Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
권호 표지

Evaluation of DNA Fragments on Boar Sperm by Ligation-mediated Quantitative Real Time PCR

  • Lee, Eun-Soo (Division of Biological Science, Gachon University of Medicine and Science) ;
  • Choi, Sun-Gyu (Division of Biological Science, Gachon University of Medicine and Science) ;
  • Yang, Jae-Hun (Division of Biological Science, Gachon University of Medicine and Science) ;
  • Bae, Mun-Sook (Konkuk University School of Medicine) ;
  • Park, Jin-Young (Division of Biological Science, Gachon University of Medicine and Science) ;
  • Park, Hong-Min (Division of Biological Science, Gachon University of Medicine and Science) ;
  • Han, Tae-Kyu (Division of Biological Science, Gachon University of Medicine and Science) ;
  • Hwang, You-Jin (Division of Biological Science, Gachon University of Medicine and Science) ;
  • Kim, Dae-Young (Division of Biological Science, Gachon University of Medicine and Science)
  • Received : 2010.05.18
  • Accepted : 2010.05.28
  • Published : 2010.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

Sperm chromatin integrity is essential for successful fertilization and development of an embryo. Reported here is a quantification of DNA fragments which is intimately associated with reproductive potential to provide one of criteria for sperm chromatin integrity. Three sperm populations were considered: CONTROL (no treatment), UV irradiation (48mW/$cm^2$, 1h) and $H_2O_2$ (oxidative stress induced by hydrogen peroxide, 10 mM, 50 mM and 100 mM). DNA fragments in boar sperm were evaluated by using ligation-mediated quantitative real-time polymerase chain reaction (LM-qPCR) assay, which relies on real-time qPCR to provide a measure of blunt 5' phosphorylated double strand breaks in genomic DNA. The results in agarose gel electrophoresis showed no significant DNA fragmentation and no dose-dependent response to $H_2O_2$. However, the remarkable difference in shape and position was observed in melting curve of LM-qPCR. This result supported that the melting curve analysis of LM-qPCR presented here, could be more sensitive and accurate than previous DNA fragmentation assay method.

Keywords

References

  1. Agarwal A and Said TM. 2003. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum. Reprod. Update 9:331-345. https://doi.org/10.1093/humupd/dmg027
  2. Bee-Hansen GB, Christensen P, Vibjerg D, Nielsen MB and Hedeboe AM. 2008. Sperm chromatin structure integrity in liquid stored boar semen and its relationships with field fertility. Theriogenology 69:728-736. https://doi.org/10.1016/j.theriogenology.2007.12.004
  3. Boe-Hansen GB, Ersb$\phi$ll AK, Greve T and Christensen T. 2005. Increasing storage time of extended boar semen reduces sperm DNA integrity. Theriogenology 63:2006-2019. https://doi.org/10.1016/j.theriogenology.2004.09.006
  4. Bryla M, Trzcinska M, Wieczorek J, Slomski R and Smorag Z. 2010. Effect of semen quality in transgenic boars on the developmental competence of preimplantation embryos. Anim. Reprod. Sci. 118:77-82. https://doi.org/10.1016/j.anireprosci.2009.06.008
  5. Byun JW, Choo SH, Kim HH, Kim YJ, Hwang YJ and Kim DY. 2008. Evaluation of boar sperm viability by MTT reduction assay in beltsville thawing solution extender. Asian-Aust. J. Anim. Sci. 21:494-498. https://doi.org/10.5713/ajas.2008.70480
  6. De Ambrogi M, Spinaci M, Galeati G and Tamanini C. 2006. Viability and DNA fragmentation in differently sorted boar spermatozoa. Theriogenology 66:1994-2000. https://doi.org/10.1016/j.theriogenology.2006.05.017
  7. De la Torre J, Lopez-Fernandez C, Pita M, Fernandez JL, Johnston SD and Gosalvez J. 2007. Simultaneous observation of DNA fragmentation and protein loss in the boar spermatozoon following application of the sperm chromatin dispersion (SCD) test. J. Androl. 28:533-540. https://doi.org/10.2164/jandrol.106.002246
  8. Didion BA, Kasperson KM, Wixon RL and Evenson DP. 2009. Boar fertility and sperm chromatin structure status: A retrospective report. J. AndroI. 30:655-660. https://doi.org/10.2164/jandrol.108.006254
  9. Enciso M, Lopez-Fernandez C, Fernandez JL, Garcia P, Gosalbez A and Gosalvez J. 2006. A new method to analyze boar sperm DNA fragmentation under bright-field or fluorescence microscopy. Theriogenology 65:308-316. https://doi.org/10.1016/j.theriogenology.2005.05.044
  10. Evenson DP, Larson KJ and Jost LK. 2002. Sperm chromatin structure assay: Its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J. Androl, 23:25-43.
  11. Flores E, Cifuentes D, Fernandez-Novell JM, Medrano A, Bonet S, Briz MD, Pinart E, Pena A, Rigau T and RodriguezGil JE. 2008. Freeze-thawing induces alterations in the protamine- l/DNA overall structure in boar sperm. Theriogenology 69:1083-1094. https://doi.org/10.1016/j.theriogenology.2008.01.022
  12. Fraser L and Strzezek J. 2007a. Effect of different procedures of ejaculate collection, extenders and packages on DNA integrity of boar spermatozoa following freezing-thawing. Anim. Reprod. Sci. 99:317-329. https://doi.org/10.1016/j.anireprosci.2006.06.003
  13. Fraser L and Strzezek J. 2007b. Is there a relationship between the chromatin status and DNA fragmentation of boar spermatozoa following freezing-thawing. Theriogenology 68:248-257. https://doi.org/10.1016/j.theriogenology.2007.05.001
  14. Gadella BM and Harrison RA. 2002. Capacitation induces cyclic adenosine 3',5'-monophosphate-dependent, but apoptosis-unrelated, exposure of aminophospholipids at the apical head plasma membrane of boar sperm cells. Biol. Reprod. 67: 340-350. https://doi.org/10.1095/biolreprod67.1.340
  15. Hernandez M, Roca J, Ballester J, Vazquez JM, Martinez EA, Johannisson A, Saravia F and Rodriguez-Martinez H. 2006. Differences in SCSA outcome among boars with different sperm freezability. Int. J. Androl. 29:583-591. https://doi.org/10.1111/j.1365-2605.2006.00699.x
  16. Hu JH, Li QW, Jiang ZL and Li WY. 2008. Effects of different extenders on DNA integrity of boar spermatozoa following freezing-thawing. Cryobiology 57:257-262. https://doi.org/10.1016/j.cryobiol.2008.09.004
  17. Hwang YJ, Bae MS, Yang JH, Kim BK, Kim SO, Lee ES, Choi SG, Kwon YR, Suh MH and Kim DY. 2009. Sex ratio determination by quantitative real time PCR using amelogenin gene in porcine sperm. J. Emb. Trans. 24: 225-230.
  18. Hwang YJ, Yang JH, Kim SO, Kim BK, Choi SG, Park CK and Kim DY. 2009. Effect of dimethyIformamide on post-thaw motility, acrosome integrity and DNA structure of frozen boar sperm. J. Emb. Trans. 24:275-279.
  19. Karja NW, Wongsrikeao P, Murakami M, Agung B, Fahrudin M, Nagai T and Otoi T. 2004. Effects of oxygen tension on the development and quality of porcine in vitro fertilized embryos. Theriogenology 62:1585-1595. https://doi.org/10.1016/j.theriogenology.2004.03.012
  20. Lopez-Fernandez C, Perez-Llano B, Garcia-Casado P, Sala R, Gosalbez A, Arroyo F, Fernandez JL and Gosalvez J. 2008. Sperm DNA fragmentation in a random sample of the Spanish boar livestock. Anim. Rcprod. Sci. 103:87-98. https://doi.org/10.1016/j.anireprosci.2006.11.015
  21. Maleszewski M, Kuretake C, Evenson D, Yanagimachi H, Bjordahl J and Yanagimachi R. 1998. Behavior of transgenic mouse spermatozoa with gaIline protamine. Biol. Rcprod. 58:8-14. https://doi.org/10.1095/biolreprod58.1.8
  22. Meliska CJ and Bartke A, 1997. Copulatory behavior and fertility in transgenic male mice expressing human placental growth hormone gene. J. Androl. 18:305-311.
  23. Nakai M, Kashiwazaki N, Takizawa A, Maedomari N, Ozawa M, Noguchi J, Kaneko H, Shino M and Kikuchi K. 2007. Effects of chelating agents during freeze-drying of boar spermatozoa on DNA fragmentation and on developmental ability in vitro and in vivo after intracytoplasmic sperm head injection. Zygote. 15:15-24. https://doi.org/10.1017/S0967199406003935
  24. Perez-Llano B, Enciso M, Garcia-Casado P, Sala R and Gosalvez J. 2006. Sperm DNA fragmentation in boars is delayed or abolished by using sperm extenders. Theriogenology 66:2137-2143. https://doi.org/10.1016/j.theriogenology.2006.01.066
  25. Perez-Llano B, Lopez-Fernandez C, Garcia-Casado P, Arroyo F, Gosalbez A, Sala R and Gosalvez J. 2010. Dynamics of Sperm DNA fragmentation in the swine: Ejaculate and temperature effects. Anim. Reprod. Sci. 119:235-243. https://doi.org/10.1016/j.anireprosci.2010.01.002
  26. Salas A, Remesar X, Esteve M. 2007. Oleoyl-estrone treatment activated apoptotic mechanism in white adipose tissue. Life Science. 280(4):293-8.
  27. Saravia F, Nunez-Martmez I, Moran JM, Soler C, Muriel A, Rodriguez-Martinez H and Pena FJ. 2007. Differences in boar sperm head shape and dimensions recorded by computer- assisted sperm morphometry are not related to chromatin integrity. Theriogenology 68:196-203. https://doi.org/10.1016/j.theriogenology.2007.04.052
  28. Silva PNF and Gadella BM. 2006. Detection of damage in mammalian sperm cells. Theriogenology 65:958-973. https://doi.org/10.1016/j.theriogenology.2005.09.010
  29. Staley K, Blaschke AJ, Chun J. 1997. Apoptotic DNA fragmentation is deetected by a semi-quantitative ligation-mediated PCR of blunt DNA ends. Cell Death Differentiation. 4(1):66-75. https://doi.org/10.1038/sj.cdd.4400207