INVOLVEMENT OF TYROSINE PHOSPHORYLATION IN CAPACITATION-INDUCED CALCIUM INCREASE IN HUMAN SPERMATOZOA

PUGA MOLINA L; DA ROS VG; BRUKMAN N; DARSZON A; CUASNICU PS; NUÑEZ S; BUFFONE M

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Sperm capacitation involves an increase in protein tyrosine phosphorylation(pTyr) and intracellular Ca2+ levels. However, the crosstalkbetween these two signaling cascades is poorly understood.Based on this and considering our previous results showing that humansperm pTyr is blocked by inhibiting proline-rich tyrosine kinase2 (PYK2), we investigated the effect of the PYK2 inhibitor PF431396(PF) on the intracellular Ca2+ increase during human sperm capacitation.Using the Ca2+ indicator Fluo-4 AM, we analyzed Ca2+ levelsin sperm incubated for 6 h in capacitating media with or without PF.Interestingly, fluorescence intensityevaluated by both flow cytometryand single cell imaging was significantly reduced at 10 μM PF, aconcentration that also affects pTyr. Although PYK2 was reportedto play a role in Store Operated Calcium Entry (SOCE) in other celltypes, thapsigargin (10 μM), a compound that induces release ofCa2+ from intracellular stores and activates SOCE, produced similareffect in PF-treated sperm compared to controls. Another explanationfor the PF-induced decrease in Ca2+ is that PYK2 is involved inthe activation of CatSper, the main sperm Ca2+ channel essential formale fertility. In this regard, our observations showing that spermtreated with the CatSper inhibitor HC-056456 (10 μM) preventedCa2+ increase, confirm the key role of CatSper in the Ca2+ cascadeleading to capacitation. Based on this, we analyzed the effect ofPF on cytoplasm alkalinization and membrane hyperpolarization,two events required for CatSper activation. Flow cytometry resultsshowed a reduced alkalinization in PF-treated sperm which could bereverted by NH4Cl, suggesting that pTyr plays a role in Ca2+ signalingthrough regulation of cytoplasmic pH. Together, these observationsreveal a role for PYK2 in the regulation of intracellular Ca2+, contributingto a better understanding of the cross-talk between pTyr andCa2+ signaling pathways during human sperm capacitation.