Protein kinase C activation during progesterone-stimulated acrosomal exocytosis in human spermatozoa.

The involvement of protein kinase C (PKC) in exocytosis of the mammalian sperm acrosome is still a controversial issue. Work carried out thus far has failed to provide direct evidence for the activation of this enzyme upon stimulation with natural agonists of acrosomal exocytosis. We have therefore used progesterone stimulation of the acrosome reaction in human spermatozoa to clarify this issue. In spermatozoa preincubated under conditions known to support capacitation and fertilization in vitro, treatment with progesterone caused a time-dependent stimulation of phosphorylation of at least eight proteins ranging in size from approximately 20-220 kDa. The inclusion of the PKC inhibitors chelerythrine chloride or calphostin C reduced the observed phosphorylation in a concentration-dependent manner. Exogenously supplied phorbol 12-myristate-13-acetate (PMA) or the permeant diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol (OAG), synthetic activators of PKC, also stimulated phosphorylation in preincubated spermatozoa, but inclusion of calphostin C diminished the response. Furthermore, the prior inclusion of the 1,4-dihydropyridine Ca2+ channel antagonist nifedipine also inhibited phosphorylation, suggesting that PKC is activated downstream of Ca2+ channel opening. Exocytosis triggered by progesterone was significantly inhibited by chelerythrine chloride or calphostin C. Both PMA and OAG triggered exocytosis, but the inclusion of chelerythrine chloride significantly inhibited the response; exocytotic responses were seen only in capacitated cells. These results provide the first direct evidence that PKC activation plays a role in the signal transduction pathway underlying acrosomal exocytosis in progesterone-stimulated capacitated spermatozoa.