Role of PI3-kinase and PI4-kinase in actin polymerization during bovine sperm capacitation

We have recently demonstrated the involvement of phospholipase D (PLD) in actin polymerization during mammalian sperm capacitation. In the present study, we investigated the involvement of phosphatidylinositol 3- and 4-kinases (PI3K and PI4K) in actin polymerization, as well as the production of PIP2(4,5), which is a known cofactor for PLD activation, during bovine sperm capacitation. PIK3R1 (p85 alpha regulatory subunit of PI3K) and PIKCB (PI4K beta) in bovine sperm were detected by Western blotting and immunocytochemistry. Wortmannin (WT) inhibited PI3K and P14K type III at concentrations of 10 nM and 10 VM, respectively. P14K activity and PIP2((4,5)) production were blocked by 10 mu M WT but not by 10 nM WT, whereas PI3K activity and PIP3(3,4,5) production were blocked by 10 nM WT. Moreover, spermine, which is a known P14K activator and a component of semen, activated sperm P14K, resulting in increased cellular PIP2((4,5)) and F-actin formation. The increases in PIP2((4,5)) and F-actin intracellular levels during sperm capacitation were mediated by P14K but not by PI3K activity. Activation of protein kinase A (PKA) by dibutyryl cAMP enhanced PIP2(4,5), PIP3(3 4 5), and F-actin formation, and these effects were mediated thr jh PI3K. On the other hand, activation of PKC by phorbol myristate acetate enhanced PIP2((4,5)) and F-actin formation mediated by P14K activity, while the PI3K activity and intracellular PIP3(3 4 5) levels were reduced. These results suggest that two alternave pathways lead to P14K activation: indirect activation by PKA, which is mediated by PI3K; and activation by PKC, which is independent of PI3K activity. Our results also suggest that spermine, which is present in the ejaculate, regulates P14K activity during the capacitation process in vivo.