Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/13262
標題: 利用蛋白質體學來分析豬精蟲特異性蛋白之研究
Analysis of sex-specific proteins from porcine sperm membrane by a Proteomics approach
作者: 李芝穎
Lee, Chih-Ying
關鍵字: sperm;精蟲;sex-specific proteins;性特異性蛋白
出版社: 獸醫學系暨研究所
引用: 1. 鄭秋虹。利用性別差異免疫法篩選豬X/Y精子特異性膜蛋白之研究。碩士論文。國立中興大學獸醫學研究所。台灣省。中華民國。2003。 2. Aasen E, Medrano JF. Amplification of the ZFY and ZFX genes for sex identification in humans, cattle, sheep and goats. Biotechnology 8: 1279-1281, 1990. 3. Ali JI, Eldridge FE, Koo GC, Schanbacher BD. Enrichment of bovine X- and Y-chromosome-bearing sperm with monoclonal H-Y antibody - fluorescence- activated cell sorter. Arch Androl 24: 235-245, 1990. 4. Anderson GB. Identification of embryonic sex by detection of H-Y antigen. Theriogenology 27: 81-97, 1987. 5. APPa KB, Rao C, Pawshe H, Totey SM. Sex determination of in vitro developed buffalo (Bualus bubalis) embryos by DNA amplification. Mol Reprod Dev 36: 291-296, 1993. 6. Avery B, Schmidt M. Sex determination of bovine embryos using H-Y antibodies. Acta Vet Scand 30: 155-164, 1989. 7. Beernink FJ, Dmowski WP, Ericsson RJ. Sex preselection through albumin separation of sperm. Fertil Steril 59: 382-386, 1993. 8. Bennett D, Boyse EA. Sex ratio in progeny of mice inseminated with sperm treated with H-Y antiserum. Nature 246: 308-309, 1973. 9. Blecher SR, Howie S, Li S, Detmar J, Blahut LM. A new approach to immunological sexing of sperm. Theriogenology 52: 1309-1321, 1999. 10. Bondioli KR. Embryo sexing: a review of current techniques and their potential for commercial application in livestock production. J Anim Sci 70: 219, 1992. 11. Bodmer M, Janett F, Hassig M, Dass Nd, Reichert P, Thun R. Fertility in hifers and cows after low dose insemination with sex-sorted and non-sorted sperm under field conditions. Theriogenology 64: 1647-1655, 2005. 12. Bradbury MW, Isola LM, Gordon JW. Enzymatic amplification of a Y chromosome repeat in a single blastomere allows identification of the sex of preimplantation mouse embryos. Proc Natl Acad Sci 87: 4053-4057, 1990. 13. Bradley MP, Heslop BF. The distribution of sex-specific (H-Y) antigens within the seminiferous tubules of the testis: an immunohistochemical study. Hum Genet 79: 347-351, 1988. 14. Braithwaite A, Smith FJ. Chromatographic Methods,5thed. Chapman and Hall, chapter 4, London, 1996. 15. Claassens OE, Oosthuizen CJ, Brusnicky J, Franken DR, Kruger TF. Fluorescent in situ hybridization evaluation of human Y-bearing spermatozoa separated by albumin density gradients. Fertil Steril 63: 417-418, 1995. 16. Cong J, Thompson VF, Goll DE. Immunoaffinity purification of the calpains. Protein Expr Purif 25: 283-290, 2002. 17. Domon B, Aebersold R. Mass spectrometry and protein analysis. Science 312: 212-217, 2006. 18. Eichwald EJ, Silmser CR. Untitled communication. Transplant Bull 2:148-149, 1955. 19. Ericsson RJ, Langevin CN, Nishino M. Isolation of fractions rich in human Y sperm. Nature 229: 421, 1973. 20. Faber DC, Molina JA, Ohlrichs CL, Vander Zwaag DF, Ferre LB. Commercialization of animal biotechnology. Theriogenology 59:125-138, 2003. 21. Gardner RL, Edwards RG. Control of the sex ratio at full term in the rabbit by transferring sexed blastocysts. Nature 218: 346-349, 1968. 22. Garner DL. Flow cytometric sexing of mammalian sperm. Theriogenology (in press), 2005. 23. Garner DL, Gledhill BL, Pinkel D, Lake S, Stephenson D, Van Dilla MA, Johnson LA. Quantification of the X- and Y-chromosome-bearing spermatozoa of domestic animals by flow cytometry. Biol Reprod 28: 312-321, 1983. 24. Goldburg EH, Boyse EA, Bennett D, Scheid M, Carswell EA. Serological demonstration of H-Y (male) antigen on mouse sperm. Nature 232: 478-480, 1971. 25. Havlis J., Thomas H., Sebela M., and Shevchenko A. Fast-response proteomics by accelerated in-gel digestion of proteins. Anal. Chem. 75: 1300-1306, 2003. 26. Hendriksen PJ. Do X and Y spermatozoa differ in proteins? Theriogenology 52: 1295-1307, 1999. 27. Hendriksen PJM, Tieman M, van der Lende T, Johnson LA. Binding of H-Y monoclonal antibodies to separated X and Y chromosome bearing porcine and bovine sperm. Molec Reprod Dev 35: 189-196, 1993. 28. Hendriksen PJM, Welch GR, Grootegoed JA, van der Lende T, Johnson LA. Comparison of detergent- solubilized membrane and soluble proteins from flow cytometrically sorted X- and Y- chrosome bearing porcine spermatozoa by high resolution 2-D electrophoresis. Molec Reprod Dev 45: 342-350, 1996. 29. Herr CM, Matthaei KI, Petrzak U, Reed KC. A rapid Y-chromosome-detecting ovine embryo sexing assay. Theriogenology 33: 245, 1990. 30. Hoppe PC, Koo GC. Reacting mouse sperm with monoclonal H-Y antibodies does not influence sex ratio of eggs fertilized in vitro. J Reprod Immunol 6: 1-9, 1984. 31. Horvat S, Medrano JF, Behboodi E, Anderson GB, Murray JD. Sexing and detection of gene construct in microinjected bovine blastocysts using the polymerase chain reaction. Transgenic Res 2: 134-140, 1993. 32. Howes EA, Miller NG, Dolby C, Hutchings A, Butcher GW, Jones R. A search for sex-specific antigens on bovine spermatozoa using immunological and biochemical techniques to compare the protein profiles of X and Y chromosome-bearing sperm populations separated by fluorescence-activated cell sorting. J Reprod Fertil 110: 195-204, 1997. 33. Huang S.Y., Lin J.H., Chen Y.H., Chuang C.K., Lin E.-C., Huang M.C., Sun H.S., and Lee W.C. A reference map and identification of porcine testis proteins using two-dimensional gel electrophoresis and mass spectrometry. Proteomics 5: 4205-4212, 2005. 34. Iizuka R, Kaneko S, Aoki R, Kobayashi T. Sexing of human sperm by discontinuous Percoll density gradient and its clinical application.Hum Reprod 2: 573-575, 1987. 35. Jafar SI, Flint APF. Sex selection in mammals: A review. Theriogenology 46: 191-200, 1995. 36. Johnson LA, Flook JP, Hawk HW. Sex preselection in rabbits: live births from X and Y sperm separated by DNA and cell sorting. Biol Reprod 41: 199-203, 1989. 37. Johnson LA, Flook JP, Look MV, Pinkel D. Flow sorting of X and Y chromosome-bearing spermatozoa into two populations. Gamete Res 16: 1-9, 1987. 38. Johnson LA, Rath D, Vazquez JM, Maxwell W.MC, Dobrinsky JR. Preselection of sex of offspring in swine for production: current status of the process and its application. Theriogenology 63: 615-624, 2005. 39. Kaneko S, Oshio S, Kobayashi T, Iizuka R, Mohri H. Human X- and Y-bearing sperm differ in cell surface sialic acid content. Biochem Biophys Res Commun 124:950-955, 1984. 40. Kashino Y. Separation methods in the analysis of protein membrane complexs. J Chomatogr. B 797: 191-216, 2003. 41. King WA. Sexing embryos by cytological methods. Theriogenology 21: 7-17, 1984. 42. Lyon MF. Gene action in the X-chromosome of the mouse (Mus musculus L.). Naturwissenschaften 190: 372-373, 1961. 43. Maxwell W.MC, Evans G, Hollinshead FK, Bathgate R, de Graaf SP, Eriksson BM, Gillan L, Morton KM, O’Brien JK. Integration of sperm sexing technology into the ART toolbox. Anim Reprod Sci 82-83:79-95, 2004. 44. Monk M, Harper M. X-chromosome activity in preimplantation mouse embryos from XX and XO mothers. J Embryol Exp Morphol 46: 53-64, 1978. 45. Ohno S, Wachtel SS. On the selective elimination of Y bearing sperm. Immunogenetics 7: 13-16, 1978. 46. Ohno S. Sex Chrosomes and Sex-Linked Genes. Berlin: Springer-Verlag 46-73, 1967. 47. Perkins, D.N., Pappin, D.J.C., Creasy, D.M., and Cottrell, J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20: 3551–3567, 1999. 48. Pinkel D, Garner DL, Gledhill BL, Lake S, Stephenson D, Johnson LA. Flow cytometric determination of the proportions of X- and Y-chromosome-bearing sperm in samples of purportedly separated bull sperm. J Anim Sci 60: 1303-1307, 1985. 49. Pinkel D, Gledhill BL, Lake S, Stephenson D, Van Dilla MA. Sex preselection in mammals? Separation of sperm bearing Y and "O" chromosomes in the vole Microtus oregoni. Science 218: 904-906, 1982. 50. Pollevick GD, Giambiagi S, Mancardi S, de Luca L, Burrone O, Frasch AC, Ugalde RA. Sex determination of bovine embryos by restriction fragment polymorphisms of PCR amplified ZFX/ZFY loci. Biotechnology 10: 805-807, 1992. 51. Roe S. Protein purification application. New York: OXFORD, 2001a. 52. Roe S. Protein purification technique. New York: OXFORD, 2001b. 53. Roberts AM. Gravitational separation of X and Y spermatozoa. Nature 238: 223-225, 1972. 54. Santoni V, Molloy M, Rabilloud T. Membrane proteins and proteomics: un amour impossible ? Electrophoresis 21: 1054-1070, 2000. 55. Scopes BK. Protein purification: principles and practice. New York: Springer-Verlag, 1987. 56. Scopes RK. Protein purification in the nineties. Biotechnol Appl Biochem 23: 197-204, 1996. 57. Seddon AM, Curnow P, Booth PJ. Membrane proteins, lipids and detergents: not just a soap opera. Biochim Biophys Acta 1666:105-117, 2004. 58. Seidel GE Jr. Economics of selecting for sex: the most important genetic trait. Theriogenology 59: 585-598, 2003. 59. Seidel GE Jr., Johnson LA. Sexing mammalian sperm--overview. Theriogenology 52: 1267-1272, 1999. 60. Shelton JA, Goldberg EH. Male-restricted expression of H-Y antigen on preimplantation mouse embryos. Transplantation 37: 7-8, 1984. 61. Shorr RG, Heald SL, Jeffs PW, Lavin TN, Strohsacker MW, Lefkowitz RJ, Caron MG. The beta-adrenergic receptor: rapid purification and covalent labeling by photoaffinity crosslinking. Proc Natl Acad Sci 79: 2778-2782, 1982 62. Simpson E, Chandler P, Goulmy E, Disteche CM, Ferguson-Smith MA, Page DC. Separation of the genetic loci for the H-Y antigen and for testis determination on human Y chromosome. Nature 326: 876-878, 1987. 63. Smeaton DC, Harris BL, Xu ZZ, Vivanco WH. Factors affecting commercial application of embryo technologies in New Zealand: a modeling approach. Theriogenology 59: 617-634, 2003. 64. Trowbridge IS, Omary MB. Human cell surface glycoprotein related to cell proliferation is the receptor for transferrin. Proc Natl Acad Sci 78: 3039-3043, 1981. 65. Utsumi K, Iritani A. Embryo sexing by male specific antibody and by PCR using male specific (SRY) primer. Mol Reprod Dev 36: 238-241, 1993. 66. van Munster EB, Stap J, Hoebe RA, te Meerman GJ, Aten JA. Difference in sperm head volume as a theoretical basis for sorting X- and Y-bearing spermatozoa: potentials and limitations. Theriogenology 52:1281-1293, 1999. 67. Vidal F, Moragas M, Catala V, Torello MJ, Santalo J, Calderon G, Gimenez C, Barri PN, Egozcue J, Veiga A. Sephadex filtration and human serum albumin gradients do not select spermatozoa by sex chromosome: a fluorescent in-situ hybridization study. Hum Reprod 8: 1740-1743, 1993. 68. Wachtel GM, Wachtel SS, Nakamura D, Moreira-Filho CA, Brunner M, Koo GC. H-Y antibodies recognize the H-Y transplantation antigen. Transplantation 37: 8-13, 1984. 69. Wachtel S, Nakamura D, Wachtel G, Felton W, Kent M, Jaswaney V. Sex selection with monoclonal H-Y antibody. Fertil Steril 50: 355-360, 1988. 70. Wang HX, Flaherty SP, Swann NJ, Matthews CD. Assessment of the separation of X- and Y-bearing sperm on albumin gradients using double-label fluorescence in situ hybridization. Fertil Steril 61: 720-726, 1994. 71. White KL, Anderson GB, Pashen RL, BonDurant RH. Detection of histocompatibility-Y antigen: identification of sex of pre-implantation ovine embryos. J Reprod Immunol 10: 27-32, 1987. 72. Williams TJ. A technique for sexing mouse embryos by a visual colorimetric assay of the X-linked enzyme, glucose 6-phosphate dehydrogenase. Theriogenology 25: 733-743, 1986. 73. Wood TC, White KL, Thompson DL Jr, Garza F Jr. Evaluation of the expression of a male-specific antigen on cells of equine blastocysts. J Reprod Immunol 14: 1-8, 1988. 74. Zavos PM. Preconception sex determination via intra-vaginal administration of H-Y antisera in rabbits. Theriogenology 20: 235-240, 1983.
摘要: 
直至目前為止,X-/Y-精子分離技術對於應用於經濟動物產業仍無法滿足不傷胚胎、低花費、高效能及方便性的技術需求。本研究實際應用Ohno’s law,以哺乳動物之性特異性蛋白 (sex-specific proteins, SSPs) 較其他non-SSPs在演化上具高度保留性為基礎理論,來進行X、Y精子表面特異性蛋白 (SSPs) 篩選,以期做為X、Y精子免疫分離法之基礎。

實驗先以豬精子細胞膜表面蛋白作為抗原,進行雌雄兔性別差異免疫法以產生相對應抗體。再應用雌雄兔產生的抗體製備性別特異性親和管柱,通入豬精子細胞膜蛋白,進行 SSPs 之純化。實驗結果發現將經過一次純化的性特異性蛋白質利用二維電泳 (two-dimensional electrophoresis) 配合鍍銀染色法分析,分別在電泳膠片上可見13個 X-SSPs 點及9個 Y-SSPs 點。進行ㄧ次重覆性實驗後由於無法得到完整的蛋白質分佈情形,故以區域性做為劃分;X-SSPs分別為 pI值位於3,分子量位10-20 KDa間及 pI值4,分子量40-50二個區域;Y-SSPs區域為pI值4,分子量30-40、 pI值5,分子量50、 pI值6-7,分子量30-40及 pI值6-7,分子量20四個區域。進行二、三次重覆性實驗後分別在電泳膠片上可見5個 X-SSPs 點、9個 Y-SSPs 點以及1個X-SSPs點及14個Y-SSPs點。以MALDI-TOF質譜儀及MASCOT比對工具進行 SSPs 身份鑑定。其中由雄兔抗體管柱所辨識到的hypothetical蛋白質(X-SSP)證實來源為Hereford種母牛的第七對染色體所轉譯出的X-染色體性聯蛋白質。其他以本研究理論認定之X-SSPs 及Y-SSPs,則因蛋白質資料庫之目前資料不足暫時無法成功鑑定其身份。

綜言之, 本研究已證實Ohno''s law 之存在性,亦即精子細胞膜表面存有與X-/Y- 染色體性聯之特有蛋白質。本研究所認定之精子性特異性蛋白質 (hypothetical proteins及/或其他) 的存在,提供了未來進行預選子代精蟲免疫篩選法之研究基礎。

Current techniques of sexing sperm have not been widely available for agriculture breeding industry, due to its high costs, low efficacy and inconvenience. A non-invasive, immunological method for sexing sperm would be satisfactory to above-mentioned needs. This study was designed, that is based on the Ohno's law that sex-specific proteins (SSPs) are evolutionary more highly conserved than non-SSPs, to isolate SSPs from X or Y sperm and create a foundation for further immunological sperm sexing. Opposite-sex immunizations (antigens: porcine sperm membrane crude proteins) of rabbits were applied to raise the corresponding antibodies for each sex. Sex-specific affinity columns (M-, F-columns) prepared from the antibodies of respective sex were used to purify putative SSPs. Sequential studies demonstrated that with application of high resolution two-dimensional electrophoresis (2-DE) and the silver stain after one-cycle affinity chromatography column (ACC), 13 X-SSPs and 9 Y-SSPs protein spots were detected on 2-DE display (experiment 1); multiple X-SSPs (pI=3, 10-20 KDa; pI=4, 40-50 KDa) and Y-SSPs spots (pI=4, 30-40 KDa; pI=5, 50 KDa, pI=6-7, 30-40 KDa; pI=6-7, 20 KDa) were detected (exp. 2); 5 X-SSPs and 9 Y-SSPs spots were detected (exp. 3); 1 X-SSPs and 14 Y-SSPs spots were detected (exp. 4). Selected spots were subject to MALDI-TOF mass spectrometry and database search. One of the hypothetical proteins from M-column (putative X-SSP) preparation was recognized to be the female protein that is encoded from Hereford cow sex chromosome 7. Identification of putative proteins was unsuccessful due to limited data base of porcine proteins. In conclusions, this study verifies the existing of Ohno's law. The putative SSPs are present at a detectable form under these delicate preparations, and that are minor components in the membrane. These results indicate the feasibility of immunological sperm sexing techniques.
URI: http://hdl.handle.net/11455/13262
其他識別: U0005-2308200616314700
Appears in Collections:獸醫學系所

Show full item record
 

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.