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Signal transduction mechanisms for the regulation of fowl sperm motility

调节家禽精子活力的信号转导机制

基本信息

批准号：
08660348
负责人：
ASHIZAWA Koji
金额：
$ 1.54万
依托单位：
MIYAZAKI UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

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

在ATP存在的情况下，鸡去膜精子在30ºC、-70ºC或-196ºC快速冷冻前和冷冻后的活力都很旺盛。经过冷冻和解冻处理的去膜精子的ATP消耗速率约为200 nmol ATP水解/10^9个精子/mm，与未处理的精子相似。在400℃时，冻融精子和对照精子均不具有运动能力，但将温度降低至30℃或添加蛋白磷酸酶抑制剂可使精子恢复运动能力。在Mn^2^+的基础上添加重组蛋白磷酸酶I型（PP-1），可显著降低脱膜鸡精子的活力。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）技术，分析了[γ -^3^2P]ATP在30℃温度下对脱膜鸡精子蛋白的磷酸化作用。在116、86、79、50和29 kda蛋白中，磷酸化状态存在显著差异。与对照样品相比，这些蛋白在PP-1和Mn^2^+的存在下被去磷酸化。在有或没有Ca^2^+的情况下，完整的鸡精子在30°C的温度下保持活跃的运动。与此相反，在40ºC时，不加Ca^2^+，其运动受到可逆抑制，但加入Ca^2^+后，其运动可以立即恢复。然而，在加入Ca^2^+之前，加入特异性Ca^2^+通道阻滞剂维拉帕米，不能在40ºC的温度下获得完全运动的精子。在这些条件下，通过荧光Ca^2^+指示剂fura-2测量，细胞内游离Ca^2^+浓度低于对照组（未添加维拉帕米）。添加Sr^2^+，可以刺激线粒体中Ca^2^+的释放，也可以有效地刺激运动，并诱导细胞内游离Ca^2^+浓度随之增加。这些观察结果表明，参与禽类精子温度依赖性固定的物质与轴突蛋白密切相关，pp - i介导的轴突蛋白和/或禽类精子的辅助细胞骨架成分的一些蛋白质的去磷酸化可能参与了运动的抑制。此外，细胞内游离Ca^2^+在调节鸡精子运动中起关键作用。少