Reproductive and Developmental Biology

  • 제29권1호
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  • Pages.1-7
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  • 2005
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  • 1738-2432(pISSN)
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  • 2288-0151(eISSN)
한국동물번식학회 (The Korean Society of Animal Reproduction)
권호 표지

마우스 수정란의 초기 배 발생단계별 Telomeric DNA의 양적 분석과 Telomerase 활성도 분석

Analysis of the Amount of Telomeric DNA and Telomerase Activity on Preimplantation Mouse Embryoic Cells

  • 강민영 (진주산업대학교 동물생명과학과·동물생명산업연구센터) ;
  • 한명숙 (진주산업대학교 동물생명과학과·동물생명산업연구센터) ;
  • 이상찬 (세화병원) ;
  • 김종흥 (세화병원) ;
  • 손시환 (진주산업대학교 동물생명과학과·동물생명산업연구센터)
  • Kang M. Y. (Department of Animal Science and Biotech. & RAIRC, Jinju National University) ;
  • Han M. S. (Department of Animal Science and Biotech. & RAIRC, Jinju National University) ;
  • Lee S. C. (SeaWha Hospital) ;
  • Kim J. H. (SeaWha Hospital) ;
  • Sohn S. H. (Department of Animal Science and Biotech. & RAIRC, Jinju National University)
  • 발행 : 2005.03.01
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초록

텔로미어란 염색체 말단부에 (TTAGGG)n의 반복 염기서열이 단백질과 결합된 형태를 말하는데 이의 역할은 염색체의 안정성에 본질적으로 작용하여 세포의 노화, 사멸 및 암의 발생과 관련이 있다고 알려져 있다. 반면 텔로머레이스는 telomeric DNA 합성에 직접 관여하는 ribonucleoprotein이다. 본 연구에서는 마우스 염색체의 텔로미어 분포 양상을 제시하고, 초기 배 발생단계별 수정란의 텔로미어 함량과 이들 수정란의 텔로머레이스 활성도를 분석하고자 하였다. 본 분석에는 마우스의 섬유아세포, 생식세포, 정자, 난자 및 1세포기, 2세포기, 4세포기, 8세포기, 상실배와 배반포배의 각 단계별 수정란을 대상으로 하였다. 텔로미어의 양적 분석은 human telomeric DNA probe를 이용한 Q-FISH 방법을 이용하였고, 텔로머레이스 활성도는 TRAP 방법을 이용하였다. 분석 결과 마우스 염색체의 텔로미어는 성 염색체를 포함한 모든 염색체의 앙 말단부에 분포되어 있고, 염색체별 다소의 양적 차이를 보이나 대부분의 염색체에서 q-arm 말단이 p-arm 말단에 비해 높은 텔로미어의 함량을 나타내었다. Q-FISH를 이용한 마우스 초기 배 발생단계별 수정란의 텔로미어의 양적 분석에서 수정 직후 1세포기에서부터 상실배까지 거의 비슷한 텔로미어 함유율을 나타내고 있으나 배반포기에서 월등히 증가된 양상을 나타내었다. TRAP 분석을 이용한 초기배아의 텔로머레이스 활성도는 초기 배 발생 모든 단계에서 이의 활성도를 나타내었으며, 특히 상실배 및 배반포기에서 점진적으로 강한 활성을 보였다. 이상의 분석 결과로부터 마우스의 초기 배 분열단계의 각 세포들에 있어 텔로미어의 함유율과 텔로머레이스 활성도는 높은 상관관계가 있는 것으로 나타났다. 따라서 포유동물의 초기 배자에 있어 텔로미어의 함유율과 텔로머레이스 활성도는 배 발생 및 배자의 세포 분화와 매우 밀접한 관련이 있는 것으로 사료되어 텔로미어의 양적 분석 및 텔로머레이스 활성도 분석은 발생학적 연구를 위한 또 다른 좋은 자료로 생각된다.

Telomeres consisting of (TTAGGG)n tandem repeat DNA sequences and associated proteins are essential for chromosome stability and related with cell senescence, apoptosis and cancer. The telomerase is a ribonucleoprotein which act as a template for the synthesis of telomeric DNA. This study was carried out to identify the distribution of telomeres on mouse chromosomes and also to analyze the amount of telomeres and telomerase activity of mouse embryos at early embryonic stages. Germ cells and early embryos from 1 cell to blastocyst stage were analyzed. The amount of telomeres was analyzed by quantitative fluorescence in situ hybridization technique(Q-FISH) using a human telomeric DNA probe, and telomerase activity was measured by telomeric repeat amplification protocol assay(TRAP). In results, the telomeres on mouse chromosomes were distributed at the ends of all autosomes and sex chromosomes. Although the quantity of telomeres varied among chromosomes, most of chromosomes had higher amount in q-arm telomeres than in p-arm telomeres. The results of Q-FISH indicated that the relative amount of telomeres of mouse embryos in each embryonic stage was approximately the same except the higher amount in blastocysts. Using TRAP assay on mouse embryos, telomerase activity was detected in all preimplantation stages from mature oocytes to blastocysts. Especially the telomerase activity was significantly increased at the morula and blastocyst stage. In conclusion, there may be a close association between the amount of telomeres and telomerase activity in early embryonic stages, and analysis of telomere quantity and telomerase activity on early development will be helpful for the investigation of embryogenesis and embryonic cell differentiation in mice.

키워드

참고문헌

  1. Artandi SE, Alson S, Tietze MK, Sharpless NE, Ye S, Greenberg RA, Castrillon DH, Horner JW, Weiler SR, Carrasco RD, DePinho RA (2002): Constitutive telomerase expression promotes mammary carcinomas in aging mice. Genetics 99:8191-8196
  2. Betts DH, King WA (1999): Telomerase activity and telomere detection during early bovine development Dev Genet 25:397-403 https://doi.org/10.1002/(SICI)1520-6408(1999)25:4<397::AID-DVG13>3.0.CO;2-J
  3. Blackburn EH (1991): Structure and function of telomeres. Nature 350:569-573 https://doi.org/10.1038/350569a0
  4. Blasco MA (2003): Telomerase in cancer and aging: lessons from the mouse. Cancer Letters 194:183-184 https://doi.org/10.1016/S0304-3835(02)00705-X
  5. Bryant E, Hutchings KG, Moyzis RK, Griffith JK (1997): Measurement of telomeric DNA content in human tissues. Biotech 23:476-478
  6. Buys C (2000): Telomeres, telomerase, and cancer. New England J Med 342:1282-1283 https://doi.org/10.1056/NEJM200004273421710
  7. Chang JY (2004): Telomerase: A potential molecular marker and therapeutic target for cancer. J Sur Oncology 87:1-3 https://doi.org/10.1002/jso.20076
  8. Cheong C, Hong KU, Lee HW (2003): Mouse models for telomere and telomerase biology. Exp Mol Med 3:141-153
  9. Cottliar AS, Slavutsky IR (2001): Telomeres and telomerase activity: their role in aging and in neoplastic development. Medicina 61:335-342
  10. Dolci S, Levai L, Pellegrini M, Faraoni L, Graziani G, Carlo AD, Geremia R (2002): Stem cell factor activates telomerase in mouse mitotic spermatogonia and in primordial germ cells. J Cell Sci 115:1643-1649
  11. Eisenhauer KM, Gerstein RM, Chiu CP, Conti M, Hsueh AJ (1997): Telomerase activity in female and male rat germ cells undergoing meiosis and in early embryos. Biol Reprod 56:1120-1125 https://doi.org/10.1095/ biolreprod56.5.1120
  12. Ferlicot S, Youssef NF, Delhommeau F, Paradis V, Bedossa P (2003): Measurement of telomere length on tissue sections using quantitative fluorescence in situ hybridization (Q-FISH). J Pathology 200:661-666 https://doi.org/10.1002/path.1392
  13. Gan Y, Engelke KJ, Brown CA, Au JLS (2001): Telomere amount and length assay. Pharmaceutical Research 18: 1655-1659 https://doi.org/10.1023/A:1013306109801
  14. Greider CW (1991): Telomeres. Curr. Opin. Cell Biol 3:444-451 https://doi.org/10.1016/0955-0674(91)90072-7
  15. Greider CW (1993): Telomerase and telomere-length regulation: lessons from small eukaryotes to mammals [Review]. Cold Spring Harbor Symp Quant Biol 58:719-723. https://doi.org/10.1101/SQB.1993.058.01.079
  16. Greider CW, Blackburn EH (1985): Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43:405-413 https://doi.org/10.1016/0092-8674(85)90170-9
  17. Han MS, Cho EJ, Ha HB, Park HS, Sohn SH (2004): Sex determination of in vitro fertilized bovine embryos by fluorescence in situ hybridization technique. Reprod Dev Biol 28:133-137
  18. Harley CB (1991): Telomere loss: mitotic clock or genetic time bomb? [Review]. Mutat Res 256:271-282. https://doi.org/10.1016/0921-8734(91)90018-7
  19. Herrera E, Samper E, Blasco MA (1999): Telomere shortening in mTR-/- embryos is associated with failure to close the neural tube. EMBO 18:1172-1181 https://doi.org/10.1093/emboj/18.5.1172
  20. Holt SE, Shay JW, Wright WE (1996): Refining the telomere-telomerase hypothesis of aging and cancer. Nat Biotech 14:836-839 https://doi.org/10.1038/nbt0796-836
  21. Kim NW, Wu F (1997): Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP). Nucleic Acids Res 25:2595-2597 https://doi.org/10.1093/nar/25.13.2595
  22. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW (1994): Specific association of human telomerase activity with immortal cells and cancer. Science 266:2011-2015 https://doi.org/10.1126/science.7605428
  23. Kipling D (1997): Telomerase structure and telomerase expression during mouse development and tumorigenesis. Eur J Cancer 33:792-800 https://doi.org/10.1016/S0959-8049(97)00060-9
  24. Kipling D, Cooke HJ (1990): Hypervariable ultra-long telomeres in mice. Nature 347:400-402 https://doi.org/10.1038/347400a0
  25. Kobayashi J, Sekimoto A, Uchida H, Wada T, Sasaki K, Sasada H, Umezu M, Sato E (1998): Rapid detection of male-specific DNA sequence in bovine embryos using fluorescence in situ hybridization. Mol Reprod Develop 51:390-394 https://doi.org/10.1002/(SICI)1098-2795(199812)51:4<390::AID-MRD5>3.0.CO;2-F
  26. Lansdorp PM, Verwoerd NP, van de Rijke FM, Dragowska V, Little MT, Dirks RW, Raap AK, Tanke HJ (1996): Heterogeneity in telomere length of human chromosomes. Hum Mol Genet 5:685-691 https://doi.org/10.1093/hmg/5.5.685
  27. Law HKW, Lau YU (2001): Validation and development of quantitative flow cytometry-based fluorescence in situ hybridization for intercenter comparison of telomere length measurement. Cytometry 43:150-153 https://doi.org/10.1002/1097-0320(20010201)43:2<150::AID-CYTO1030>3.0.CO;2-H
  28. Lingner J, Cech TR (1998): Telomerase and chromosome end maintenance. Curr Opin Genet Dev 8:226-232 https://doi.org/10.1016/S0959-437X(98)80145-7
  29. Liu J (1999): Studies of the molecular mechanisms in the regulation of telomerase activity. FASEB 13:2091-2104 https://doi.org/10.1096/fasebj.13.15.2091
  30. Liu L, Blasco MA, Trimarchi JR, Keefe DL (2002): An essential role for functional telomeres in mouse germ cells during fertilization and early development. Dev Biol 249:74-84 https://doi.org/10.1006/dbio.2002.0735
  31. Oexle K (1998): Telomere length distribution and southern blot analysis. J Theor Biol 190:369-377 https://doi.org/10.1006/jtbi.1997.0559
  32. Pathak S, Multani AS, Furlong CL, Sohn SH (2002). Telomere dynamics, aneuploidy, stem cells and cancer. Int J Oncology 20:637-641
  33. Poon SS, Martens UM, Ward RK, Lansdorp PM (1999): Telomere length measurements using digital fluorescence microscopy. Cytometry 36:267-278 https://doi.org/10.1002/(SICI)1097-0320(19990801)36:4<267::AID-CYTO1>3.0.CO;2-O
  34. Prowse KR, Greider CW (1995): Developmental and tissue-specific regulation of mouse telomerase and telomere length. Cell Biol 92:4818-4822
  35. Ravindranath N, Dalal R, Solomon B, Djakiew D, Dym M (1997): Loss of telomerase activity during male germ cell differentiation. Endocrinology 138: 4026-4029 https://doi.org/10.1210/en.138.9.4026
  36. Robinson MO (2000): Telomerase and cancer. Genet Eng 22:209-222
  37. Shay JW, Bacchetti S (1997): A survey of telomerase activity in human cancer. Eur J Cancer 33:787-791 https://doi.org/10.1016/S0959-8049(97)00062-2
  38. Slijepcevic P (2001): Telomere length measurement by Q-FISH. Methods in Cell Science 23:17-22 https://doi.org/10.1023/A:1013177128297
  39. Starling JA, Maule J, Hastie ND, Allshire RC (1990): Extensive telomere repeat arrays in mouse are hypervariable. Nucleic Acids Res 18:6881-6888 https://doi.org/10.1093/nar/18.23.6881
  40. Weng NP, Hodes RJ (2000). The role of telomerase expression and telomere length maintenance in human and mouse. J Clinical Immunology 20:257-267 https://doi.org/10.1023/A:1017223602293
  41. Wright WE, Piatyszek MA, Rainey WE, Byrd W, Shay JW (1996): Telomerase activity in human germline and embryonic tissues and cells. Dev Genet 18: 173-179 https://doi.org/10.1002/(SICI)1520-6408(1996)18:2<173::AID-DVG10>3.0.CO;2-3
  42. Xu J, Yang X (2000): Telomerase activity in bovine embryos during early development. Biol Reprod 63:1124-1128 https://doi.org/10.1095/biolreprod63.4.1124
  43. Yen CH, Pazik J, Elliott RW (1996): A polymorphic interstitial telomere array near the center of mouse chromosome 8. Mamm Genome 7:218-221 https://doi.org/10.1007/s003359900059
  44. Yen CH, Pazik J, Zhang Y, Elliott RW (1997): An interstitial telomere array proximal to the distal telomere of mouse chromosome 13. Mamm Genome 8:411-417 https://doi.org/10.1007/s003359900458
  45. Zijlmans JM, Martens UM, Poon SS, Roap AK, Tanke, HJ, Ward RK, Lansdorp PM (1997): Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats. Proc Natl Acad Sci U.S.A. 94:7423-7428 https://doi.org/10.1073/pnas.94.14.7423
  46. 손시환, 박충생, 송상현 (1996): 염색체 분석 기법에 의한 소 체외수정란의 성 조절. 한국가축번식학회지 20: 179-190