绵羊精子冻融前后差异蛋白质组学研究

doi:10.3864/j.issn.0578-1752.2011.24.015

摘要/Abstract

摘要： 【目的】检测和分析绵羊精子冻融前后的差异蛋白质。【方法】用二维凝胶电泳分离精子蛋白，凝胶用考马斯亮蓝染色，用PDQuest8.0软件检测分析差异蛋白质斑点并经液相色谱串联离子阱质谱鉴定。【结果】鲜冻精凝胶图谱分析后得到 14个差异蛋白斑点，在国际生物信息学中心数据库搜索到相对应的5种蛋白质：推定的热休克蛋白70.1、转录因子AP-2α蛋白、烯醇酶、烯醇酶1和血清白蛋白前体。在解冻精子中，推定的热休克蛋白70.1、烯醇酶和烯醇酶1含量减少，转录因子AP-2α蛋白含量增加，血清白蛋白前体来源于精浆。【结论】绵羊精子经冷冻、解冻后,蛋白质存在差异，对差异蛋白质的研究有利于探索绵羊精子在冷冻过程中的生理状态，为进一步研究绵羊精子蛋白质组学及揭示精子冻伤机理提供依据。

关键词: 绵羊, 精子蛋白质, 二维凝胶电泳分析, 蛋白质组

Abstract: 【Objective】To investigate the differential proteins between fresh and frozen-thawed sheep sperm. 【Method】The current research presents the protein changes using two-dimensional gel electrophoresis (2-DE) after stained with commassie blue, differential proteins were detected by PDQuest 8.0 software and subjected to ion trap mass spectrometer equipped with a Surveyor HPLC system, and differential spots of protein were identified. 【Result】A search against protein sequences in the National Center for Biotechnology Information databases indicated that the differential fourteen proteins correspond to five proteins including putative heat shock protein 70.1, transcription factor AP-2-alpha, enolase, enolase1 and serum albumin precursor that protein content of putative heat shock protein 70.1, enolase, enolase1 were decreased and protein content of transcription factor AP-2-alpha was increased in frozen-thawed spermatozoa, serum albumin precursor was seminal plasma protein. 【Conclusion】 It was concluded there was a significant difference at protein level in sheep sperm during frozen and frozen-thawed. Differential proteins analysis may be useful to clarify the physiology state of sheep sperm during cryopreservation for further studies on sheep sperm proteomic and revealing the cryodamage mechanism of sperm cryopreservation.

Key words: sheep, sperm protein, two-dimensional polyacrylamide gel electrophoresis analysis, proteome

李鸿岩, 赵兴绪, 张勇, 贺钰烜, 杨永新. 绵羊精子冻融前后差异蛋白质组学研究[J]. 中国农业科学, 2011, 44(24): 5092-5099.

LI Hong-Yan, ZHAO Xing-Xu, ZHANG Yong, HE Yu-Hui, YANG Yong-Xin. Analysis of the Differential Proteomics Between Fresh and Frozen-Thawed Sheep Sperm[J]. Scientia Agricultura Sinica, 2011, 44(24): 5092-5099.

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参考文献

[1]Ramalho-Santos J, Amaral A, Sousa A P, Rodrigues A S. Probing the structure and function of mammalian sperm using optical and fluorescence microscopy. Modern Research and Educational Topics in Microscopy, 2007, (1):394-402.

[2]Lee H L, Kim S H, Ji D B, Kim Y J. A comparative study of sephadex, glass wool and percoll separation techniques on sperm quality and IVF results for cryopreserved bovine semen. Journal of Veterinary Science, 2009, 10(3):249-255.

[3]Charles O G, Michael V W, Jane S F. Conserving, distributing and managing genetically modified mouse lines by sperm cryopreservation. Public Library of Science One, 2008, 3(7):e2792.

[4]Lessard C, Parent S, Leclerc P, Bailey J L, Sullivan R. Cryopreservation alters the levels of the bull sperm surface protein P25b. Journal of Andrology, 2000, 5(21):700-707.

[5]Mason C E, Seringhaus M R, Sattler C. Personalized genomic medicine with a patchwork, partially owned genome. Yale Journal of Biology and Medicine, 2007, 80(4):145-151.

[6]Peddinti D, Nanduri B, Kaya A, Feugang J M, Burgess S C, Memili E. Comprehensive proteomic analysis of bovine spermatozoa of varying fertility rates and identification of biomarkers associated with fertility. BMC Systems Biology, 2008, 2(19):1-14.

[7]Martin G, Cagnon N, Sabido O, Sion B, Grizard G, Durand P, Levy R. Kinetics of occurrence of some features of apoptosis during the cryopreservation process of bovine spermatozoa. Human Reproduction, 2007, 22(2): 380-388.

[8]Cao W L, Wang Y X, Xiang Z Q, Li Z. Cryopreservation-induced decrease in heat-shock protein 90 in human spermatozoa and its mechanism. Asian Journal of Andrology, 2003, 5(1):43-46.

[9]Zilli L, Schiavone R, Zonno V, Rossano R, Storelli C, Vilella S. Effect of cryopreservation on sea bass sperm proteins. Biology of Reproduction, 2005, 72(5):1262-1267.

[10]Miarelli M, Signorelli F. Differential expression of liver proteins in Chianina and Holstein young bulls. Journal of Animal Science, 2009, 88:593-598.

[11]Hafez B. Reproduction in Farm Animals. 7th Edition. Oxford，UK : Wiley-Blackwell, 2000: 385.

[12]Triwulanningsih E, Situmorang P, Sugiarti T, Sianturi R G, Kusumaningrum D A. Effect of glutathione addition to the sperm diluent medium on quality of bovine chilled semen. Indonesian Journal of Agriculture, 2010, 3:60-65.

[13]O'Connell M, McClure N, Lewis S E M. The effects of cryopreservation on sperm motility and mitochondrial function. Human Ruprodution, 2008, 3(17):704-709.

[14]Varela J A S, Corcini C D, Ulguim R R, Alvarenga M V F, Bianchi I, Corrêa M N, Lucia Jr T, Deschamps J C. Effect of low density lipoprotein on the quality of cryopreserved dog semen. Animal Reproduction Science, 2009, 115: 323-327.

[15]Helfenstein F, Podevin M, Richner H. Sperm morphology, swimming velocity, and longevity in the house sparrow Passer domesticus. Behavioral Ecology and Sociobiology, 2010, 64: 557-565.

[16]Kirkland P A, Busby J, Stevens S J, Maupin-Furlow J A. Trizol-based method for sample preparati on and isoelectric focusing of halophilic proteins. Analytical Biochemistry, 2006, 351: 254-259.

[17]Bradford M M A. Arapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976, 72: 248-254.

[18]Candiano1 G, Bruschi M, Musante L, Santucci L, Ghiggeri G M, Carnemolla B, Orecchia P, Zardi L, Righetti P G. Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis, 2004, 25, 1327-1333.

[19]Tan Z, Wortman M, Lu S, Dong Z. Regulation of heat shock protein 70-1 expression by androge receptor and its signaling in human prostate cancer cells. International Journal of Oncology, 2010, 36(2): 459-467.

[20]Daugaard M, Rohde M, Ja M. The heat shock protein 70 family: Highly homologous proteins with overlapping and distinct functions. Federation of European Biochemical Societies Letters, 2007, 581: 3702-3710.

[21]Wei J G, Kent G G. Genomic Deletions of the Drosophila melanogaster HSP70 Genes. Genetics, 2004, 168: 1467-1476.

[22]Cheng Y H, Bruce J A, Hossain S, Trapnell B, Kong S, Handwerger S. Critical role for transcription factor AP-2α in human trophoblast differentiation. Physiology Genomics, 2004, 18: 99-107.

[23]Zhang J, Brewer S, Huang J, Williams T. Overexpression of transcription factor AP-2alpha suppresses mammary gland growth and morphogenesis. Developmental Biology, 2003, 256(1): 127-145.

[24]Liu K J, Shi N Y. The role of enolase in tissue invasion and metastasis of pathogens and tumor cells. Journal of Cancer Molecules, 2007, 3(2): 45-48.

[25]Plow E F, Das R. Enolase-1 as a plasminogen receptor. Blood, 2009, 113: 5371-5372.

[26]Andre F, Jean-l V, Genevieve G, Boucher D. Enolase isoforms activities in spermatozoa from men normospermia and abnormospermia. Asian Journal of Andrology, 2002, 23: 202-210.

[27]Chang G C, Liu J K, Hsieh C L, Hu T S, Liu H K, Luh K T. Identification of α-Enolase as an autoantigen in lung cancer: Its overexpression is associated with clinical outcomes. Clinical Cancer Research, 2006, 12: 5746-5754.

[28]Chang Y S, Wu W, Walsh G, Hong W K, Mao L. Enolase-α Is Frequently Down-Regulated in non-small cell lung cancer and predicts aggressive biological behavior. Clinical Cancer Research, 2003, 9: 3641-3644.

[29]Huang Y H, Kuo S P, Lin M H, Shih C M, Chu S T, Wei C C, Wu T J, Chen Y H. Signals of seminal vesicle autoantigen suppresses bovine serum albumin-induced capacitation in mouse sperm. Biochemical and Biophysical Research Communications, 2005, 338:1564-1571.

[30]Cabrita E, Anel L, Herraez M P. Effect of external cryoprotectants as membrane stabilizers on cryopreserved rainbowtrout sperm. Theriogenology, 2001, 56 (4):623-635.