PROGESTERONE-STIMULATED INTRACELLULAR CALCIUM INCREASE IN HUMAN SPERMATOZOA IS PROTEIN-KINASE C-INDEPENDENT

Indirect studies suggested that protein kinase C (PKC) has a role in s perm motility and the acrosome reaction. Physiological inducers of the sperm acrosome reaction include progesterone, which can increase intr acellular calcium ([Ca2+](i)), tyrosine phosphorylation of proteins an d chloride efflux in human spermatozoa. PKC may be involved in progest erone-stimulated acrosome reaction, although controversial results hav e been obtained concerning the effect of PKC inhibition on progesteron e-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 [Ca 2+](i) increase and tyrosine phosphorylation of proteins. We found tha t progesterone stimulates sperm PKC activity and that PKC inhibition w ith staurosporine and bisindolylmaleimide partially reversed the effec t of progesterone on acrosome reaction, indicating an involvement of t he enzyme in the effect of the steroid. We next evaluated the effect o f three different PKC inhibitors (sangivamycin, staurosporine and bisi ndolylmaleimide) on progesterone-stimulated [Ca2+](i) increase. Neithe r short-term (15 min) nor long-term (90 min) preincubation with any of the three compounds had a substantial effect on the stimulatory effec t of progesterone on sperm [Ca2+](i). Nor was responsiveness to proges terone affected by either short-term (determining activation of PKC) o r long-term (determining down-regulation of PKC) incubation with the t umour promoter phorbol myristate acetate (PMA), a known non-physiologi cal stimulator of PKC. These results indicate that progesterone-stimul ated 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 wi th the appropriate control, the effect of progesterone on tyrosine pho sphorylation was slightly (but not significantly) reduced by the inhib itors, sangivamycin, staurosporine and bisindolylmaleimide, but was si gnificantly inhibited by calphostin C. These results do not permit a f inal conclusion on the involvement of PKC in progesterone-stimulated t yrosine 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 indic ate that PKC plays a role in progesterone-induced acrosome reaction an d that progesterone-stimulated PKC activation is downstream to stimula tion of calcium influx by the steroid.