Protein tyrosine phosphorylation in boar sperm capacitation induced by a novel relaxin-like protein

本研究计划阐明松弛素样蛋白（精松素）介导的猪精子获能和顶体反应中蛋白酪氨酸磷酸化的作用。本研究的结果如下。本研究表明，精松素确实能诱导猪精子获能和顶体反应，具有较高的精子存活率。在松弛素处理的猪精子中，蛋白酪氨酸激酶的活性随着孵育时间的延长而增加，在孵育4 h时达到平稳水平（10U/10^8个细胞）。此外，Western blot分析显示，松弛素诱导的能化/顶体反应的猪精子中存在一个分子大小为30 kDa的磷酸化蛋白条带，表明松弛素诱导了猪精子中酪氨酸的磷酸化。蛋白质组学分析也将其描述为有趣和不寻常的。对大肠杆菌中表达的重组磷酸化蛋白- mbp融合蛋白产生多克隆抗体后，免疫组化分析明确了磷酸化蛋白在精子头部和中段的定位。

This research project was planed to elucidate protein tyrosine phosphorylation during boar sperm capacitation and acrosome reaction mediated by relaxin-like protein (seminal relaxin). The outcome of this study as follows.The present study demonstrated that seminal relaxin actually induced boar sperm capacitation and acrosome reaction with high sperm viability. In relaxin-treated boar sperm, the activity of protein tyrosine kinase was increased with progress of the incubation time, which reached a plateau (10U/10^8 cells) at 4 h of incubation. Furthermore, Western blot analysis revealed a specific band of phosphorylated protein corresponding to the molecular size of 30 kDa in the capacitated/acrosome reacted boar sperm induced by relaxin, suggesting that relaxin induces tyrosine phosphorylation in boar sperm. Also proteome analysis characterized it as interesting and unusual. When polyclonal antibody was generated to the recombinant phosphorylated protein-MBP fusion protein expressed in E. coli, immunohistochemical analysis provided clear-cut evidence of the localization of phosphorylated protein in the sperm head and midpiece.