Essential Role of CFTR in PKA‐Dependent Phosphorylation, Alkalinization, and Hyperpolarization During Human Sperm Capacitation

Abstract

Mammalian sperm require to spend a limited period of time in the female reproductive tract to become competent to fertilize in a process called capacitation. It is well established that HCO3− is essential for capacitation because it activates the atypical soluble adenylate cyclase ADCY10 leading to cAMP production, and promotes alkalinization of cytoplasm and membrane hyperpolarization. However, how HCO3− is transported into the sperm is not well understood. There is evidence that CFTR activity is involved in the human sperm capacitation but how this channel is integrated in the complex signaling cascades associated with this process remains largely unknown. In the present work we have analyzed the extent to which CFTR regulates different events in human sperm capacitation. We observed that inhibition of CFTR affects HCO3− -entrance dependent events resulting in lower PKA activity. CFTR inhibition also affected cAMP/PKA-downstream events such as the increase in tyrosine phosphorylation, hyperactivated motility and acrosome reaction. In addition, we demonstrated for the first time, that CFTR and PKA activity are essential for the regulation of intracellular pH and membrane potential in human sperm. Addition of permeable cAMP partially recovered all the PKA-dependent events altered in the presence of inh-172 which is consistent with a role of CFTR upstream of PKA activation.

CFTR/ENaC-dependent regulation of membrane potential during human sperm capacitation is initiated by bicarbonate uptake through NBC

“…NBC cotransporters are necessary for activating the cAMP/PKA pathway-We have previously demonstrated the role of CFTR in the uptake of HCO 3 -during capacitation (9). However, because CFTR requires phosphorylation by PKA to be active, we postulate that an initial HCO 3 -transport occurs in human sperm to stimulate ADCY10 and produce the cAMP-dependent activation of PKA.…”

“…Previous evidence suggests the participation of both SLO1 and SLO3 channels in the hyperpolarization associated with capacitation in human sperm (20-23). Conversely, we observed that inhibition of CFTR results in Em depolarization that can be partially reversed by cAMP permeable analogs (9). It is reported in many cell types that CFTR regulates epithelial Na + channels (ENaC) (24-27).…”

“…At the molecular level, once sperm enter the seminal plasma and the female reproductive tract, they are exposed to higher HCO 3 -concentration (~15-25 mM) (5,6) which results in activation of the atypical soluble adenylyl cyclase (ADCY10) that in turn leads to cAMP synthesis and activation of PKA (7). The activation of this signaling pathway is followed by downstream events such as hyperpolarization of the plasma membrane (8)(9)(10). In addition to stimulating ADCY10, the uptake of HCO 3 -mice, it is postulated that a Na + -dependent electrogenic HCO 3 -incorporation through members of the Na + /HCO 3 cotransporter (NBC) is responsible for the initial anion entrance (11).…”

“…On the other hand, the mechanism of HCO 3 -entrance in human sperm is completely unknown. Recent evidence from our group demonstrated that inhibition of CFTR affects HCO 3 -uptake resulting in low PKA activity and inhibition of cAMP/PKA-downstream events such as the increase in tyrosine phosphorylation, hyperactivated motility and acrosome reaction (9). It is well established in several systems that CFTR phosphorylation by PKA is required for its activation (15)(16)(17).…”

“…Previously, we showed that CFTR and PKA activity are necessary for Em regulation during human sperm capacitation (9). The regulation of membrane potential (Em) is an important event for fertility (18,19).…”