Localization of cyclooxygenase and production of prostaglandins in bovine spermatozoa

Abstract

Ejaculated bovine spermatozoa were examined for their capacity to synthesize prostaglandins E2 and F(2α) (PGE2, PGF(2α)). It was found that in the absence of exogenous substrate, arachidonic acid basal PGF(2α) production was less than that of PGE2. However, addition of 61 μmol arachidonic acid l-1 resulted in at least a twofold increase in PGE2 and PGF(2α) above control values (1.3 ng and 0.3 ng per 108 spermatozoa, respectively). Addition of calcium and the calcium ionophore A23187 to the incubation medium did not cause a significant increase in the production of either PG. The presence of indomethacin (100-200 μg ml-1) caused a 50-70% inhibition of the production of both PGs. Activity of cyclooxygenase was determined by western blot analysis, using a specific polyclonal antiserum, and by fluorescence immunohistochemistry using a monoclonal antibody. The western blot displayed a clear signal for the presence of cyclooxygenase in ejaculated and epididymal spermatozoa. The immunohistochemical studies showed that the enzyme is localized in the apical region of the head, the post-acrosomal region and the mid-piece of the tail. Since the synthesis of PGs in the absence of exogenous arachidonic acid is low, the effect of melittin, a known phospholipase A2 activator, on PG production was examined. Incubation of spermatozoa with melittin produced a threefold increase in PGE2 and a sixfold increase in PGF(2α). Staurosporine, a protein kinase C inhibitor, inhibited the effect of melittin indicating that activation of phospholipase A2 by protein kinase C is an obligatory step in PG synthesis by bovine spermatozoa. To determine the physiological role of PG synthesis by bovine spermatozoa the effect of PGE2 and PGF(2α) on Ca2+ uptake was examined. PGE2, but not PGF(2α) increased the Ca2+ uptake linearly during the first 10 min of incubation. These data provide evidence that bovine spermatozoa can synthesize prostaglandins in the presence of arachidonic acid or melittin. Furthermore, it was demonstrated that the spermatozoa contain the key enzyme for prostaglandin synthesis, cyclooxygenase.