한국가축번식학회지 (Korean Journal of Animal Reproduction)

한국동물번식학회 (The Korean Society of Animal Reproduction)
권호 표지

소 동결-융해 정자에 있어서 체외수정능력과 정자 기능 및 성상 분석법간의 상관관계

Correlations between the Capacity of In Vitro Fertilization and the Assays of Sperm Function and Characteristics in Frozen-thawed Bovine Spermatozoa

  • 류범용 (서울대학교 의학연구원 인구의학연구소) ;
  • 정영채 (중앙대학교 동물자원과학과) ;
  • 김창근 (중앙대학교 동물자원과학과) ;
  • 신현아 (중앙대학교 동물자원과학과) ;
  • 한정호 (서울대학교 의학연구원 인구의학연구소) ;
  • 김석현 (서울대학교 의학연구원 인구의학연구소, 서울대학교 의과대학 산부인과학교실) ;
  • 문신용 (서울대학교 의학연구원 인구의학연구소, 서울대학교 의과대학 산부인과학교실) ;
  • 김흥률 (농협중앙회 젖소개량부) ;
  • 최한 (농협중앙회 젖소개량부)
  • Ryu, B.Y. (Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University) ;
  • Chung, Y.C. (Department of Animal Science and Technology, Chung-Ang University) ;
  • Kim, C.K. (Department of Animal Science and Technology, Chung-Ang University) ;
  • Shin, H.A. (Department of Animal Science and Technology, Chung-Ang University) ;
  • Han, J.H. (Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University) ;
  • Kim, S.H. (Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Department of Obstetrics and Gynecology, College of Medicine, Seoul National University) ;
  • Moon, S.Y. (Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Department of Obstetrics and Gynecology, College of Medicine, Seoul National University) ;
  • Kim, H.R. (National Agriculture Cooperation Federation, Dairy Cattle Improvement Center) ;
  • Choi, H. (National Agriculture Cooperation Federation, Dairy Cattle Improvement Center)
  • 발행 : 2002.09.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

본 연구는 종모우의 정자수정능력 평가방법의 개발과 정자 기능 및 성상 변화에 영향을 미치는 요인을 알아보기 위하여 시행하였다. 동결-융해된 종모우 정액을 대상으로 정자의 운동성과 정자의 형태를 분석하였고, 정자의 기능 검 사 항목으로서 체외수정(IVF), HOST, Ca-ionophore에 의한 첨체반응율, 정자의 ROS 측정을 위한 luminol, lucigenin-dependent chemiluminescence, LPO 분석을 위한 malondialdehyde의 측정 및 TUNEL (terminal deoxynucleotidyl transferase(TdT) dUTP nick end labelling) 기법을 이용한 정자의 DNA fragmentation를 측정하였으며 이들 각각의 조사 항목들의 분석치들과 체외수정율 및 배발생율과의 상관관계를 조사하여 다음과 같은 결과를 얻었다. 1. 고수정군과 저수정군의 체외수정율과 배반포 발생율의 평균은 각각 64.4%와 34.3%, 18.50%와 6.2%였으며 두 군간에 통계학적으로 유의한 차이를 보였다(P<0.05). 고수정군과 저수정군의 정자운동성과 첨체반응률은 각각 평균79.0 %와 66.2%, 40.7%와 22.9%로 두 군간에 통계학적으로 유의한 차이를 보였으나(P<0.05), 정상형태 정자의 비율과 HOST는 각각 평균 94.6%와 92.7%, 69.4%와 59.8%로 두 군간 유의한 차이를 보이지 않았다. 2. Luminol dependent chemiluminescence, LPO 및 DNA fragmentation의 평균은 고수정군과 저수정군에 있어서 각각 6.4와 6.5, 2.Onmol와 3.Inmol 및 2.6%와 7.4%로 두 군간 통계학적으로 유의한 차이를 보였으나(P<0.05), lucigenin dependent chemiluminescence는 4.7와 4.6로 두 군간 유의한 차이를 보이지 않았다. 3. 체외 수정율은 정자의 운동성 및 첨체반응율과 통계학적으로 유의한 정(positive)의 상관관계(r=0.87, p<0.01; r=0.81, p<0.05)를 나타내었으며, luminol dependent chemiluminescence, lipid peroxldation 및 DNA fragmentation과는 통계학적으로 유의한 부(negative)의 상관관계 (r= -0.81, p<0.05; r: -0.74, p<0.05; r : 0.81, p<0.05)를 나타내었다. 그러나 체외수정율은 정상형태 정자의 비율, HOST 및 lucigenin dependent chemiluminescence와는 유의한 상관 관계를 나타내지 않았다. 4. 배반포 발생율은 첨체반응율과 통계학적으로 유의한 정의 상관관계(r=0.71, p<0.05)를 나타내었으며, luminol dependent chemiluminescence, lipid peroxidation 및 DNA fragmentation과는 통계학적으로 유의한 부의 상관관계(r= -0.71, p<0.05; r= -0.89, p<0.01; r= -0.71, P<0.05)를 나타내었다. 배반포 발생율은 정자의 운동성, 정상형태 정자의 비율 및 HOST, lucigenin dependent chemilumihescence와는 유의한 상관관계를 나타내지 않았다. 이상의 결과를 종합해 보면 정액질의 저하에 ROS의 영향이 밀접히 연관되어 있음을 알 수 있으며, 또한 본 연구에서 적용된 기법들은 정액질의 평가 및 정자 수정능력 향상을 위한 기술개발에 있어서 유용한 평가 방법으로 이용될 수 있을 것으로 사료된다.

The objective of this study was to develop an in vitro assessment of sperm fertilizing capacity of bulls and investigate the factors influencing sperm function and characteristics of frozen-thawed bovine spermatozoa. in vitro fertilization (IVF), the evaluation of motility and normal morphology, HOST (hypoosmotic swelling test), Ca-ionophore induced acrosome reaction, luminol and lucigenin-dependent chemiluminescence for the measurement of reactive oxygen species (ROS), the measurement of malondialdehyde formation for the analysis of lipid peroxidation (LPO), and the evaluation of DNA fragmentation using the method of 747-mediated nick end labelling (TUNEL) by flow cytometry were performed in frozen-thawed bovine spermatozoa. Correlations between the rates of fertilization, blastocyst formation after IVF and the values of respective assays were investigated. 1. IVF rate and blastocyst formation rate averaged 64.4% and 34.3% for spermatozoa from high -fertility bull group and averaged 18.5% and 6.2% for spermatozoa from low-fertility bull group, respectively. There were significantly different between two bull groups. Sperm motility and percentage acrosome reaction averaged 79.0% and 66.2% for spermatozoa from high-fertility bull group and averaged 40.7% and 22.9% for spermatozoa from low-fertility bull group, respectivitely. There were not different between two bull groups. 2. Luminol depenent chemiluminescence, LPO and DNA fragementation averaged 6.4, 2.0 nmol and 2.6% from spermatozoa from high-fertility bull group and averaged 6.5, 3.1 nmol and 7.4% for spermatozoa from low-fertility bull group, respectively. There were significantly different between two bull groups. There was no significant difference in lucigenin dependent chemiluminescence between two bull groups. 3. Fertilization rate was positively correlated with motility and the rate of Ca-ionophore induced acrosome reaction, but negatively correlated with the frequency of luminol-dependent chemiluminescence, the rate of LPO, and the percentage of sperm with DNA fragmentation. There was no correlation between fertilization rate and the percentage of swollen spermatozoa, normal morphology, and the frequency of lucigenin-dependent chemiluminescence. 4. Blastocyst formation rate was positively correlated with the rate of Ca-ionophore induced acrosome reaction, but negatively correlated with the frequency of luminol-dependent chemiluminescence, the rate of LPO, and the percentage of sperm with DNA fragmentation. There was no correlation between blastocyst formation rate and motility, the percentage of swollen spermatozoa, normal morphology, and the frequency of lucigenin-dependent chemiluminescence. In conclusion, these data suggest that ROS significantly impact semen quality. The assays of this study may provide a basis fur improving in vitro assessment of sperm fertilizing capacity.

키워드

참고문헌

  1. Aitken, R. J., Clarkson, J. S., Hargreave, T. B., Irvine, D. S. and Wu. F. C. 1989. Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia. J. Androl., 10:214-220 https://doi.org/10.1002/j.1939-4640.1989.tb00091.x
  2. Aitken, R. J., Buckingham, D. W. and Katrine, M. W. 1992. Reactive oxygen species and human spermatozoa: Analysis of the cellular mechanism involved in luminol- and lucigenin-dependent chemiluminescence. J. Cell. Physiol., 151:466-477 https://doi.org/10.1002/jcp.1041510305
  3. Aitken, R. J., Harkiss, D. and Buckingham. D. W. 1993. Analysis of lipid peroxidation mechanism in human spermatozoa. Mol. Reprod. Dev., 35:302-315 https://doi.org/10.1002/mrd.1080350313
  4. Alkan, I., Sirnsek, F., Haklar, G., Kervancioglu, E., Ozveri, H., Yalcin, S. and Akdas, A. 1997. Reactive oxygen species production by the spermatozoa of patients with idiopathic infertility: relationship to seminal plasma antioxidants. J. Urol., 157:140-143 https://doi.org/10.1016/S0022-5347(01)65307-2
  5. Alvarez, J. G., Touchstone, J. C., Blasco, L. and Storey, B. T. 1987. Spontaneous lipid peroxidation and production of hydrogen peroxide in human spermatozoa: superoxide dismutase as major enzyme protectant against oxygen toxicity. J. Androl., 8:338-348 https://doi.org/10.1002/j.1939-4640.1987.tb00973.x
  6. Alvarez, J. G. and Storey, B. T. 1992. Evidence for increased peroxidative damage and loss of super oxide dismutase activity as a model of sublethal cryodamage to human sperm during cryopreservation. J. Androl., 13:232-241
  7. Bennet, P. J., Moatti, J. P., Mansat, A., Ribbes, H., Cayrac, J. C., Pontonnier, F., Chap, H. and Douste-Blazy, L. 1987. Evidence for the activation of phospholipase during acrosome reaction of human sperm elicited by calcium ionophore A23187. Biochim. Biophys. Acta., 919:255-265 https://doi.org/10.1016/0005-2760(87)90265-7
  8. Beorlegui, N., Cetica, P., Trinchero, M., Cordoba, M. and Beconi, M. 1997. Comparative study of functional and biochemical parameters in frozen bovine sperm. Andrologia, 29:37-42. https://doi.org/10.1111/j.1439-0272.1997.tb03146.x
  9. Biggers, J. S., Whitten, W. K. and Whittingham, D. G. 1971. The culture of mouse embryos in vitro. In: Method in mammalian embryology, J.C. Daniel (Ed.), Freeman, San Francisco, pp. 86-116
  10. Blom, E. 1973. The ultrastructure of some characteristic sperm defects and a proposal for a new classification of the bull spermiogram. Nord. Vet. Med., 28:511
  11. Blondin, P., Coenen, K. and Sirard, M. A. 1997. The impact of reactive oxygen species on bovine sperm fertilizing ability and oocyte maturation. J. Androl., 18:454-460
  12. Brackett, B. G. and Oliphant, G. 1975. Capacitation of rabbit spermatozoa in vitro. Biol. Reprod., 12:260-274 https://doi.org/10.1095/biolreprod12.2.260
  13. Calamera, J. C., Doncel, G. F., Olmedo, S. B., Kolm, P. and Acosta, A. A. 1998. Modified sperm stress test: a simple assay that predicts sperm -related abnormal in-vitro fertilization. Reprod., 13:2484-2488
  14. Comporti, M. 1989. Three models of free radical induced cell injury. Chem. Biol. Interactions, 72:1-56.
  15. Cummins, J. M., Pember, S. M., Jequier, A. M., and Yovich, J. L. 1991. A test of the human sperm acrosome reaction following ionophore challenge. J. Androl., 12:98-103
  16. De Lamirande, E. and Gagnon, C. 1992. Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axoneme; and II. Depletion of adenosine triphosphate plays an important role in the inhibition of sperm motility. J. Androl., 13:368-386
  17. De Lamirande, E. and Gagnon, C. 1995. Impact of reactive oxygen species on spermatozoa; a balancing act between beneficial and detrimental effects. Hum. Reprod., 10(suppl 1):15-21
  18. De Leeuw, A. M., Den Daas, J, H. and Woelders, H. 1991. The fix vital stain method. Simultaneous determination of viability and acrosomal status of bovine spermatozoa. J. Androl., 12:112-118
  19. Goldman, R., Ferber, E. and Zort, U. 1992. Reactive oxygen species are involved in the activation of cellular phospholipase A2. FEBS Lett., 309:190-192 https://doi.org/10.1016/0014-5793(92)81092-Z
  20. Hammitt, D. G., Martin, P. A. and Callanan, T. 1989. Correlation between heterospermic fertility and assays of porcine seminal quality before and after cryopreservation. Theriogenology, 32: 385-399
  21. Howard, T. H. and Pace, M. M. 1988. Seminal evaluation and artificial insemination. In: Fertility and infertility in veterinary practice, J.A. Laing, W. J. B. Morgan, W. C. Wagner (Eds.), 4 th ed., Bailliere Tindall, Philadelphia, USA, pp. 39-51
  22. Janny, L. and Menezo, Y. 1994. Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation. Mol. Reprod. Dev., 38:36-42
  23. Jeyendran, R. S., Van der Ven, H. H., Perez-Pelaez, M., Crabo, B. G. and Zaneveld, L. J. 1984. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod. Fertil., 70:219-228 https://doi.org/10.1530/jrf.0.0700219
  24. Jeyendran, R. S., Van der Ven, H. H. and Zaneveld, L. J. 1992. The hypoosmotic swelling test: an update. Arch. Androl., 29:105-116 https://doi.org/10.3109/01485019208987714
  25. Kelso, K. A., Redpath, A., Noble, R. C. and Speake, B. K. 1997. Lipid and antioxidant changes in spermatozoa and seminal plasma throughout the reproductive period of bulls. J. Reprod. Fertil., 109:1-6 https://doi.org/10.1530/jrf.0.1090001
  26. Kiefer, D., Check, J. H. and Katsoff, D. 1996. The value of motile density, strict morphology, and the hypoosmotic swelling test in in vitro fertilization and embryo transfer. Arch. Androl., 37:57-60
  27. Koukoulis, G. N., Vantman, D. J., Dennison, L., Banks, S. M. and Sherins, R. J. 1989. Low acrosin activity in a subgroup of men with idiopathic infertility does not correlate with sperm density, percent motility, curvilinear velocity of linearity. Fertil. Steril., 52: 120-127 https://doi.org/10.1016/S0015-0282(16)60800-2
  28. Lopes, S., Jurisicova, A., Sun, J. G. and Casper, R. F. 1998. Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum. Reprod., 13:896-900 https://doi.org/10.1093/humrep/13.4.896
  29. Mordel, N., Dano, I., Epstein-Eldan, M., Shemesh, A., Schenker, J. G. and Laufer, N. 1993. Novel parameters of human sperm hypoosmotic swelling test and their correlation to standard spermatogram, total motile sperm fraction, and sperm penetration assay. Fertil. Steril., 59:1276-1279
  30. Munne, S. and Estop, A. 1991. The effect of in-vitro ageing on mouse sperm chromosomes. Hum. Reprod., 6:703-708. https://doi.org/10.1093/oxfordjournals.humrep.a137412
  31. Naz, R. K. 1992. Effects of antisperm antibodies on early cleavage of fertilized ova. Biol. Reprod., 7(suppl. 1):101-106
  32. Oehninger, S., Coddington, C. C., Scott, R., Franken, D. A., Burkman, L. J., Acosta, A. A. and Hodgen, G. D. 1989. Hemizona assay: assessment of sperm dysfunction and prediction of in vitro fertilization outcome. Fertil. Steril., 51:665-670 https://doi.org/10.1016/S0015-0282(16)60618-0
  33. Sakkas, D., Batt, P. A. and Cameron, A. W. N. 1989. Development of preimplantation goat (Capra hircus) embryos in vivo and in vitro. J. Reprod. Fertil., 87:359-365 https://doi.org/10.1530/jrf.0.0870359
  34. Slater, T. F. and Sawyer, B. C. 1971. The stimulatory effects of carbon tetrachloride and other halogenoalkanes on peroxidative in rat liver fractions in vitro. Biochem. J., 123:805-814 https://doi.org/10.1042/bj1230805
  35. Smith, R., Vantman, D., Ponce, J., Escobar, J. and Lissi, E. 1996. Total antioxidant capacity of human seminal plasma. Hum. Reprod., 11:1655-1660
  36. Sun, J. G., Jurisicova, A. and Casper, R. F. 1997. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol. Reprod., 56:602-607 https://doi.org/10.1095/biolreprod56.3.602
  37. Tesarik, J. 1989. Appropriate timing of the acrosome reaction is a major requirement for the fertilizing spermatozoon. Hum. Reprod., 4:957-961 https://doi.org/10.1093/oxfordjournals.humrep.a137020
  38. Twigg, J., Fulton, N., Gomez, E., Irvine, D. S. and Aitken, R. J. 1998. Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants. Hum. Reprod., 13:1429-1436
  39. Woelders, P. F. 1991. Overview of in vitro methods for evaluation of semen quality. In: Boar semen preservation II, L.A. Johnson, D. Rath (Eds.), Paul Parey Scientific Publishers, Berlin, German, pp. 145-164
  40. Yanagimachi, R., Yanagimach, H. and Rogers, B. J. 1976. The use of zona-free animal ova as a test system for the assessment of the fertilizing capacity of human spermatozoa. Biol. Reprod., 15:471-476 https://doi.org/10.1095/biolreprod15.4.471
  41. Yovich, J. M., Edirisinghe, W. R. and Yovich, J. L. 1994. Use of the acrosome reaction to ionophore challenge test in managing patients in an assisted reproduction program: a prospective, double-blind, randomized controlled study. Fertil. Steril., 61:902-910 https://doi.org/10.1016/S0015-0282(16)56704-1
  42. 류범용, 김석현, 박서영, 지병철, 정병준, 김회선, 방명걸, 오선경, 서창석, 최영민, 김정구, 문신용, 이진용, 손영수. 1998. 인간 정자의 수정 능력 평가에 있어서 ARlC 검사의 유용성에 관한 연구. 대한산부인과학회잡지. 41:2562-2570