title: Inhibitors of phosphoinositide 3-kinase, LY294002 and wortmannin, affect sperm capacitation and associated phosphorylation of proteins differently: Ca2+-dependent divergences.
abstract: Sperm capacitation is regulated by multiple pathways that also control sperm motility and tyrosine (Tyr) phosphorylation of several sperm proteins. Among the reported pathways, phosphoinositide 3-kinase (PI3K) signaling and its role in modulating sperm postejaculatory changes and motility remain elusive. It was shown that wortmannin, a selective inhibitor of PI3K, prevents human sperm acrosome reaction. Using LY294002 (2-(4-morphlinyl)-8-phenyl-4H-1-benzopyran-4-one), another chemically different inhibitor of PI3K, it was suggested that this enzyme inhibits human sperm motility. In this study, we used the 2 known inhibitors of PI3K to investigate their effect on sperm capacitation and associated protein phosphorylation events. Our data show that sperm incubated with LY294002 undergo capacitation and increased Tyr phosphorylation of specific sperm proteins in a manner similar to that promoted by the capacitation inducer fetal cord serum ultrafiltrate (FCSu), as well as double phosphorylation of the threonine (Thr)-glutamine (Glu)-Tyr motif. Under similar conditions, wortmannin did not affect these sperm functions on its own, although it did prevent the effect induced by FCSu. Consistently, wortmannin decreased the phospho (P)-Tyr content of sperm proteins and prevented the phosphorylation of their Thr-Glu-Tyr motif. We also show by means of immunoblotting and cell fractionation experiments the presence of PI3K and its downstream effector Akt (protein kinase B) at the membrane level, as well as sperm heads and flagella. Our data show that human spermatozoa contain a consensus motif usually phosphorylated by Akt and that its P-serine (Ser)/Thr content is increased by both LY294002 and FCSu, while it is decreased by wortmannin. In addition, the 2 inhibitors differently affected the intracellular calcium concentration, [Ca(2+)](i). While LY294002 increased [Ca(2+)](i), wortmannin did not affect its content and did not prevent the LY294002 effect. Thus, we propose that the LY294002-promoted increase in [Ca(2+)](i) operates independently of PI3K. In conclusion, we suggest that special care be taken when using LY294002 to investigate the role that PI3K plays in a cellular phenomenon.
authors.affiliation: Urology Research Laboratory, Royal Victoria Hospital and McGill University, Montréal, Canada.
authors.forename: Veronica
authors.initials: V
authors.lastname: Nauc
authors.forename: Eve
authors.initials: E
authors.lastname: De Lamirande
authors.forename: Pierre
authors.initials: P
authors.lastname: Leclerc
authors.forename: Claude
authors.initials: C
authors.lastname: Gagnon
mesh_terms: D000730:Androstadienes / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology*; D002867:Chromones / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008297:Male; D009025:Morpholines / Q000494:pharmacology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D012661:Semen / Q000187:drug effects / Q000502:physiology; D013075:Sperm Capacitation / Q000187:drug effects*; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology*; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D002118:Calcium; D000077191:Wortmannin