocysts. Biol. Reprod. 55:1383-1389 https://doi.org/10.1095/biolreprod55.6.1383
  19. Lim JM, Lee BC, Lee ES, Chung HM, Ko JJ, Park SE, Cha KY and Hwang WS. 1999. In vitro maturation and in vitro fertilization of bovine oocytes cultured in a chemically defined, protein- free medium: effects of carbohydrates and amino acids. Reprod. Fertil. Dev., 11:127-132 https://doi.org/10.1071/RD99001
  20. Linder GE and Wright RW. 1983. Bovine embryo morphology and evaluation. Theriogenology, 20:407-416 https://doi.org/10.1016/0093-691X(83)90201-7
  21. Lonergan P, Monaghan P, Rizos D, Boland MP and Gordon I. 1994. Effect of follicle 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
  22. Nagai T. 2001. The improvement of in vitro maturation systems for bovine and porcine oocytes. Theriogenology, 55:1291-1301 https://doi.org/10.1016/S0093-691X(01)00483-6
  23. Papaioannou VE and Ebert KM. 1988. The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro. Development, 102:793-803
  24. Park YS, Kim SS, Choi SH, Park NC, Byun MD and Park HD. 2004. Effects of amino acid in in-vitro maturation medium on nuclear maturation and embryo development of Korean Native Cow. Reprod. Deve. Biol., 28:29-38
  25. Park YS, Choi SH, Han JC, Park HD and Byun MD. 2002. Effects of maturation time on in-vitro production of Korean Native Cow embryos. Korean J. Anim. Reprod., 27:35-44
  26. Pinyopummintr T and Bavister BD. 1991. In vitro matured/in vitro fertilized bovine oocytes can develop into morulae/blastocysts in chemically defined protein-free culture medium. Biol. Reprod., 45:736-742 https://doi.org/10.1095/biolreprod45.5.736
  27. Pinyopummintr T and Bavister BD. 1994. Development of bovine embryos in a cell-free culture mdrium: effects of type of serum, timing of its inclusion and heat inactivation. Theriogenology, 41:1241-1249 https://doi.org/10.1016/0093-691X(94)90481-W
  28. Rebecca LK, Michelle L and Barry DB. 1999. Developmental competence and metabolism of bovine embryos cultured in semi defined and defined culture media. Biol. Reprod., 60:1345- 1352 https://doi.org/10.1095/biolreprod60.6.1345
  29. Sirard MA, Florman HM, Leibfried-Rutledge ML, Barnes FL, Sims ML and First NL. 1989. Timing for nuclear progression and protein synthesis necessary for meiotic maturation of bovine oocytes. Biol. Reprod., 40:1257-1263 https://doi.org/10.1095/biolreprod40.6.1257
  30. Steeves TE and Gardner DK. 1999. Temporal and differential effects of amino acids on bovine embryo development in culture. Biol. Reprod., 61:731-740 https://doi.org/10.1095/biolreprod61.3.731
  31. Trounson A, Anderiesz C and Jones G. 2001. Maturation of human oocytes in vitro and their developmental competence. Reproduction, 121: 51-75 https://doi.org/10.1530/rep.0.1210051
  32. Ward F, Enright B, Riaos D, Boland M and Lonergan P. 2002. Optimization of in vitro bovine embryo production: effect of duration of maturation, length of gamete co-incubation, sperm concentration and sire. Theriogenology, 57:2105-2117 https://doi.org/10.1016/S0093-691X(02)00696-9
  33. Watson AJ, Sousa P, Caveney A, Barcroft LC, Natale D, Urquhart J and Westhusin ME. 2000. Impact of bovine oocyte maturation media on oocyte transcript levels, blastocyst development, cell number, and apoptosis. Biol. Reprod., 62:355-364 https://doi.org/10.1095/biolreprod62.2.355
  34. Yang NS, Lu KH and Gordon I. 1990. In vitro fertilization (IVF) and culture(IVC) of bovine oocytes from stored ovaries. Theriogenology, Abst., 33:352 https://doi.org/10.1016/0093-691X(90)90776-P