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Review

Sperm Ion Channels: Molecular Targets for the Next Generation of Contraceptive Medicines?

Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois

Correspondence to: Dr Di Zhang, Neuroscience Research, Dept R4ND, Bldg AP9-1125, Abbott Laboratories, 100 Abbott Park Rd, Abbott Park, IL 60064-6118 (e-mail: di.zhang{at}abbott.com).

Received for publication January 14, 2005; accepted for publication June 13, 2005.

Attention to ion channels as drug targets for contraception has grown with the realization that certain channel subunits are localized exclusively in sperm, that selective knockdown of subunits can lead to infertility without un-toward effects, and the rationale that selective inhibitors and/or openers of ion channels could interfere with sperm function. The state of current research includes more recently discovered ion channel subunits and derived combinations, elucidation of their physiological roles in relation to sperm biology and proof-of-principle studies using pharmacological and/or gene knockout approaches. Several publications have reviewed in detail the physiological basis of sperm function, including the role of ion channels (Darszon et al, 1999; Jagannathan et al, 2002). The scope of this review is to update recent advances in sperm ion channel biology in mammals, current directions in target validation, and proof-of-concept studies that provide enhanced understanding toward more expedient identification of key ion channels of therapeutic relevance and to highlight examples where ion channel modulators may have potential as contraceptive medications.

Contraceptive Approaches for Men

Although men and women are considered to share comparable responsibility in family planning and birth control, the primary role of prevention of pregnancy is, to a large extent, borne by women. Although this situation may, in part, be due to social, cultural, and/or economic factors and poor understanding of factors controlling male fertility, an important determinant is, perhaps, the limited range of contraceptive options available for men compared with women. For example, although the oral pill is quite popular as a contraceptive for women, no comparable pharmaceutical dosage forms has been successfully developed for men. At present, the only effective methods for contraception in men are those that prevent sperm transport, such as condoms and vasectomy (Anderson and Baird, 2002). Several public-opinion surveys have indicated that most men would use new contraceptive techniques, such as a pill or other oral dosage form if made available (Anderson and Baird, 1997; Martin et al, 2000). An ideal medication should be efficient, safe, economical, and convenient for use, providing rapid onset of action and quick recovery of fertility upon termination of the drug. Considering the essential roles of several steroid and peptide hormones in spermatogenesis, several studies have focused on suppression of sperm production in the testis by hormonal methods. One of the key hurdles, however, stems from the fact that, while complete hormonal suppression is not required for the inhibition of ovulation in women, complete hormonal suppression of spermatogenesis is needed for a comparable degree of contraception in men. In addition, there is a long period between the onset of treatment and the occurrence of desired infertility, and side effects, like hormonal imbalances, may occur during this prolonged treatment.

Considering the drawbacks of hormonal methods, there is a growing interest in the development of nonhormonal methods for male contraceptives, and several approaches are being pursued at present. One approach is the development of contraceptive vaccines against antigenic proteins on sperm surface based on the rationale that such vaccines may prevent sperm-egg interactions during fertilization (Delves et al, 2002). Development of compounds targeting sperm stored in the epididymis is another approach, considering that the long latency to suppress spermatogenesis is not required for efficacy and, in principle, normal sperm would resume quickly upon withdrawal of the drug (Cooper and Yeung, 1999). Third, investigators showed that plants used in Chinese traditional medicine, such as gossypol and several compounds isolated from the plant Tripterygium wilffordii, exhibit antifertility effects. Although nonhormonal methods with minimal side effects and quick onset of action are appealing in principle, agents targeting such mechanisms have not yet emerged in the market place.

Sperm Physiology, Maturation, and Fertilization

Sperm are highly specialized cells, composed of a head, a short neck, and a long tail, intended for the task of fertilizing an egg. The head contains the haploid nucleus in a highly compact form and the acrosome, a specialized secretary vesicle with hydrolytic enzymes, whereas the tail is a long flagellum that is composed of a middle piece, principal piece, and end piece. The tail contains the motile apparatus necessary for the movement and penetration of sperm into the egg at fertilization. Motility is generated by a highly organized microtubule-based structure called the axoneme that is surrounded by outer dense fibers in the middle and principal pieces of the tail. A mitochondria sheath in the middle piece and a fibrous sheath in the principal piece cover the outer dense fibers.

The generation of sperm occurs continuously within the seminiferous tubules of the testes by a complex process known as spermatogenesis (see Figure). At the beginning of this process, spermatogonia, the most immature germ cells, withdraw from cell cycle and differentiate into spermatocytes. The spermatocytes undergo meiosis and become haploid round spermatids. These spermatids undergo extensive morphological changes to become elongated spermatids and further differentiate into mature spermatozoa. Once formed within the seminiferous tubules, the immotile spermatozoa are released into the luminal fluid and are transported to the epididymis. In humans, the transit of spermatozoa through the epididymis takes 2-6 days. During this period, spermatozoa undergo a series of essential maturation changes to attain the ability to swim and fertilize the egg. Upon maturation, spermatozoa are stored in cauda epididymis until ejaculation, when the spermatozoa with the surrounding fluid, along with alkaline secretions of the male accessory sex glands, are released.

View larger version (28K): [in this window] [in a new window]   Ion channels implicated in sperm function. Depicted are critical steps during sperm generation, maturation, and fertilization process along with ion channel types and/or subunits implicated in these processes.

Sperm are unable to fertilize the egg immediately after ejaculation and require a period of incubation in the female reproductive tract to acquire the ability of fertilization. During this period, sperm undergo a series of biochemical transformations, collectively known as capacitation, which include changes in the plasma membrane composition and fluidity, intracellular ions, and cellular metabolism. The motility machinery becomes activated by both extra- and intracellular factors and sperm become progressively more motile. Further, sperm make significant changes in swimming patterns and display a unique hyperactivated motility, which is required for sperm to swim in the viscous oviduct fluid and to penetrate the zona pellucida (Yanagimachi, 1994). Sperm capacitation process is triggered by the elevation of intracellular Ca²⁺ and bicarbonate, which results in activation of adenylate cyclase, elevation of cyclic-AMP, and phosphorylation of specific proteins (Breitbart, 2002). Upon contact with the glycoproteins on the surface of the egg, sperm undergo the acrosome reaction, resulting in the fusion of the plasma membrane and the outer acrosomal membrane and the release of stored hydrolytic enzymes. After infiltration of the egg coat, the sperm membrane fuses with that of the egg, triggering Ca²⁺ oscillations, which is a prerequisite for activation of the fertilization process.

Diversity of Ion Channels in Sperm

Ion channels serve as principal integrators of cellular excitability. This fundamental role is achieved by the exquisite selectivity for permeating ions of appropriate size and charge with appropriate spatial, temporal, and kinetic resolutions. Ion channels differ from each other in several ways, including the nature of ions they conduct, gating properties, primary amino acid sequence, subunit composition, and regulatory mechanisms. Voltage-gated ion channels are generally classified based on the selective conductivity of ions, such as Na⁺, Ca²⁺, and K⁺ channels, whereas ligand-gated ion channels are classified on the basis on the primary signaling transmitter, such as acetylcholine, 5-hydroxy tryptamine (5-HT), and -amino butyric acid (GABA). Access to the ion channel pore is governed by gates, which may be opened or closed by electrical, chemical, or mechanical forces. For instance, voltage-gated channels sense transmembrane potential change, ligand-gated channels open in response to specific ligands, and cyclic nucleotide or Ca²⁺-activated channels respond to their appropriate second messengers.

Numerous studies have demonstrated that Ca²⁺ ions are a primary determinant of sperm cell function, including capacitation, progressive motility, hyperactivated motility, and acrosome reaction; accordingly, a number of Ca²⁺-permeable channels and transporters have been reported (see Table). Functional studies have suggested that N- and R-type voltage-gated Ca²⁺ channels are present in mouse sperm (Wennemuth et al, 2000). Low-voltage-activated T-type Ca²⁺ currents have also been measured in mouse spermatogenic cells (Hagiwara and Kawa, 1984). Consistent with these observations, the transcripts of both high voltage-gated (Cav1.2, 2.1, 2.3) and low voltage-gated (Cav3.1, 3.2, 3.3) Ca²⁺ channels were detected in rodent spermatogenic cells. Immunostaining studies of mature spermatozoa further confirmed the expression of these subunits along with Cav2.2 (Goodwin et al, 1997; Espinosa et al, 1999; Serrano et al, 1999; Westenbroek and Babcock, 1999; Wennemuth et al, 2000; Carlson et al, 2003). In human sperm, the expression of many voltage-gated Ca²⁺ channel subunits was documented (Park et al, 2003). In addition, mammalian sperm also express putative 6 transmembrane ion channel-like proteins, termed CatSper (cation channel of sperm), and 4 members—CatSper1, CatSper2, CatSper3, and CatSper4—have been reported (Quill et al, 2001; Ren et al, 2001; Lobley et al, 2003).

Although many Ca²⁺ channel subunits exist in sperm, their precise contributions to sperm physiology remain largely undefined. Except for CatSper, the expression of most Ca²⁺ channel subunits is not confined to sperm alone. Knockout studies revealed that mice lacking Cav2.2, Cav2.3, Cav3.1 subunits are viable and male fertile, indicating that these channels may not be essential for sperm behavior and they may function redundantly (Saegusa et al, 2000; Kim et al, 2001; Beuckmann et al, 2003). In the case of Cav1.2 and Cav2.1, the early lethal phenotypes of mice lacking these channels preclude analyses of their contributions to sperm behavior (Jun et al, 1999; Seisenberger et al, 2000). Knockout studies have revealed that CatSper1 and CatSper2 are specifically required for sperm motility and male fertility (Ren et al, 2001; Quill et al, 2003).

Besides Ca²⁺ channels, other cation channels are also expressed in sperm (see Table). Preliminary evidence for the involvement of K⁺ channels in sperm-volume regulation and capacitation processes have emerged from studies with nonselective blockers (Munoz-Garay et al, 2001; Yeung and Cooper, 2001). The presence of K⁺ channels in human and rat sperm was revealed by electrophysiological studies of artificially reconstituted membranes (Chan et al, 1997). However, the molecular identity of these channels in mammalian testis and sperm has been limited thus far. Only few K⁺ channels or currents have been reported. These include a delayed rectifier K⁺ channel expressed in rat testis (Jacob et al, 2000), a pH-sensitive K⁺ channel (slo3) in mouse and human spermatocyte (Schreiber et al, 1998), and an inwardly rectifying K⁺ current (Munoz-Garay et al, 2001). The expression of transient receptor potential (TRP) channels and cyclic nucleotide-gated (CNG) channels has also been detected in both spermatogenic cells and mature sperm (Weyand et al, 1994; Jungnickel et al, 2001; Castellano et al, 2003). TRP channels were shown to play a role in sperm motility and acrosome reaction. CNG channels, present on the flagellum, may serve as a Ca²⁺ entry pathway in sperm (Wiesner et al, 1998).

Sperm Ion Channels as Drug Targets

Although there is increasing evidence that ion channels are fundamental to sperm physiology, how these integral membrane proteins influence sperm function remains to be fully understood. During the past decade, diverse ion channels in sperm have been investigated using a variety of methodologies, including fluorescence measurements, immunohistochemistry, and, to a lesser extent, by pharmacological and electrophysiological approaches, particularly patch clamp measures (reviewed in Darszon et al, 1999). The critical roles of ion channels in sperm function, together with the observation that certain subunits and/or subtypes showed restricted expression on sperm, offers an attractive premise for drug targeting (Ren et al, 2001; Quill et al, 2003). Selective inhibitors of ion channel function could, in principle, inhibit sperm function and prevent fertilization. Indeed, there have been a few examples where ion channel modulators have been found to influence contraception in humans. For instance, nifedipine, a commonly prescribed Ca²⁺ channel blocker in the treatment of high blood pressure and migraine, was shown to have reversible contraceptive effects (Benoff et al, 1994). Male patients on nifedipine experience difficulties in fertility but are able to initiate a pregnancy in about 3 months after switching to a different antihypertensive drug that is not an ion channel blocker (Hershlag et al, 1995). Gossypol and other compounds isolated from Chinese medicine, traditionally used as male contraceptives, also inhibit Ca²⁺ channels in mouse spermatogenic cells and acrosome reaction, suggesting that the inhibition of Ca²⁺ channels might be a general mechanism of the antifertility effects of these agents (Shi et al, 2003). The sections below describe current understanding of functional roles of select ion channels in the context of sperm physiology and hurdles in pursuing these ion channels as targets for the development of male contraceptive drugs.

Function Roles of Ion Channels in Sperm

     Volume Regulation— Spermatozoa produced in the testis have the ability to regulate their volume to cope with the dramatic changes in the environmental osmolality during their journey from epididymis to female reproductive tract. The epididymal fluid provides sperm with a unique milieu of high osmolality (approximately 340 mOsmol/kg) compared with that in testicular fluid due to increased K⁺ concentration and organic molecules in the epididymal fluid (Hinton et al, 1981). This increase in osmolality induces uptake of osmolytes by sperm to counteract cell shrinkage. Consequently, sperm cells have a high intracellular concentration of osmolytes at the time of ejaculation. However, the osmolality in the female tract is only 280-290 mOsmol/kg and, therefore, sperm are subjected to abrupt hypo-osmotic challenge upon ejaculation. To counteract the risk of cell swelling, sperm have developed a mechanism to efficiently efflux certain osmolytes accumulated in the epididymis (Yeung et al, 2003).

In an in vitro study, human sperm incubated in medium containing L-carnitine, myo-inositol, and taurine did not change volume, while the presence of glutamate and K⁺ in the medium decreased the efficiency of forward progression of sperm, which is indicative of volume increase (Yeung et al, 2003). These observations suggested that glutamate and K⁺, rather than small organic molecules, are potential osmolytes pumped out by human sperm to regulate volume. In a separate study with mouse sperm, K⁺ and glutamate as osmolytes for volume regulation was confirmed (Yeung et al, 2004). However, in contrast with human sperm, it was observed that taurine, L-carnitine, and myo-inositol were also effective osmolytes.

Volume regulation in the female tract is crucial for sperm function, as demonstrated by studies with c-ros tyrosine kinase knockout mice (Yeung et al, 1999, 2002). Mice lacking c-ros were found to be male sterile due to defects in sperm-volume regulation, leading to swelling and tail angulations, thereby making sperm unable to penetrate and migrate through the mucus. The importance of volume regulation in human sperm was confirmed in a separate study where an increase in sperm cell volume resulted in decreased efficiency of forward progression (Yeung et al, 2003).

Studies with quinine, a nonselective ion channel blocker of voltage-gated and Ca²⁺-activated K⁺ channels, revealed the importance of K⁺ channels in sperm-volume regulation (Petrunkina et al, 2001; Yeung and Cooper, 2001; Yeung et al, 2002, 2003). In the presence of quinine, ejaculated sperm incubated in medium with lower osmolality failed to adjust volume and the size of sperm increased as measured by flow cytometry. The increase in sperm volume was accompanied by reduced straight-line velocity and linearity of the swim path but increased lateral head displacement and curvilinear velocity, as measured by computer-aided sperm analysis. Consequently, the fat sperm had marked reduction of mucus penetration and migration. This effect of quinine on sperm volume and kinetics was reduced or abolished by K⁺ ionophores, such as valinomycin and gramicidin, but largely preserved in a Ca²⁺-free medium (Yeung and Cooper, 2001). Another K⁺ channel blocker, 4-aminopyridine, also mimicked the effects of quinine. In contrast, other K⁺ channel blockers, such as tetraethylammonium chloride (TEA), margatoxin, and charybdotoxin, did not affect sperm volume and kinetics (Yeung and Cooper, 2001). It should be noted that, besides K⁺ channels, quinine can also influence volume-sensitive anion channels and, thus, the possibility that anion channels might also participate in sperm-volume regulation cannot be eliminated (Yeung and Cooper, 2001; Petrunkina et al, 2004).

As discussed in the preceding paragraphs, the importance of sperm volume regulation in fertility, inferred from c-ros knockout mice and pharmacological studies, suggests that the ion channels responsible for volume regulation may serve as targets for the development of male contraceptive medicine. In principle, inhibitors of these channels could block the efflux of sperm osmolytes in the female tract, leading to morphologic changes that prevent the ability of sperm to travel. However, the precise molecular identities of ion channels involved in this process need to be elucidated.

     Sperm Capacitation— The induction of sperm acrosome reaction requires Ca²⁺ entry through low voltage-activated Ca²⁺ channels (Arnoult et al, 1996a). These channels are thought to be unavailable for opening in uncapacitated sperm due to voltage-dependent steady-state inactivation (Arnoult et al, 1999). This inhibition can be relieved by hyperpolarization of the sperm membrane potential during the sperm capacitation process. Membrane hyperpolarization depends on activation of certain K⁺ channels (Arnoult et al, 1999), the molecular nature of which remains to be established. Darszon and colleagues described a novel K⁺-selective inwardly rectifying current in mouse spermatogenic cells that can be effectively blocked by addition of Ba²⁺ to the external solution (Munoz-Garay et al, 2001). Interestingly, application of Ba²⁺ during capacitation inhibited sperm hyperpolarization and decreased the subsequent acrosome reaction induced by zona pellucida. These data implied that opening of an inwardly rectifying K⁺ channel might be responsible for hyperpolarization during capacitation. The finding that activation of this K⁺ channel can also be modulated by pH_i led to the speculation that this channel may be activated during sperm capacitation when pH_i increases, thus enhancing K⁺ permeability leading to hyperpolarization. However, it remains to be explored whether this K⁺ channel is identical to that responsible for membrane-potential change during the capacitation process in human sperm.

     Progressive Motility— Sperm must swim long distances in the female reproductive tract to reach the site of fertilization. Newly formed spermatozoa that just leave the testis are incapable of progressive motility and consequently are unable to fertilize an egg. They gradually gain progressive motility during transit through the epididymis. Ca²⁺ is vital for the activation of sperm motility and, accordingly, several studies have been undertaken to identify Ca²⁺ regulation pathways. Of the various voltage-gated channel subunits, Cav2.3 may play a role in sperm motility (Sakata et al, 2002). The expression of Cav2.3 was detected along the dorsal and ventral sides of the proximal segment of the principal piece of mouse sperm, suggesting that Cav2.3 may control Ca²⁺ influx necessary for asymmetric flagella movement (Westenbroek and Babcock, 1999). Consistent with this idea, a computer-assisted sperm-motility assay revealed that straight-line velocity and linearity were greater in Cav2.3^-/- sperm than those in Cav2.3^+/+ sperm (Sakata et al, 2002). However, this phenotype of the Cav2.3 channel deficiency was evident only under certain in vitro conditions, and male mice lacking Cav2.3 were fertile (Saegusa et al, 2000), indicating that Cav2.3 might not be essential for sperm motility.

Members of the more recently identified transient receptor potential (TRP) channel family have been reported to play a role in the regulation of flagella motility (Castellano et al, 2003). Immunolocalization studies revealed expression of TRPC1, 3, 6, and 7 on the surface of the flagellum of human sperm. Furthermore, TRP channel blockers, such as SKF96365, inhibit sperm motility in a concentration-dependent fashion, suggesting that TRP channels can influence sperm motility.

In many species, sperm travel directionally in the female reproductive tract to reach the site of fertilization. Recent studies have revealed that the Drosophila homolog of PKD2, which in humans encodes a Ca²⁺-activated, nonselective cation channel with 6-membrane domains, might be involved in this directional movement (Gao et al, 2003). Targeted disruption of PKD2 gene resulted in nearly complete male sterility without affecting spermatogenesis in the Drosophila. The mutant sperm were motile but unable to reach the female storage organ. Rare mutant sperm that indeed reached the storage organs were able to fertilize the egg. In humans, PKD2 is expressed on primary cilia of renal tubules, where it is an important component of the mechanosensor that senses tubular fluid flow and responds by inducing Ca²⁺ influx from intracellular stores. Mutations in PKD2 genes cause polycystic kidney disease. Considering that sperm tail is akin to cilia, it is possible that PKD2 cation channel may sense directional cues in the female reproductive tract and facilitate directional movement of sperm. Consistent with this notion, male infertility was observed in patients with polycystic kidney disease (Van der Linden et al, 1995). Should PKD2-related proteins in humans play a significant role in directional movement, as hypothesized, inhibitors of this family of cation channels would be appealing for developing male contraceptive agents.

     Hyperactivated Motility— Penetration through the gelatinous zona pellucida layer of the oocyte requires sperm to swim in a hyperactivated state at the time and site of fertilization. Sperm attain the power of hyperactivated motility during the capacitation process within the female reproductive tract (Yanagimachi, 1994). Several studies indicated that Ca²⁺ not only serves a role in motility but is also a key regulator in the initiation and maintenance of sperm hyperactivation (Yanagimachi, 1982; Suarez et al, 1987). The Ca²⁺ concentration in the cytoplasm increases during hyperactivation, and this regulates the movement of axoneme (Lindemann and Goltz, 1988).

In bovine sperm, hyperactivation can be induced rapidly by thapsigargin, an inhibitor of Ca²⁺-ATPase, in the absence of external Ca²⁺ (Ho and Suarez, 2001). Because thapsigargin depletes intracellular stores and thereby elevates cytoplasmic Ca²⁺ levels, this observation suggests that the release of Ca²⁺ from internal Ca²⁺ stores is critical to hyperactivated motility. Several potential internal calcium stores exist in sperm, including the redundant nuclear envelope (RNE), mitochondria, and acrosome. Of these, the RNE and mitochondria were found in the neck region, where flagella beating initiates. Blocking Ca²⁺ efflux from mitochondria does not inhibit hyperactivated motility, implying that Ca²⁺ release from RNE, rather than mitochondria, may trigger hyperactivated motility (Ho and Suarez, 2003). Immunostaining studies revealed the presence of inositol 1,4,5-trisphosphate receptor (IP₃R) in the RNE, suggesting that IP₃R might mediate the release of Ca²⁺. However, it remains to be explored whether this mechanism holds true in humans.

The increase of Ca²⁺ concentration can result from either efflux of stored Ca²⁺ from membrane-bounded internal stores or influx through the plasma membrane. One candidate gene potentially responsible for the influx of extracellular Ca²⁺ is CatSper1 (Ren et al, 2001). Immunocytochemistry studies revealed the expression of CatSper1 specifically in the plasma membrane above the fibrous sheath in the principal piece of the sperm tail but not in the middle piece or sperm head (Ren et al, 2001). Targeted disruption of CatSper1 gene led to male sterile phenotype in otherwise normal mice. While sperm counts, shape, and mating behavior of mutant mice were indistinguishable from those of wild type, the mutant sperm displayed reduced basal velocity and lacked vigorous beating and bending in the tail region. Further studies revealed the lack of hyperactivated motility in these mutant sperm (Carlson et al, 2003). In vitro fertilization assays revealed that mutant sperm could not fertilize eggs with intact zona pellucida layer but could fertilize eggs whose outer layers had been enzymatically removed, suggesting that CatSper1^-/- mice retained the ability for egg activation but could not penetrate the egg outer layer due to impaired hypermotility (Ren et al, 2001). These findings demonstrated that CatSper1 is required for hyperactivated motility needed for sperm to penetrate the zona pellucida.

CatSper1 represents a unique class of putative ion channel-like proteins with 6 transmembrane segments, akin to voltage-gated K⁺ channels (Ren et al, 2001). However, the ion-selectivity pore domain sequence is closest to a single domain of the 4-repeat voltage-gated Ca²⁺-selective channels. As is characteristic for voltage-gated channels, the fourth transmembrane segment of CatSper1 has positively charged amino acids interspersed between every 3 amino acids. Depolarization could evoke Ca²⁺ increase in CatSper1^+/+ sperm but not in CatSper1^-/- sperm (Carlson et al, 2003). CatSper1 also contains a remarkable abundance of histidine residues in its amino terminus that might be involved in the pH regulation of sperm motility. Examination of intracellular Ca²⁺ dynamics in the sperm tail using Ca²⁺ indicators revealed that neither cAMP nor cGMP elicited a significant Ca²⁺ influx in CatSper1^-/- sperm, in contrast with the wild-type sperm, suggesting that CatSper1 may participate in cyclic nucleotide-stimulated Ca²⁺ influx (Ren et al, 2001). Because CatSper1 lacks the putative cyclic-nucleotide-binding region, it is unclear whether cyclic nucleotides gate directly to facilitate the opening of CatSper1 channel or may involve indirect mechanisms.

In agreement with the observed phenotype of CatSper1 knockout mice, a study in humans revealed significantly reduced expression of CatSper1 among subfertile men with deficient sperm motility (Nikpoor et al, 2004) and further supports the notion that selective CatSper1 inhibitors may attenuate or curtail sperm motility and consequently impair fertilization. The restricted localization of CatSper1 in mature sperm implies that a selective blocker should not affect other tissues, thus minimizing any potential side effects. This notion is supported by the normal development and behavior of the mutant mice (Ren et al, 2001).

Although CatSper1 was validated based on knockout studies, the biophysical and pharmacological properties of this target yet remain elusive. Attempts to measure whole-cell currents of both wild-type and mutant sperm under a variety of conditions and configurations were unsuccessful, in part due to the poor accessibility of sperm to conventional electrophysiology methodologies (Ren et al, 2001). The functional expression of recombinant CatSper1 has not been yet accomplished in heterologous expression systems, including Xenopus oocytes, HEK293 and CHO-K1 cells. No currents could be elicited by changes in voltage, pH, osmolality, and/or cyclic nucleotide concentrations, although the expression of protein itself could be detected (Ren et al, 2001). The failure of functional expression of CatSper1 in these cell types may be attributed to the lack of accessory proteins necessary for a putative ion channel complex or alternatively due to the lack of structural attributes of the sperm principal piece for their functional organization.

Other proteins with homology to CatSper1, viz, CatSper 2, 3, and 4, have been identified (Quill et al, 2001; Lobley et al, 2003). Like CatSper1, these proteins all contain a single ion transport domain comprised of 6 transmembrane spanning regions, where the fourth transmembrane region resembles a voltage sensor and a pore between transmembrane regions 5 and 6. CatSper2 protein is also localized in the sperm flagellum. Mice lacking CatSper2 were also male sterile, which was attributed to the absence of hyperactivated motility needed for penetration of the extracellular matrix of the egg (Quill et al, 2003). Interestingly, a genetic linkage study demonstrated the involvement of CatSper2 in asthenoteratozoospermia, a human male infertility disease characterized by poor sperm motility (Avidan et al, 2003). These observations point to the notion that CatSper2, like CatSper1, may serve as another drug target. High levels of expression of CatSper3 and CatSper4 were reported in the testis (Lobley et al, 2003) and, accordingly, it would be of considerable interest to understand the role of these novel subunits in sperm function and male fertility.

Like CatSper1, attempts to measure CatSper2 activity in sperm and functional expression of CatSper2 in heterologous systems were unsuccessful (Quill et al, 2001). Considering the similar structure of CatSper1 and CatSper2 and that these 2 channels are both involved in sperm hyperactivated motility, it is possible that they may interact directly or indirectly to form a functional oligomer. Indeed, coiled-coil protein-protein interaction domains were identified in the C-terminal tails of each of the CatSper subunits. However, coexpression of CatSper1 and CatSper2 in mammalian cells failed to yield a functional channel and coimmunoprecipitation of these 2 proteins failed to demonstrate any direct physical interaction (Quill et al, 2001). It may be speculated that specific combination of all 4 CatSper proteins may be required to form a functional channel or additional unidentified accessory proteins are required to facilitate generation of a functional complex either with individual CatSper proteins or a combination thereof.

     Acrosome Reaction— Sperm acrosome reaction is initiated by binding of ZP3, a glycoprotein component of the zona pellucida, to the sperm head. ZP3 binding causes sperm to undergo rapid membrane depolarization. Several studies indicated that the initial membrane depolarization relies on the activation of a poorly selective cation channel, which is likely to be a rapidly activating nicotinic acetylcholine receptor (nAChR) (Florman, 1994; Arnoult et al, 1996b; Bray et al, 2002b; Son and Meizel, 2003). It was observed that acetylcholine and nicotine can initiate the acrosome reaction in capacitated human and mouse sperm and selective antagonists of 7 nAChR subtype inhibit acrosome reaction initiated by both acetylcholine and ZP3. Further studies suggest that 7 nAChR may play a role upstream from the sustained Ca²⁺ influx and is a candidate as the cation channel involved in the initial membrane depolarization (Son and Meizel, 2003). Besides cation channels, Cl^- flux via the sperm glycine receptor/Cl^- channel may also be involved in the early signal transduction cascades associated with ZP3-initiated acrosome reaction (Bray et al, 2002a).

The initial membrane depolarization leads to the opening of voltage-gated and other types of Ca²⁺ channels sequentially (as discussed below), resulting in the rise of intracellular Ca²⁺ levels. Ca²⁺ influx is essential for the onset of the acrosome reaction because it was shown that the induction of acrosome reaction was greatly attenuated by the reduction of extracellular Ca²⁺ (Singh et al, 1978). Besides, Ca²⁺ channel antagonists disrupted the acrosome reaction (Kazazoglou et al, 1985; Florman et al, 1992). By probing intracellular Ca²⁺ concentration with ion-selective fluorescent dyes, 2 phases of ZP3-evoked Ca²⁺ influx were observed (Florman, 1994; Arnoult et al, 1999). The first phase is a transient Ca²⁺ increase that occurs during initial seconds of ZP3 signaling followed by a second phase of sustained Ca²⁺ influx.

During the initial transient Ca²⁺ increase phase, the response has a time course of activation and pharmacological sensitivity anticipated of low voltage-activated T-type Ca²⁺ channels (Arnoult et al, 1999). Electrophysiological studies revealed that T-type current is the only functional voltage-gated Ca²⁺ current recorded from mouse spermatogenic cells, even at the elongating spermatid stage (Arnoult et al, 1996a). Blockade of T-type Ca²⁺ channels during gamete interaction inhibits zona pellucida-dependent Ca²⁺ elevations, as demonstrated by ion-selective fluorescent probes, and this process also inhibits acrosome reaction. The involvement of T-type Ca²⁺ channels in acrosome reaction is also supported by studies with toxins from the scorpion, Parabuthus granulatus, kurtoxin-like I and II (Lopez-Gonzalez et al, 2003). Both toxins decreased T-type Ca²⁺ channel activity in mouse spermatogenic cells and inhibited acrosome reaction in mature sperm. These indicate that the activation of T-type Ca²⁺ channels is responsible for the initial Ca²⁺ influx during acrosome reaction. In human sperm, all three T-type Ca²⁺ channels are present in the sperm head (Trevino et al, 2004). Recent characterization of Ca²⁺ currents in spermatogenic cells derived from Cav3.1^-/- mice suggest that Cav3.2 may serve as a major T-type Ca²⁺ channel involved in acrosome reaction (Stamboulian et al, 2004).

The influx of Ca²⁺ through the T-type Ca²⁺ channels is transient and usually takes effect within less than 500 milliseconds. However, the Ca²⁺ response to zona pellucida is prolonged and the acrosome reaction occurs some minutes later. The T-type channel is unlikely able to support a sustained Ca²⁺ response and a secondary Ca²⁺ influx mediated by other channels must occur for the acrosome reaction. Two closely-related mechanisms are involved in the second sustained Ca²⁺ influx—an efflux of Ca²⁺ from acrosome followed by influx of Ca²⁺ through the plasma membrane (Jagannathan et al, 2002). In the first mechanism, the binding of zona pellucida on the sperm head triggers the production of IP₃, which activates the IP₃ receptors expressed on the acrosomal membrane, leading to the efflux of Ca²⁺ from acrosome to the cytosol and eventual depletion of this internal Ca²⁺ store. This scenario is supported by the observation that thapsigargin, an inhibitor of the Ca²⁺ pump, could trigger acrosome reaction (Dragileva et al, 1999; O'Toole et al, 2000). Thapsigargin can attenuate the transport of Ca²⁺ into the acrosome but has no effect on the passive efflux of Ca²⁺ from the acrosome. This essentially mimics the effect of activation of IP₃ receptor, resulting in the depletion of Ca²⁺ from the acrosome.

Mobilization of Ca²⁺ from internal stores is not sufficient to initiate acrosome reaction, considering that acrosome reaction cannot be induced in the absence of extracellular Ca²⁺. Rather, the depletion of the internal Ca²⁺ store triggers activation of store-operated Ca²⁺ channels localized in the plasma membrane, leading to sustained Ca²⁺ entry from extracellular medium and the acrosome reaction (O'Toole et al, 2000). Treatment of mouse spermatozoa with an antibody directed against TRPC2 inhibited the sustained Ca²⁺ influx induced by zona pellucida. Concurrently, the zona pellucida-induced acrosome reaction was reduced by more than 75% (Jungnickel et al, 2001). This finding not only demonstrates the pivotal importance of the sustained component of Ca²⁺ influx for the acrosome reaction but also suggests TRPC2 as the store-operated Ca²⁺ channel mediating the sustained Ca²⁺ influx. However, mice lacking TRPC2 are fertile, suggesting a possible redundant role for other TRPC channels expressed in mouse sperm (Leypold et al, 2002; Stowers et al, 2002). In humans, TRPC2 is a pseudogene (Vannier et al, 1999), while the expression of TRPC1, 3, 4, and 6 were detected in the sperm head, which may participate in generating sustained Ca²⁺ influx during acrosome reaction (Castellano et al, 2003).

As discussed above, acrosome reaction is required for sperm to fertilize the egg, defects of which lead to male infertility. Approximately 25% of infertile men with normal semen parameters showed disordered zona pellucida-induced acrosome reaction (Liu et al, 2001). Therefore, it could be speculated that ion channels involved in acrosome reaction may also serve as potential targets for male contraceptive agents.

Conclusions

There has been considerable progress in the field of ion channels and sperm biology over the years, with novel ion channel subunits and ion channel-like proteins emerging as potential drug targets. Although their roles need to be further defined, it is becoming clearer that ion channels, especially Ca²⁺ channels, are essential in sperm maturation, function, and fertilization, as demonstrated by pharmacological and gene knockout studies. For example, the critical roles revealed for the members of the CatSper family of proteins in sperm hyperactivated motility endow these proteins as likely drug targets, although their ion channel properties have not been thus far established. CatSpers are expressed in a sperm-specific manner and, accordingly, selective modulators may be anticipated to have minimal side effects. Molecular and pharmacological dissection of the role of ion channel subunits in sperm and their interactions with other components of the sperm motility machinery to influence sperm function and behavior is expected to continue as an emerging area of research.

To identify and advance ion channel-based contraceptive drugs, it is essential to screen and identify small molecules that are specific blockers of the target sperm ion channel. An ideal agent would block ion channels that affect posttesticular sperm function without affecting sperm production in the testis, thereby facilitating the drug to act quickly and allowing sperm to be regenerated upon withdrawal of the drug. Several robust cell-based assays to screen for ion channel blockers in high through-put fashion have recently been available along with diverse compound libraries, including those from natural sources. One potential hurdle is that ionic currents from mature sperm are often difficult to record directly by conventional patch-clamp techniques due to the small size, high differentiation, complex geometry, and motility of mammalian sperm. To circumvent this problem, one approach could be to study ion channels in spermatogenic cells based on the assumption that sperm are terminally differentiated cells and the ion-channels expressed during spermatogenesis are retained in the mature sperm. Alternatively, a defined heterologous system that faithfully expresses the ion channel of interest may serve as an appropriate cell-based assay system for compound evaluation and characterization. Known ion channel-active compound libraries may also be directly evaluated in assays tailored to measure effects of various aspects of sperm physiology, including motility, hypermotility, acrosome reaction and capacitation, albeit with lower assay through-put, and with less precise definition of ion channel target per se.

In summary, although multiple contraceptive options are available for women, recent research efforts are beginning to explore a number of approaches in the area of male contraception. An ideal ion channel-active contraceptive could be taken either by men or women to prevent fertilization, with a success rate comparable with oral contraceptives and with far fewer side effects than a hormone-based contraceptive. Current research progress in elucidation of roles of ion channels in sperm biology lends hope to the realization that targeting such mechanisms in a selective manner may yield a novel class of drugs in the contraceptive market place of tomorrow.

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