Adeoya-Osiguwa S A, Fraser L R
Anatomy and Human Biology, King's College London, Strand, United Kingdom.
Mol Reprod Dev. 1996 May;44(1):111-20. doi: 10.1002/(SICI)1098-2795(199605)44:1<111::AID-MRD13>3.0.CO;2-7.
Membrane preparations from mouse sperm heads and tails were used in a gamma 32P-ATP hydrolysis assay to investigate Ca(2+)-dependent ATPase activity. In membranes from sperm heads, but not tails, a Ca2(+)-dependent ATPase that was further stimulated by calmodulin was detected. The addition of partially purified mouse sperm decapacitation factor (DF) to head membrane preparations significantly stimulated Ca(2+)-ATPase activity, this effect being further increased in the presence of DF plus calmodulin; in contrast, no response was observed when the same treatment was applied to tail membranes. Sperm preincubated in the presence of trifluoperazine (TFP), a calmodulin antagonist, were significantly more fertile than cells from the same males incubated in the absence of TFP, indicating that inhibition of calmodulin accelerates capacitation. When sperm cells were preincubated briefly, then gently centrifuged to remove DF and resuspended in medium containing 45Ca2+ +/- DF, their ability to accumulate 45Ca2+ was significantly lower in the early stages after resuspension in the presence of DF than in its absence. These data correlated with chlortetracycline analysis of the sperm functional state. When cells were centrifuged and resuspended in medium only, there was a noticeable shift from the F pattern (characteristic of uncapacitated cells) to the B pattern (characteristic of capacitated cells), but the reintroduction of DF caused a significant reversion to the F pattern. Finally, using a monoclonal antibody to somatic cell Ca2(+)-ATPase, we have obtained evidence that the enzyme is particularly localized to the postacrosomal region of the mouse sperm head; specific binding was observed only in permeabilized cells, indicating that the epitope involved in the binding has an intracellular location. Based on these various pieces of evidence, we propose that when present on mouse sperm, DF stimulates calmodulin-sensitive Ca(2+)-ATPase activity and thus ensures maintenance of a low intracellular Ca2+ concentration. As capacitation proceeds, DF is lost and Ca2(+)-ATPase activity declines, allowing intracellular Ca2+ to rise and promoting capacitation-related changes. The fact that inhibitors of Ca(2+)-ATPase and calmodulin appear to accelerate capacitation in several mammalian species, as determined by chlortetracycline analysis, suggests that Ca(2+)-ATPase activity may play an important role in modulating capacitation in many or even all mammals.
从小鼠精子头部和尾部制备的膜用于γ32P - ATP水解试验，以研究钙依赖性ATP酶活性。在精子头部而非尾部的膜中，检测到一种受钙调蛋白进一步刺激的钙依赖性ATP酶。向头部膜制剂中添加部分纯化的小鼠精子去能因子（DF）可显著刺激钙ATP酶活性，在DF加钙调蛋白存在时这种效应进一步增强；相反，对尾部膜进行相同处理时未观察到反应。在钙调蛋白拮抗剂三氟拉嗪（TFP）存在下预孵育的精子比在无TFP条件下孵育的同一只雄性小鼠的精子生育力显著更高，这表明抑制钙调蛋白可加速获能。当精子细胞短暂预孵育，然后轻轻离心以去除DF并重悬于含有45Ca2 + ± DF的培养基中时，在重悬于有DF存在的培养基后的早期阶段，它们积累45Ca2 +的能力显著低于无DF时。这些数据与精子功能状态的金霉素分析相关。当细胞离心并重悬于仅含培养基的溶液中时，有一个明显的从F模式（未获能细胞的特征）向B模式（获能细胞的特征）的转变，但重新引入DF会导致显著地变回F模式。最后，使用针对体细胞钙ATP酶 的单克隆抗体，我们获得证据表明该酶特别定位于小鼠精子头部顶体后区域；仅在透化细胞中观察到特异性结合，这表明参与结合的表位位于细胞内。基于这些各种证据，我们提出当存在于小鼠精子上时，DF刺激钙调蛋白敏感的钙ATP酶活性，从而确保维持低细胞内钙浓度。随着获能的进行，DF丢失且钙ATP酶活性下降，使细胞内钙升高并促进与获能相关的变化。通过金霉素分析确定，钙ATP酶和钙调蛋白抑制剂似乎在几种哺乳动物物种中加速获能，这一事实表明钙ATP酶活性可能在调节许多甚至所有哺乳动物的获能中起重要作用。
