Triphan Jörg, Aumüller Gerhard, Brandenburger Timo, Wilhelm Beate
Department of Anatomy and Cell Biology, Philipps-University, Robert-Koch-Str. 8, D-35037 Marburg, Germany.
Eur J Cell Biol. 2007 May;86(5):265-73. doi: 10.1016/j.ejcb.2007.02.003. Epub 2007 Mar 29.
Calcium (Ca(2+)) signals, produced by the opening of plasma membrane entry channels, regulate a number of functions in spermatozoa such as capacitation and motility. The mechanisms of Ca(2+) removal from the sperm, required to restore resting Ca(2+), include plasma membrane Ca(2+)-dependent ATPase (PMCA) isoenzymes as well as a plasma membrane Na(+)-Ca(2+) exchanger. We have recently shown that bovine sperm PMCA is stimulated by PDC-109, a secretory protein of bovine seminal vesicles. To demonstrate the subcellular localization and regulation of bovine sperm PMCA, we have performed cell fractionation, enzyme activity determination and Western blotting studies of PMCA in spermatozoa removed from the cauda epididymidis of bull. Fractionation of sperm heads and tails resulted in a distinct association of ATPase activity with the tail membrane fraction. In vitro stimulation studies with PDC-109 using intact and fractionated sperm showed an increase in enzyme activity up to 105% in sperm tail membranes. Furthermore, thapsigargin inhibition did not alter the stimulatory effect of PDC-109 on ATPase activity, indicating that no sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), but only PMCA isoenzymes are involved in this effect. Western blotting studies using a polyvalent PMCA antibody showed the exclusive presence of a 135 kDa band in the tail plasma membrane fraction. To elucidate whether or not the stimulatory effect was a direct one or indirectly mediated through PKA and PKC activation, PKA and PKC inhibitors, respectively, were used in the Ca(2+)-ATPase activity assays, which was followed by PDC-109 stimulation. The stimulatory effect of PDC-109 on PMCA was still observed under these conditions, while no phosphotyrosine proteins could be detected by Western blotting in sperm extracts following PDC-109 treatment. Co-immunoprecipitation studies, PDC-109 affinity chromatography as well as overlay blots failed to show a strong association of both PMCA and PDC-109, pointing to an indirect, perhaps phospholipid-mediated effect.
由质膜进入通道开放产生的钙（Ca(2+)）信号调节精子中的许多功能，如获能和运动。精子中恢复静息Ca(2+)所需的Ca(2+)去除机制包括质膜Ca(2+)依赖性ATP酶（PMCA）同工酶以及质膜Na(+)-Ca(2+)交换体。我们最近发现，牛精子PMCA受到牛精囊分泌蛋白PDC-109的刺激。为了证明牛精子PMCA的亚细胞定位和调节，我们对从公牛附睾尾取出的精子进行了细胞分级分离、酶活性测定和PMCA的蛋白质印迹研究。精子头部和尾部的分级分离导致ATP酶活性与尾膜部分有明显关联。使用完整和分级分离的精子对PDC-109进行的体外刺激研究表明，精子尾膜中的酶活性增加高达105%。此外，毒胡萝卜素抑制并未改变PDC-109对ATP酶活性的刺激作用，表明此效应中不涉及肌浆网/内质网Ca(2+)-ATP酶（SERCA），而仅涉及PMCA同工酶。使用多价PMCA抗体进行的蛋白质印迹研究表明，尾质膜部分仅存在一条135 kDa的条带。为了阐明刺激作用是直接的还是通过PKA和PKC激活间接介导的，分别在Ca(2+)-ATP酶活性测定中使用PKA和PKC抑制剂，随后进行PDC-109刺激。在这些条件下仍观察到PDC-109对PMCA的刺激作用，而在PDC-109处理后的精子提取物中通过蛋白质印迹未检测到磷酸酪氨酸蛋白。免疫共沉淀研究、PDC-109亲和色谱以及覆盖印迹均未显示PMCA和PDC-109有强关联，表明可能是间接的、也许是磷脂介导的效应。
