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.

Inhibition of phosphatidylinositol 3-kinase modifies boar sperm motion parameters

“…We additionally show that both inhibition of the PI3-K pathway and activation of the cAMP pathway have similar effects on boar sperm motion parameters without any synergic effects, suggesting a close cross-talk between both signaling pathways. The presence of PI3-K regulatory subunit has been reported in human and hamster spermatozoa as an unique band of 85 kDa and identified as the p85 regulatory subunit of class IA PI3-K (NagDas et al 2002, Nauc et al 2004. However, immunoblotting experiments strongly suggest the presence of at least two isoforms of the PI3-K regulatory subunit in boar spermatozoa (Fig.…”

Regulation of hydrogen peroxide release in circulating hemocytes of the planorbid snail Biomphalaria glabrata

“…Recent publications suggest a need for caution in interpreting results obtained using these inhibitors, as the same drugs have been found to differ in their effects within a single study. To illustrate, Nauc et al [26] found that inhibition of sperm capacitation and associated phosphorylation of proteins using LY294002 may actually be independent of PI 3 kinase based on the differential effects of wortmannin and LY294002. Therefore in the current study it cannot be concluded with certainty that PI 3 kinase also plays a role in PMA-induced H 2 O 2 release as only wortmannin had an inhibitory effect.…”

B-Type Natriuretic Peptide-Induced Cardioprotection against Reperfusion Is Associated with Attenuation of Mitochondrial Permeability Transition

“…LY294002, another inhibitor of PI3K, promoted increase in intracellular calcium concentration operates independently of PI3K. 23) So wortmannin was used in the study. As illustrated in Fig.…”