The Study on Apoptosis and Expression of Fas, Fas-ligand, Bax, and Bcl-2 in Human Fragmented Embryos

분절화된 인간 배아에서 세포자연사와 Fas, Fas-ligand, Bax, Bcl-2 발현에 관한 연구

  • Kim, Jong-Sik (Infertility Research Laboratory, Seoul Women's Hospital) ;
  • Kim, Myoung-Shin (Infertility Research Laboratory, Seoul Women's Hospital) ;
  • Yang, Hyun-Won (Department of OB/GYN, Eulji University School of Medicine) ;
  • Yu, Chai-Hyeock (Department of Life Science, College of Natural Sciences, Inha University) ;
  • Yoon, Yong-Dal (Department of Life Science, College of Natural Sciences, Hanyang University) ;
  • Bae, In-Ha (Department of Biology, College of Natural Sciences, Sungsin Women's University) ;
  • Jung, Byeong-Jun (Infertility Clinic, Seoul Women's Hospital) ;
  • Song, Hyun-Jin (Infertility Clinic, Seoul Women's Hospital)
  • 김종식 (서울여성병원 불임연구실) ;
  • 김명신 (서울여성병원 불임연구실) ;
  • 양현원 (을지대학교 의과대학 산부인과학교실) ;
  • 류재혁 (인하대학교 생물학과) ;
  • 윤용달 (한양대학교 생명과학과) ;
  • 배인하 (성신여자대학교 생명과학과) ;
  • 정병준 (서울여성병원 불임클리닉) ;
  • 송현진 (서울여성병원 불임클리닉)
  • Published : 2002.09.30

Abstract

Objective : The present study was performed to investigate whether apoptosis occur in human embryos by annexin staining and detect the expression of Fas, Fas-ligand (FasL), Bax, and Bcl-2 in human fragmented embryos derived from IVF-ET by immunofluorescence and Western blot analysis. Materials and Methods: Using annexin staining, immunofluorescence and Western blot analysis on normal and fragmented embryos, we were able to detect apoptotsis and apoptotic gene products in fragmented embryos. Result: Phosphatidylserine (PS) translocation, the marker for apoptosis, were detected frequently in fragmented embryos. Bcl-2 and Bax protein were detected in both fragmented and non-fragmented embryos. When fragmented embryos compared to normal embryos, immunofluorescent intensity of Bcl-2 tended to be lower in fragmented embryos. Bax gene expression increased in the fragmented embryos compared to the normal embryos. This result supports a model in which the molar ratio of Bcl-2 to Bax determines whether apoptosis induced or inhibited in human embryo. Fas was highly expressed in human preimplantation embryos but not FasL. It suggests that embryo may undergo apoptosis by binding with FasL produced by follicular or immune cells. Conclusion: The over expression of Bax and Fas will trigger apoptosis to lead embryo fragmentation and change embryo to be nonviable.

Keywords

References

  1. Andres S, Christel HG, Maria SA, Aila T, Timo T. The predictive value of pronuclear morphology of zygotes in the assessment of human embryo quality. Hum Reprod 2001; 16: 2177-81 https://doi.org/10.1093/humrep/16.10.2177
  2. Scott L, Alvero R, Leonides M, Miller B. The morphology of human PN embryos is positively related to blastocyst development and implantation. Hum Reprod 2000; 15: 2394-403 https://doi.org/10.1093/humrep/15.11.2394
  3. Perez G, Tao XJ, Tilly J. Fragmentation and death (a.k.a. apoptosis) of ovulated oocytes. Mol Hum Reprod 1999; 5: 414-20 https://doi.org/10.1093/molehr/5.5.414
  4. Alikani M, Cohen J, Tomkin G. Human embryo fragmentation in vitro and its implications for preg nancy and implantation. Fertil Steril 1999; 71: 836-42 https://doi.org/10.1016/S0015-0282(99)00092-8
  5. Flechon JE, Kopecny V. The nature of the 'nucleolus precursor body' in early preimplantation embryos: a review of fine-structure cytochemical, immunocytochemical and autoradiographic data related to nucleolar function. Zygote 1998; 6: 183-91 https://doi.org/10.1017/S0967199498000112
  6. Antczak M, Van Blerkom J. Temporal and spatial aspects of fragmentation in early human embryos: possible effects on developmental competence and association with the differential elimination of regulatory proteins from polarized domains. Hum Reprod 1999; 14: 429-47 https://doi.org/10.1093/humrep/14.2.429
  7. Levy R, Benchaib M, Cordonier H. Annexin V labelling and terminal transferase-mediated DNA end labelling (TUNEL) assay in human arrested embryos. Mol Hum Reprod 1998; 4: 775-86 https://doi.org/10.1093/molehr/4.8.775
  8. Fujino Y, Ozaki K, Yamamasu S, Ito F, Matsuoka I, Hayashi E, et al. DNA fragmentation of oocytes in aged mice. Hum Reprod 1996; 11: 1480-3 https://doi.org/10.1093/oxfordjournals.humrep.a019421
  9. Collins MK, Perkins GR, Rodriguez TG, Nieto MA, Lopez RA. Growth factors as survival factors: regulation of apoptosis. Bioessays 1994; 16: 133-8 https://doi.org/10.1002/bies.950160210
  10. Yang HW, Hwang KJ, Kwon HC, Kim HS, Choi KW, Oh KS. Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos. Hum Reprod 1998; 13: 998-1002 https://doi.org/10.1093/humrep/13.4.998
  11. Aitken RJ, Clakson JS. Significance of reactive oxygen species and antioxidant in defining the efficacy of sperm preparation technigues. J Andrology 1998; 9: 367-71
  12. Allan DJ, Harmon BV, Roberts SA. Spermatogonial apoptosis has three morpholgically recognizable phases and sows no circadian rhythm during normal spermatogenesis in the rat. Cell Prolif 1992; 25: 241-50 https://doi.org/10.1111/j.1365-2184.1992.tb01399.x
  13. Jurisicova A, Ian R, Alessandro F, Robert FC, Sue V. Effect of maternal age and conditions of fertilization on programmed cell death during murine preimplantation embryo development. Mol Hum Reprod 1998; 4: 139-45 https://doi.org/10.1093/molehr/4.2.139
  14. Jurisicova A, Keith EL, Robert FC, Susannah LV. Expression and regulation of genes associated with cell death during murine preimplantation embryo development. Mol Reprod Dev 1998; 51: 243-53 https://doi.org/10.1002/(SICI)1098-2795(199811)51:3<243::AID-MRD3>3.0.CO;2-P
  15. Kypianou N, Issacs JT. Activation of programmed cell death in the rat ventural prostate after castration. Endocrinology 1998; 122: 552-62 https://doi.org/10.1210/endo-122-2-552
  16. Tapanainen JS, Tilly JL, Vihko KK, Hsueh AJW. Hormonal control of apoptotic cell death in the testis: gonadotropins and androgens as testicular cellsurvival factors. Mol Endocrinol 1993; 7: 643-50 https://doi.org/10.1210/me.7.5.643
  17. Palumbo A, Yeh J. Apoptosis as a basic mechanism in the ovarian cycle: follicular atresia and luteal regression. J Soc Gynecol Invest 1995; 2: 565-73 https://doi.org/10.1016/1071-5576(94)00062-6
  18. Kokawa K, Shikone T, Nakano R. Apoptosis in the human uterine endometrium during the menstrual cycle. J Clin Endocrinol Metab 1996; 81: 4144-7 https://doi.org/10.1210/jc.81.11.4144
  19. 이병석, 최은아, 장경환, 김진영, 배상욱, 박기현, 조동제, 이국, 김재욱, 송찬호. 황체화된 인간 과립세포에서 Apoptosis 관련 유전자인 bcl-2와 T-RPM-2의 발현. 대한불임회지 1997; 24: 267-71
  20. Reed JC. Bcl-2 and the regulation of programmed cell death. J Cell Biol 1997; 124: 1-6 https://doi.org/10.1083/jcb.124.1.1
  21. Katsuji K, Toshihiko S, Tsutomu O, Rika N, Yuki I, Shigetaka Y, et al. Apoptosis and the expression of Bax and Bcl-2 in hyperplasia and adenocarcinoma of the uterine endometrium. Hum Reprod 2001; 16: 2211-8 https://doi.org/10.1093/humrep/16.10.2211
  22. Kokawa K, Shikone T, Otani T. Apoptosis and the expression of Bcl-2 and Bax in patients with endometrioid, clear cell and serous carcinomas of the uterine endometrium. Gynecol Oncol 2001; 81: 178-83 https://doi.org/10.1006/gyno.2001.6138
  23. Palumbo A, Yeh J. In situ location of apoptosis in the rat ovary during follicular atresia. Biol Reprod 1994; 51: 888-95 https://doi.org/10.1095/biolreprod51.5.888
  24. Ratts VS, Flaws JA, Kolp R, Sorenson CM, Tilly JL. Ablation of bcl-2 gene expression decreases the number of oocytes and primordial follicles established in the post-natal female mouse gonad. Endocrinology 1995; 136: 3665-8 https://doi.org/10.1210/en.136.8.3665
  25. Liu HC, He ZY, Mele CA, Veeck LL, Davis O, Rosenwaks Z. Expression of apoptosis-related genes in human oocytes and embryos. J Assist Reprod Genet 2000; 17: 521-33 https://doi.org/10.1023/A:1009497925862
  26. Yonehara S, Ishii A, Yonehara M. A cell killing monoclonal antibody (anti-Fas) to a cell surface antigen co-down reulated with the receptor of tumor necrosis factor. J Exp Med 1989; 169: 1747 https://doi.org/10.1084/jem.169.5.1747
  27. Hsueh AJW, Billig H, Tsafriri A. Ovarian follicle atresia: A hormonally controlled apoptotic process. Endocrine Reviews 1994; 15: 707-24
  28. World Health Organization. Laboratory manual for the examination of human semen and sperm cervical mucus interaction. 3rd ed., Cambridge, Cambridge University Press; 1992
  29. Veeck LL. Preembryo grading: In atlas of the human oocyte and early embryo conceptus. Vol 2, Baltimore: Williams and Wilkins 1991; 121-49
  30. Jurisicova A, Varmuza S, Casper RF. Programmed cell death and human embryo fragmentation. Mol Hum Reprod 1996; 2: 93-8 https://doi.org/10.1093/molehr/2.2.93
  31. Flach G, Jhonson MH, Braude PR, Taylor RS, Bolton VN. The transition from maternal to embryonic control in the 2-cell mouse embryo. EMBO J 1982; 1: 681-6
  32. Jarrell VL, Day BN, Prather RS. The trancription from maternal to zygotic control of development occurs during the 4-cell stage in the domestic pig, Sus scrofa: Quantitative and qualitative aspacts of protein synthesis. Biol Reprod 1991; 44: 62-8 https://doi.org/10.1095/biolreprod44.1.62
  33. Gloria IP, Xiao JT, Jonathan LT. Fragmentation and death (a.k.a. apoptosis) of ovulated oocytes. Mol Hum Reprod 1999; 5: 414-20 https://doi.org/10.1093/molehr/5.5.414
  34. Aoki F, Worrad DM, Schultz RM. Regulation of transcriptional activity during first and second cell cycles in the preimplantation embryo. Dev Biol 1997; 181: 296-307 https://doi.org/10.1006/dbio.1996.8466
  35. Arends ML, Morris RG, Wyllie AH. Apoptosis: The role of the endonuclease. Am J Pathol 1990; 136: 593-608
  36. Jonathan VB, Patrick D, Samuel A. A microscopic and biochemical study of fragmentation phenotypes in stage-appropriate human embryos. Hum Reprod 2001; 16: 719-29 https://doi.org/10.1093/humrep/16.4.719
  37. Jonathan VB, Patrick WD. DNA strand breaks and phosphatidylserine redistribution in newly ovulated and cultured mouse and human oocytes: occurrence and relationship to apoptosis. Hum Reprod 1998; 13: 1317-24 https://doi.org/10.1093/humrep/13.5.1317
  38. Owens GP, Hahn WE, Cohen J. Identification of mRNAs associated with programmed cell death in immature thymocytes. Mol Cell Biol 1991; 11: 4177-88 https://doi.org/10.1128/MCB.11.8.4177
  39. Kokawa K, Shikone T, Otani T. Apoptosis and the expression of Bax and Bcl-2 in squamous cell carcinoma and adenocarcinoma of the uterine cervix. Cancer 1999; 85: 1799-809 https://doi.org/10.1002/(SICI)1097-0142(19990415)85:8<1799::AID-CNCR21>3.0.CO;2-M
  40. Hockenbery DM, Nunez G, Milliman C, Schreiber RD, Korsmeyer SJ. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 1990; 348: 334-6 https://doi.org/10.1038/348334a0
  41. Kamada S, Shimono A, Shinto Y, Tsujimura T, Takahashi T, Nada T, et al. Bcl-2 deficiency in mice leads to pleiotropic abnormalities: Accelerated lymphoid cell death in thymus and spleen, polycystic kidney, hairhypopigmentation, and distorted small intestine. Cancer Res 1995; 55: 354-9
  42. Kunson CM, Korsmeyer SJ. Bcl-2 and bax function independently to regulate cell death. Nat Genet 1997; 16: 358-63 https://doi.org/10.1038/ng0897-358
  43. Tilly JL, Tilly KI, Kenton ML, Johnson AL. Expression of members of the bcl-2 gene family in the mature rat ovary: Equine chorionic gonadotropin-mediated inhibition of granulosa cell apoptosis is a associated with decreased bax and constitutive bcl-2 and bcl-xong messenger ribonucleic acid levels. Endocrinolgy 1995; 136: 232-41 https://doi.org/10.1210/en.136.1.232
  44. Ginger EE, Chaoyu T, Abigail SM, Carol MW. Expression of caspase and Bcl-2 apoptotic family members in mouse preimplantation embryos. Biol Reprod 1999; 61: 231-9 https://doi.org/10.1095/biolreprod61.1.231
  45. Kondo H, Maruo T, Peng X, Mochizuki M. Immunological evidence for the expression of the Fas antigen in the infant and adult human ovary during follicular regression and atresia. J Clin Endocrinol Metab 1996; 81: 2702-10 https://doi.org/10.1210/jc.81.7.2702
  46. Kawamura K, Fukuda J, Kodama H, Kumagai J, Kumagai A, Tanaka T. Expression of Fas and Fas ligand mRNA in rat and human preimplantation embryos. Mol Hum Reprod 2001; 7: 431-6 https://doi.org/10.1093/molehr/7.5.431
  47. Rodriguez IM, Matsuura K, Ody C, Nagata S, Vassalli P. Systemin injection of a tripeptide inhibits the intracellular activation of CPP32-like proteases in vivo and fully protects mice against Fas -mediated fulminant liver destruction and death. J Exp Med 1996; 184: 2067-72 https://doi.org/10.1084/jem.184.5.2067