Functional human sperm capacitation requires both bicarbonate-dependent PKA activation and down-regulation of Ser/Thr phosphatases by Src family kinases

Abstract

In all mammalian species studied so far, sperm capacitation correlates with an increase in protein tyrosine (Tyr) phosphorylation mediated by a bicarbonate-dependent cAMP/protein kinase A (PKA) pathway. Recent studies in mice revealed, however, that a Src family kinase (SFK)-induced inactivation of serine/threonine (Ser/Thr) phosphatases is also involved in the signaling pathways leading to Tyr phosphorylation. In view of these observations and with the aim of getting a better understanding of the signaling pathways involved in human sperm capacitation, in the present work we investigated the involvement of both the cAMP/PKA and SFK/phosphatase pathways in relation to the capacitation state of the cells. For this purpose, different signaling events and sperm functional parameters were analyzed as a function of capacitation time. Results revealed a very early bicarbonate-dependent activation of PKA indicated by the rapid (1 min) increase in both phospho-PKA substrates and cAMP levels (P < 0.05). However, a complete pattern of Tyr phosphorylation was detected only after 6-h incubation at which time sperm exhibited the ability to undergo the acrosome reaction (AR) and to penetrate zona-free hamster oocytes. Sperm capacitated in the presence of the SFK inhibitor SKI606 showed a decrease in both PKA substrate and Tyr phosphorylation levels, which was overcome by exposure of sperm to the Ser/Thr phosphatase inhibitor okadaic acid (OA). However, OA was unable to induce phosphorylation when sperm were incubated under PKA-inhibitory conditions (i.e. in the absence of bicarbonate or in the presence of PKA inhibitor). Moreover, the increase in PKA activity by exposure to a cAMP analog and a phosphodiesterase inhibitor did not overcome the inhibition produced by SKI606. Whereas the presence of SKI606 during capacitation produced a negative effect (P < 0.05) on sperm motility, progesterone-induced AR and fertilizing ability, none of these inhibitions were observed when sperm were exposed to SKI606 and OA. Interestingly, different concentrations of inhibitors were required to modulate human and mouse capacitation revealing the species specificity of the molecular mechanisms underlying this process. In conclusion, our results describe for the first time the involvement of both PKA activation and Ser/Thr phosphatase down-regulation in functional human sperm capacitation and provide convincing evidence that early PKA-dependent phosphorylation is the convergent regulatory point between these two signaling pathways.

Cholecystokinin receptors regulate sperm protein tyrosine phosphorylation via uptake of HCO3−

“…Even for sperm, the dosage of a certain inhibitor is deferent between the species. The IC 50 of the SFK (Src family kinase) inhibitor SK1606 in human sperm was five times lower than the CCK receptors regulate protein phosphorylation one observed in mouse sperm (Krapf et al 2010, Battistone et al 2013). Our present results showed that 5 mM CCK1 or CCK2 receptor antagonist could stimulate the increase of tyrosine phosphorylation ( Fig.…”

“…The serine/threonine phosphorylation of PKA substrate proteins is a critical event on the upstream of sperm capacitation-associated protein tyrosine phosphorylation (Battistone et al 2013). To examine whether CCK receptor antagonists have the influence on PKA substrate phosphorylation, we determined the change of PKA substrate phosphorylation in sperm treated with CCK receptor antagonists.…”

Compartmentalization of Distinct cAMP Signaling Pathways in Mammalian Sperm

“…This plot was then used to determine the sperm intracellular cAMP concentration by interpolation. This assay was used recently for human sperm (39). Alternatively, cAMP levels were measured using the direct cyclic AMP ELISA kit (Enzo Life Sciences/Biomol, Farmingdale, NY).…”

Protein kinase C activity in boar sperm

“…5). In addition to calcium, BSA and bicarbonate in the capacitating medium are wellknown to affect membrane fluidity either directly or indirectly through activation of sperm adenylyl cyclase and increase in intracellular cAMP concentration in species such as mouse and human (Wang et al 2007;Battistone et al 2013). Phorbol esters have been reported as capable of initiating acrosomal exocytosis in capacitated human sperm (De Jonge et al, 1991;Rotem et al, 1992) and in zona-induced mouse sperm (Lee et al, 1987); as well as of producing early events leading to acrosomal exocytosis in bull (Lax et al, 1997).…”