A novel relaxin-like protein discovered in boar seminal vesicle : Binding on sperm plasma membrane and physiological function

本研究计划确定松弛素样蛋白在猪精子上的结合位点，以澄清精子上结合位点的性质，并检查精子上结合的可能生理作用。研究结果如下：1.开发TR-FIA作为一种新的检测系统：本研究成功地开发了一种新的猪松弛素检测系统，采用时间分辨荧光免疫分析法（TR-FIA）最近推出的非RIA格式。该测定系统是基于铕（Eu）标记的松弛素和测试样品之间的多克隆抗松弛素抗体的竞争的固相TR-FIA。该方法具有灵敏度高、测定时间短、不需要采取特殊的安全措施、精密度、重现性和准确度高等优点.猪精子结合位点的鉴定及性质：本研究明确表明松弛素样蛋白主要与猪精子顶体结合，中段和尾部结合较弱，结合位点为蛋白质和脂质的结合，提示该蛋白通过特异性受体激活精子功能。3.松弛素样蛋白与猪精子结合的可能生理作用：本研究表明松弛素样蛋白作为胆固醇对接蛋白诱导精子获能，提示该蛋白可能作为预测公猪精子获能性的潜在标志物。

This research project was planned to identify the binding sites on boar sperm for relaxin-like protein, to clarify the nature of the binding sites on the sperm, and to examine possible physiological roles of the binding on the sperm. The outcome of this research is as follows.1. Development of TR-FIA as a novel assay system : This research was successful in developing a new assay system for porcine relaxin by adapting time-resolved fluoroimmunoassay (TR-FIA) introduced recently as a non-RIA format. The assay system was a solid-phase TR-FIA based on competition for a polyclonal anti-relaxin antibody between europium (Eu) -labeled relaxin and test samples. This assay we established had several advantages : the assay had a high sensitivity, the measurement time was completed in only 5 h with one second per sample, no special safety precautions had to be taken, and the precision, reproducibility and accuracy of the assay were satisfactory.2. Identification and nature of the binding sites on boar sperm : This research has clearly demonstrated that relaxin-like protein binds mainly to the acrosomal region of boar spermatozoa, with weaker binding at the midpiece and tail, and the binding sites are the nature of both protein and lipid, suggesting that the protein activates sperm function through a specific receptor.3.Possible physiological roles of the binding on boar sperm : This research has shown that relaxin-like protein acts as cholesterol-docking protein and induces sperm capacitation, suggesting that the protein may have value as a potential marker for predicting sperm fertilizing ability of boars.