精子蛋白32的表达及酪氨酸磷酸化调控猪精子顶体蛋白的活化
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摘要
为了探究猪精子蛋白32(Sperm protein,sp32)表达及酪氨酸磷酸化调控猪精子顶体蛋白活化的关系,本研究对不同处理(鲜精、冷冻-解冻、获能、顶体反应)的猪精子顶体膜蛋白进行分离,通过考马斯亮蓝染色、SDS-PAGE电泳和Western blot检测和分析。结果表明,猪精子经过获能处理、冷冻-解冻处理以及顶体反应处理后,前顶体蛋白(Proacrosin)和顶体蛋白(Acrosin)在转化过程中,sp32表达有所差异,获能和顶体反应处理的sp32的表达量略高于冷冻-解冻处理组,而显著高于鲜精处理组。与其他处理组相比,冷冻-解冻精子处理组sp32酪氨酸磷酸化水平产生显著的差异。但在SDS-PAGE电泳中,猪鲜精处理组在蛋白质分子质量38～170ku区间的蛋白表达条带较其他对比组明显,这表明猪精子在受精前所必需经历的获能和顶体反应过程中,顶体膜蛋白伴随着大分子的蛋白质修饰和降解。结论:sp32作为一种前顶体蛋白结合蛋白,在前顶体蛋白活化过程中它的表达水平及酪氨酸磷酸化水平上调。
The aim of this study was to investigate the relationship between the expression level,tyrosine phosphorylation of sp32 and the activation of the boar proacrosin/acrosin system,the acrosomal membrane proteins of boar sperm for different treatments(fresh sperm,freezing-thawing,capacitation,acrosome reaction) were separated,and stained by CBB,assayed using SDS-PAGE and Western blot analysis.The results showed that the expression level of sp32 was different in conversion of proacrosin/acrosin system after treatment of boar sperm capacitation,freezing-thawing and acrosome reaction,the expression of sp32 in experiment groups of capacitation and acrosome reaction were slightly higher than the freezing-thawing experiment group,and significantly higher than that of fresh semen group.The level of sp32 tyrosine phosphorylation had a significant difference between the freezing and thawing experiment group and other experiment groups.But bands with molecular mass of 38-170 ku in the fresh semen group were more obvious;it showed that the acrosomal membrane proteins were accompanied by modification and degradation of big molecular proteins when sperm underwent capacitation and the acrosome reaction.sp32 as a proacrosin binding protein,its expression and tyrosine phosphorylation level were up-regulated in the activation of the boar proacrosin/acrosin system.
The aim of this study was to investigate the relationship between the expression level,tyrosine phosphorylation of sp32 and the activation of the boar proacrosin/acrosin system,the acrosomal membrane proteins of boar sperm for different treatments(fresh sperm,freezing-thawing,capacitation,acrosome reaction) were separated,and stained by CBB,assayed using SDS-PAGE and Western blot analysis.The results showed that the expression level of sp32 was different in conversion of proacrosin/acrosin system after treatment of boar sperm capacitation,freezing-thawing and acrosome reaction,the expression of sp32 in experiment groups of capacitation and acrosome reaction were slightly higher than the freezing-thawing experiment group,and significantly higher than that of fresh semen group.The level of sp32 tyrosine phosphorylation had a significant difference between the freezing and thawing experiment group and other experiment groups.But bands with molecular mass of 38-170 ku in the fresh semen group were more obvious;it showed that the acrosomal membrane proteins were accompanied by modification and degradation of big molecular proteins when sperm underwent capacitation and the acrosome reaction.sp32 as a proacrosin binding protein,its expression and tyrosine phosphorylation level were up-regulated in the activation of the boar proacrosin/acrosin system.
引文
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[15]叶华虎,苟本富,刘卢生,等.考马斯亮蓝染色评价哺乳动物精子顶体反应发生的效果[J].第三军医大学学报,2003,25(7):582-582,587.
[16]GONZLEZ-FERNNDEZ L,EGA-FERRUSOLAC O,MACIAS-GARCIA B,et al.Identification ofprotein tyrosine phosphatases and dual-specificityphosphatases in mammalian spermatozoa and theirrole in sperm motility and protein tyrosine phospho-rylation[J].Biol Reprod,2009,80:1239-1252.
[17]张媛媛,胡启蒙,王亮亮,等.哺乳动物精子磷酸化蛋白质组学研究进展[J].中国生物工程杂志,2012,32(4):76-82.
[18]李大吉.猪精子前顶体蛋白动力学活化作用的研究[D].延吉:延边大学,2010.
[19]张旭成.不同处理对猪精子蛋白磷酸化的位点分布、水平及质量的影响[D].延吉:延边大学,2010.
[20]KALAB P,PEKNICOVA J,GEUSSOVA G,et al.Regulation of protein tyrosine phosphorylation inboar sperm through a cAMP-dependent pathway[J].Mol Reprod Dev,1998,51:304-314.
[21]BRAVO M M,APARICIO I M,GARCIA-HERRE-ROS M,et al.Changes in tyrosine phosphorylationassociated with true capacitation and capacitation-likestate in boar spermatozoa[J].Mol Reprod Dev,2005,71(1):88-96.
[22]BABA T,MCHIKAWAL Y,KASHIWABARA S,et al.Proacrosin activation in the presence of a 32kuprotein from boar spermatozoa[J].Biochem Bio-phys Res Commun,1989,160(3):1026-1032.
[23]BABA T,NIIDA Y,MICHIKAWA Y,et al.An ac-rosomal protein,sp32,in mammalian sperm is abinding protein specific for two proacosins and an ac-rosin intermediate[J].J Biol Chem,1994,269:10133-10140.
[2]ARCELAY E,SALICIONI A M,WERTHEIMERE,et al.Identification of proteins undergoing tyro-sine phosphorylation during mouse sperm capacitation[J].Int J Dev Biol,2008,52:463-472.
[3]TAKASHI W I,MAHBUB HASAN A K M,SATOK.Protein-tyrosine kinase signaling in the biologicalfunctions associated with sperm[J].J Signal Trans-duct,2012,10(1):1-18.
[4]FICARRO S,CHERTIHIN O,WESTBROOK V A,et al.Phosphoproteome analysis of capacitated hu-man sperm.Evidence of tyrosine phosphorylation of akinase-anchoring protein 3 and valosin-containingprotein/p97during capacitation[J].J Biol Chem,2003,278:11579-11589.
[5]HARAYAMA H,NISHIJIMA K,MURASE T,etal.Relationship of protein tyrosine phosphorylationstate with tolerance to frozen storage and the poten-tial to undergo cyclic AMP-dependent hyperactivationin the spermatozoa of Japanese Black bulls[J].MolReprod Dev,2010,77:910-921.
[6]HARRISON R,GAD ELLA B M.Bicarbonate in-duced membrane processing in sperm capacitation[J].Theriogenology,2005,63:342-351.
[7]ARCELAY E,SALICIONIA M,WERTHEIMERE,et al.Identification of proteins undergoing tyro-sine phosphorylation during mouse spermcapacitation[J].Int J Dev Biol,2008,52:463-472.
[8]AMINARDE D E,FLAHERTY C.Sperm activa-tion:role of reactive oxygen species and kinases[J].Biochim Biophys Acta,2008,1784:106-115.
[9]BAILEY J L,TARDIF S,DUBC,et al.Use ofphosphoproteomics to study tyrosine kinase activityin capacitating boar sperm kinase activity and capaci-tation[J].Theriogenology,2005,63:599-614.
[10]DUBC,LECLERC P,BABA T,et al.Theproacrosin binding protein,sp32,is tyrosine phospho-rylated during capacitation of pig sperm[J].J An-drol,2005,26(4):519-528.
[11]TARDIF S,DUBC,BAILEY J L.Porcine spermcapacitation and tyrosine kinase acticity are dependenton bicarbonate and calcium but protein tyrosine phos-phorylation is only associated with calcium[J].BiolReprod,2003,68:207-213.
[12]范蓉,罗彬,谢小薰,等.精子蛋白32研究进展[J].解剖学报,2007,30(4):503-505.
[13]韩明铭,金一,方南洙,等.不同获能条件对猪精子体外获能和体外受精的影响[J].中国畜牧杂志,2009,45(19):18-21.
[14]MOLLER C C,BLEIL J D,KINLOCH R A,et al.Structural and functional relationships between andhamster zonapellucida glycoprotein[J].Dev Biol,1990,137(1):276-286.
[15]叶华虎,苟本富,刘卢生,等.考马斯亮蓝染色评价哺乳动物精子顶体反应发生的效果[J].第三军医大学学报,2003,25(7):582-582,587.
[16]GONZLEZ-FERNNDEZ L,EGA-FERRUSOLAC O,MACIAS-GARCIA B,et al.Identification ofprotein tyrosine phosphatases and dual-specificityphosphatases in mammalian spermatozoa and theirrole in sperm motility and protein tyrosine phospho-rylation[J].Biol Reprod,2009,80:1239-1252.
[17]张媛媛,胡启蒙,王亮亮,等.哺乳动物精子磷酸化蛋白质组学研究进展[J].中国生物工程杂志,2012,32(4):76-82.
[18]李大吉.猪精子前顶体蛋白动力学活化作用的研究[D].延吉:延边大学,2010.
[19]张旭成.不同处理对猪精子蛋白磷酸化的位点分布、水平及质量的影响[D].延吉:延边大学,2010.
[20]KALAB P,PEKNICOVA J,GEUSSOVA G,et al.Regulation of protein tyrosine phosphorylation inboar sperm through a cAMP-dependent pathway[J].Mol Reprod Dev,1998,51:304-314.
[21]BRAVO M M,APARICIO I M,GARCIA-HERRE-ROS M,et al.Changes in tyrosine phosphorylationassociated with true capacitation and capacitation-likestate in boar spermatozoa[J].Mol Reprod Dev,2005,71(1):88-96.
[22]BABA T,MCHIKAWAL Y,KASHIWABARA S,et al.Proacrosin activation in the presence of a 32kuprotein from boar spermatozoa[J].Biochem Bio-phys Res Commun,1989,160(3):1026-1032.
[23]BABA T,NIIDA Y,MICHIKAWA Y,et al.An ac-rosomal protein,sp32,in mammalian sperm is abinding protein specific for two proacosins and an ac-rosin intermediate[J].J Biol Chem,1994,269:10133-10140.