Evaluation of Sexing in Boar Sperm Using Chromogenic In Situ Hybridization

Chromogenic In Situ Hybridization을 이용한 돼지 정자의 성 선별

  • Kim, H.H. (Equal contributors) ;
  • Roh, D.E. (Equal contributors) ;
  • Jo, T.K. (Division of Biological Science, Gachon University of Medicine and Science) ;
  • Byun, J.W. (Division of Biological Science, Gachon University of Medicine and Science) ;
  • Lee, J.W. (Division of Biological Science, Gachon University of Medicine and Science) ;
  • Kim, Y.S. (Division of Biological Science, Gachon University of Medicine and Science) ;
  • Hwang, Y.J. (Division of Biological Science, Gachon University of Medicine and Science) ;
  • Kim, D.Y. (Division of Biological Science, Gachon University of Medicine and Science)
  • Published : 2007.09.29


돼지 정자의 성 선별에는 일반적으로 유속 세포 분석기를 이용한다. 유속 세포 분석은 DNA량의 차이에 기초하여 정자를 분리하는 기술로써 X 정자와 Y 정자를 90% 정확도로 분리할 수 있다. 그러나 이러한 유속 세포분석 기술은 정자의 손상을 야기해 정자의 기능과 수정능에 영향을 미치므로, 본 연구에서는 특정한 핵산 서열을 탐지할 수 있는 Chromogenic in situ hybridization(CISH)을 그와 비교하여 평가하였다. 유속 세포 분석을 수행하기 위해 정자를 SYBR 14와 PI로 염색하였고, histogram, dotplot, density, contour를 측정하였다. Y 염색체 특이적인 primer를 이용한 PCR로 유속 세포 분석의 정확도를 검사하였다. HRP/DAB 시스템에 기초한 CISH 분석에는 X 또는 Y 염색체에 상보적으로 결합하는 probe가 사용되었다. CISH 분석은 기존의 방법들보다 빠르고 쉬우며 비용이 적게 든다는 장점이 있다. 또한, CISH는 보다 정화한 정자의 선별을 가능하게 하는 것으로 나타났다. 본 연구에 따르면 CISH가 기존의 선별 방법들을 평가하는 기술로서만이 아니라 특정한 성별을 가진 포유동물의 생산에도 사용될 수 있을 것이다.


  1. Abeydeera LR, Johnson LA, Welch GR, Wang WH, Boquest AC, Cantley TC, Rieke A and Day BN. 1998. Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry. Theriogenology, 50:981-988 https://doi.org/10.1016/S0093-691X(98)00201-5
  2. Amina Vocaturo, Flavia Novelli, Maria Benevolo, Giulia Piperno, Ferdinando Marandino, Anna Maria Cianciulli, Roberta Merola, Raffaele Perrone Donnorso, Isabella Sperduti, Simonetta Buglioni and Marcella Mottolese. 2007. Chromogenic in situ hybridization to detect HER-2/neu gene amplification in histological and thinPrep ${\circledR}$-Processed breast cancer fine-needle aspirates: A sensitive and practical method in the trastuzumab era. Oncologist, 11 :878-886 https://doi.org/10.1634/theoncologist.11-8-878
  3. Beckett TA, Martin RH and Hoar DI. 1989. Assessment of the sephadex technique for selection of X-bearing sperm by analysis of sperm chromosomes, deoxyribonucleic acid and Y bodies. Fertil. Steril., 52:829-835 https://doi.org/10.1016/S0015-0282(16)53048-9
  4. Chandler JE, Canal AM, Paul JB and Moser EB. 2002. Collection frequency affects percent Y-chromosome bearing sperm, sperm head area and quality of bovine ejaculates. Theriogenology, 57:1327-1346 https://doi.org/10.1016/S0093-691X(01)00721-X
  5. Chandler JE, Steinholt-Chenevert HC, Adkinson RW and Moser EB. 1998. Sex ratio variation between ejaculates within sire evaluated by polymerase chain reaction, calving and farrowing records. J. Dairy Sci., 81:1855-1867 https://doi.org/10.3168/jds.S0022-0302(98)75756-X
  6. Checa ML, Dunner S and Canon J. 2002. Prediction of X- and Y-chromosome content in bovine sperm by using DNA pools through capillary electrophoresis. Theriogenology, 58:1579-1586 https://doi.org/10.1016/S0093-691X(02)01077-4
  7. Cran DG, Johnson LA and Polge C. 1995. Sex preselectionin cattle: A field trial. Vet. Ret., 135:495-496
  8. Fugger EF, Black SH, Keyvanfar K and Schulman JD. 1998. Births of normal daughters after MicroSort sperm separation and intrauterine insemination, in-vitro fertilization, or intracytoplasmic sperm injection. Hum. Reprod., 13:2367-2370 https://doi.org/10.1093/humrep/13.9.2367
  9. Gamer DL and Johnson LA. 1995. Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol. Reprod., 53:276-284 https://doi.org/10.1095/biolreprod53.2.276
  10. Gamer DL. 2001. Sex-sorting mammalian sperm: concept to application in animals. J. Androl., 22:519-526
  11. Gamer DL. 2006. Flow cytometric sexing of mammalian sperm. Theriogenology, 65:943-957 https://doi.org/10.1016/j.theriogenology.2005.09.009
  12. Gimenez C, Egozcue J and Vidal F. 1994. Sexing sibling mouse blastomeres by polymerase chain reaction and fluorescent in-situ hybridization. Hum. Reprod., 9:2145-2149 https://doi.org/10.1093/oxfordjournals.humrep.a138407
  13. Inmaculada Parrilla, Juan M. Vazquez, Maria A. Gil, Ignacio Caballero, Carmen Almin ana, Jordi Roca and Emilio A. Martinez. 2005. Influence of storage time on functional capacity of flow cytometrically sex-sorted boar spermatozoa. Theriogenology, 64:86-98 https://doi.org/10.1016/j.theriogenology.2004.11.004
  14. Joerg H, Asai M, Graphodaskaya D, Janett F and Stranzinger G. 2004. Validating bovine sexed semen samples using quantitative PCR. J. Anim. Breed. Genet., 121:209-215 https://doi.org/10.1111/j.1439-0388.2004.00455.x
  15. Johnson LA. 2000. Sexing mammalian sperm for production of offspring: the state of the art. Anim. Reprod. Sci., 60-61:93-107 https://doi.org/10.1016/S0378-4320(00)00088-9
  16. Johnson LA, Flook JP and Hawk HW. 1989. Sex preselectionin rabbits: Live births from X and Y sperms eparated by DNA and cell sorting. Biol. Reprod., 411: 99-203
  17. Kawarasaki T, Welch GR, Long CR, Yoshida M and Johnson L. 1998. A verification of flow cytometrically-sorted X- and Y-bearing porcine spermatozoa and reanalysis of spermatozoa for DNA content using the fluorescence in situ hybridization (FISH) technique. Theriogenology, 50:625-635 https://doi.org/10.1016/S0093-691X(98)00167-8
  18. Lobel SM, Pomponio RJ and Mutter GL. 1993. The sex ratio of normal and manipulated human sperm quantitated by polymerase chain reaction. Fertil. Steril., 59:387-392 https://doi.org/10.1016/S0015-0282(16)55682-9
  19. Maxwell WMC, Evans G, Hollinshead FK, Bathgate R, deGraaf SP and Eriksson BM. 2004. Integration of sperm sexing technology into the art toolbox. Anim. Reprod. Sci., 83:79-85 https://doi.org/10.1016/j.anireprosci.2004.04.013
  20. Minna Tanner, David Gancberg, ba Angelo Di Leo, Denis Larsimont, Ghizlane Rouas, Martine J. Piccart, and Jorma Isola. 2000. A practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer sample. Am. J. Pathol., 157:1467-1472 https://doi.org/10.1016/S0002-9440(10)64785-2
  21. Ogawa S, Yamakawa H, Yamanoi J, Nishida S, Kano Y and Takeshima T. 1988. Are fluorescent bodies of Y-spermatozoa detectable in common with mammalian species? Theriogenology, 29: 1 083-1089 https://doi.org/10.1016/S0093-691X(88)80032-3
  22. Parati K, Bongioni G, Aleandri R and Galli A. 2006. Sex ratio determination in bovine semen: A new approach by quantitative real time PCR. Theriogenology, 66:2202-2209 https://doi.org/10.1016/j.theriogenology.2006.07.007
  23. Probst S and Rath D. 2003 Production of piglets using intracytoplasmic sperm injection (ICSI) with flowcytometrically sorted boar semen and artificially activated oocytes. Theriogenology, 59:961-973 https://doi.org/10.1016/S0093-691X(02)01135-4
  24. Rath D, Johnson LA, Dobrinsky JR, Welch GR and Niemann H. 1997. Production of piglets preselected for sex following in vitro fertilization with X and Y chromosome-bearing spermatozoa sorted by flow cytometry. Theriogenology,47: 795-800 https://doi.org/10.1016/S0093-691X(97)00035-6
  25. Rath D, Long CR, Dobrinsky JR, Welch GR, Schreier LL and Johnson LA. 1999. In vitro production of sexed embryos for gender preselection: high-speed sorting of X-chromosome-bearing sperm to produce pigs after embryo transfer. J. Anim. Sci., 77:3346-3352
  26. Sato T, Ikuta K, Sherlock J, Adinolfi M and Suzumori K. 2003. Comparison between fluorescence in situ hybridization (FISH) and quantitative-fluorescent polymerase chain reaction (QF-PCR) for the detection of aneuploidies in single blastomeres. Prenat. Diagn., 23 :678-684 https://doi.org/10.1002/pd.660
  27. Seidel Jr GE. 2007. Overview of sexing sperm. Theriogenology, 68:443-446 https://doi.org/10.1016/j.theriogenology.2007.04.005
  28. Seidel Jr GE and Gamer DL. 2002. Sexing mammalian sperm by flow cytometry. Reproduction, 124:733-743 https://doi.org/10.1530/rep.0.1240733
  29. Shimada K, Valdez MB Jr, Mizutani M and Namikawa T. 2007 Potential application of sperm bearing female-specific chromosome in chickens. Cytogenet. Genome Res., 117: 240-247 https://doi.org/10.1159/000103185
  30. Szyda J, Simianer H and Lien S. 2000. Sex ratio distortion in bovine sperm correlates to recombination in the pseudoautosomal region. Genet Res. Camb., 75:53-59 https://doi.org/10.1017/S0016672399004085
  31. Welch GR, Waldbieser GC, Wall RJ and Johnson LA. 1995. Flow cytometric sperm sorting and PCR to confirm separation of X-and Y-chromosome bearing bovine sperm. Anim. Biotechnol., 6:131-139 https://doi.org/10.1080/10495399509525842
  32. Wyrobek AJ, Schmid TE and Marchetti F. 2005. Cross-species sperm-FISH assays for chemical testing and assessing paternal risk for chromosomally abnormal pregnancies. Environ. Mol. Mutagen., 45:271-283 https://doi.org/10.1002/em.20121