Bonaccorsi L, Krausz C, Pecchioli P, Forti G, Baldi E
Dipartimento di Fisiopatologia Clinica, Università di Firenze, Italy.
Mol Hum Reprod. 1998 Mar;4(3):259-68. doi: 10.1093/molehr/4.3.259.
Indirect studies suggested that protein kinase C (PKC) has a role in sperm motility and the acrosome reaction. Physiological inducers of the sperm acrosome reaction include progesterone, which can increase intracellular calcium ([Ca2+]i), tyrosine phosphorylation of proteins and chloride efflux in human spermatozoa. PKC may be involved in progesterone-stimulated acrosome reaction, although controversial results have been obtained concerning the effect of PKC inhibition on progesterone-stimulated [Ca2+]i increase. In the present study, we investigated the direct effect of progesterone on the activity of PKC, as well as the effect of a panel of PKC inhibitors on progesterone-stimulated [Ca2+]i increase and tyrosine phosphorylation of proteins. We found that progesterone stimulates sperm PKC activity and that PKC inhibition with staurosporine and bisindolylmaleimide partially reversed the effect of progesterone on acrosome reaction, indicating an involvement of the enzyme in the effect of the steroid. We next evaluated the effect of three different PKC inhibitors (sangivamycin, staurosporine and bisindolylmaleimide) on progesterone-stimulated [Ca2+]i increase. Neither short-term (15 min) nor long-term (90 min) preincubation with any of the three compounds had a substantial effect on the stimulatory effect of progesterone on sperm [Ca2+]i. Nor was responsiveness to progesterone affected by either short-term (determining activation of PKC) or long-term (determining down-regulation of PKC) incubation with the tumour promoter phorbol myristate acetate (PMA), a known non-physiological stimulator of PKC. These results indicate that progesterone-stimulated calcium influx is independent of PKC activation. In addition, we found that preincubation with PKC inhibitors had a stimulatory effect per se on tyrosine phosphorylation of sperm proteins. When compared with the appropriate control, the effect of progesterone on tyrosine phosphorylation was slightly (but not significantly) reduced by the inhibitors, sangivamycin, staurosporine and bisindolylmaleimide, but was significantly inhibited by calphostin C. These results do not permit a final conclusion on the involvement of PKC in progesterone-stimulated tyrosine phosphorylation of sperm proteins. However, the lack of effect of PMA on tyrosine phosphorylation indicates that PKC stimulation is not sufficient to induce this effect. In conclusion, our results indicate that PKC plays a role in progesterone-induced acrosome reaction and that progesterone-stimulated PKC activation is downstream to stimulation of calcium influx by the steroid.
间接研究表明，蛋白激酶C（PKC）在精子活力和顶体反应中起作用。精子顶体反应的生理诱导剂包括孕酮，它可以增加细胞内钙（[Ca2+]i）、蛋白质的酪氨酸磷酸化以及人类精子中的氯离子外流。PKC可能参与孕酮刺激的顶体反应，尽管关于PKC抑制对孕酮刺激的[Ca2+]i增加的影响已获得有争议的结果。在本研究中，我们研究了孕酮对PKC活性的直接影响，以及一组PKC抑制剂对孕酮刺激的[Ca2+]i增加和蛋白质酪氨酸磷酸化的影响。我们发现孕酮刺激精子PKC活性，并且用星形孢菌素和双吲哚马来酰胺抑制PKC部分逆转了孕酮对顶体反应的影响，表明该酶参与了类固醇的作用。接下来，我们评估了三种不同的PKC抑制剂（桑吉瓦霉素、星形孢菌素和双吲哚马来酰胺）对孕酮刺激的[Ca2+]i增加的影响。用这三种化合物中的任何一种进行短期（15分钟）或长期（90分钟）预孵育对孕酮对精子[Ca2+]i的刺激作用均无实质性影响。用肿瘤启动子佛波醇肉豆蔻酸酯（PMA）进行短期（确定PKC的激活）或长期（确定PKC的下调）孵育对孕酮的反应性也没有影响，PMA是一种已知的非生理性PKC刺激剂。这些结果表明，孕酮刺激的钙内流独立于PKC激活。此外，我们发现用PKC抑制剂预孵育本身对精子蛋白质的酪氨酸磷酸化有刺激作用。与适当的对照相比，抑制剂桑吉瓦霉素、星形孢菌素和双吲哚马来酰胺使孕酮对酪氨酸磷酸化的作用略有（但不显著）降低，但被钙磷蛋白C显著抑制。这些结果不允许就PKC是否参与孕酮刺激的精子蛋白质酪氨酸磷酸化得出最终结论。然而PMA对酪氨酸磷酸化没有影响，这表明PKC刺激不足以诱导这种作用。总之，我们的结果表明PKC在孕酮诱导的顶体反应中起作用，并且孕酮刺激的PKC激活在类固醇刺激钙内流的下游。
