title: 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 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 PIP(2(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 PI4K type III at concentrations of 10 nM and 10 microM, respectively. PI4K activity and PIP(2(4,5)) production were blocked by 10 microM 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, which is 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 increases in PIP(2(4,5)) and F-actin intracellular levels during sperm capacitation were mediated by PI4K but not by PI3K activity. Activation of protein kinase A (PKA) by dibutyryl cAMP 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 phorbol myristate acetate enhanced PIP(2(4,5)) and F-actin formation mediated by PI4K activity, while the 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 results also suggest that spermine, which is present in the ejaculate, regulates PI4K activity during the capacitation process in vivo.
authors.affiliation: The Mina & Everard Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.
authors.forename: Nir
authors.initials: N
authors.lastname: Etkovitz
authors.forename: Sara
authors.initials: S
authors.lastname: Rubinstein
authors.forename: Limor
authors.initials: L
authors.lastname: Daniel
authors.forename: Haim
authors.initials: H
authors.lastname: Breitbart
mesh_terms: D019870:1-Phosphatidylinositol 4-Kinase / Q000032:analysis / Q000502:physiology*; D000199:Actins / Q000032:analysis / Q000378:metabolism*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002417:Cattle / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D019869:Phosphatidylinositol 3-Kinases / Q000032:analysis / Q000502:physiology*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000032:analysis; D018129:Phosphatidylinositol Phosphates / Q000032:analysis; D010716:Phosphatidylinositols / Q000494:pharmacology; D011108:Polymers / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013075:Sperm Capacitation / Q000502:physiology*; D013094:Spermatozoa / Q000737:chemistry / Q000201:enzymology; D013096:Spermine / Q000494:pharmacology; D000077191:Wortmannin
chemical_list: D000199:Actins; D000730:Androstadienes; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D011108:Polymers; D047428:Protein Kinase Inhibitors; C060974:phosphatidylinositol 3,4,5-triphosphate; D013096:Spermine; D019869:Phosphatidylinositol 3-Kinases; D019870:1-Phosphatidylinositol 4-Kinase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D000077191:Wortmannin