Developmental Biology 240, 1–14 (2001)doi:10.1006/dbio.2001.0387, available online at http://www.idealibrary.com on

REVIEW

Ion Transport in Sperm Signaling

Alberto Darszon,* ,1 Carmen Beltran,* Ricardo Felix,†Takuya Nishigaki,* and Claudia L. Trevino**Departamento de Genetica y Fisiologıa Molecular, Instituto de Biotecnologıa, UniversidadNacional Autonoma de Mexico (UNAM), Cuernavaca, Morelos 62250, Mexico; and†Departamento de Fisiologıa, Biofısica y Neurociencias, CINVESTAV-IPN, Mexico D. F.

Ion channels and transporters, key elements in sperm–egg signaling and environmental sensing, are essential forfertilization. External cues and components from the outer envelopes of the egg influence sperm ion permeability andbehavior. Combining in vivo measurements of membrane potential, intracellular ions, and second messengers with newmolecular approaches and reconstitution strategies are revealing how sperm ion channels participate in motility, spermmaturation, and the acrosome reaction. Sperm are tiny differentiated terminal cells unable to synthesize proteins anddifficult to characterize electrophysiologically. Spermatogenic cells, the progenitors of sperm, have become useful tools forprobing sperm ion channels since they are larger and molecular biology techniques can be applied. These complementarystrategies are opening new avenues to determine how sperm ion channels function in gamete signaling. © 2001 Academic Press

Key Words: acrosome reaction; capacitation; fertilization; ion channels; ion transport; sperm; spermatogenic cells.

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INTRODUCTION

Fertilization is essential for sexual reproduction and forgenerating a new individual. Although sperm–egg signalinghas been studied for almost a century (Lillie, 1919), themolecular entities involved have only now begun to beunveiled. During gametogenesis, sperm are produced in thetestis and eggs in the ovaries. Mature and competent maleand female gametes are required to achieve fusion andfertilization. Components from the external layers of theegg prime spermatozoa for fertilization by altering its mem-brane permeability and second messenger levels.

The potential for motility develops only after sperm leavethe testis. For example, a sea urchin spawns as many as 40billion sperm into the sea. Upon release, these cells startswimming, powered by a microscopic flagellar engine,exquisitely regulated by chemical signals from the environ-ment and the egg. In contrast, sperm from internal fertiliz-ers (reptiles, birds, and mammals) develop the potential formotility as they pass through the vas deferens (a duct from

1 To whom correspondence should be addressed. Fax: 152-73-

172388. E-mail: darszon@ibt.unam.mx. c

0012-1606/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

he epididymis to the urethra) and the epididymis (Mori-awa, 1994; Maddocks and Setchell, 1988).Motility ensues when sperm are released into the repro-

uctive ground or ejaculated into the female reproductiveract. Activation is triggered by environmental cueshrough transduction events involving sperm ion channelsMorisawa, 1994; Darszon et al., 1999; Kraznai et al., 2000).n mammals, sperm undergo a further maturation processithin the female reproductive tract, named capacitation.rom the millions of sperm released only a few reach theirarget (Vacquier, 1998; Wassarman et al., 2001). Manyascinating questions still surround the role of sperm che-

otaxis in nature and the mechanisms involved (Miller andogt, 1996; Eisenbach, 1999).After maturing, swimming sperm from all species pos-

essing an acrosome, including mammals, must undergohe acrosome reaction (AR)2 to fertilize the homologous egg

2 Abbreviations used: AC, adenylyl cyclase; AR, acrosome reac-tion; BLMs, black lipid membranes; [Ca21]i, intracellular Ca21

concentration; [cAMP]i, intracellular cyclic AMP; [cGMP]i, intra-ellular cyclic GMP; DHPs, dihydropyridines; DIDS, 4,49-iisothiocyanatostilbene-2,29-disulfonic acid; EJ, egg jelly; Em,embrane potential; FSP, fucose sulfate polymer; GC, guanylyl

yclase; GPI, glycosylphosphatidylinositol; HCN, hyperpolariza-

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(McLeskey et al., 1998; Vacquier, 1998; Wassarman et al.,001). Jean C. Dan discovered this exocytotic process in thearly 1950s. The acrosome is a single, large, Golgi-derived,ecretory vesicle found in the anterior head of sperm fromany animal species. During AR, the acrosomal and

lasma membranes fuse at multiple sites, yielding spermembrane elements required for penetration of the egg coat

nd subsequent fusion with the egg plasma membraneYanagimachi, 1998). The AR requires the presence ofxternal Ca21 (Dan, 1954). The determinate influence thatxternal ions have on sperm motility and AR brings to lighthe importance of ion channels in these crucial cell pro-esses. Ion channels are key elements in cell signalingHille, 1992; Jan and Jan, 1997); sperm motility, maturation,nd AR are inhibited by certain ion channel blockersFlorman et al., 1998; Publicover and Barrat, 1999).

Ion channels are incredible catalyzers of ion transporthrough the nonconducting lipid bilayer. Because each onellows the flow of millions of ions per second, a few of theman induce substantial electric and concentration changesn a small cell, such as the sperm, in milliseconds (Hille,992). It is no wonder that nature selected these amazingransducers to achieve fast information exchanges betweenells and the external world. Ionic gradients across cells notnly determine membrane potential (Em) through ion-

selective channels, permeant ions can modulate enzymesand further influence channel activity, causing additionalEm changes and ion flow. Em governs the rates and directionf ion flow through channels and exchangers and its fluc-uations modulate intracellular pH (pHi), Ca21 ([Ca21]i), and

other second messengers that importantly influence spermflagellar motility and AR.

Although ion channels are known to be important insperm physiology, learning how these integral membraneproteins operate and are regulated in sperm is essential tounderstand fertilization. Voltage and ion-sensitive dyes,bilayer reconstitution, electrophysiology, DNA recombi-nant techniques, RNA expression in heterologous systems,immunocytochemistry, and pharmacology are revealing thefascinating properties of these molecules. Sperm are tinydifferentiated cells unable to synthesize proteins. Sper-matogenic cells, the progenitors of sperm, are larger andactively synthesize channels. These latter cells are facili-tating the study of sperm ion channels by using molecularbiology and patch–clamp techniques. Correlating ion chan-nel distribution and function through spermatogenesis willenable us to understand how these proteins modulate

tion activated and cyclic nucleotide-gated K1 channel; IP3, inositol1,4,5-trisphosphate; [K1]e, extracellular K1 concentration; [Na1]i,intracellular Na1 concentration; pHi, intracellular pH; PKDREJ, a

ammalian homologue of REJ; PTx, pertussis toxin; REJ, receptoror EJ; SAPs, sperm-activating peptides; SNAREs, soluble-ethylmaleimide-sensitive attachment protein receptors; SOCs,

tore operated Ca21 channels; TEA1, tetraethylammonium; ZP,

ona pellucida.

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differentiation and AR, a meticulously choreographed sig-naling process.

Due to space limitations, we will summarize the avail-able information and highlight key questions in workingmodels of the essential steps required by sperm to fertilizeeggs: motility, capacitation, and AR. We will refer mostly tosea urchin and mouse sperm since these are the best-characterized systems. We apologize for leaving out impor-tant contributions and for inevitably being partial. Thereare several interesting and helpful reviews on general as-pects of gamete interaction and function (Hoshi et al., 1994;uzuki, 1995; Florman et al., 1998; Benoff, 1998; Darszon etl., 1999; Flesch and Gadella, 2000; Publicover and Barrat,999; Vacquier, 1998; Wassarman et al., 2001; Baldi et al.,000).

MARINE INVERTEBRATE SPERM

Responses to the Ionic Environment and to SmallPeptides from the Outer Layer of the Egg

Diffusible compounds from the outer layer of eggs influ-ence sperm swimming trails in many marine invertebrates(Miller, 1985; Morisawa, 1994). Though close to 100 sperm-activating peptides (SAPs) have been identified from seaurchin (Suzuki, 1995) and starfish (Nishigaki et al., 1996)egg jelly, only a few have been structurally characterized inother species (Oda et al., 1998). In general, SAPs producesimilar biological effects on spermatozoa. They are believedto have a chemoactivation function and, in some cases,attract sperm to the egg, accelerate sperm penetrationthrough the jelly coat, and promote AR (Suzuki, 1995).

Signaling by speract (or SAP-1), the first purified andstructurally identified SAP, involves ion channels andtransporters (reviewed in Darszon et al., 1999). Figure 1illustrates a working model of its transduction cascade:speract binding to its receptor(s) (Dangott and Garbers,1984; Yoshino and Suzuki, 1992) activates guanylyl cyclase(GC) (Bentley et al., 1988). In Arbacia punctulata, thepeptide (resact or SAP-IIA) binds directly to GC (Singh etal., 1988). Speract signaling then causes K1 efflux throughGMP-dependent K1 channels (Babcock et al., 1992; Gal-ndo et al., 2000), causing a decrease in sperm Em. Thisyperpolarization may enhance Na1/Ca21 exchange to

maintain low [Ca21]i (Bridge, 2001). Some phosphatases andphosphodiesterases may be pHi-sensitive and rapidly inac-tivate GC, decreasing [cGMP]i (Garbers, 1989). High K1

seawater blocks all sperm responses to speract except forthe large [cGMP]i increase (Harumi et al., 1992). Themolecular characterization of the cGMP-regulated K1 chan-

el will provide an important piece of the echinodermperm chemotaxis puzzle.A K1-dependent hyperpolarization in freshwater fish

(Krasznai et al., 2000) and the subsequent increase incAMP]i in salmonid fish (Morisawa, 1994) are essential forsperm motility initiation. This hyperpolarization is also

fundamental in the sperm response to the sperm-activating

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and -attracting factor in ascidians (Izumi et al., 1999). Theseresults suggest that common mechanisms may underlie theregulation of sperm flagellar movement.

The speract-induced hyperpolarization also stimulatesNa1/H1 exchange (Lee and Garbers, 1986), adenylyl cyclase(AC) (Beltran et al., 1996; see AR section), and possibly acation channel named SPIH (Gauss et al., 1998). Thesechanges lead to increases in pHi, [cAMP]i, and Na1 influx.SPIH has been cloned and belongs to the hyperpolarization-activated and cyclic nucleotide-gated K1 channel (HCN)amily (Gauss et al., 1998). The HCN family encompassesacemaker channels that control heart beat, regulate rhyth-ic firing in individual neurons, contribute to the restingembrane potential, and modulate periodicity in neural

FIG. 1. The speract signaling cascade in S. purpuratus sea urchin(GC, 2), promoting K1 efflux through a cGMP-dependent K1 chanhyperpolarization activates Na1/H1 exchange (4), adenylyl cyclase (channel named SPIH (6), leading to intracellular alkalinization, eleddition, Em hyperpolarization can enhance Na1/Ca21 exchange (7ll-defined Ca21 channel (8). Also, the increase in [Na1]i and Em dephosphatases and phosphodiesterases may be pHi-sensitive and ra

speract (100 nM) measured by fluorescent indicators DiSC3(5) (Em), B

etworks (Kaupp and Seiffert, 2001). SPIH is activated by c

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yperpolarizing potentials and potently up-regulated byAMP. Its K1 selectivity is poor (PK1/PNa1 ;5), thus allow-

ing Na1 influx under physiological conditions and possiblyontributing to the re- or depolarization of sperm. BecausePIH is found mainly in the flagellum and HCN channelsre involved in periodicity, this channel could modulateagellar beating and participate in sea urchin sperm che-otaxis (Kaupp and Seiffert, 2001). Indeed, channels acti-

ated by cAMP and hyperpolarizing potentials sharingelectivity properties with SPIH have been recorded inlanar bilayers containing flagellar sperm plasma mem-ranes (Labarca et al., 1996) and by patch–clamp techniquesn swollen sea urchin sperm (Sanchez et al., 2001).

Resact, the only sea urchin SAP with demonstrated

. (1) Binding of speract to its receptor activates a guanylyl cyclase) causing a decrease in sperm membrane potential (Em). This Em

), and a hyperpolarization-activated and cyclic nucleotide-gated K1

n of [cAMP]i, and Na1 influx (Em depolarization), respectively. Inmaintain [Ca21]i low. The increase in [cAMP]i may stimulate anization could lead to reversal of the Na1/Ca21 exchange (79). Some

inactivate GC and decrease [cGMP]i. Inset: Sperm responses toF (pHi), and Fluo-3 ([Ca21]i). All upward changes indicate increases.

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hemotactic capacity, requires external Ca21 to alter sperm

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FIG. 2. The sea urchin and mouse sperm acrosome reaction. In both species, ligand binding to the receptor(s) (1) induces a transientincrease in [Ca21]i (2) and alkalinization (4). These changes are indispensable for the following sustained increase in [Ca21]i (7) that is afundamental signal to trigger the acrosome reaction. The transient increase in [Ca21]i through voltage-dependent Ca21 channels maytimulate phospholipase C (PLC, 5) and produce inositol 1,4,5-trisphosphate (IP3). Ca21 release through IP3 receptors (6) depletes thentracellular Ca21 deposit, presumably the acrosome, leading to the activation of store-operated Ca21 channels (SOC, 7) in the plasma

embrane. The increase in pHi may directly and/or indirectly (by rising affinity of IP3 receptor for IP3) stimulate SOC activity. The detailedechanisms in the two species display differences. In sea urchin, when the fucose sulfate polymer (FSP) binds to its sperm receptor (REJ,

), a dihydropyridine (DHP)-sensitive Ca21 channel (2) and a TEA1-sensitive K1 channel (3) are transiently activated by an unknownechanism. Possibly, REJ itself may have channel activity. Hyperpolarization as a result of K1 efflux stimulates voltage- and

Ca21-dependent Na1/H1 exchange (4), raising pHi. The increase in cAMP could regulate various channels. DIDS-sensitive Cl2 channels mayparticipate in setting the resting Em. In mouse, several receptors for ZP3 may exist. One of them (possibly PKDREJ) may activate a cationchannel to increase sperm Em and activate T-type voltage-dependent Ca21 channel (2). Another candidate, b-galactosyltransferase (GalT),s believed to couple with pertussis toxin (PTx)-sensitive G proteins (Gi, 3), which controls pHi regulators, Na1-dependent Cl2/HCO3

2

exchange, and flufenamic acid-sensitive acid export (4). Niflumic acid-sensitive Cl2 channels may cause Em depolarization to activate the

T-type voltage-dependent Ca21 channels, which also could be involved in the pathway of progesterone-induced AR.

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.

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motility (Ward et al., 1985). It is worth noting that S.purpuratus and A. punctulata are separated by ;200 mil-lion years in evolution (Smith, 1988); therefore, there couldbe differences in the way SAPs modulate sperm motility.High Ca21 concentrations trigger asymmetric flagellar beat-ng in demembranated sperm (Brokaw, 1979) and intactperm (Cook et al., 1994). Though [Ca21]i is crucial inagellar movement regulation, little is known about SAP-ctivated Ca21 uptake. A Na1/Ca21 exchanger working ineverse was proposed to participate in the [Ca21]i increaseuring this process (Schackmann and Chock, 1986). How-ver, consistent with the Ca21-dependent depolarizationaused by speract (Reynaud et al., 1993), a cAMP-regulateda21 channel may contribute to this uptake (Cook and

Babcock, 1993).Under physiological conditions, sperm may effectively

contact SAPs only in the vicinity of the egg (,0.5 mm).Considering the linear velocity of swimming sperm (;0.2

FIG. 3. Mammalian sperm capacitation. During sperm transit throhe plasma membrane by acceptor molecules (such as albumin) cperm pHi increase possibly mediated by a Na1-dependent Cl2HCO

HCO32 stimulates a bicarbonate-dependent AC (AC, 3) and activate

through a poorly characterized Ca21 influx pathway (4). These chyperactivation. The pHi increase may activate inwardly rectifying

small but significant K1 efflux through these channels contribuT-type voltage-gated Ca21 channels from the “inactivated state” (6

mm/s), SAPs may affect their trajectory just for a few 1

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seconds and will saturate their receptors upon arrival at theegg (Nishigaki and Darszon, 2000). Time-resolved measure-ments of sperm response to SAPs using stopped-flow tech-niques (Nishigaki et al., 2001), photolysis of caged com-pounds, and fast imaging techniques are needed tounderstand sperm motility regulation.

The Acrosome Reaction

Sea urchin sperm are released into the sea, where inter-action with the outer layer of the egg (egg jelly; EJ) triggersAR. The AR-inducing component in EJ of S. purpuratus is aucose sulfate polymer (FSP) (Alves et al., 1998), while inchinometra lucunter (Alves et al., 1997) it is a galactoseulfate polymer (sulfated galactan). In the starfish Asteriasmurensis, a pentasaccharide repeat containing xylose, sul-ated fucose, and galactose is the AR inducer (Koyota et al.,

the female reproductive tract, cholesterol is partially removed fromg membrane reorganization (1). This process somehow leads to achange (2) and flufenamic acid-sensitive acid-export (29). Influx of

AMP-dependent protein kinase (PKA). Sperm [Ca21]i also increaseses stimulate a protein tyrosine kinase (PTK) and initiate spermchannels (5), which are inactive (or closed) before capacitation (59).

gradually decrease sperm Em. This hyperpolarization may reliefbe ready for channel activation (6) by ZP3 (69).

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polysaccharides in EJ contribute to species specificity offertilization in marine animals.

The AR involves fusion between the outer acrosomal andplasma membranes, exposing the inner acrosomal mem-brane specialized for fusion with the egg (Garbers, 1989;Vacquier, 1998; Darszon et al., 1999). As in neurons andendocrine cells, membrane fusion during sea urchin andmammalian sperm AR appears to be governed by SNAREs(soluble N-ethylmaleimide-sensitive attachment protein re-ceptors) and small G proteins like Rabs (Schulz et al., 1998;Ramalho-Santos et al., 2000; Michaut et al., 2000; Katafu-chi et al., 2000; Kierszenbaum, 2000).

Seconds after FSP binds to the sperm receptor for EJ (REJ,now suREJ1, a 210-kDa membrane glycoprotein), Na1 andCa21 influx and H1 and K1 efflux activate with concomitantchanges in Em, and increases in [Ca21]i and pHi (Fig. 2).Furthermore, FSP elevates [cAMP] and inositol 1,4,5-trisphosphate (IP3) and stimulates PKA, phospholipase D(Darszon et al., 1999; Mengerink et al., 2000), and nitricoxide synthase (Kuo et al., 2000).

REJ shares extensive homology with human polycystin-1(Moy et al., 1996), a membrane protein encoded by PKD1that coassembles at the plasma membrane withpolycystin-2, the gene product of PKD2, to produce achannel that is proposed to regulate renal tubular morphol-ogy and function (Hanaoka et al., 2000). Mutations in eitherPKD1 or PKD2 are associated with autosomal dominantpolycystic kidney disease (Arnaout, 2001). PKD2 has aminoacid similarity to voltage-gated Ca21 and other cation chan-

els, especially within their S3–S6 segments and the loopetween S5 and S6 (Nomura et al., 1998). This protein itselfay form Ca21-permeable nonselective channels (Hanaoka

et al., 2000; Gonzalez-Perrett, et al., 2001). The mostconserved sequence among PKD domains is WDFGDGS,but its specific role is still unknown. Considering theIg-like fold of this sequence, it has been suggested that itcould be involved in ligand binding. Conservation couldalso reflect constraints on structure rather than on function(Bycroft et al., 1999).

Several isoforms of REJ exist; SuREJ3, an 11-transmembrane protein, is found in the sperm head plasmamembrane over the acrosome. This protein shares severalfeatures with latrophilins, G-protein-coupled receptors in-volved in exocytosis. For example, the SUEL domain (seaurchin egg lectin) of approximately 105 aa is the mostconserved domain of latrophilins. In addition, the three seaurchin REJ proteins have the conserved region where latro-philins are posttranslationally cleaved. Interestingly, thereis also homology in the carboxyl terminus among suREJ3,huPKD1, PKDREJ, huPKD2, voltage-gated Ca21 channels,and Drosophila transient receptor potential (trp) channels(Mengerink et al., 2000). The REJ proteins could be subunitsof a ligand-gated channel that triggers AR in sea urchinsperm or regulators of a Ca21 channel that induces thisreaction (Vacquier and Moy, 1997; Mengerink et al., 2000)(Fig. 2). Notably, a high-conductance, Ca21-permeable chan-

el, whose properties resemble some of those displayed by

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he PKDL and PKD1–PKD2 channels, has been recorded inlack lipid membranes (BLMs) containing sea urchin andouse sperm plasma membranes (Lievano et al., 1990;

eltran et al., 1994). The sea urchin sperm channel has a70-pS main conductance state in 50 mM CaCl2 and poorlyiscriminates between monovalent and divalent cationsPCa/PNa 5 5.9) (Lievano et al., 1990).

[Ca21]i determinations indicate the participation of twoifferent Ca21 channels in the sea urchin sperm AR (Guer-

rero and Darszon, 1989a,b; Schackmann, 1989). FSP bindingto REJ transiently opens a Ca21-selective channel that isblocked by verapamil and dihydropyridines (DHPs). Fiveseconds later, a second channel, insensitive to the latterblockers, activates and leads to the AR. The second channeldoes not inactivate, is permeable to Mn21, and is pHi-dependent (Guerrero et al., 1998). The two Ca21 channelsare somehow linked, since blocking the first channel inhib-its the second, and inhibition of the FSP-induced pHi

increase associated with the AR prevents activation of thesecond channel and AR.

Evidence indicates that the second channel belongs to thefamily of store-operated Ca21 channels (SOCs) (Gonzalez-Martınez et al., 2001; Parekh and Penner, 1997; Barritt,1999) and may be important in the AR of many species(Santi et al., 1998; O’Toole et al., 2000). Since sperm do nothave endoplasmic reticulum, the internal store is likely tobe the acrosome (Trevino et al., 1998). S. purpuratus spermproduce IP3 (Domino et al., 1989) during AR and possessIP3 receptors (Zapata et al., 1997).

In L. pictus sea urchin sperm, FSP induces a transientK1-dependent hyperpolarization probably mediated by K1

channels, followed by a depolarization. This hyperpolariza-tion may remove inactivation from voltage-gated Ca21

channels (Gonzalez-Martınez and Darszon, 1987; Lievanoet al., 1990). The AR and the increases in Ca21 uptake andHi associated with this reaction are blocked by elevatedK1]e. FSP increases pHi, at least in part, by activating aa21-dependent Na1/H1 exchange stimulated by the hyper-olarization (Gonzalez-Martınez et al., 1992). In S. purpu-atus sperm, tetraethylammonium (TEA1), a K1 channel

blocker, inhibits AR (Schackmann, 1989) and TEA1-ensitive K1 channels from sea urchin sperm plasma mem-rane have been recorded in planar bilayers (Lievano et al.,985). Sperm Ca21 and Cl2 channels have also been detectedn BLMs and in patch–clamp recordings, some of which areensitive to blockers that inhibit Ca21 uptake and AR (seeable in Darszon et al., 1999). These observations empha-ize that AR is an ion channel-regulated event.Submicromolar Zn21 induces AR and modulates pHi in L.

ictus sperm (Clapper et al., 1985). It increases [Ca21]i andpHi and changes Em (our unpublished results). Does Zn21

trigger AR by directly modulating ion transport and/orindirectly by activating a metalloprotease (Farach et al.,987; our unpublished results)? Solving the Zn21 riddle will

unveil clues about sea urchin sperm ion transport regula-tion and AR.

All conditions leading to AR increase cAMP; however,

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this increase depends on Ca21 uptake (Garbers, 1989; Dars-zon et al., 1999). How AC is activated after triggering seaurchin sperm AR and its role in this process are stillunknown. The sperm AC is different from that of somaticcells; it is not modulated by G proteins or forskolin but byCa21, pHi, and membrane potential (Garbers, 1989; Beltrant al., 1996).Cl2 channels may also be involved in the AR since

4,49-diisothiocyanatostilbene-2,29-disulfonic acid (DIDS),an inhibitor of anion channels and transporters, blocks thesea urchin sperm AR (Morales et al., 1993). DIDS-sensitiveanion channels from sperm plasma membranes have beenrecorded in BLMs. Cl2 channels may influence spermresting Em, or may be modulated during AR (Morales et al.,1993).

Recently, suREJ1, the speract receptor, a 63-kDa glyco-sylphosphatidylinositol (GPI)-anchored protein, Gs, AC,GC, and PKA were found in sea urchin sperm lipid rafts.From these, only the speract receptor, the GPI-anchoredprotein, and Gs coimmunoprecipitated, suggesting theirfunctional association in the speract response and possiblyin AR (Ohta et al., 2000). Probably suREJ1, AC, GC, andPKA may interact in different microdomains. The func-tional characterization of signal transduction microdo-mains should shed light on the mechanism of AR and mayhelp to elucidate the role of G proteins in sperm physiology.

MAMMALS

Spermatogenic Cells, Molecular Cloning, andHeterologous Expression

In addition to being very small and difficult to studyelectrophysiologically, sperm are differentiated terminalcells unable to make proteins. Thus, gene expression andprotein assembly have to be studied in the large progenitorspermatogenic cells (Florman et al., 1998; Darszon et al.,1999).

Spermatogenic cells express a wide variety of ion chan-nels and transporters (Darszon et al., 1999; Herrera et al.,000). Electrophysiology indicates that the inward currentbserved in these cells has a rapid time course and repre-ents Ca21 influx through channels of the T-type. Thesehannels have a low activation threshold and exhibitoltage-dependent inactivation (Hagiwara and Kawa, 1984;ievano et al., 1996; Arnoult et al., 1996a; Santi et al.,

1996). They are subjected to positive modulation by albu-min and dephosphorylation as well as to negative modula-tion by estradiol and a tyrosine kinase-dependent phosphor-ylation (Arnoult et al., 1997; Espinosa et al., 2000). Ni21 and

HPs are potent inhibitors of the spermatogenic cell-current (Santi et al., 1996; Arnoult et al., 1998). In

addition, several K1 currents have been identified in sper-matogenic cells, including a TEA1-sensitive, noninactivat-ing outward current (Hagiwara and Kawa, 1984) and arapidly activating and sustained inwardly rectifying current

(Munoz-Garay et al., 2001). In Xenopus oocytes injected

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with RNAs from rat spermatogenic cells, ATP evokesanother class of K1 current which is dependent on [Ca21]i

and blocked by charybdotoxin and TEA1 (Chan et al., 1998;u et al., 1998). Similarly, spermatogenic cells display Cl2

channels that activate at voltages above 250 mV and areinhibited by niflumic acid (Espinosa et al., 1998). Interest-ingly, mice deficient in plasma membrane Cl2 channelsundergo massive degeneration of male germ cells (Bosl etal., 2001).

Considering that cyclic nucleotides are involved in spermphysiology, it is not surprising that the first sperm channelto be cloned using a bovine testis library was a cyclicnucleotide gated (CNG) channel (Weyand et al., 1994).These channels are formed from at least two subunits: thea subunit displays the channel activity and the b subunitlone is not functionally active (Kaupp, 1995). Expression ofhe bovine testis a subunit in Xenopus oocytes results in

single channel transitions of 20 pS. The channel is cationic,permeable to Ca21, and has a much higher affinity for cGMP.100-fold) than for cAMP. Small cGMP-induced currentsere recorded from inside–out patches from human andovine sperm (Weyand et al., 1994).Several b subunit isoforms of CNG channels were iden-

tified in bovine testis (Wiesner et al., 1998). Immunodetec-tion revealed that while the a subunit is present along theentire sperm flagellum, the b subunits are distinctly local-zed. Since various combinations of as and bs yield chan-els with different Ca21 permeability, Ca21 microdomainsay exist and participate in flagellar bending control

Wiesner et al., 1998). However, the physiological role ofhis CNG channel is still unknown.

Transcripts for a number of voltage-gated Ca21 channelon conducting (a1) subunits have been identified in sper-

matogenic cells including a1A, a1E, a1C, a1G, and a1H (Lievanot al., 1996; Goodwin et al., 1997; Benoff, 1998; Espinosa etl., 1999). Consistent with this, immunocytochemical ex-eriments indicate that three Ca21 channel a1 subunits (A,

C, and E) are present and regionally localized in sperm(Goodwin et al., 1997; Westenbroek and Babcock, 1999),and that b1–3 auxiliary subunits are also expressed in bothspermatogenic cells and sperm (Serrano et al., 1999). Moreecently, considering the pharmacological profile ofepolarization-evoked [Ca21]i increases in epididymal

sperm and patch–clamp recordings in spermatogenic cells,it was proposed that N-type Ca21 channels contribute to thebserved activity. Consistent with these findings, a1B sub-

units were detected by Western blotting of rodent spermmembranes (Wennemuth et al., 2000); however, detectionof the RNA messenger is still pending.

A unique type of channel, mSlo3, has been cloned frommouse testis and heterologously expressed (Schreiber et al.,1998). It belongs to the Ca21-dependent K1 channel familyut it is regulated by pHi and membrane voltage. Likewise,

a member of the inward rectifier K1 channel family (Kir 5.1)and various sequences homologous to a delayed rectifier K1

channel (Kv1.3) have been amplified based on PCR analysis

on rat testis (Salvatore et al., 1999; Jacob et al., 2000).

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Sperm Capacitation

Mammalian sperm are morphologically differentiated af-ter leaving the testis, but still have to acquire the ability tofertilize an egg. This occurs after completing in the femaletract a series of dynamic events called “capacitation”(Yanagimachi, 1994). This process primes sperm to respondto the zona pellucida (ZP), the extracellular egg coat thatinduces AR. The molecular mechanisms and the signaltransduction pathways mediating capacitation are onlypartially defined and appear to involve modification of[Ca21]i and other ions, plasma membrane lipid transfer, andemodeling, as well as changes in protein phosphorylationVisconti et al., 1999; Baldi et al., 2000; Flesh and Gadella,000) (Fig. 3). As in other cells, Na1/Ca21 and Ca21/H1

exchangers, and Ca21-ATPases regulate sperm [Ca21]i

(Fraser, 1995). High-amplitude and asymmetric flagellarbends, known as hyperactivation, accompany the [Ca21]i

increase (Suarez, 1996). An increase in [cAMP]i is associatedith sperm maturation through the epididymis and capaci-

ation (White and Aitken, 1989; Visconti and Tezon, 1989;isconti et al., 1995).In most somatic cells, extracellular signals modulate a

ransmembrane G-protein-regulated adenylyl cyclase thatynthesizes cAMP. However, in sperm, this may be carriedut by a different form of AC that was recently clonedSinclair et al., 2000). This AC is directly regulated byicarbonate but not by G proteins or pHi (Okamura et al.,

1985; Chen et al., 2000). High levels of RNA message forthe soluble form of this AC (sAC) are unique to male germcells from rat. This message accumulates in round sperma-tids and sAC is present in mature sperm. These findingssuggest that this AC may play an important role in spermmaturation through the epididymis, capacitation, hypermo-tility, and/or the AR (Sinclair et al., 2000). It is likely thatthe AC of sea urchin and other marine sperm is related tothis new sAC.

During capacitation, the sperm plasma membrane hyper-polarizes, at least in part due to an enhanced K1 permeabil-ty (Zeng et al., 1995; Arnoult et al., 1999). It is unclearhich of the biochemical modifications that occur during

perm capacitation affect K1 channels; however, a pHi-ependent inwardly rectifying K1 channel has been identi-

fied very recently in spermatogenic cells (Munoz-Garay etal., 2001). Before capacitation, sperm pHi is relatively acidicand may impose a functionally quiescent state. An acidicpHi may negatively regulate sperm inwardly rectifyinghannels, thereby maintaining membrane potential depo-arized and indirectly preventing unregulated Ca21 entry,nd thus AR (Vredenburgh-Wilberg and Parrish, 1995; Zengt al., 1996). The pHi changes that occur during capacita-ion could activate these channels, permitting K1 efflux andonsequently hyperpolarizing sperm. Membrane potentialn sperm can become as negative as 280 mV, a valueufficient to relieve steady-state, voltage-dependent inacti-ation of voltage-gated Ca21 T-channels (Arnoult et al.,

996a; Santi et al., 1996). Ca21 influx through these chan-

Copyright © 2001 by Academic Press. All right

els, activated by ZP via an unknown mechanism, mayrive AR (Fig. 3). Though several types of voltage-gated Ca21

channels are present in sperm, their role in capacitation isstill unknown.

Interest is now being focused on the role of signalingcomplexes found in specialized lipid domains such as raftsduring fertilization (Ohta et al., 2000). Because spermmaturation and capacitation involve reorganization ofmembrane components and lipid removal (Flesh andGadella, 2000), exploring ion channel and protein redistri-bution during these processes is important.

Mammalian Acrosome Reaction

As mentioned earlier, the AR is an exocytotic eventwhere the acrosome and plasma membrane fuse in thesperm head. In mammals, it is the equatorial segment of thesperm head that fuses with eggs. This region is present onthe cell surface before exocytosis and becomes fusogeniconly after AR. ZP3, one of the ZP glycoproteins, inducesAR. Fusion releases proteases and/or glycosidases that al-low sperm penetration through the ZP (Wassarman et al.,2001).

In spite of much effort, including the use of knockoutmice, the identity of the sperm surface receptor(s) for ZP3 isstill unclear. Many candidates have been proposed, amongthem zonadhesin (Gao and Garbers, 1998) andb-galactosyltransferase (Shi et al., 2001). Multiple, con-certed, and cooperative interactions between ZP3 and thesperm surface, possibly involving receptor aggregation, maybe needed to achieve AR. How these receptors conveyinformation to initiate signal transduction in mammaliansperm is not known. ZP and other agents like progesteronetrigger AR by influencing ion fluxes, phospholipid metabo-lism, cAMP levels, and protein phosphorylation via mecha-nisms that are not well understood (McLeskey et al., 1998;Roldan, 1998; Breitbart and Naor, 1999; Darszon et al.,1999; Baldi et al., 2000).

Ca21 is essential for fusion during AR; Ca21 ionophorestrigger this process in vitro and some Ca21 channel blockersinhibit it (Yanagimachi, 1994; Darszon et al., 1999). ZPinduces changes in sperm membrane potential (Arnoult etal., 1996a), as well as [Ca21]i and pHi increases, that precedeexocytosis in sperm (Fig. 2) (Florman et al., 1989, 1992,1998). ZP activates pertussis toxin-sensitive Gi proteins(Endo et al., 1988) that transiently increase pHi and lead toAR (Arnoult et al., 1996b). A Gi protein interacts withb-galactosyltransferase (Shi et al., 2001). Although there isevidence for Na1-dependent Cl2/HCO3

2 (Zeng et al., 1996)nd a Na1/H1 exchange (Garca and Meizel, 1999), many

questions remain unsolved about sperm pHi regulationduring the AR. Are some sperm ion channels modulated byZP3-activated Gi proteins? How are pHi and [Ca21]i relatedand tuned during AR?

Ion-selective fluorescent probes have revealed two phasesof the ZP3-induced increase in [Ca21]i, consistent with the

participation of at least two different types of Ca21 channels

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9Ion Transport in Sperm Signaling

in the mouse sperm AR (Florman, 1994). Upon ZP3 activa-tion, [Ca21]i transiently elevates to micromolar levels

ithin 40–50 ms and relaxes to resting values within theext 200 ms (Arnoult et al., 1999). The pharmacology andinetics of this transient are consistent with the propertiesf T-type Ca21 channels. T-type Ca21 currents are the only

voltage-dependent Ca21 currents expressed during the laterstages of rodent spermatogenesis (Lievano et al., 1996;Arnoult et al., 1996a). These channels, the AR, and thetransient increase in [Ca21]i are inhibited by micromolaroncentrations of DHPs, pimozide, and Ni21. Although it isikely that the fast transient increase in sperm [Ca21]i is

mediated by a ZP3-dependent activation of T-type Ca21

channels (Florman et al., 1998; Darszon et al., 1999), themolecular identity of the AR relevant voltage-gated Ca21

channels remains to be established (Benoff, 1998; Serrano etl., 1999; Wennemuth et al., 2000).The fast [Ca21]i transient is followed by a slower and

ustained increase which is necessary for the AR. ZP3ignaling may achieve the sustained [Ca21]i elevation by

releasing Ca21 from an IP3-sensitive intracellular store(Walensky and Snyder, 1995) and subsequent Ca21 entryhrough the plasma membrane (Fig. 2). There are severalsoforms of phospholipase C in sperm (Vanha-Perttula andasurinen, 1989; Baldi et al., 2000; Breitbart and Naor,999) and ZP3 stimulates IP3 production in these cellsTomes et al., 1996). The IP3 receptor (Walensky andnyder, 1995; Trevino et al., 1998) and a Ca-ATPase (Dragi-eva et al., 1999) are present in the acrosomal membrane.

oreover, recently it was shown that PLCd4 is present inouse sperm and that it is involved in the early events of

he ZP3-induced AR (Fukami et al., 2001). Interestingly, initro all PLC isoenzymes are activated by Ca21, but PLCdsre the most sensitive to Ca21. This isoform can bind to

phosphatidylinositol 4,5-bisphosphate (PIP2) through itsPH domain in the absence of other signals (Rhee and Bae,1997). Thus, possibly a transient [Ca21]i increase is suffi-cient to activate this PLCd independently of receptor acti-ation.In general, the emptying of Ca21 stores generates a gating

ignal that couples intracellular Ca21 release to the openingof SOCs in the plasma membrane (Parekh and Penner,1997). Both mouse spermatogenic cells and sperm possesspHi-dependent Ca21 channels and SOCs, which could par-ticipate in the sustained [Ca21]i elevation necessary for the

R (Santi et al., 1998). Moreover, in mouse sperm, thapsi-argin, an endoplasmic reticulum Ca21-ATPase inhibitor,

induces the AR (Meizel and Turner, 1993). This inhibitorstimulates external Ca21 uptake (Blackmore, 1993) with

inetics and sensitivity to Ni21 and DHPs similar to thosef the second phase of Ca21 influx induced by ZP3 (O’Toolet al., 2000).Antagonists of T-type Ca21 channels added before ZP3

also inhibit the sustained elevation in [Ca21]i (Arnoult et al.,996b). Therefore, the transient increase in [Ca21]i appearso be necessary to open the second Ca21 pathway that keeps

[Ca21]i elevated to allow AR (O’Toole et al., 2000). How-

Copyright © 2001 by Academic Press. All right

ver, the molecular identity of this SOC has yet to bestablished. Some members of the trp gene family haveeen proposed to encode SOCs. Seven mammalian trpomologues have been identified (Putney and McKay, 1999;oulay et al., 1999). Notably, all seven genes of this familyre present in spermatogenic cells (Vannier et al., 1999; ournpublished results). Interestingly, trp2 has been recentlyhown to regulate ZP3-induced Ca21 entry into mouseperm (Jungnickel et al., 2001).How ZP3 opens voltage-gated Ca21 channels is still aystery. Interestingly, PKDREJ, a mammalian homologue

f REJ, the sea urchin sperm receptor for the EJ, is expressedn human and mouse only in the testis, in a pattern thatoincides with sperm maturation (Hughes et al., 1999). TheKDREJ transcript has sequence similarity to theembrane-associated region of polycystin-1. Recently itas shown that coexpression of polycystin-1 and -2 yieldsa21-permeable channels (Hanaoka et al., 2000). The PK-

DREJ protein does not contain the lectins found in REJ,making its interaction with ZP unclear. It could be part ofthe channel machinery that initiates AR, possibly by depo-larizing sperm.

Intracellular Cl2 is high enough in sperm so that anionhannel activation could depolarize sperm and trigger ARGarca-Soto et al., 1987; Garcıa and Meizel, 1999). TheP-induced AR in boar, porcine, and human sperm islocked by antagonists of the neuronal glycine receptor/Cl2

channel, while glycine, its agonist, induces AR (Melendrezand Meizel, 1995; Llanos et al., 2001). In addition, niflumicacid and other stilbenes that block sperm anion channelsinhibit the mouse sperm AR (Espinosa et al., 1999). Spermrom mice defective in the glycine receptor/Cl2 channel are

unable to undergo the ZP induced-AR (Sato et al., 2000).The physiological relevance of AR agonists distinct from

ZP is still a matter of debate. The transduction cascadesinitiated by some of these agonists may operate duringspermatogenic differentiation; some may participate in che-motaxis, facilitate capacitation, and/or the AR (Darszon etal., 1999; Baldi et al., 2000). Progesterone, the best studiedof these agonists, increases [Ca21]i in a biphasic mannerMeizel et al., 1997; Blackmore and Eisoldt, 1999; Baldi etl., 2000; Kirkman-Brown et al., 2000) as well as [Na1]i

(Patrat et al., 1999). It also causes Cl2 efflux (Meizel, 1997)nd membrane depolarization (Foresta et al., 1993). Sinceholesterol activates phospholipase C, it may also inducea21 depletion from sperm stores and activate SOCs (Black-

more, 1999). This would explain why progesterone and ZP3activate distinct upstream signaling elements (Murase andRoldan, 1996) but can act cooperatively in driving secretion(Roldan et al., 1994).

CONCLUDING REMARKS

Ion channels and transporters are essential instrumentsin the symphony of fertilization. Pressing questions need to

be answered before we can fully understand the momentous

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event of generating a new life. The molecular identity of thereceptors to the egg ligands, which lead to the acrosomereaction and the ion channels and transporters activatedduring this elaborate process, must be determined before wecan learn how they are orchestrated. Certainly genomicsand proteomics will soon contribute to solving some ofthese questions. However, electrophysiology and intracel-lular ion imaging, together with ion transport reconstitu-tion, will still be necessary to unravel why and how ionchannels and transporters play a cardinal role in spermmaturation, the acrosome reaction, and fertilization. Thespecific location and composition of the transduction com-plexes involved in gamete signaling will be established inthe coming years.

ACKNOWLEDGMENTS

We thank V. Vacquier, M. Whitaker, C. Leaky, C. Wood, and P.Visconti for critically reading the manuscript. This work waspartially supported by grants from CONACyT: 27707-N (to A.D.);32052-N (to C.B.); 31735-N (to R.F.); and DGAPA IN201599 (toA.D. and C.T.). Part of this work was carried out during thesabbatical of A.D. at the University of Newcastle upon Tyne in thelaboratory of Dr. Michael Whitaker. The support of The WelcomeTrust and BBSRC is acknowledged.

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Received for publication April 25, 2001Revised July 14, 2001

Accepted July 14, 2001

Published online September 26, 2001

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