小鼠精子获能过程中FSCB蛋白磷酸化研究  

FSCB phosphorylation during mouse spermatozoa capacitation

作　　者：刘顺利[1] 陈铭锐[1] 崔庆斌 李彦锋[3] 

机构地区：[1]济南军区总医院烧伤整复科,山东省济南市250031 [2]南京理工大学医院外科 [3]第三军医大学大坪医院泌尿外科

出　　处：《中华生物医学工程杂志》2015年第4期303-307,共5页Chinese Journal of Biomedical Engineering

基　　金：国家自然科学基金（30872573、81401250）

摘　　要：目的：研究精子鞭毛纤维鞘CABYR结合蛋白（FSCB）在小鼠精子获能过程中发生磷酸化的分子机制。方法取重组FSCB蛋白进行蛋白激酶A（PKA）体外磷酸化实验，反应产物电泳后分别用抗磷酸化PKA底物抗体和抗磷酸化酪氨酸抗体进行免疫印迹分析。取小鼠未获能与获能2 h精子，分别应用抗磷酸化PKA底物抗体、抗磷酸化酪氨酸抗体或FSCB抗体进行免疫共沉淀，免疫共沉淀产物使用FSCB抗体行免疫印迹分析。使用FSCB抗体和磷酸化PKA底物抗体进行免疫荧光定位分析。结果重组FSCB可被PKA催化亚单位体外磷酸化。FSCB在精子获能过程中可以被PKA磷酸化，并可同时发生酪氨酸磷酸化。免疫荧光定位显示FSCB与磷酸化PKA底物共定位于精子主段。结论 FSCB作为PKA作用底物之一参与小鼠精子的获能过程。Objective To investigate the molecular mechanism underlying the phosphorylation of fibrous sheath CABYR binding protein （FSCB） during the process of mouse sperm capacitation. Methods Recombinant FSCB was phosphorylated with the presence of protein kinase A（PKA）in-vitro. The products were electrophoresed,followed by Western blot using anti-phospho-PKA substrate antibody and anti-phosphotyrosine antibody,respectively. Mouse spermatozoa before and at 2 h after capacitation were lysed and subjected to immunoprecipitation using anti-phospho-PKA substrate antibody,anti-phosphotyosine or anti-FSCB antibodies,respectively,followed by Western blot using anti-FSCB antibody. Immunofluorescence localization using FSCB antibody and phospho-PKA substrate antibody were performed. Results The recombinant FSCB could be phosphorylated in-vitro in the presence of PKA catalytic subunit. FSCB can be phosphorylated by PKA with simultaneous tyrosine phosphorylation during the process of sperm capacitation. Immunofluorescence analysis showed that FSCB and PKA phosphorylated substrate were co-localized to the spermatic principal piece. Conclusion FSCB is involved in the process of sperm capacitation as one of the PKA substrates.

关 键 词：精子获能 纤维鞘 蛋白激酶A 磷酸化 

分 类 号：R321[医药卫生—人体解剖和组织胚胎学]