LOCALIZATION OF CYCLOOXYGENASE AND PRODUCTION OF PROSTAGLANDINS IN BOVINE SPERMATOZOA

Ejaculated bovine spermatozoa were examined for their capacity to synt hesize prostaglandins E(2) and F-2 alpha (PGE(2), PGF(2 alpha)). It wa s found that in the absence of exogenous substrate, arachidonic acid, basal PGF(2 alpha) production was less than that of PGE(2). However, a ddition of 61 mu mol arachidonic acid I--I resulted in at least a twof old increase in PGE(2) and PGF(2 alpha) above control values (1.3 ng a nd 0.3 ng per 10(8) spermatozoa, respectively). Addition of calcium an d 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 mu g ml(-1)) caused a 50-70% inhibition of the production of both PGs. Activity of cyclooxygenase was determined by w estern blot analysis, using a specific polyclonal antisenun, and by fl uorescence immunohistochemistry using a monoclonal antibody. The weste rn blot displayed a clear signal for the presence of cyclooxygenase in ejaculated and epididymal spermatozoa. The immunohistochemical studie s 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 s ynthesis of PGs in the absence of exogenous arachidonic acid is low, t he effect of melittin, a known phospholipase A(2) activator, on PG pro duction was examined. Incubation of spermatozoa with melittin produced a threefold increase in PGE(2) and a sixfold increase in PGF(2 alpha) . Staurosporine, a protein kinase C inhibitor, inhibited the effect of melittin indicating that activation of phospholipase A(2) by protein kinase C is an obligatory step in PG synthesis by bovine spermatozoa. To determine the physiological role of PG synthesis by bovine spermato zoa the effect of PGE(2) and PGF(2 alpha) on Ca2+ uptake was examined. PGE(2), but not PGF(2 alpha), increased the Ca2+ uptake linearly duri ng the first 10 min of incubation. These data provide evidence that bo vine spermatozoa can synthesize prostaglandins in the presence of arac hidonic acid or melittin. Furthermore, it was demonstrated that the sp ermatozoa contain the key enzyme for prostaglandin synthesis, cyclooxy genase.