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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
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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. 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Theriogenology 20: 235-240, 1983.
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.
其他識別: U0005-2308200616314700
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