Rodríguez C, Killian G
Dairy Breeding Research Center, Department of Dairy and Animal Science, The Pennsylvania State University, University Park 16802, USA.
Anim Reprod Sci. 1998 Dec 1;54(1):1-12. doi: 10.1016/s0378-4320(98)00139-0.
The objective of this study was to define the complement of ampullary and isthmic oviductal fluid proteins that associate with the sperm membrane during the non-luteal and luteal stages of the oestrous cycle. Oviductal fluid was obtained from three dairy cows via indwelling cannulae in the ampulla and isthmus of the same oviduct. Daily samples of oviductal fluid were combined by region and stage of the cycle to create pools consisting of non-luteal isthmic, luteal isthmic, non-luteal ampullary, and luteal ampullary oviductal fluid. An aliquot from each oviductal fluid pool was biotinylated by incubation in biotin reagent. Spermatozoa from three fertile bulls were pooled, washed and incubated for 4 h in each of the four pools of biotinylated oviductal fluid. After incubation, sperm membrane proteins were solubilized, and proteins subjected to one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The biotinylated oviductal fluid proteins that associated with the sperm membranes were detected by avidin-horseradish peroxidase and diaminobenzidine substrate. Eight oviductal fluid proteins with apparent molecular weights of 97, 75, 66, 55, 48, 34, 28 and 24 kDa were consistently detected to associate with the sperm membrane. Differences in ampullar and isthmic fluid proteins that associated with sperm during the luteal and non-luteal stage of the oestrous cycle were not detected.
本研究的目的是确定在发情周期的非黄体期和黄体期与精子膜结合的壶腹部和峡部输卵管液蛋白质的组成。通过在同一输卵管的壶腹部和峡部留置套管，从三头奶牛获取输卵管液。按周期阶段和区域将每日采集的输卵管液样本合并，形成由非黄体期峡部、黄体期峡部、非黄体期壶腹部和黄体期壶腹部输卵管液组成的样本池。每个输卵管液样本池的一份等分试样在生物素试剂中孵育进行生物素化。将来自三头可育公牛的精子合并、洗涤，并在四个生物素化输卵管液样本池中各孵育4小时。孵育后，溶解精子膜蛋白，对蛋白质进行一维十二烷基硫酸钠聚丙烯酰胺凝胶电泳和蛋白质印迹分析。通过抗生物素蛋白 - 辣根过氧化物酶和二氨基联苯胺底物检测与精子膜结合的生物素化输卵管液蛋白。 consistently detected to associate with the sperm membrane. Differences in ampullar and isthmic fluid proteins that associated with sperm during the luteal and non-luteal stage of the oestrous cycle were not detected. 始终检测到八种表观分子量分别为97、75、66、55、48、34、28和24 kDa的输卵管液蛋白与精子膜结合。未检测到发情周期黄体期和非黄体期与精子结合的壶腹部和峡部液蛋白的差异。 （注：原文最后一句英文表述不太完整，翻译时尽量按照已有内容准确翻译）
