Gamete Biology

Role of PI3-Kinase and PI4-Kinase in Actin Polymerization During Bovine Sperm Capacitation

Abstract
We have recently demonstrated the involvement of phospholipase D (PLD) in actin polymerization during mammalian spe rm capacitation. In this study, we investigated the involvement of phosphatidylinositol-3 and 4-kinases (PI3K, PI4K) in actin polymerization, and production of PIP_2(4,5), which is a known cofactor for PLD activation, during bovine sperm capacitation. The presence of PIK3R1 (p85 alpha regulatory subunit of PI3K) and PIKCB (PI4K beta) in bovine sperm was detected by western blot and by immunocytochemistry. Wortmannin (WT) inhibits PI3K and PI4K type III at 10 nM and 10 µM, respectively. Here, we showed that PI4K activity and PIP_2(4,5) production were blocked by 10 µM WT but not by 10 nM WT, whereas PI3K activity and PIP_3(3,4,5) production were blocked by 10 nM WT. Moreover, spermine, a known PI4K activator and a component of semen, activated sperm PI4K, resulting in increased cellular PIP_2(4,5) and F-actin formation. The increase in PIP_2(4,5) and F-actin intracellular levels during sperm capacitation which were mediated by PI4K but not by PI3K activity. Activation of PKA by dbcAMP enhanced PIP_2(4,5), PIP_3(3,4,5) and F-actin formation and these effects were mediated through PI3K. On the other hand, activation of PKC by PMA enhanced PIP_2(4,5) and F-actin formation, mediated by PI4K activity, while PI3K activity and intracellular PIP_3(3,4,5) levels were reduced. These results suggest that two alternative pathways lead to PI4K activation: indirect activation by PKA, which is mediated by PI3K, and activation by PKC, which is independent of PI3K activity. Our data also suggest that spermine, which is present in the ejaculate, might regulate PI4K activity during the capacitation process in vivo.

Key words: Gamete Biology • Developmental biology • Kinases • Signal transduction • Sperm capacitation