Involvement of a Na+/HCO 3−Cotransporter in Mouse Sperm Capacitation

Abstract

Mammalian sperm are incapable of fertilizing eggs immediately after ejaculation; they acquire fertilization capacity after residing in the female tract for a finite period of time. The physiological changes sperm undergo in the female reproductive tract that render sperm able to fertilize constitute the phenomenon of "sperm capacitation." We have demonstrated that capacitation is associated with an increase in the tyrosine phosphorylation of a subset of proteins and that these events are regulated by an HCO 3 ؊ /cAMP-dependent pathway involving protein kinase A. Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. Here we present evidence that, in addition to its role in the regulation of adenylyl cyclase, HCO 3 ؊ has a role in the regulation of plasma membrane potential in mouse sperm. Addition of HCO 3 ؊ but not Cl ؊ induces a hyperpolarizing current in mouse sperm plasma membranes. This HCO 3 ؊ -dependent hyperpolarization was not observed when Na ؉ was replaced by the nonpermeant cation choline ؉ . Replacement of Na ؉ by choline ؉ also inhibited the capacitation-associated increase in protein tyrosine phosphorylation as well as the zona pellucida-induced acrosome reaction. The lack of an increase in protein tyrosine phosphorylation was overcome by the presence of cAMP agonists in the incubation medium. The lack of a hyperpolarizing HCO 3 ؊ current and the inhibition of the capacitation-dependent increase in protein tyrosine phosphorylation in the absence of Na ؉ suggest that a Na ؉ /HCO 3 ؊ cotransporter is present in mouse sperm and is coupled to events regulating capacitation.Upon ejaculation, mammalian sperm are not able to fertilize; they become fertilization-competent during transit through the female reproductive tract (1). The molecular, biochemical, and physiological changes that occur in sperm while in the female tract are collectively referred to as capacitation. During capacitation, changes in membrane dynamics and properties, enzyme activities, and motility render spermatozoa responsive to stimuli that induce the acrosome reaction and prepare these cells for penetration of the egg vestments prior to fertilization. Mammalian sperm capacitation is also accompanied by the hyperpolarization of the sperm plasma membrane (3). Hyperpolarization is observed as an increase in the intracellular negative charges when compared with the extracellular environment. Although it is not clear how sperm plasma membrane potential is regulated during capacitation, it appears that membrane hyperpolarization may be partially because of an enhanced K ϩ permeability as a result of a decrease in inhibitory modulation of K ϩ channels (3). Recently, Muñ oz-Garay et al. (4) demonstrated with patch clamp techniques that inward rectifying K ϩ channels are expressed in mouse spermatogenic cells and proposed that these channels may be responsible for the capacitation-associated membrane hyperpolarization. Interestingly, Ba 2ϩ blocks these K ϩ channels with an IC 50 similar to that shown to inhibit ...

KSper, a pH-sensitive K + current that controls sperm membrane potential

“…Capacitation refers to a series of biochemical and physiological changes in the sperm cell that enable it to reach and fertilize the egg. Among these changes is an increase in the intracellular pH (pH i ) (2) and hyperpolarization of the sperm plasma membrane potential (V m ) (3,4). Resting pH i of mammalian sperm is Ϸ6.5 as determined by pH-sensitive fluorescent probes (2,(5)(6)(7).…”

Cl− Channels and Transporters in Sperm Physiology

“…The specifi c carriers responsible for HCO 3 − transport have not yet been fully defi ned. Experiments from our group suggest that Na + / HCO 3 − cotransporters allow HCO 3 − infl ux into mouse sperm (Demarco et al 2003 ). However, other HCO 3 − transport systems can also play a role in the control of HCO 3 − levels in sperm.…”

“…Indeed, hyperpolarization is important in mouse, bovine, and equine sperm capacitation (Arnoult et al 1996 ;Arnoult et al 1999 ;De La Vega-Beltran et al 2012 ;Demarco et al 2003 ;McPartlin et al 2011 ;Munoz-Garay et al 2001 ;Zeng et al 1995 ), although it has not been demonstrated in human sperm. The sperm resting E m is relatively depolarized in most mammalian sperm (between −30 and −40 mV) (De La Vega-Beltran et al 2012 ;Demarco et al 2003 ;Espinosa and Darszon 1995 ;Hernandez-Gonzalez et al 2006 ;McPartlin et al 2011 ;MunozGaray et al 2001 ;Santi et al 2010 ;Zeng et al 1995 ). Sperm populations are very heterogeneous and only a fraction of the cells capacitate in vitro (~30 %); therefore, the average E m values must be cautiously considered.…”

“…Altogether the set of changes required for this maturation process is called capacitation and involves the development of a distinctive sperm motility pattern known as hyperactivation, and the sperm capacity to undergo the AR, an exocytotic event that allows the sperm to fertilize the egg. During sperm capacitation PKA is activated (Harrison 2004 ), leading to tyrosine phosphorylation increases (Visconti et al 1995a , b ); pH i (Zeng et al 1995 ) and [Ca 2+ ] i elevate (Baldi et al 1991 ;Breitbart 2003 ;DasGupta et al 1993 ;Suarez et al 1993 ;Xia and Ren 2009 ); plasma membrane composition and organization are modifi ed (Cross 1998 ;Davis 1981 ;Gadella and Harrison 2000 ;Go and Wolf 1983 ;Travis and Kopf 2002 ;Visconti et al 1999 ); and the cell E m is hyperpolarized in the mouse and other species (Arnoult et al 1999 ;Demarco et al 2003 ;Munoz-Garay et al 2001 ;Zeng et al 1995 ).…”

Transmembrane Cell Signaling for the Initiation of Trout Sperm Motility: Roles of Ion Channels and Membrane Hyperpolarization for Cyclic AMP Synthesis

“…As sperm AC is activated by extracellular bicarbonate ions (Okamura et al, 1985), production of cAMP in sperm is likely to be carried out, at least in part, by soluble AC. A Na + /HCO 3 -cotransporter is present in mouse sperm and could participate in the transport of HCO 3 -into sperm (Demarco et al, 2003). Sperm hyperactivation appears to be modulated by reactive oxygen species (ROS) (Aitken and Fisher, 1994;de Lamirande et al, 1997).…”