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Identification and analysis of temperature-sensitive proteins : the, temperature dependent immobilization of fowl spermatozoa at body temperature as a model

温度敏感蛋白的鉴定和分析：以鸡精子在体温下的温度依赖性固定为模型

基本信息

批准号：
11460130
负责人：
ASHIZAWA Koji
金额：
$ 7.3万
依托单位：
Miyazaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

The motility of spermatozoa is controlled by phosphorylation-dephosphorylation of specific proteins existing in flagella. Protein phosphorylation is mediated by protein kinases and dephosphorylation is mediated by protein phosphatases. Although it has been proposed that the reversible temperature-dependent immobilization of fowl spermatozoa is related to the activation of protein phosphatase, it has not been clarified in detail yet. The motility of spermatozoa in TES/NaCl buffer with or without IPVL at 40 ℃ was almost negligible, due to the temperature-dependent immobilization of fowl spermatozoa. However, sperm motility was restored at 40 ℃ following the addition of calyculin A, but not by fenvalerate or deltamethrin. The AR was stimulated by IPVL extracts at 40 ℃, but only in the presence of Ca^<2+>, calyculin A, fenvalerate or deltamethrin, the latter three phosphatase inhibitors stimulating the AR in a dose-dependent manner in the range of 10-1000 nM. At 30 ℃, the effects of PP2A activators (C2 - Ceramide and C6 - Ceramide) on the motility of spermatozoa, were not clear. At 40 ℃, both intact and demembranated sperm motility were almost negligible, regardless of the presence of Ceramides. Furthermore, stimulation of motility by Ca^<2+> was gradually inhibited following the addition of Ceramides. The same tendency was observed even when calyculin A was used instead of Ca^<2+>.These results suggest that Ca^<2+> may act via activation of protein phosphorylation pathways and that the Ca^<2+> -activated intracellular molecular mechanisms for the regulation of AR are different from those for restoration of sperm motility ; i.e., protein dephosphorylation by PP2B, in addition to PP1 and/or PP2A, in the former and PP1 and/or PP2A alone in the latter case.

精子的运动力由鞭毛中特定蛋白质的磷酸化-去磷酸化控制。蛋白磷酸化由蛋白激酶介导，去磷酸化由蛋白磷酸酶介导。虽然有人提出鸡精子可逆的温度依赖性固定与蛋白磷酸酶的激活有关，但尚未得到详细阐明。由于家禽精子的固定具有温度依赖性，因此在40 ℃下，在有或没有IPVL的TES/NaCl缓冲液中，精子的活力几乎可以忽略不计。然而，在40 ℃下，添加calyculin A后，精子活力恢复，而不是氰戊菊酯或溴氰菊酯。在40 ℃下，IPVL提取物可刺激AR，但仅在Ca^<2+>、calyculin A、氰戊菊酯或溴氰菊酯存在的情况下，后三种磷酸酶抑制剂在10-1000 nM范围内以剂量依赖性方式刺激AR。在30 ℃时，PP 2A激活剂（C2 - Ceramide和C6 - Ceramide）对精子活力的影响尚不清楚。在40 ℃下，无论是否存在神经酰胺，完整和脱膜精子活力几乎可以忽略不计。此外，加入神经酰胺后，Ca^<2+>对运动的刺激逐渐受到抑制。甚至当用calyculin A代替Ca^<2+>时也观察到同样的趋势。这些结果表明Ca^<2+>可能通过激活蛋白磷酸化途径起作用，并且Ca^<2+>激活的调节AR的细胞内分子机制与恢复精子运动力的机制不同;即，在前一种情况下，除了PP 1和/或PP 2A之外，还通过PP 2B进行蛋白质去磷酸化，而在后一种情况下，仅通过PP 1和/或PP 2A进行蛋白质去磷酸化。