Tyrosine phosphorylation signaling regulates Ca 2+ entry by affecting intracellular pH during human sperm capacitation

Capacitation is a mandatory process for the acquisition of mammalian sperm fertilization competence and involves the activation of a complex and still not fully understood system of signaling pathways. Under in vitro conditions, there is an increase in both protein tyrosine phosphorylation (pTyr) and intracellular Ca levels in several species. In human sperm, results from our group revealed that pTyr signaling can be blocked by inhibiting proline-rich tyrosine kinase 2 (PYK2). Based on the role of PYK2 in other cell types, we investigated whether the PYK2-dependent pTyr cascade serves as a sensor for Ca signaling during human sperm capacitation. Flow cytometry studies showed that exposure of sperm to the PYK2 inhibitor N-[2-[[[2-[(2,3-dihydro-2-oxo-1 H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]- N-methyl-methanesulfonamide hydrate (PF431396) produced a significant and concentration-dependent reduction in intracellular Ca levels during capacitation. Further studies revealed that PF431396-treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca and pH levels after 2 hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH Cl, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca entry in human sperm through regulation of cytoplasmic pH. These results contribute to a better understanding of the modulation of the polymodal CatSper and signaling pathways involved in human sperm capacitation.