title: Progesterone-stimulated intracellular calcium increase in human spermatozoa is protein kinase C-independent.
abstract: 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.
authors.affiliation: Dipartimento di Fisiopatologia Clinica, Università di Firenze, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Bonaccorsi
authors.forename: C
authors.initials: C
authors.lastname: Krausz
authors.forename: P
authors.initials: P
authors.lastname: Pecchioli
authors.forename: G
authors.initials: G
authors.lastname: Forti
authors.forename: E
authors.initials: E
authors.lastname: Baldi
mesh_terms: D002118:Calcium / Q000032:analysis*; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis; D006801:Humans; D008297:Male; D019887:Peanut Agglutinin; D010766:Phosphorylation; D011374:Progesterone / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D011506:Proteins / Q000378:metabolism; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000201:enzymology / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D019887:Peanut Agglutinin; D011506:Proteins; D014443:Tyrosine; D011374:Progesterone; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium