Cross-talk between different signaling pathways in human sperm capacitation

BATTISTONE MA; DA ROS VG; SALICIONI AM; VISCONTI PE; CUASNICU PS

Simposio; 17th Annual Frontiers in Reproduction Symposium; 2014

Sperm capacitation correlates with an increase in protein tyrosine (Tyr) phosphorylation mediated by PKA activation. With the aim of getting a better understanding of the molecular mechanisms that lead to this process, in the present work we investigated different intracellular signaling pathways involved in human sperm capacitation. First of all, we analyzed the kinetic of cAMP/PKA pathway during capacitation. Results revealeda very early bicarbonate-dependent activation of PKA indicated by the rapid (1 min) increase in both PKA substrates phosphorylation and cAMP levels. However, a complete pattern of Tyr phosphorylation was detected only after 6 h-incubation at which time sperm exhibited the ability to undergo the spontaneous and progesterone-induced acrosome reaction (AR) and to penetrate zona-free hamster eggs. After that, we studied the involvement of the SRC family Tyr kinases (SFKs) and Ser/Thr phosphatases in human sperm capacitation. 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. In addition, the presence of SKI606 during capacitation significantly inhibited sperm motility, progesterone-induced AR and the sperm ability to fertilize zona-free hamster oocytes in vitro. None of these inhibitions were observed when sperm were exposed to SKI606 and OA. These observations strongly indicate 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 betwen these two signaling pathways. Considering that in the absence of extracellular Ca2+ in the capacitating medium, human sperm exhibit an increase in Tyr phosphorylation, we also investigated the role of Ca2+ as a modulator of kinases and/or phosphatases. Results showed that a decrease in extracellular Ca2+ did not modify PKA substrate phosphorylation. Moreover, using specific inhibitors, we observed that the effect of the absence of Ca2+ in Tyr phosphorylation depended on the activity of Ser/Thr phosphatase PP2B and calmodulin, a Ca2+ binding protein, and occurs dependently of SFK/phosphatase. In conclusion, our results support the participation of different pathways in human sperm Tyr phosphorylation: one depending on PKA activation, one involving Ser/Thr phosphatase down-regulation and one regulated by extracellular Ca2+, which cross-talk at different levels.