特发性弱精子症精浆蛋白差异表达研究
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摘要
目的探讨精浆蛋白在成年男性特发性弱精子症中的差异表达情况。方法纳入2017年1-5月我院泌尿外科门诊特发性弱精子症患者15例作为试验组,年龄24~47岁。同时,纳入精液正常成年男性15例作为对照组,年龄21~44岁。用等重同位素多标签相对定量蛋白质组学(isobaric tags for relative and absolute quantitation,i TRAQ)的方法研究成年男性正常精浆蛋白以及特发性弱精子症患者的精浆蛋白表达情况。结果共发现精浆中2 784个蛋白质。其中,与正常组相比,特发性弱精子症患者精浆中表达上调蛋白数为14个,精浆中表达下调蛋白数为79个(表达差异倍数>1.2倍,且P<0.05)。经斑点印迹技术验证蛋白INF2表达水平与iTRAQ结果一致。经GO(gene ontology)蛋白功能注释及富集分析,特发性弱精子症精浆中差异蛋白功能主要包括调节钙离子跨膜转运、胞浆内转运以及螯合、蝶呤钼辅因子合成及代谢、调节铜离子跨膜转运等。经KEGG蛋白通路注释及富集分析,特发性弱精子症精浆中差异蛋白主要富集于叶酸生物合成、金黄色葡萄球菌感染等通路。结论特发性弱精子症患者精浆蛋白同正常成年男性精浆蛋白存在差异,其可能是引起特发性弱精子症的原因。
Objective To explore the differential expression profile of the seminal plasma proteome in idiopathic asthenospermia. Methods Fifteen patients(24 ~ 47 years old) with identified idiopathic asthenospermia admitted in our outpatient department from January to May 2017 were enrolled as the experimental group. Fifteen adult males(21 ~ 44 years old) with normal semen were enrolled as the control group. Isobaric tags for relative and absolute quantitation(iTRAQ) labeling was used to determine the expression of seminal plasma proteins in the 2 groups of males. Results There were 2 784 seminal plasma proteins found in this study. Compared with the normal group,there were 14 proteins up-regulated and 79 down-regulated in seminal plasma of idiopathic asthenospermia patients(the difference was more than 1. 2 times,P < 0. 05). Dot blotting confirmed that INF2 expression level was consistent with the result of i TRAQ.According to the Gene Ontology(GO) annotation and enrichment analysis,the differential proteins were mainly involved in the regulation of calcium ion transmembrane transport,calcium ion intracellular transport and calcium ion sequestering, molybdopterin cofactor metabolic/biosynthetic process, and copper ion transmenbrane transport. According to the results of KEGG protein pathway annotation and enrichment analysis,the differential proteins were mainly involved in folate biosynthesis,Staphylococcus aureus infection and other pathways. Conclusion There are proteomic differences in seminal plasma between idiopathic asthenospermia and normal semen,which may be the cause of idiopathic asthenospermia.
Objective To explore the differential expression profile of the seminal plasma proteome in idiopathic asthenospermia. Methods Fifteen patients(24 ~ 47 years old) with identified idiopathic asthenospermia admitted in our outpatient department from January to May 2017 were enrolled as the experimental group. Fifteen adult males(21 ~ 44 years old) with normal semen were enrolled as the control group. Isobaric tags for relative and absolute quantitation(iTRAQ) labeling was used to determine the expression of seminal plasma proteins in the 2 groups of males. Results There were 2 784 seminal plasma proteins found in this study. Compared with the normal group,there were 14 proteins up-regulated and 79 down-regulated in seminal plasma of idiopathic asthenospermia patients(the difference was more than 1. 2 times,P < 0. 05). Dot blotting confirmed that INF2 expression level was consistent with the result of i TRAQ.According to the Gene Ontology(GO) annotation and enrichment analysis,the differential proteins were mainly involved in the regulation of calcium ion transmembrane transport,calcium ion intracellular transport and calcium ion sequestering, molybdopterin cofactor metabolic/biosynthetic process, and copper ion transmenbrane transport. According to the results of KEGG protein pathway annotation and enrichment analysis,the differential proteins were mainly involved in folate biosynthesis,Staphylococcus aureus infection and other pathways. Conclusion There are proteomic differences in seminal plasma between idiopathic asthenospermia and normal semen,which may be the cause of idiopathic asthenospermia.
引文
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[15]CHAKRABARTI R,JI W K,STAN R V,et al.INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake,inner membrane constriction,and division[J].J Cell Biol,2018,217(1):251-268.DOI:10.1083/jcb.201709111.
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[17]刘睿智,江露,许宗革,等.精浆一氧化氮与精液主要参数之间关系探讨[J].中国妇幼保健,2006,21(9):1245-1247.LIU R Z,JIANG L,XU Z G,et al.Study of the relationsllip between seminal parameters and concentrations of Ilitric oxide in semen plasma[J].Matern Child Health Care China,2006,21(9):1245-1247.
[18]HELLSTROM W J,BELL M,WANG R,et al.Effect of sodium nitroprusside on sperm motility,viability,and lipid peroxidation[J].Fertil Steril,1994,61(6):1117-1122.DOI:10.1016/s0015-0282(16)56766-1.
[19]吴世木,王培书,郑金川,等.男性不育患者精浆NO水平及其与抗精子抗体的关系[J].生殖与避孕,2001,21(5):304-306.WU S M,WANG P S,ZHEN J C,et al.The study on relat ions between NO level in seminal plasma and anti-sperm antibody in infertile patients[J].Reprod Contracept,2001,21(5):304-306.
[20]WONG W Y,MERKUS H M,THOMAS C M,et al.Effects of folic acid and zinc sulfate on male factor subfertility:a double-blind,randomized,placebo-controlled trial[J].Fertil Steril,2002,77(3):491-498.DOI:10.1016/s0015-0282(01)03229-0.
[21]EBISCH I M,THOMAS C M,PETERS W H,et al.The importance of folate,zinc and antioxidants in the pathogenesis and prevention of subfertility[J].Hum Reprod Update,2007,13(2):163-174.DOI:10.1093/humupd/dml054.
[22]DIAO R,FOK K L,CHEN H,et al.Deficient humanβ-defensin 1 underlies male infertility associated with poor sperm motility and genital tract infection[J].Sci Transl Med,2014,6(249):249ra108.DOI:10.1126/scitranslmed.3009071.
[2]李维娜,朱文兵,唐章明,等.16835例中国不育男性的精液质量分析[J].中南大学学报(医学版),2014,39(2):157-160.DOI:10.11817/j.issn.1672-7347.2014.02.008.LI W N,ZHU W B,TANG Z M,et al.Semen quality of16 835 infertile men in China[J].J Cent South Univ(Med Sci),2014,39(2):157-160.DOI:10.11817/j.issn.1672-7347.2014.02.008.
[3]黎兴盛,鲍琳,杨春,等.216例男性不育症精液检验与血清中抗精子抗体的关系[J].实验与检验医学,2011,29(2):121-122,148.DOI:10.3969/j.issn.1674-1129.2011.02.007.LI X S,BAO L,YANG C,et al.The correlation between the semen routine indicators and serum anti-sperm antibody level in infertility patients[J].Exp Lab Med,2011,29(2):121-122,148.DOI:10.3969/j.issn.1674-1129.2011.02.007.
[4]ROSS P L,HUANG Y N,MARCHESE J N,et al.Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents[J].Mol Cell Proteomics,2004,3(12):1154-1169.DOI:10.1074/mcp.M400129-MCP200.
[5]VGVRI,REZELI M,SIHLBOM C,et al.Molecular microheterogeneity of prostate specific antigen in seminal fluid by mass spectrometry[J].Clin Biochem,2012,45(4/5):331-338.DOI:10.1016/j.clinbiochem.2011.11.018.
[6]WIS'NIEWSKI J R,ZOUGMAN A,NAGARAJ N,et al.Universal sample preparation method for proteome analysis[J].Nat Methods,2009,6(5):359-362.DOI:10.1038/nmeth.1322.
[7]GTZ S,GARCA-G MEZ J M,TEROL J,et al.Highthroughput functional annotation and data mining with the Blast2GO suite[J].Nucleic Acids Res,2008,36(10):3420-3435.DOI:10.1093/nar/gkn176.
[8]QUEVILLON E,SILVENTOINEN V,PILLAI S,et al.InterPro Scan:protein domains identifier[J].Nucleic Acids Res,2005,33(Web Server issue):W116-W120.DOI:10.1093/nar/gki442.
[9]MORIYA Y,ITOH M,OKUDA S,et al.KAAS:an automatic genome annotation and pathway reconstruction server[J].Nucleic Acids Res,2007,35(Web Server issue):W182-W185.DOI:10.1093/nar/gkm321.
[10]周青松,曾凡春,张祯雪,等.复方氨基酸胶囊联合维生素E治疗特发性弱精子症的临床研究[J].中华男科学杂志,2016,22(4):343-346.DOI:10.13263/j.cnki.nja.2016.04.012.ZHOU Q S,ZENG F C,ZHANG Z X,et al.Compound amino acid combined with vitamin E for idiopathic asthenospermia[J].Nat J Androl,2016,22(4):343-346.DOI:10.13263/j.cnki.nja.2016.04.012.
[11]万学红,卢雪峰.诊断学[M].8版.北京:人民卫生出版社,2013:336.WAN X H,LU X F.Diagnostics[M].8th Ed.Beijing:People’s Medical Publishing House,2013:336.
[12]CHIU W W,CHAMLEY L W.Human seminal plasma antibody-binding proteins[J].Am J Reprod Immunol,2003,50(3):196-201.DOI:10.1034/j.1600-0897.2003.00067.x.
[13]徐建新,朱涛,吕胜启,等.应用蛋白质芯片飞行时间质谱仪研究弱精子症患者精浆差异蛋白表达[J].中国男科学杂志,2015,29(10):12-16.DOI:10.3969/j.issn.1008-0848.2015.10.003.XU J X,ZHU T,LV S Q,et al.Differential protein profile analysis in seminal plasma of asthenospermia patients by SELDI-TOF-MS[J].Chin J Androl,2015,29(10):12-16.DOI:10.3969/j.issn.1008-0848.2015.10.003.
[14]SARASWAT M,JOENVRS,JAIN T,et al.Human spermatozoa quantitative proteomic signature classifies normo-and asthenozoospermia[J].Mol Cell Proteomics,2017,16(1):57-72.DOI:10.1074/mcp.M116.061028.
[15]CHAKRABARTI R,JI W K,STAN R V,et al.INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake,inner membrane constriction,and division[J].J Cell Biol,2018,217(1):251-268.DOI:10.1083/jcb.201709111.
[16]ESPINO J,MEDIERO M,LOZANO G M,et al.Reduced levels of intracellular calcium releasing in spermatozoa from asthenozoospermic patients[J].Reprod Biol Endocrinol,2009,7:11.DOI:10.1186/1477-7827-7-11.
[17]刘睿智,江露,许宗革,等.精浆一氧化氮与精液主要参数之间关系探讨[J].中国妇幼保健,2006,21(9):1245-1247.LIU R Z,JIANG L,XU Z G,et al.Study of the relationsllip between seminal parameters and concentrations of Ilitric oxide in semen plasma[J].Matern Child Health Care China,2006,21(9):1245-1247.
[18]HELLSTROM W J,BELL M,WANG R,et al.Effect of sodium nitroprusside on sperm motility,viability,and lipid peroxidation[J].Fertil Steril,1994,61(6):1117-1122.DOI:10.1016/s0015-0282(16)56766-1.
[19]吴世木,王培书,郑金川,等.男性不育患者精浆NO水平及其与抗精子抗体的关系[J].生殖与避孕,2001,21(5):304-306.WU S M,WANG P S,ZHEN J C,et al.The study on relat ions between NO level in seminal plasma and anti-sperm antibody in infertile patients[J].Reprod Contracept,2001,21(5):304-306.
[20]WONG W Y,MERKUS H M,THOMAS C M,et al.Effects of folic acid and zinc sulfate on male factor subfertility:a double-blind,randomized,placebo-controlled trial[J].Fertil Steril,2002,77(3):491-498.DOI:10.1016/s0015-0282(01)03229-0.
[21]EBISCH I M,THOMAS C M,PETERS W H,et al.The importance of folate,zinc and antioxidants in the pathogenesis and prevention of subfertility[J].Hum Reprod Update,2007,13(2):163-174.DOI:10.1093/humupd/dml054.
[22]DIAO R,FOK K L,CHEN H,et al.Deficient humanβ-defensin 1 underlies male infertility associated with poor sperm motility and genital tract infection[J].Sci Transl Med,2014,6(249):249ra108.DOI:10.1126/scitranslmed.3009071.