Tyrosine phosphorylation signaling regulates Ca2+ 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 Ca2+ 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 (2+) 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-1H-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 (2+) levels during capacitation. Further studies revealed that PF431396-treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca (2+) channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca (2+) and pH levels after 2hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca (2+) levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH Cl-4, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca (2+) 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.