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Ejaculated bovine spermatozoa were examined for their capacity to synthesize prostaglandins E2and F2α(PGE2, PGF2α). It was found that in the absence of exogenous substrate, arachidonic acid, basal PGF2αproduction was less than that of PGF2. However, addition of 61 μmol arachidonic acid l−1resulted in at least a twofold increase in PGF2and PGF2αabove control values (1.3 ng and 0.3 ng per 108spermatozoa, 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 A2activator, on PG production was examined. Incubation of spermatozoa with melittin produced a threefold increase in PGE2and a sixfold increase in PGF2α. Staurosporine, a protein kinase C inhibitor, inhibited the effect of melittin indicating that activation of phospholipase A2by 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 PGE2and PGF2αon Ca2+uptake was examined. PGE2, but not PGF2α, 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.

Exogenous arachidonic acid induces the acrosome reaction and the production of the prostaglandins PGE2 and PGF2 alpha in bovine spermatozoa. Exogenous PGE2 also induces the acrosome reaction and PGF2 alpha synthesis. To understand better the role of PGE2 in the induction of PGF2 alpha synthesis through modulation of phospholipase A2, inhibitors of this enzyme were used. The effects of PGE2 were blocked by phospholipase A2 inhibitors and this inhibition was reversed by addition of arachidonic acid. These data indicate that PGE2 activates phospholipase A2 to produce arachidonic acid. To determine whether protein kinase C modulates phospholipase A2 activity in this process, staurosporin, an inhibitor of protein kinase C, was used. The effect of PGE2 on PGF2 alpha production is inhibited by staurosporin and this inhibition was reversed by addition of arachidonic acid indicating that protein kinase C is involved in phospholipase A2 activation. The effect of exogenous arachidonic acid or PGE2 on the acrosome reaction is blocked by lipoxygenase inhibitors but not by inhibitors of cyclo-oxygenase, indicating that lipoxygenase products are involved in the mechanism of the acrosome reaction. The presented data shed light on the cross-talk between cyclo-oxygenase and lipoxygenase and their involvement in the sperm acrosome reaction. It is suggested that cyclo-oxygenase products modulate the activity of lipoxygenase which is a key enzyme in the mechanism leading to the acrosome reaction. Stimulation of cyclo-oxygenase to synthesize PGE2 activates phospholipase A2 to release arachidonic acid which is the substrate for lipoxygenase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Exogenous arachidonic acid induces the acrosome reaction and the production of the prostaglandins PGE2 and PGF2α in bovine spermatozoa. Exogenous PGE2 also induces the acrosome reaction and PGF2α synthesis. To understand better the role of PGE2 in the induction of PGF2α synthesis through modulation of phospholipase A2, inhibitors of this enzyme were used. The effects of PGE2 were blocked by phospholipase A2 inhibitors and this inhibition was reversed by addition of arachidonic acid. These data indicate that PGE2 activates phospholipase A2 to produce arachidonic acid. To determine whether protein kinase C modulates phospholipase A2 activity in this process, staurosporin, an inhibitor of protein kinase C, was used. The effect of PGE2 on PGF2α production is inhibited by staurosporin and this inhibition was reversed by addition of arachidonic acid indicating that protein kinase C is involved in phospholipase A2 activation. The effect of exogenous arachidonic acid or PGE2 on the acrosome reaction is blocked by lipoxygenase inhibitors but not by inhibitors of cyclo-oxygenase, indicating that lipoxygenase products are involved in the mechanism of the acrosome reaction. The presented data shed light on the cross-talk between cyclo-oxygenase and lipoxygenase and their involvement in the sperm acrosome reaction. It is suggested that cyclo-oxygenase products modulate the activity of lipoxygenase which is a key enzyme in the mechanism leading to the acrosome reaction. Stimulation of cyclo-oxygenase to synthesize PGE2 activates phospholipase A2 to release arachidonic acid which is the substrate for lipoxygenase activity. Thus, the role of cyclo-oxygenase products is probably to reactivate the phospholipase A2 which was initially activated via the signal transduction cascade.

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