Signal transduction mechanisms for the regulation of fowl sperm motility

在ATP存在下，去膜鸡精子在30 ℃时，在-70 ℃或-196 ℃快速冷冻前和冷冻后（不加冷冻保护剂）的运动都很活跃。经冷冻和解冻处理的脱膜精子的ATP消耗率约为200 nmol ATP水解/10^9个精子/mm，也与未处理的精子相似。在400 ℃时，冻融精子和对照精子都不能运动，但通过将温度降至30 ℃或加入蛋白磷酸酶抑制剂，可以恢复运动能力，加入含Mn ^2 ^+的重组蛋白磷酸酶I（PP-1）后，去膜鸡精子的运动能力明显降低。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析了在30 ℃与[γ-^3^2P]ATP孵育过程中脱膜鸡精子蛋白的磷酸化。磷酸化状态的显著差异为obs ...更多信息 在大约116、86、79、50和29-kDa蛋白质中服务。与对照组相比，PP-1和Mn^2^+存在时，这些蛋白质被脱磷酸化，完整的鸡精子在30 ° C下有或没有Ca^2^+时都能保持旺盛的运动。与此相反，在40 ℃下，没有Ca^2^+时，运动受到可逆性抑制，但加入Ca^2^+后，运动可以立即恢复。然而，在加入Ca^2^+之前加入特异性Ca^2^+通道阻断剂维拉帕米，在40 ℃下不能获得完全活动的精子。在这些条件下，细胞内游离Ca^2^+浓度低于对照组（未加入维拉帕米），这是用荧光Ca^2^+指示剂fura-2测得的。Sr^2^+的加入似乎能刺激线粒体释放Ca^2^+，它也能有效地刺激40 ℃下的运动，并诱导细胞内游离Ca^2^+浓度的增加。这些观察结果表明，参与鸡精子温度依赖性固定的物质与轴丝密切相关，PP-I介导的家禽精子轴丝和/或辅助细胞骨架成分中某些蛋白质的去磷酸化可能与运动抑制有关。此外，细胞内游离Ca ^2 ^+在鸡精子运动中起着重要的调节作用。少

In the presence of ATP, the motility of demembranated fowl spermatozoa was vigorous at 30ﾟC, both before and after rapid freezing at -70ﾟC or -196ﾟC without cryoprotectants. The rate of ATP consumption of the demembranated spermatozoa treated by freezing and thawing was, at approximately 200 nmol ATP hydrolysis/10^9 spermatozoa/mm, also similar to that of the untreated spermatozoa. At 400 C, neither frozen-thawed spermatozoa nor control spermatozoa were motile, but motility could be restored by the decreasing the temperature to 30ﾟC or by the addition of inhibitors of protein phosphatases.The motility of demembranated fowl spermatozoa decreased markedly following the addition of recombinant protein phosphatase type I (PP-1) supplemented with Mn^2^+. Phosphorylation of demembranated fowl sperm proteins during incubation with [gamma-^3^2P]ATP at 30ﾟC was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A marked difference in phosphorylation status was obs … More erved in approximately 116, 86, 79, 50 and 29-kDa proteins. These proteins were dephosphorylated in the presence of PP-1 and Mn^2^+ compared with those in control samples.Intact fowl spermatozoa maintained vigorous movement at 30ﾟC with or without Ca^2^+. In contrast, the motility was reversibly inhibited at 40ﾟC without Ca^2^+, but could be immediately restored by the addition of Ca^2^+. However, the addition of verapamil, a specific Ca^2^+ channel blocker, before the addition of Ca^2^+, could not obtain fully motile spermatozoa at 40ﾟC.Under these conditions, the intracellular free Ca^2^+ concentration was lower than that of the control (no addition of verapamil), as measured by a fluorescent Ca^2^+ indicator, fura-2. The addition of Sr^2^+, which appears to stimulate the release of Ca^2^+ from the mitochondria, was also effective for the stimulation of motility at 40ﾟC, and induced a concomitant increase in the intracellular free Ca^2^+ concentrations.These observations suggest that the substance(s) involved in the temperature-dependent immobilization of fowl spermatozoa are tightly associated with the axoneme, and that PP-I-mediated dephosphorylation of some of these proteins of the axoneme and/or accessory cytoskeletal components of fowl spermatozoa may be involved in the inhibition of motility. Furthermore, it is suggested that intracellular free Ca^2^+ play a pivotal role in regulating fowl sperm motility. Less