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Sperm channel diversity and functional multiplicity

Department of Genetics of Development and Molecular Physiology, Institute of Biotechnology, UNAM, Cuernavaca, Mexico, ¹ Department of Physiology and Pathophysiology, School of Medicine, UAEM, Cuernavaca, Mexico and ² Department of Cell Biology, Center for Research and Advanced Studies, Instituto Politécnico Nacional, Mexico City, Mexico

Correspondence should be addressed to A Darszon, Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Adpo. Postal 510-3, Cuernavaca, Morelos 62250, Mexico; Email: darszon{at}ibt.unam.mx

	Abstract

Top Abstract Introduction Motility Sperm capacitation Acrosome reaction in marine... Acrosome reaction in mammalian... Concluding remarks Acknowledgements References

 
Ion channels are extraordinarily efficient machines that move ions in diversely controlled manners, allowing cells to rapidly exchange information with the outside world and with other cells. Communication is the currency of fertilization, as it is of most fundamental cell signaling events. Ion channels are deeply involved in the dialogue between sperm, its surroundings, and the egg. How sperm swim, find the egg and fertilize it depend on ion permeability changes modulated by environmental cues and components of the egg outer layer. Different ion channels distinctly localized in these tiny, amazing cells perform specific decoding functions that shape the sophisticated behavior of sperm. It is not surprising that certain sperm ion channels are turning out to be unique. New strategies to characterize sperm ion transport have opened exciting possibilities to dissect sperm–egg signaling and unveil novel contraception targets.

	Introduction

Top Abstract Introduction Motility Sperm capacitation Acrosome reaction in marine... Acrosome reaction in mammalian... Concluding remarks Acknowledgements References

 
Ion channels are amazing membrane proteins that efficiently catalyze ion transport through the non-conducting lipid bilayer. A single ion channel, one single protein, allows millions of ions per second to flow across the membrane. Only a few of these channels are necessary to trigger, in milliseconds, substantial electric and concentration changes in a small cell like sperm. It is not surprising that such remarkable transducers were evolutionarily selected to perform fast information exchanges between the external world and cells or within them. Ionic gradients across cells not only determine membrane potential (E_m) through ion-selective channels, permeant ions can modulate enzymes and further influence channel activity causing additional E_m changes and ion flow. E_m governs the rates and direction of ion-flow through channels and exchangers – its fluctuations modulate intracellular pH (pH_i), intracellular Ca²⁺([Ca²⁺]_i) and other second messengers that influence sperm maturation, motility and the acrosome reaction (AR).

Mature, competent male and female gametes produced in the testis or ovaries are needed to achieve fusion and fertilization. Identifying the key mechanisms and molecules involved in the sperm–egg dialog remains incomplete. Sea urchins spawn tens of billions of sperm into the sea. Upon release, motility ensues, powered by a microscopic flagellar engine that is exquisitely regulated by chemical signals from the environment and the egg. On the other hand, the potential for motility in sperm from internal fertilizers (reptiles, birds and mammals) develops during their journey through the vas deferens (a duct from the epididymis to the urethra) and the epididymis (Morisawa 1994, Yanagimachi 1994). Activation proceeds when sperm are ejaculated into the female reproductive tract.

Environmental cues activate motility through transduction events involving sperm ion channels (Darszon et al. 2005, Quill et al. 2006). Mammalian sperm have to further mature within the female reproductive tract through a process named capacitation (Visconti et al. 2002). Though millions of sperm are released, only a few reach their goal (Suarez & Ho 2003). The mechanisms of sperm chemotaxis and its importance for some species still possess intriguing questions (Eisenbach 1999).

Motile mature sperm from all species possessing an acrosome – a large, Golgi-derived secretory vesicle located in the anterior region of the sperm – must undergo the AR to fertilize the homologous egg (Yanagimachi 1994, Neill & Vacquier 2004), during which the acrosomal and plasma membranes fuse at multiple sites. This reaction releases certain components needed for the sperm to penetrate the egg coat and subsequently fuse with the egg plasma membrane. External ions, particularly Ca²⁺, are remarkably influential on sperm maturation, motility and the AR (Yanagimachi 1994, Jungnickel et al. 2003). These features of sperm function emphasize the essential role ion channels play in the physiology of this important cell.

In this brief review we have attempted to highlight sperm functional aspects where ion channels play a key role (motility, capacitation and AR), and used working models to illustrate testable hypotheses that can contribute to a better understanding of how sperm fertilize eggs. We regret having to be so concise and have had to leave out some important contributions. Excellent recent reviews may make up for these limitations (Baldi et al. 2002, Visconti et al. 2002, Jungnickel et al. 2003, Neill & Vacquier 2004, Zhang & Gopalakrishnan 2005, Jimenez-Gonzalez et al. 2006).

	Motility

Top Abstract Introduction Motility Sperm capacitation Acrosome reaction in marine... Acrosome reaction in mammalian... Concluding remarks Acknowledgements References

 
Motility is perhaps the most easily observed function of sperm, and is yet arguably the least well understood. Ion channels have long been suspected to critically influence sperm motility. However, until recently, direct evidence of involvement in this process has been lacking due to the difficulties in performing experiments with individual motile sperm.

An early hint that ion channels regulate motility came from observations that the composition of the external milieu may have profound effects on this process. Often sperm are maintained immotile in the testes prior to release, in for example, sea urchins, this immotile state is principally maintained by a high extracellular K⁺ ([K⁺]_e) (Darszon et al. 2005). Activation of sperm motility occurs upon spawning, owing to the reduction of [K⁺]_e. Manipulating the [Ca²⁺] to which permeabilized or demembranated invertebrate or mammalian sperm are exposed alters flagellar bending asymmetry, and high [Ca²⁺] inhibits flagellar motility (Brokaw 1979, Tash & Means 1982). Ca²⁺ also plays a critical role in chemotaxis, whereby a sperm responds to a gradient of chemoattractant surrounding the egg, and reorientates its trajectory to swim up the gradient (see Fig. 1). This process has been observed most frequently in the sperm of invertebrate marine animals, but exists also in mammalian and plant (bracken) sperm. In all studied cases, chemotaxis is dependent on the presence of external Ca²⁺, suggesting that Ca²⁺-permeant pathways participate in this process.

View larger version (41K): [in this window] [in a new window]   Figure 1 Summary of the different ion channels and their spatial distribution in sperm. The figure includes channels that have been immunolocalized to the region indicated. Additional evidence for the presence of many of these channels such as RT-PCR, in situ hybridization and western blots is available (Darszon et al. 2005). Sea urchin flagella do not have principal and mid pieces, channels found in the flagella of this species are indicated as present in the principal piece. Channels that participate in functions such as volume regulation (not described in the text) have also been included (Yeung et al. 2006).

Hyperactivated motility is observed in sperm isolated from the upper regions of the female reproductive tract. Initiation of hyperactivated motility often coincides with the onset of capacitation, although the two processes are independent (Ho & Suarez 2003). It consists of exaggerated, large amplitude flagellar beats that cause the sperm to swim in characteristic ‘figure-of-eight’ formation in low-viscosity medium. In high-viscosity medium, hyperactivated motility is more progressive (Suarez & Ho 2003). Hyperactivation is thus thought important for sperm progression through the higher-viscosity environment of the oviduct and possibly for penetration of the egg cumulus.

Ca²⁺ is required for the initiation and maintenance of hyperactivated motility by directly regulating components of the axonemal machinery. In hyperactivated hamster sperm, transitory increases in [Ca²⁺]_i were recorded that temporally correlated with the flagellar beat frequency (Suarez et al. 1993). The source for the Ca²⁺ appears to be the redundant nuclear envelope (RNE), a reticular structure at the flagellum neck. In bull sperm, agonists of the inositol 1,4,5-trisphosphate (IP₃) receptor induced hyperactivation, and IP₃ receptors (IP₃R) were located on the RNE (Ho & Suarez 2003). In immobilized human sperm, a gradient of progesterone stimulated long-lasting oscillations in [Ca²⁺]_i that originated from a sperm head region containing the RNE (Harper et al. 2004). The oscillations correlated with increases in flagellar activity that are comparable to those occurring during hyperactivation. The Ca²⁺ oscillations were dependent on ryanodine receptor (RyR) activity rather than that of IP₃Rs, in contrast to bull sperm in which RyRs were not detected (Suarez & Ho 2003, Jimenez-Gonzalez et al. 2006). In both bull sperm and human sperm, the increases and oscillations in [Ca²⁺]_i and the motility changes were dependent on external Ca²⁺, but not immediately, suggesting that this effect is due to an eventual failure to replenish intracellular stores with external Ca²⁺.

A number of voltage-gated Ca²⁺ (Ca_v) channel subunits have been detected in mammalian sperm (Fig. 2), and Ca_v channel activity has also been measured in spermatogenic cells and sperm (Arnoult et al. 1996, 1999, Wennemuth et al. 2000). Ca_v1.2, 2.1 and 2.3 subunits were localized to the proximal piece of mouse sperm flagella (Westenbroek & Babcock 1999), as was the auxiliary ß3 subunit (Serrano et al. 1999). Sperm from Ca_v2.3 knockout mice displayed increased linearity in their swimming behavior, although mice were normally fertile (Sakata et al. 2002). Additionally, all three T-type Ca_v3 channel subunits have also been localized to the flagellum of mouse and human sperm (Trevino et al. 2004). Other channels possibly involved in motility belong to the cyclic nucleotide-gated channel family (CNG). The first sperm ion channel cloned and heterologously expressed was from this family (Weyand et al. 1994). These channels were immunolocalized along the length of the flagellum (CNGA3) or confined to the principal piece alone (CNGB1) of bovine sperm (Wiesner et al. 1998) however CNGA3 knockout mice are normally fertile (Turner 2006). Furthermore, members of the transient receptor potential (TRP) channel family have been localized to the flagella of both human (transient receptor potential canonical; TRPC1, C3 and C6) and mouse sperm (TRPC1, C3, C4 and C6), inhibitors of TRP channels blocked human sperm motility (Castellano et al. 2003). The most convincing evidence for the role of ion channels in sperm motility comes from a novel class of Ca²⁺ channels, named CatSpers, whose expression is confined to the testes. Four members of this family have been described so far, and two of them, Cat-Spers 1 & 2, localize to the flagella (Quill et al. 2001, Ren et al. 2001, Lobley et al. 2003, Jin et al. 2005). Knockout mice for catsper1 are infertile, due to a specific inability to undergo hyperactivation (Ren et al. 2001, Carlson et al. 2003). These knockout mice also show defects in cAMP and depolarization-induced Ca²⁺ entry (Ren et al. 2001, Carlson et al. 2003). catsper1 and catsper2 knockout mice have identical phenotypes because neither protein alone localizes to the flagellum without co-expression of the other (Quill et al. 2003, Carlson et al. 2005). Whole-cell patch-clamp recordings sealing on cytoplasmic droplets of sperm revealed a constitutively active, weakly voltage-dependent Ca²⁺ current that is strongly potentiated by intracellular alkalinization (Kirichok et al. 2006). This current is absent in catsper1 knockout mice, and is localized to the flagella principal piece, demonstrating that Cat-Sper1 is a component of a flagellar Ca²⁺ channel. Interestingly, simultaneous depolarization and internal alkalinization induced by external K⁺ and NH₄Cl addition trigger a Ni²⁺ sensitive [Ca²⁺]_i elevation. This voltage dependent channel could contribute to the increase in [Ca²⁺]_i that occurs during capacitation (Fraire-Zamora & Gonzalez-Martinez 2004) and possibly during hyperactivation. The channel is permeable to Na⁺ in the absence of external Ca²⁺ (Espinosa & Darszon 1995, Gonzalez-Martinez 2003).

View larger version (14K): [in this window] [in a new window]   Figure 2 (A) The normal circular trajectory of a sea urchin sperm is interrupted by a sharp turn, caused by a Ca2+-dependent increase in flagellar asymmetry. B) Uncaging of cGMP induces an increase in intracellular Ca2+ and a transient increase in flagellar asymmetry (0–50 ms) that rapidly recedes (75–100 ms). For experimental details, please see (Wood et al. 2005). Sea urchin sperm swim in a circular pattern because their flagella beat with an inherently asymmetrical sinusoidal waveform (see A). As the plane of the flagellar waveform is slightly skewed, the sperm swim in a helical path in ‘open water’ that converts to a two-dimensional circular pattern once the sperm encounter a surface, such as the base of a microscopic imaging chamber. This fortuitous detail permits extended observation of motility, as the sperm swim in circles within the microscope field-of-view for prolonged periods. In many species of urchin, egg-derived peptides stimulate the sperm to alter its trajectory by undergoing a transient sharp turn – red trajectory in (A). These turns are necessary, but not sufficient, for chemotaxis, as they are generated in response to egg peptides derived from chemotactic (Arbacia punctulata; Bohmer et al. 2005) and non-chemotactic (S. purpuratus; C Wood unpublished data) species alike. The turn itself results from a transient increase in flagellar asymmetry, which depends on the presence of Ca2+ in the extracellular medium. Recently it was shown that these turns occur in response to a transient increase in intracellular Ca2+ in the flagellum (Bohmer et al. 2005, Wood et al. 2005), although as yet it is not known how such increases are generated. Upon binding to sperm, egg peptides activate a guanylate cyclase, and cGMP levels rise rapidly. Indeed, in the absence of egg peptides, uncaging cGMP inside a swimming sperm is sufficient to stimulate sperm to undergo sharp turns which depend on a rapid increase in intracellular Ca2+ (see (B) and Bohmer et al. 2005, Wood et al. 2005). The link between the increase in cGMP and flagellar Ca2+ is unknown, but one suggestion is that cGMP acts indirectly to increase intracellular Ca2+ by altering another intracellular parameter such as pH or membrane potential (Darszon et al. 2005, Hildebrand & Kaupp 2005). Reports of the pH changes occurring after egg peptide binding or release of cGMP are contradictory, with results from S. purpuratus showing that a pH increase precedes the increase in Ca2+, while data from A. punctulata indicate that the pH increase occurs later (see Darszon et al. 2005, Hildebrand & Kaupp 2005). Measurements of membrane potential changes in S. purpuratus show a rapid hyperpolarization event occurring before a larger depolarization, which has been proposed to activate a hyperpolarization-sensitive Na+/H+ exchanger to lead to the increase in pH, and remove inactivation from voltage-sensitive Ca2+ channels which open upon depolarization (see Darszon et al. 2005, Hildebrand & Kaupp 2005). Antibodies to Cav channels label the flagella of sea urchin sperm, and inhibitors of Cav channels such as nimodipine and Ni2+ are able to block both the rapid increase in Ca2+ and the turning event upon liberation of cGMP. The main evidence pointing to the importance of the hyperpolarization event is the effect of increasing the K+ concentration of the extracellular medium; under such conditions, binding of egg peptides increases the concentration of cGMP, but blocks all downstream changes in pH, membrane potential, Ca2+ and cAMP. Uncaging of cGMP under these conditions produces similar results, unlike uncaging of cAMP which results in increases in intracellular Ca2+ (see Darszon et al. 2005, Hildebrand & Kaupp 2005). This evidence has been taken to propose a model whereby increases in cGMP activate K+-permeant channels in the membrane by an unknown mechanism, leading to hyperpolarization of the membrane.

Additionally, in sea urchin sperm, two hyperpolarization-activated and cyclic nucleotide-gated channels (HCN) have been immunolocalized to the flagella compartment, namely SpHCN1 (Gauss et al. 1998) and SpHCN2 (Galindo et al. 2005). These channels could open in response to the hyperpolarization event(s) and/or increases in cyclic nucleotides that follow binding of egg peptides, and an inhibitor of HCN channels alters the kinetics of the early [Ca²⁺]_i changes induced by such peptides (Nishigaki et al. 2004).

In ascidian sperm, an egg-derived factor both activates sperm motility and stimulates chemotaxis. Sperm activation is sensitive to inhibitors of Ca_v channels, and chemotaxis is blocked by an inhibitor of store-operated Ca²⁺ (SOC) channels (Yoshida et al. 2003) indicating a role for these channel families in differing motility phenomena.

	Sperm capacitation

Top Abstract Introduction Motility Sperm capacitation Acrosome reaction in marine... Acrosome reaction in mammalian... Concluding remarks Acknowledgements References

 
Mature epididymal sperm acquire competence to fertilize during the sperm transit through the female reproductive tract. This complex process, called capacitation, involves several changes in sperm. These include reorganizing the plasma membrane as a consequence of cholesterol removal, extensive protein tyrosine phosphorylation, hyperpolarization of the E_m and increases in pH_i and [Ca²⁺]_i (reviewed by Visconti et al. 2002, Darszon et al. 2005). Capacitation is also associated with the appearance of hyperactivated motility, which some authors still consider as part of capacitation (Visconti et al. 2002, Suarez & Ho 2003). All these changes prime sperm to respond to the zona pellucida (ZP), the extracellular egg coat that induces the AR. Although the molecular mechanisms and the signal transduction events are only partially defined, capacitation and tyrosine-phosphorylation have been shown to be regulated by a cAMP-dependent pathway involving PKA (Visconti et al. 2002; Fig. 3).

View larger version (37K): [in this window] [in a new window]   Figure 3 Diagram of ion fluxes and signaling events of mammalian sperm capacitation. Uptake of bicarbonate (HCO3–) through the Na+–HCO3– cotransporter and other transporters stimulates sAC and cAMP elevation and lipid reorganization in the plasma membrane, respectively. Cholesterol acceptors such as albumin also promote the process of lipid reorganization. cAMP has several targets such as CNG channels, PKA and ENaC possibly indirectly. Also, an increase in intracellular Ca2+ may directly activate sAC and an increase in pHi can promote Ca2+ influx through CatSper1 and 2 channels. Therefore, there is a cAMP, Ca2+ and pHi synergism. Furthermore, the changes in these intracellular parameters lead to tyrosine phosphorylation involving PKA activation, hyperactivation of sperm motility and the hyperpolarization by opening K+ channels (Kir) and closing ENaC. PTK; protein tyrosine kinase.

Numerous studies have demonstrated that capacitation is dependent on different ions present in the capacitation medium, such as Ca²⁺, K⁺, HCO₃^–and Na⁺ (Visconti et al. 2002, Darszon et al. 2005). In vitro capacitation requires the presence of three key components: Ca²⁺, HCO₃^– and bovine serum albumin (BSA). Several types of ion channels and transporters have been reported in mammalian sperm (Darszon et al. 2005; Fig. 2), but how they participate in the regulation of E_m, pH_i and [Ca²⁺]_i during capacitation is not well established. Non-capacitated sperm maintain a relatively depolarized E_m (~ –35 to –45 mV) that hyperpolarizes (–80 mV) during capacitation (Arnoult et al. 1999, Munoz-Garay et al. 2001). This has been proposed to remove inactivation from T-type Ca_v channels and recruit them to a closed state, from which they are available for opening during the induction of the AR by ZP3 (Zona Pellucida Protein 3) (discussed further below in the mammalian acrosome reaction section; Arnoult et al. 1999). The identity of the ion transport system(s) responsible for the [Ca²⁺]_i increase observed during capacitation is not known. Ca_v channels other than T-type as well as several members of the TRP family have also been identified in sperm (Fig. 2) and their precise role during capacitation, if any, remains to be established.

Since external K⁺ and K⁺-channel blockers influence the hyperpolarization that accompanies mouse sperm capacitation, it is thought that the K⁺ permeability contributes to this process (Arnoult et al. 1999, Munoz-Garay et al. 2001, Acevedo et al. 2006). Molecular and functional evidence indicate the presence of voltage-gated K⁺ channels (Salvatore et al. 1999), Ca²⁺-activated K⁺ channels (Chan et al. 1998) and inwardly rectifying K⁺ (K_ir) channels (Munoz-Garay et al. 2001, Felix et al. 2002, Acevedo et al. 2006) in spermatogenic and sperm cells. Before capacitation, sperm pH_i is relatively acidic and may down-regulate sperm K_ir channels, thereby maintaining E_m depolarized and indirectly preventing unregulated Ca²⁺ entry, and thus the AR (Zeng et al. 1996). The pH_i increase and ATP decrease (Baker & Aitken 2004) that accompany capacitation could activate pH and ATP dependent K_ir channels, allowing K⁺ efflux and the consequent hyperpolarization of sperm.

An Na⁺/ HCO₃^– cotransporter has also been shown to hyperpolarize mouse sperm E_m upon the increase in external HCO₃^– that occurs during ejaculation (Demarco et al. 2003). On the other hand, inhibition of an electrogenic Na⁺ sperm permeability would also result in a hyperpolarization. Epithelial Na⁺ channels (ENaCs) are amiloride-sensitive channels that contribute to the resting E_m by displacing it towards the Na⁺ equilibrium potential (Awayda et al. 2000). Recently, the presence of ENaCs ( and subunits) was reported in mouse sperm (Hernandez-Gonzalez et al. 2006). The activity of these channels could explain the depolarized state observed in non-capacitated sperm. Possibly, mouse sperm ENaCs are constitutively active in non-capacitated sperm and closed during capacitation, resulting in hyperpolarization of E_m.

In conclusion, the data indicate that the membrane hyperpolarization associated with capacitation could be regulated by at least two different channels and a cotransporter, whose activities reflect different sperm physiological states: closed K⁺ channels and active ENaCs maintain the membrane potential in a depolarized state, while active K⁺ channels and closed ENaCs produce hyperpolarization. It is important to determine the molecular mechanisms by which these channels are regulated and the consequences for capacitation. For example, the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel is an important regulator of ENaCs, among other channels. Perhaps, a signaling pathway dependent on cAMP/PKA is involved in the regulation of ENaCs during capacitation.

	Acrosome reaction in marine sperm

Top Abstract Introduction Motility Sperm capacitation Acrosome reaction in marine... Acrosome reaction in mammalian... Concluding remarks Acknowledgements References

 
Sperm from marine external fertilizers undergo the AR when encountering the outer investment of the egg (egg jelly, EJ; Yanagimachi 1994). Commonly, the species-specific AR inducing factor, a major component of the EJ, is a sulfated polysaccharide. Small structural differences determine species specificity. In many marine organisms the AR comprises the exocytosis of the acrosomal vesicle and the pH_i-dependent polymerization of actin, leading to the extension of the acrosomal tubule (reviewed in Darszon et al. 2005). In sea urchin sperm, the tubule is covered with bindin, a protein which interacts with a receptor when it fuses with the homologous egg. The fucose sulfate polymer (FSP) binds to its sperm receptor (suREJ1) localized at the tip of the head over the acrosome and along the flagellum, and triggers K⁺-dependent changes in E_m and increases in [Ca²⁺]_i, pH_i, [Na⁺]_i, cAMP and IP₃. In addition, FSP stimulates PKA, phospholipase D and nitric oxide synthase activities (Neill & Vacquier 2004, Darszon et al. 2005).

The fusion of plasma and acrosomal membrane during the AR produces hybrid vesicles (HVs) that contain at least three types of ionic channels. A cAMP activated cationic channel sensitive to Ba²⁺ was most frequently encountered in black lipid membrane (BLM) recordings (Schulz et al. 2004). Ionic channels from flagellar membranes with similar characteristics have been also identified in BLMs (Labarca et al. 1996) and patch-clamp techniques in sea urchin sperm (Sanchez et al. 2001). The two other channels were also cationic and had larger conductances (Schulz et al. 2004).

Besides suREJ1, sea urchin sperm contain suREJ2 and suREJ3 that have been cloned and sequenced. The three suREJ proteins possess a REJ module, shared by the human autosomal dominant polycystic kidney disease (ADPKD) protein, polycystin-1 (PC1), and by PCREJ, a testis-specific protein in mammals of unknown function. ADPKD is caused by mutations in the PKD1 and PKD2 genes, encoding the transmembrane proteins PC1 and PC2 respectively (Neill & Vacquier 2004). In mammals PC1 and PC2 associate to form non-selective cation channels (Xu et al. 2003). PC2 or TRPP2, a member of the TRP superfamily of proteins, has amino acid sequence similarity to Ca_v and other cation channels (Nilius & Voets 2005). This protein itself forms Ca²⁺-permeable nonselective channels that may be involved in ligand binding (Ikeda & Guggino 2002).

suREJ3 includes a C-terminal transmembrane region that is homologous to Ca_v channels which has been implicated in associations with PC2 (Xu et al. 2003). Indeed, sea urchin sperm possess a PC2 homolog (suPC2) associated with suREJ3 in the sperm plasma membrane in a thin band overlying the acrosomal vesicle (Mengerink et al. 2002, Neill & Vacquier 2004). Notably, a high-conductance, Ca²⁺-permeable channel, whose properties resemble some of those displayed by the PC1–PC2 channels, has been recorded in BLMs containing sea urchin or mouse sperm plasma membranes (Lievano et al. 1990, Beltran et al. 1994). The sea urchin sperm channel discriminates poorly between monovalent and divalent cations (PCa/PNa ~5; Lievano et al. 1990). This evidence suggests that REJ and PC proteins may be involved in the ion fluxes that follow binding of FSP and lead to the AR. As described later, TRP channels are present in mouse sperm where they seem to participate in the AR (Jungnickel et al. 2001, Trevino et al. 2001).

Ca²⁺ uptake during the sea urchin sperm AR precedes the changes in pH_i and [Na⁺]_i and is mediated at least by two different Ca²⁺ channels (Rodriguez & Darszon 2003). FSP triggers a fast transient increase in [Ca²⁺]_i that is sensitive to verapamil and dihydropyridines (DHPs; Ca_v channel blockers that inhibit the AR). Five seconds later, a second Ca²⁺ channel insensitive to the latter blockers, permeable to Mn²⁺ and Na⁺, and pH_i dependent, opens for minutes and leads to the sperm AR. Interestingly, one of the first two Ca_v channel orthologs (Ca_v 2.3) recently identified in Strongylocentrotus purpuratus sea urchin sperm is preferentially localized in the acrosome area (Granados-Gonzalez et al. 2005). On the other hand, as in the mammalian sperm AR (Santi et al. 1998, O’Toole et al. 2000, Jungnickel et al. 2001), the second channel appears to be a SOC channel (Gonzalez-Martinez et al. 2001). Indeed, IP₃ receptors have been detected in sea urchin sperm, possibly in the acrosome (Zapata et al. 1997). It is known that IP₃-mediated release of Ca²⁺ from intracellular stores activate SOCs (Nilius & Voets 2005). Though not a physiological event, the second channel can be opened by a lower molecular weight (~60 kDa) hydrolyzed form of FSP, which can increase [Ca²⁺]_i but does not induce AR by itself (Hirohashi & Vacquier 2002). Namely, activation of this second channel alone causes acrosomal exocytosis but not acrosomal actin polymerization (Hirohashi & Vacquier 2003).

Elevation of the [K⁺]_e to 30–50 mM or addition of tetraethylammonium (TEA), a K⁺ channel blocker, inhibits the AR and the increases in [Ca²⁺]_i and pH_i associated to this reaction in S. purpuratus sperm (Darszon et al. 2005). These results suggest the participation of K⁺ channels in the AR. In L. pictus sperm, binding of FSP induces a fast K⁺-dependent hyperpolarization (Gonzalez-Martinez & Darszon 1987) which precedes and probably leads to the activation of a Ca²⁺-dependent Na⁺/H⁺ exchange that in turn increases pH_i (Gonzalez-Martinez et al. 1992). This hyperpolarization could remove inactivation from Ca_v channels (Gonzalez-Martinez & Darszon 1987, Lievano et al. 1990).

Besides Ca²⁺ channels, TEA⁺-sensitive K⁺ (Lievano et al. 1985, Gonzalez-Martinez et al. 1992, Labarca et al. 1996) and Cl^– channels have been recorded in planar bilayers with incorporated S. purpuratus sperm plasma membranes. The sea urchin AR is inhibited by 4,4'-diisothiocyanatostilbene-2-2'-disulfonic acid (DIDS), an anion channel and transporter blocker (Morales et al. 1993). The sensitivity of some of the ion channels recorded in planar bilayers to inhibitors of the AR is consistent with their involvement in this fundamental event (Darszon et al. 2005).

	Acrosome reaction in mammalian sperm

Top Abstract Introduction Motility Sperm capacitation Acrosome reaction in marine... Acrosome reaction in mammalian... Concluding remarks Acknowledgements References

 
After traveling to find the egg, sperm must recognize its extracellular coat, the zona pellucida (ZP) and bind to it. The AR allows sperm to penetrate this layer and fuse with the egg. This process is accompanied by the release of lytic enzymes and the fusion of the acrosome and plasma membranes in the sperm head. Among the components of ZP, ZP3 is recognized as the natural AR inducer. The ZP3 receptor has not been unequivocally identified in the sperm plasma membrane though several candidates exist (Jovine et al. 2005). The signaling cascade that culminates with the AR is not fully understood but intensive research has shed light on this crucial phenomenon (Fig. 4). ZP and other agents like progesterone trigger AR by influencing ion fluxes, phospholipid metabolism, cAMP levels, and protein phosphorylation. During the AR there are increases in [Ca²⁺]_i and pH_i, the latter being sensitive to pertussis toxin (PTx) that inactivates G_i proteins. The current model proposes that ZP3 binding generates a biphasic Ca²⁺ response involving at least three separate yet sequentially-linked Ca²⁺ channels. In mouse sperm, initially [Ca²⁺]_i elevates to micromolar levels within 50 ms and relaxes back to resting values within 200 ms. The pharmacology and kinetics of this excursion are consistent with opening of T-type Ca_v channels (Ca_v3; Arnoultet al. 1999). Furthermore, the AR can be inhibited by several Ca_v3 channel antagonists such as DHPs, pimozide and Ni²⁺. Molecules such as urocortin, fenvalerate and gossypol have been shown to inhibit T-type currents in spermatogenic cells and may have contraceptive applications (Bai & Shi 2002, Tao et al. 2005, Xiao et al. 2006). This family of Ca_vs has three members, all of them have been immunolocalized in mammalian sperm. Ca_v3.1 and Ca_v3.2 localize to the acrosomal region, however, knockout mice for these two Ca_v isoforms are fertile (Chen et al. 2003, Stamboulian et al. 2004), and it could be that double Ca_v channel knockouts would help clarify the role of these channels. The participation of Ca_v3 in the human sperm AR is much less clear. Additionally, other members of the Ca_v superfamily and CatSpers (Lobley et al. 2003) have been identified in mammalian sperm (Fig. 2) but their function remains unknown (Guzman-Grenfell & Gonzalez-Martinez 2004).

View larger version (37K): [in this window] [in a new window]   Figure 4 (A) Intracellular changes elicited upon ligand binding to sperm receptors during the acrosome reaction (AR). Initially there are increases in Em, pHi and a Ca2+ transient. Production of IP3 causes Ca2+ store depletion that results in SOC channel opening responsible for the sustained Ca2+ increase that precedes the AR. (B) Channels and molecules involved in the changes that lead to the mammalian sperm AR. ZP3 binding to its receptor: activates a Gi protein involved in the pHi change possibly mediated by Na+/H+ transporters; causes membrane depolarization (possibly by GABA, ACh and Gly receptors or an unidentified cation channel) that opens Cav channels which cause the initial Ca2+ transient influx; Ca2+ activation of PLC generates diacylglycerol (DAG) and IP3 consuming its substrate, PIP2. IP3 thereafter binds to its receptor and causes Ca2+-store depletion which in turn (possibly in conjunction with DAG) activates SOC channels at the plasma membrane. Ca2+, cAMP and small G proteins act together to activate the SNARE machinery required for membrane fusion. SNAREs, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; PTx, pertussis toxin; EPAC, exchange protein directly activated by; Cyt, Synaptotagmin.

An unresolved question is how the TRP channels involved in the AR are activated? There are several hypotheses and the diversity of this family means it is unlikely that a single mechanism exists. Recently several TRP regulating molecules have been identified and it is worthwhile exploring their participation in the AR signaling cascade. STIM (Stromal Interaction Molecule) is a TRP-interacting protein that translocates from the ER to the plasma membrane to function either as the communication factor between the Ca²⁺-store and the plasma membrane or forming part of the channel itself (Zhang et al. 2005). TRP channels can also be regulated by phosphatidylinositol biphosphate (PIP₂; Suh & Hille 2005) and by newly discovered regulatory proteins such as junctate (Stamboulian et al. 2005) and enkurin (Sutton et al. 2004) that bind specifically to certain TRP isoforms modulating their activity.

There are other agonists such as progesterone, gamma-aminobutyric acid (GABA), acetylcholine (ACh) and glycine that can induce the AR but their physiological significance remains to be established. The presence of iono-tropic receptors for GABA (GABA_AR) (Wistrom & Meizel 1993) ACh (Kumar & Meizel 2005) and glycine (GlyR) (Sato et al. 2000) in sperm has been documented and mutant mice for nAChR (null) and GlyR (point mutation) showed reduced ZP-induced AR and in vitro fertility (Meizel & Son 2005). Considering that intracellular Cl^– was estimated to be 40 mM in capacitated human sperm, opening of Cl^– channels, such as the GABAAR receptor or the GlyR, should result in Cl^– efflux, leading to depolarization of the E_m (Garcia & Meizel 1999). The proposal is that these neurotransmitter receptors are responsible for the initial depolarization required to activate the Ca_v channels involved in the initial Ca²⁺ rise.

In human sperm, progesterone also induces a biphasic Ca²⁺ response similar to that of ZP3. However, when applied as a gradient (simulating the physiological conditions), the initial Ca²⁺ rise is replaced by oscillations proposed to be mediated by RyR-like receptors (Harper et al. 2004). These results suggest progesterone may help sperm to penetrate through the egg coat by inducing hyperactivation-like flagellar movement as well as the AR.

	Concluding remarks

Top Abstract Introduction Motility Sperm capacitation Acrosome reaction in marine... Acrosome reaction in mammalian... Concluding remarks Acknowledgements References

 
Recent developments in the tools available to study ion channels are improving our understanding of how these integral membrane proteins operate and are regulated in the sperm. Voltage and ion-sensitive fluorescent dye imaging, novel patch-clamp approaches, bilayer reconstitution, molecular biology and the growing number of available genomes are unveiling the fascinating and often unique properties of sperm ion channels. Correlating ion channel location and function will enable us to understand how these proteins participate in the finely orchestrated sperm functions involved in generating a new individual.

	Acknowledgements

Top Abstract Introduction Motility Sperm capacitation Acrosome reaction in marine... Acrosome reaction in mammalian... Concluding remarks Acknowledgements References

 
This work was supported by: DGAPA (IN225406-3 to A Darszon, IN225806-3 to C Beltrán and IN227806-3 to C Treviño), CONACyT (39908-Q to A Darszon and 39939-Q to C Beltrán) and by the Wellcome Trust and FIRCA RO3 TW 006121 to A Darszon. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Footnotes

 
Received 1 March 2006
First decision 28 March 2006
Revised manuscript received 31 March 2006
Accepted 18 March 2006

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