Involvement of Cystic Fibrosis Transmembrane Conductance Regulator in Mouse Sperm Capacitation

Abstract

Mammalian sperm acquire fertilizing ability in the female tract during a process known as capacitation. In mouse sperm, this process is associated with increases in protein tyrosine phosphorylation, membrane potential hyperpolarization, increase in intracellular pH and Ca 2؉, and hyperactivated motility. The molecular mechanisms involved in these changes are not fully known. Present evidence suggests that in mouse sperm the capacitation-associated membrane hyperpolarization is regulated by a cAMP/protein kinase A-dependent pathway involving activation of inwardly rectifying K ؉ channels and inhibition of epithelial sodium channels (ENaCs). The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl ؊ channel that controls the activity of several transport proteins, including ENaCs. Here we explored whether CFTR is involved in the regulation of ENaC inhibition in sperm and therefore is essential for the capacitation-associated hyperpolarization. Using reverse transcription-PCR, Western blot, and immunocytochemistry, we document the presence of CFTR in mouse and human sperm. Interestingly, the addition of a CFTR inhibitor (diphenylamine-2-carboxylic acid; 250 M) inhibited the capacitation-associated hyperpolarization, prevented ENaC closure, and decreased the zona pellucida-induced acrosome reaction without affecting the increase in tyrosine phosphorylation. Incubation of sperm in Cl ؊ -free medium also eliminated the capacitationassociated hyperpolarization. On the other hand, a CFTR activator (genistein; 5-10 M) promoted hyperpolarization in mouse sperm incubated under conditions that do not support capacitation. The addition of dibutyryl cyclic AMP to noncapacitated mouse sperm elevated intracellular Cl ؊ . These results suggest that cAMPdependent Cl ؊ fluxes through CFTR are involved in the regulation of ENaC during capacitation and thus contribute to the observed hyperpolarization associated with this process.

Activation of GABAAReceptor/Cl−Channel and Capacitation in Rat Spermatozoa: HCO3−and Cl−are Essential

“…In addition, it is known that Cl À and its channels play important roles in the maintenance of sperm cellular volume [Yeung et al 2004], intracellular pH [Zeng et al 1996], resting membrane potential and excitability [Chan et al 1997;Zhang and Shi 1998]. For example, the sperm cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl À channel which is involved in the transport of HCO 3 À that is important for sperm capacitation and fertilization [Hernandez-Gonzalez et al 2007;Xu et al 2007]. In addition, Cl À has been reported to be required for the progesterone-, GABA-and zona pellucid-initiated acrosome reaction in mouse, porcine, hamster and human spermatozoa [Espinosa et al 1998;Melendrez and Meizel 1995;Shi et al 1997;Wistrom and Meizel 1993;Yashimatsu and Yanagimachi 1988].…”

Cl− Channels and Transporters in Sperm Physiology

“…Supporting this notion, it has been found that fertility in uremic patients may be reduced through alterations in CFTR expression (Xu et al 2012 ). CFTR has been shown to be present in both human and mouse sperm by our group and others, using specifi c antibodies (Chan et al 2006 ;Hernandez-Gonzalez et al 2007 ;Li et al 2010 ;Xu et al 2007 ). Xu et al ( 2007 ) showed, using CFTR inhibitors and specifi c antibodies, as well as heterozygous CFTR mutants, that sperm capacitation and the associated HCO 3 − transport are signifi cantly reduced when compared to mice in normal fertilizing conditions.…”

“…As the sperm resting E m is mildly depolarized (~ −35 mV) and thus shifted from the K + equilibrium potential, it cannot be explained mainly by a high K + permeability through K + channels, as it usually happens in many cells. Just a 10 % contribution of Na + permeability would explain the observed resting E m in noncapacitated sperm, and their inhibition during capacitation would contribute to the observed hyperpolarization (see Hernandez-Gonzalez et al 2006 ;Hernandez-Gonzalez et al 2007 ). CFTR can downregulate ENaCs through mechanisms still not fully understood (Konig et al 2001 ), and the CFTR agonist genistein hyperpolarizes sperm E m (Hernandez-Gonzalez et al 2007 ) and diminishes [Na + ] i (Escoffi er et al 2012 ).…”

“…As CFTR is mainly a Cl − channel, it possibly participates in regulation of the resting E m ; however, Cl − substitution by nonpermeable anions (e.g., gluconate or methanesulfonate) does not modify it. On the other hand, this procedure inhibits hyperpolarization development during capacitation (Hernandez-Gonzalez et al 2007 ). Findings supporting this idea in mouse sperm are (1) adding 250 μM of the CFTR antagonist DPC (diphenylamine-2-carboxylic acid) inhibits hyperpolarization associated with capacitation and also the AR induced by sZP, without modifying tyrosine phosphorylation levels; (2) the CFTR agonist genistein (5-10 μM) hyperpolarizes noncapacitated spermatozoa; and (3) addition of permeable analogues of cAMP to noncapacitated sperm elevates [Cl − ] i (Hernandez-Gonzalez et al 2007 ).…”

“…On the other hand, this procedure inhibits hyperpolarization development during capacitation (Hernandez-Gonzalez et al 2007 ). Findings supporting this idea in mouse sperm are (1) adding 250 μM of the CFTR antagonist DPC (diphenylamine-2-carboxylic acid) inhibits hyperpolarization associated with capacitation and also the AR induced by sZP, without modifying tyrosine phosphorylation levels; (2) the CFTR agonist genistein (5-10 μM) hyperpolarizes noncapacitated spermatozoa; and (3) addition of permeable analogues of cAMP to noncapacitated sperm elevates [Cl − ] i (Hernandez-Gonzalez et al 2007 ). CFTR channels are also known to interact with and regulate other ion channels (i.e., epithelial Na + channels, ENaCs ) and transporters (i.e., the Cl − /HCO 3 − exchanger, SCL family) (Berdiev et al 2009 ;Konig et al 2001 ;Kunzelmann and Schreiber 1999 ;Perez-Cornejo and Arreola 2004 ).…”