不同获能方法对冻融塔里木马鹿精子体外获能及其蛋白酪氨酸磷酸化的影响
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- 英文论文题名：Effects of Different Treatment on Capacitation and Protein Tyrosine Phosphorylation of Tarim Wapiti(Cervus elaphus yarkandensis)Frozen-thawed Sperm
- 论文作者：库尔班·吐拉克 ; 陈勇 ; 王旭光 ; 迪力夏提 ; 敬斌宇 ; 李和平
- 年：2016
- 作者机构：东北林业大学野生动物资源学院;新疆农业大学动物科学学院;新疆维吾尔自治区畜牧总站;新疆厚拾生物科技有限责任公司;
- 论文关键词：塔里木马鹿 ; 冻融精子 ; 获能 ; 蛋白酪氨酸磷酸化
- 英文论文关键词：Tarim Wapiti(Cervus Elaphus Yarkandensis) ; Freeze-thaw sperm ; Capacitation ; Protein tyrosine phosphorylation
- 会议召开时间：2016-06-01
- 会议录名称：“十二五” 鹿科学研究进展
- 语种：中文
- 分类号：S825.3
- 学会代码：ZXMY
- 学会名称：中国畜牧业协会
- 页数：12
- 文件大小：1184k
- 原文格式：O
摘要
为探讨不同获能方法对塔里木马鹿精子体外获能及其蛋白酪氨酸磷酸化水平的影响,冻融塔里木马鹿精子随机分为4组,用钙离子载体、肝素、咖啡因和Percool离心4种方法进行精子获能的诱导,利用金霉素(CTC)染色法评价精子获能状态,采用SDS-PAGE分离精子膜蛋白,进行Western blot免疫印迹分析,检测酪氨酸磷酸化蛋白的表达水平。结果显示,冻融精子经4种精子获能方法处理后,肝素诱发的精子获能率显著高于IA组和Percool组(P<0.05)。IA组、肝素组和咖啡因组精子蛋白酪氨酸磷酸化水平高于Percool组和对照组。另外,冻融精子随着上游处理及肝素诱导获能的进行,检测到分子量分别为14ku、25~30ku、40 ku、47 ku、55 ku的酪氨酸磷酸化蛋白,这些蛋白的酪氨酸磷酸化水平在获能60~120min期间相对较高,而且此时精子获能率及超激活运动精子比例也显著提高(P<0.05)。结果提示,肝素可以较好地诱导马鹿精子获能,马鹿精子获能与蛋白酪氨酸磷酸化相关。
In this paper,the effects of different treatment on eapaeitation and protein tyrosine phosphorylation of Tarim Wapiti sperm were studied.Frozen- thawed Tarim Wapiti sperms were divided into 4 groups randomly and treated with A23187(IA),Heparin,caffeine or Pereool for eapaeitation.The sperm eapaeitation was evaluated by Aureomyein(CTC)method.Expression of sperm protein tyrosine phosphorylation was detected by membrane- protein separation on SDS- PAGE and Western blotting subsequently.The results indicated that sperm eapaeitation rate in treatment of heparin- induced was significantly higher than the IA treatment and Percool treatment(P < 0.05).The expression of sperm protein tyrosine phosphorylation in each group of IA,heparin and caffeine was increased than the Percool treatment and the control.With swim- up and heparin- induced,the expression of 14 ku,25- 30 ku,40 ku,47 ku and 55 ku protein tyrosine phosphorylation was detected subsequently,these proteins tyrosine phosphorylation were increased when the frozen- thawed sperms capacitated 60 ~120mins,and the capacitated sperm rate and hyper-activated sperm rate were increased significantly(P < 0.05).The results suggested that sperms of Tarim Wapiti were capacitated properly with heparin and protein tyrosine phosphorylation was associated with the eapaeitation of Tarim Wapiti sperm.
In this paper,the effects of different treatment on eapaeitation and protein tyrosine phosphorylation of Tarim Wapiti sperm were studied.Frozen- thawed Tarim Wapiti sperms were divided into 4 groups randomly and treated with A23187(IA),Heparin,caffeine or Pereool for eapaeitation.The sperm eapaeitation was evaluated by Aureomyein(CTC)method.Expression of sperm protein tyrosine phosphorylation was detected by membrane- protein separation on SDS- PAGE and Western blotting subsequently.The results indicated that sperm eapaeitation rate in treatment of heparin- induced was significantly higher than the IA treatment and Percool treatment(P < 0.05).The expression of sperm protein tyrosine phosphorylation in each group of IA,heparin and caffeine was increased than the Percool treatment and the control.With swim- up and heparin- induced,the expression of 14 ku,25- 30 ku,40 ku,47 ku and 55 ku protein tyrosine phosphorylation was detected subsequently,these proteins tyrosine phosphorylation were increased when the frozen- thawed sperms capacitated 60 ~120mins,and the capacitated sperm rate and hyper-activated sperm rate were increased significantly(P < 0.05).The results suggested that sperms of Tarim Wapiti were capacitated properly with heparin and protein tyrosine phosphorylation was associated with the eapaeitation of Tarim Wapiti sperm.
引文
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[12]Varano G,Lombardi A,Cantini G,.et al.Src activation triggers capacitation and acrosome reaction but not motility in human spermatozoa[J].Hum Reprod,2008,23(12);2 652-2 662.
[13]崔凯.马鹿精子体外获能与评价体系及其体外受精的研究[D].哈尔滨:东北林业大学.2007.
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[16]库尔班·吐拉克,敬斌宇,迪力夏提,等.塔里木马鹿不同个体冻融精子酪氨酸磷酸化蛋白的差异表达初报[J].经济动物学报,2013,17(3):136-139,145.
[17]王莹,张勇,张容姬,等.红外荧光标记蛋白检测方法的优化[J].哈尔滨医科大学学报,2009,43(2):111-117.
[18]孔丽娟.酪氨酸磷酸化蛋白在体外获能豚鼠精子上的分布与表达[D].南京:南京农业大学.2007.
[19]Yuming S,Patricia O.Mice Carrying Two t-Haplotypes:Sperm Populations with Reduced Zona Pellucide Binding Are Deficient in Capacitation[J].Biology of Reproduction,1999,61;305-311.
[20]刘烈琴,刘丑生,郭勇,等.CTC法结合体外受精比较不同方法对绵羊精子体外获能的影响[J].中国畜牧兽医,2007,34(8):31-34.
[21]梁鸿斌,刘靖清,李复煌,等.利用CTC染色进行猪精子不同体外获能方法的比较[J].中国畜牧兽医,2010,37(4):171-173.
[22]李英,侯健.肝素和羊血清对绵羊附睾精子体外获能的影响[J].内蒙古农牧学院学报,1998,19(4):20-24.
[23]Patrat C,Serres C,Jouannet P.Progesterone induces hyperpolarization after depolarization phase in human spermatozoa[J].Biol Reprod,2006,66:1 775-1 780.
[24]陈世林,李颖康,达文政,等.精子体外获能影响因素探析[J].宁夏农林科技,2002,1:43-44.
[25]孔丽娟,王根林.精子获能过程中蛋白酪氨酸磷酸化的研究进展[J].畜牧与兽医,2007,39(8):68-70.
[26]张旭成,袁慧敏,金一.猪精子获能期间蛋白质磷酸化时间依赖性的上调[J].江西农业大学学报,2010,32(3):0577-0580.
[27]Parrish J J,Susko-Parrish J,Winer M A,et al.Capacitation of bovine sperm by heparin[J].Biology of Reprod,1988,38(5):1 171-1 180.
[28]林金杏,阎萍.甲基黄嘌呤类生物碱在精子体外处理中的应用[J].黑龙江动物繁殖,2006,14(4):7-9.
[29]Breitbart H.Intracellular calcium regulation in sperm capacitation and acrosomal reaction[J].Molecul Cellul Endocrinol,2002,187:1-2,139-144.
[30]Dube C,Leclerc P,Baba T,.et al.The proacrosin binding protein,sp32,is tyrosine phosphorylated during capacitation of pig sperm[J].J Androl,2005,26(4):519-528.
[31]Bravo MM,AparicioIM,Garcia-Herreros M,.et al.Changes in tyrosine phosphorylation associated with true capacitation and capacitation-like state in boar spermatozoa[J].Mol Reprod Dev,2005,71(1):88-96.
[2]Jones R,James SP,Oxley D,.et al.The Equatorial Subsegment in Mammalian Spermatozoa Is Enriched in Tyrosine Phosphorylated Proteins[J].Biol Reprod,2008(79):421-431.
[3]Boerke A,Tsai PS,Garcia Gil N,.et al.Capacitation-dependent reorganization of microdomains in the apical sperm head plasma membrane:Functional relationship with zona binding and the zona-induced acrosome reaction[J].Theriogenology,2008,70:1 188-1 196.
[4]Bragado MJ,Aparicio IM,Gil MC,.et al.Protein kinases A and C and phosphatidylinositol 3 kinase regulate glycogen synthase kinase-3A serine 21 phosphorylation in boar spermatozoa[J].J Cell Biochem,2010,109:65-73
[5]Harayama H,Noda T,Ishikawa S,.et al.Relationship between cyclic AMP-dependent protein tyrosine phosphorylation and extracellular calcium during hyperactivation of boar spermatozoa[J].Molecular Reproduction and Development,2012,79(10):727-739.
[6]Seligman J,Zipser Y,Kosower NS,.et al.Tyrosine phosphorylation,thiol status,and protein tyrosine phosphatase in rat epididymal spermatozoa[J].Biol Reprod,2004,71;1009-1015.
[7]Mariappa D,Aladakatti RH,Dasari SK,.et al.Inhibition of tyrosine phosphorylation of sperm flagellar proteins,outer dense fiber protein-2 and tektin-2,is associated with impaired motility during capacitation of hamster spermatozoa[J].Mol Reprod Dev,2010,77;182-193.
[8]Kalab P,Peknicova J,Geussova G,.et al.Regulation of protein tyrosine phosphorylation in boar sperm through a cAMP-dependent pathway[J].Mol Reprod Dev,1998,51;604-314.
[9]Puri P,Myers K,Kline D,.et al.Proteomic analysis of bovine sperm YWHA binding partners identify proteins involved in signaling and metabolism[J].Biol Reprod,2008,79:1 183-1 191.
[10]Mahony MC,Gwathmey TY.Protein tyrosine phosphorylation during hyperactivated motility of cynomolgus monkey(Macaca fascicularis)spermatozoa[J].Biol Reprod,1999,60:1 239-1 243.
[11]Ficarro S,Chertihin O,Westbrook VA,.et al.Phosphoproteome analysis of capacitated human sperm evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation[J].J Biol Chem,2003,278:11579-11589.
[12]Varano G,Lombardi A,Cantini G,.et al.Src activation triggers capacitation and acrosome reaction but not motility in human spermatozoa[J].Hum Reprod,2008,23(12);2 652-2 662.
[13]崔凯.马鹿精子体外获能与评价体系及其体外受精的研究[D].哈尔滨:东北林业大学.2007.
[14]Parrish JJ,Susko-Parrish J,Winer MA,.et al.Capacitation of bovine sperm by heparin[J].Biol Reprod 1988,38:1 171-1 180.
[15]张士芳.体外获能马鹿精子酪氨酸磷酸化蛋白的分布与表达[D].哈尔滨:东北林业大学.2009.
[16]库尔班·吐拉克,敬斌宇,迪力夏提,等.塔里木马鹿不同个体冻融精子酪氨酸磷酸化蛋白的差异表达初报[J].经济动物学报,2013,17(3):136-139,145.
[17]王莹,张勇,张容姬,等.红外荧光标记蛋白检测方法的优化[J].哈尔滨医科大学学报,2009,43(2):111-117.
[18]孔丽娟.酪氨酸磷酸化蛋白在体外获能豚鼠精子上的分布与表达[D].南京:南京农业大学.2007.
[19]Yuming S,Patricia O.Mice Carrying Two t-Haplotypes:Sperm Populations with Reduced Zona Pellucide Binding Are Deficient in Capacitation[J].Biology of Reproduction,1999,61;305-311.
[20]刘烈琴,刘丑生,郭勇,等.CTC法结合体外受精比较不同方法对绵羊精子体外获能的影响[J].中国畜牧兽医,2007,34(8):31-34.
[21]梁鸿斌,刘靖清,李复煌,等.利用CTC染色进行猪精子不同体外获能方法的比较[J].中国畜牧兽医,2010,37(4):171-173.
[22]李英,侯健.肝素和羊血清对绵羊附睾精子体外获能的影响[J].内蒙古农牧学院学报,1998,19(4):20-24.
[23]Patrat C,Serres C,Jouannet P.Progesterone induces hyperpolarization after depolarization phase in human spermatozoa[J].Biol Reprod,2006,66:1 775-1 780.
[24]陈世林,李颖康,达文政,等.精子体外获能影响因素探析[J].宁夏农林科技,2002,1:43-44.
[25]孔丽娟,王根林.精子获能过程中蛋白酪氨酸磷酸化的研究进展[J].畜牧与兽医,2007,39(8):68-70.
[26]张旭成,袁慧敏,金一.猪精子获能期间蛋白质磷酸化时间依赖性的上调[J].江西农业大学学报,2010,32(3):0577-0580.
[27]Parrish J J,Susko-Parrish J,Winer M A,et al.Capacitation of bovine sperm by heparin[J].Biology of Reprod,1988,38(5):1 171-1 180.
[28]林金杏,阎萍.甲基黄嘌呤类生物碱在精子体外处理中的应用[J].黑龙江动物繁殖,2006,14(4):7-9.
[29]Breitbart H.Intracellular calcium regulation in sperm capacitation and acrosomal reaction[J].Molecul Cellul Endocrinol,2002,187:1-2,139-144.
[30]Dube C,Leclerc P,Baba T,.et al.The proacrosin binding protein,sp32,is tyrosine phosphorylated during capacitation of pig sperm[J].J Androl,2005,26(4):519-528.
[31]Bravo MM,AparicioIM,Garcia-Herreros M,.et al.Changes in tyrosine phosphorylation associated with true capacitation and capacitation-like state in boar spermatozoa[J].Mol Reprod Dev,2005,71(1):88-96.