Abstract
To fertilize an egg, sperm must reside in the female reproductive tract to undergo several maturational changes that are collectively referred to as capacitation. From a molecular point of view, the HCO3--dependent activation of the atypical soluble adenylyl cyclase (ADCY10) is one of the first events that occurs during capacitation and leads to the subsequent cAMP-dependent activation of protein kinase A (PKA). Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. We previously reported that PKA activation is necessary for CFTR (cystic fibrosis transmembrane conductance regulator channel) activity and for the modulation of membrane potential (Em). However, the main HCO3- transporters involved in the initial transport and the PKA-dependent Em changes are not well known nor characterized. Here, we analyzed how the activity of CFTR regulates Em during capacitation and examined its relationship with an electrogenic Na+/HCO3- cotransporter (NBC) and epithelial Na+ channels (ENaCs). We observed that inhibition of both CFTR and NBC decreased HCO3- influx, resulting in lower PKA activity, and that events downstream of the cAMP activation of PKA are essential for the regulation of Em. Addition of a permeable cAMP analog partially rescued the inhibitory effects caused by these inhibitors. HCO3- also produced a rapid membrane hyperpolarization mediated by ENaC channels, which contribute to the regulation of Em during capacitation. Altogether, we demonstrate for the first time, that NBC cotransporters and ENaC channels are essential in the CFTR-dependent activation of the cAMP/PKA signaling pathway and Em regulation during human sperm capacitation.
Highlights
To fertilize an egg, sperm must reside in the female reproductive tract to undergo several maturational changes that are collectively referred to as capacitation
Once sperm enter the seminal plasma and the female reproductive tract, they are exposed to a higher HCO3Ϫ concentration (ϳ15–25 mM) [5, 6], which results in activation of the atypical soluble adenylyl cyclase (ADCY10) that in turn leads to cAMP synthesis and activation of protein kinase A (PKA)3 [7]
Because CFTR requires phosphorylation by PKA to be active, we postulate that an initial HCO3Ϫ transport occurs in human sperm to stimulate ADCY10 and produce the cAMP-dependent activation of PKA
Summary
Sperm must reside in the female reproductive tract to undergo several maturational changes that are collectively referred to as capacitation. Once sperm enter the seminal plasma and the female reproductive tract, they are exposed to a higher HCO3Ϫ concentration (ϳ15–25 mM) [5, 6], which results in activation of the atypical soluble adenylyl cyclase (ADCY10) that in turn leads to cAMP synthesis and activation of protein kinase A (PKA)3 [7] The activation of this signaling pathway is followed by downstream events such as hyperpolarization of the plasma membrane (8 –10). Role of NBC and ENaC in human sperm capacitation our group demonstrated that inhibition of CFTR affects HCO3Ϫ uptake resulting in low PKA activity and inhibition of cAMP/ PKA-downstream events such as the increase in tyrosine phosphorylation (pTyr), hyperactivated motility, and acrosome reaction [9]. We hypothesize that CFTR activity is necessary for ENaC inhibition, and for maintaining of lower Naϩ permeability and regulation of Em during capacitation
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