Functional Human Sperm Capacitation Requires both Bicarbonate-Dependent PKA Activation and Down-regulation of Ser/Thr Phosphatases by Src Family Kinases

BATTISTONE MA; DA ROS VG; SALICIONI AM; NAVARRETE F; KRAPF D; VISCONTI PE; CUASNICU PS

MOLECULAR HUMAN REPRODUCTION.

OXFORD UNIV PRESS

Lugar: Oxford; Año: 2013 vol. 19 p. 570 - 580

1360-9947

Sperm capacitation correlates with an increase in protein tyrosine (Tyr) phosphorylation mediated by cAMP/PKA activation. Recent studies in mice suggested that the Src family tyrosine kinase (SFK) is involved in Tyr phosphorylation through the inactivation of Ser/Thr phosphatases. In the present work, we first investigated the temporal acquisition of human sperm capacitated state by analyzing hallmark capacitation-events. In addition, we studied the involvement of SFK/phosphatases in capacitation and elucidated a possible cross-talk between this pathway and the classical cAMP/PKA activation. The rapid increase in phospho-PKA substrates and the intracellular level of cAMP indicated a very early bicarbonate-dependent activation of PKA. However, Tyr phosphorylation and acrosome reaction were observed only after 6 hs of capacitation. Study of the SFK-dependent pathway showed that sperm capacitated in the presence of SKI606, a SFK inhibitor, exhibited low levels of both PKA substrate and Tyr phosphorylation, effect that was overcome when a Ser/Thr phosphatase inhibitor, okadaic acid was added to the medium. Contrary to mouse sperm, the SFK inhibitor IC50 was one order of magnitude lower and the positive effect on phosphorylation by okadaic acid required significantly higher concentrations. Pharmacological modulation of the cAMP/PKA and the SFK/phosphatase signalling pathways demonstrated that the crossroads between them took place at the phosphorylation of PKA substrates. Finally, we showed that the effect of phosphatases inactivation by SFKs had an impact on functional capacitation-dependent events such as hyperactivity motility, progesterone-induced acrosome reaction and the sperm ability to penetrate zona pellucida-free hamster oocytes in vitro. Altogether, these results indicate the involvement of SFKs in capacitation through the inactivation of Ser/Thr phosphatases, confirming the existence of two pathways converging in phospho-PKA substrates. Although this regulatory pathway appears to be conserved between mice and humans, because of the differential inhibitors IC50s, these data strongly suggest that the SFK and Ser/Thr phosphatase responsible for the regulation of capacitation in human sperm are different than those present in mouse sperm.