Lax Y, Grossman S, Rubinstein S, Magid N, Breitbart H
Department of Life Science, Bar-Ilan University, Ramat-Gan, Israel.
Biochim Biophys Acta. 1990 Mar 12;1043(1):12-8. doi: 10.1016/0005-2760(90)90104-6.
The acrosome reaction (AR) in bull spermatozoa was induced by the Ca2(+)-ionophore A23187, by dilauroylphosphatidylcholine or by arachidonic acid in the presence of Ca2+ in the incubation medium. The occurrence of AR was determined by following the release of acrosin from the cells. Nordihydroguaiaretic acid (NDGA), an inhibitor of both lipoxygenase and prostaglandin-synthetase, caused 35%, 43% and 69% inhibition of AR at concentrations of 1, 10 or 100 microM, respectively. Eicosatetraynoic acid (ETYA), an analogue of arachidonic acid, caused 17%, 61% and 77% inhibition of AR at concentrations of 20, 40 or 80 micrograms/ml, respectively. When AR was induced by arachidonic acid, ETYA, causes 36% and 58% inhibition at concentrations of 2 or 20 micrograms/ml, respectively. Under identical conditions, 100 microM indomethacin, a specific inhibitor of prostaglandin-synthetase, showed no inhibition but rather 35% stimulation at acrosin release rate. The fact that AR is inhibited by NDGA and not by indomethacin indicates that the lipoxygenase, rather than prostaglandin-synthetase, is involved in the mechanism of AR. Since the inhibition by NDGA is seen in the presence of the Ca-ionophore, we suggest that lipoxygenase activity is not involved in enhancing calcium transport into the cell, but rather at other steps in AR mechanism. A thin-layer chromatography revealed the presence of 15-HETE, the classical product of 15-lipoxygenase activity, which was identified by HPLC. Under AR conditions, there is an elevation of lipoxygenase products and the addition of NDGA caused a reduction in their levels. The inhibition of acrosin release by NDGA can be eliminated by adding 15-HETE or 15-HPETE to the incubation medium. In conclusion, we suggest here for the first time, a physiological role for 15-lipoxygenase in the mechanism of AR in mammalian spermatozoa.
在孵育介质中存在钙离子的情况下，钙离子载体A23187、二月桂酰磷脂酰胆碱或花生四烯酸可诱导公牛精子发生顶体反应（AR）。通过追踪顶体蛋白酶从细胞中的释放来确定AR的发生情况。去甲二氢愈创木酸（NDGA）是脂氧合酶和前列腺素合成酶的抑制剂，在浓度分别为1、10或100微摩尔时，对AR的抑制率分别为35%、43%和69%。花生四烯酸的类似物二十碳四烯酸（ETYA）在浓度分别为20、40或80微克/毫升时，对AR的抑制率分别为17%、61%和77%。当由花生四烯酸诱导AR时，ETYA在浓度为2或20微克/毫升时，抑制率分别为36%和58%。在相同条件下，100微摩尔的消炎痛（前列腺素合成酶的特异性抑制剂）对顶体蛋白酶释放率没有抑制作用，反而有35%的刺激作用。AR受NDGA抑制而不受消炎痛抑制这一事实表明，参与AR机制的是脂氧合酶而非前列腺素合成酶。由于在钙离子载体存在的情况下观察到了NDGA的抑制作用，我们认为脂氧合酶活性不参与增强钙离子向细胞内的转运，而是参与AR机制的其他步骤。薄层色谱显示存在15-羟基二十碳四烯酸（15-HETE），这是15-脂氧合酶活性的典型产物，通过高效液相色谱法进行了鉴定。在AR条件下，脂氧合酶产物水平升高，添加NDGA会导致其水平降低。通过向孵育介质中添加15-HETE或15-HPETE，可以消除NDGA对顶体蛋白酶释放的抑制作用。总之，我们首次在此提出15-脂氧合酶在哺乳动物精子AR机制中具有生理作用。
