Control of the low voltage-activated calcium channel of mouse sperm by egg ZP3 and by membrane hyperpolarization during capacitation

Abstract

Sperm adhesion to egg zonae pellucidae initiates sperm acrosome reactions, an exocytotic event that is an early step during fertilization. Previously, it was suggested that zona pellucida-evoked Ca 2؉ entry into sperm through low voltage-activated Ca 2؉ channels is an essential step in acrosome reactions, based on the inhibitory effects of Ca 2؉ channel antagonists. However, analysis of this channel is limited by the inability to apply electrophysiological methods directly to sperm. In this report, optical methods of determining membrane potential and internal Ca 2؉ levels were used to demonstrate that (i) contact with zonae pellucidae activates a transient Ca 2؉ response in sperm that has a time course and antagonist sensitivity anticipated of low voltage-activated Ca 2؉ channels; (ii) these channels are unavailable for opening in uncapacitated sperm because of voltage-dependent, steady state inactivation; (iii) membrane hyperpolarization during sperm capacitation is sufficient to recruit channels into a closed state, from which they are available for opening during fertilization; and (iv) channel conductance state may be a factor in determines the efficacy with which channel antagonists inhibit fertilization. This study provides evidence for the activation of sperm Ca 2؉ channels during gamete adhesion and offers a mechanism that may account for aspects of the regulation of sperm fertility during capacitation through the control of channel availability. Finally, these results suggest that channel conductance state may be a central feature in the design of channel antagonists that inhibit sperm function.

Cl− Channels and Transporters in Sperm Physiology

“…Various fi ndings suggest that hyperpolarization is essential for sperm to acquire the ability to undergo a physiological AR. For instance, carrying out capacitation in the presence of high external KCl signifi cantly reduces the ZP-induced mouse sperm AR (Arnoult et al 1999 ;De La Vega-Beltran et al 2012 ;Zeng et al 1995 ). These observations lead to the proposal that an E m hyperpolarization is important for capacitation and thus is required for the AR.…”

“…Capacitated mouse spermatozoa display an average E m approximately −60 mV. Indeed, when Arnoult et al (Arnoult et al 1999 ) measured E m in individual spermatozoa using di-8-AN-EPPS, a voltage-sensitive dye, they documented that capacitated sperm populations consisted of at least two groups: one hyperpolarized (~−80 mV), possibly representing capacitated sperm, and another of noncapacitated sperm with a resting E m approximately −43 mV. Various fi ndings suggest that hyperpolarization is essential for sperm to acquire the ability to undergo a physiological AR.…”

“…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 ).…”

“…These observations lead to the proposal that an E m hyperpolarization is important for capacitation and thus is required for the AR. Initially the role of the capacitation-associated hyperpolarization was thought to be needed to remove inactivation from T-type voltage-dependent Ca 2+ channels (Ca V 3), which could then be activated by physiological agonists (e.g., ZP) to culminate with the induction of the AR (Arnoult et al 1996 ;Arnoult et al 1999 ;Santi et al 1996 ;Zeng et al 1995 ). Recent evidence suggests that hyperpolarization of the sperm plasma membrane is necessary and suffi cient to prepare sperm for the AR (De la Vega et al 2012 ).…”

“…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 ).…”