Ashizawa K, Katayama S, Kobayashi T, Tsuzuki Y
Laboratory of Animal Reproduction, Faculty of Agriculture, Miyazaki University, Japan.
J Reprod Fertil. 1994 Aug;101(3):511-7. doi: 10.1530/jrf.0.1010511.
The presence and possible role of protein kinase C in the regulation of fowl sperm functions were investigated. Immunoblot analysis of sperm extract using antibody to protein kinase C revealed a crossreacting protein of approximately 80 kDa. As the concentration of the protein kinase C activators N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9) or 1-oleoyl-2-acetylglycerol (OAG) was increased, the motility of intact spermatozoa at 30 degrees C was reduced. However, this inhibition of motility was reversed by reducing the concentrations of activators. Even in the presence of 1 mmol CaCl2 l-1, the addition of SC-9 and OAG inhibited the motility of intact spermatozoa. In contrast, the motility of demembranated spermatozoa was not inhibited by the addition of SC-9 or OAG at 30 degrees C. However, the addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a protein kinase C inhibitor, did not appreciably affect the motility of either intact or demembranated spermatozoa at 30 degrees C. At 40 degrees C, both intact and demembranated spermatozoa were almost immotile in the presence or absence of the activators or inhibitor. Intracellular free Ca2+ concentrations, measured by means of a fluorescent Ca2+ indicator, fura-2, gradually increased after the addition of SC-9 and OAG, but no changes were observed in H-7-treated spermatozoa. These results suggest that endogenous protein kinase C is present in the cytoplasmic matrix or the membrane, but is not retained in the axoneme, and that the activation of this enzyme may contribute to a decrease in the flagellar movement of fowl spermatozoa.
研究了蛋白激酶C在禽类精子功能调节中的存在情况及其可能的作用。用抗蛋白激酶C抗体对精子提取物进行免疫印迹分析，发现一种约80 kDa的交叉反应蛋白。随着蛋白激酶C激活剂N-(6-苯基己基)-5-氯-1-萘磺酰胺（SC-9）或1-油酰基-2-乙酰甘油（OAG）浓度的增加，30℃时完整精子的活力降低。然而，通过降低激活剂浓度可逆转这种活力抑制。即使在存在1 mmol CaCl2 l-1的情况下，添加SC-9和OAG也会抑制完整精子的活力。相比之下，在30℃添加SC-9或OAG不会抑制去膜精子的活力。然而，添加蛋白激酶C抑制剂1-(5-异喹啉磺酰基)-2-甲基哌嗪二盐酸盐（H-7）在30℃时对完整或去膜精子的活力均无明显影响。在40℃时，无论有无激活剂或抑制剂，完整和去膜精子几乎都无活力。通过荧光Ca2+指示剂fura-2测量细胞内游离Ca2+浓度，添加SC-9和OAG后其逐渐升高，但在H-7处理的精子中未观察到变化。这些结果表明，内源性蛋白激酶C存在于细胞质基质或膜中，但不存在于轴丝中，并且该酶的激活可能导致禽类精子鞭毛运动的减少。
