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BOR - Papers in Press, published online ahead of print October 30, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.011163
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BIOLOGY OF REPRODUCTION 68, 1348–1353 (2003)
DOI: 10.1095/biolreprod.102.011163
© 2003 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Evidence Suggesting That the Mouse Sperm Acrosome Reaction Initiated by the Zona Pellucida Involves an {alpha}7 Nicotinic Acetylcholine Receptor1

Jung-Ho Sona, and Stanley Meizel2,a

a Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, California 95616-8643


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHOD
 RESULTS
 DISCUSSION
 REFERENCES
 
The mammalian sperm acrosome reaction (AR) is essential to fertilization and is believed to be initiated in vivo by ZP3, a glycoprotein component of the egg zona pellucida (ZP). Recently, we reported the results of antagonist studies suggesting that a nicotinic acetylcholine receptor (nAChR) containing an {alpha}7 subunit ({alpha}7nAChR) plays a role in the human sperm AR initiated by recombinant human ZP3 or by acetylcholine (ACh). Here, we show that ACh can initiate the mouse sperm AR and that antagonists of the nAChR inhibit the AR initiated by ACh or by ZP obtained from ovarian oocytes (isolated heat-solubilized mouse ZP). Preincubation with three antagonists of the nAChR, {alpha}-bungarotoxin (100 nM), {alpha}-conotoxin IMI (100 nM), and methyllycaconitine (100 nM), significantly blocked AR initiation by ACh or by isolated heat-solubilized mouse ZP (P <= 0.002). Because the only nAChR subunit known to bind all three antagonists is the {alpha}7, an {alpha}7nAChR appears to be involved in the mouse sperm AR initiated by mouse ZP or by ACh. The nAChR antagonists did not inhibit the AR initiated by calcium ionophore A23187, suggesting that the role of {alpha}7nAChR is upstream from Ca2+ influx. Pertussis toxin (PTX, 100 ng/ml) did not inhibit the AR initiated by ACh, suggesting that the {alpha}7nAChR might be a candidate for the PTX-insensitive, poorly selective cation channel shown previously to play a role in ZP-initiated mouse sperm AR. These studies with mouse sperm and ovary-derived ZP strongly support our previous conclusion that activation of an {alpha}7nAChR is important to the mammalian AR initiated by the egg ZP.

acrosome reaction, fertilization, gamete biology, sperm


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHOD
 RESULTS
 DISCUSSION
 REFERENCES
 
The mammalian sperm acrosome reaction (AR), a modified form of exocytosis involving the acrosome (a single large secretory granule in the sperm head) and the overlying plasma membrane [1, 2], is necessary to many steps of fertilization [3]. Sperm respond to AR-initiating ligands after a series of cellular changes collectively called capacitation [3, 4], and ZP3, a glycoprotein of the egg envelope, the zona pellucida (ZP), is a major physiological initiator of the AR [3, 5, 6]. Binding of sperm to ZP3 results in an extracellular Ca2+-dependent increase in the concentration of free Ca2+i required for initiation of the AR [7]. One type of voltage-operated Ca2+ channel, a T-type channel, appears to play a role in the ZP-initiated mouse AR through its involvement in a transient Ca2+ influx [8]. That transient influx may then lead to increased inositol 1,4,5-triphosphate synthesis, a release of intracellular Ca2+ stores, and activation of store-operated Ca2+ channels, resulting in a sustained increase of intracellular Ca2+ and the ZP-initiated AR [8, 9]. Ca2+ enters the bovine and murine sperm in an early important step of the ZP-initiated AR via a poorly selective cation channel [10, 11]. That channel is permeable to both Na+ and Ca2+, and the cation influx it allows contributes to membrane depolarization, which leads to activation of voltage regulated Ca2+ channels.

The nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels differentially expressed throughout the central and peripheral nervous systems and in muscle and are involved in fast synaptic transmission [12, 13]. These receptors are pentamers composed of various combinations of five different types of protein subunits ({alpha}, ß, {gamma}, {delta}, and {varepsilon}). Eight different {alpha} subunits and three different ß subunits have been identified in mammalian neurons [14]. The mammalian neuronal {alpha}7nAChR subunit can form a functional nAChR homomer in expression systems and probably in vivo [15], but studies of chick embryo suggest that there may also be in vivo heteromers containing the {alpha}7 subunit [16]. In addition to their expression in neuronal and muscle cells, nAChRs are found in several somatic epithelial cell types [17], in chromaffin cells [18], and in lymphocytes [19]. In nerve and muscle, the binding of an activating ligand to the nAChR opens the channel leading to a large influx of Na+ and a smaller influx of Ca2+ and subsequent membrane depolarization [12], but recombinant mammalian homomeric neuronal {alpha}7nAChRs can also display a particularly high permeability for Ca2+ [20, 21].

The nAChR has also been reported in mammalian sperm. Binding studies with radioactive isotope-labeled ligands and antagonists detected ram and rabbit sperm nAChRs [22, 23]. Baccetti and coworkers [24] used fluorescence microscopy and {alpha}-bungarotoxin (BTX), a nearly irreversible nAChR antagonist, coupled to a fluorescent ligand and reported detection of the nAChR in human sperm (acrosomal and flagellar regions), rabbit sperm (equatorial segment, postacrosomal, and flagellar regions), and ram sperm (postacrosomal and flagellar regions). However, the human sperm localization was actually postacrosomal rather than acrosomal (Baccetti, personal communication). The sperm of some invertebrates also appear to have nAChRs [24]. Partial transcripts for the {alpha}4, {alpha}5, and {alpha}7 subunits have been detected in human testis (National Center for Biotechnology Information database, Bethesda, MD), but of those three subunits, only the {alpha}7 binds BTX [12]. Immunohistochemical studies have detected nAChRs in the germ cells and blood vessel walls of rat testis [25]. Recently, our studies with nAChR antagonists indicated that an nAChR containing an {alpha}7 subunit plays a role in the human sperm AR initiated by acetylcholine (ACh) or by a purified recombinant human ZP3 (expressed by Chinese hamster ovary cell line), but not by progesterone [26].

The objectives of the present studies were to determine whether an nAChR is also involved in the AR initiated by ZP obtained from oocytes and to determine whether a sperm {alpha}7nAChR is important to the AR of mammalian sperm other than human. Therefore, we tested the effects of nAChR antagonists on the mouse sperm AR initiated by ovary-derived isolated heat-solubilized mouse ZP or by ACh. The results of these studies suggest that a sperm {alpha}7nAChR plays a role in both.


   MATERIALS AND METHOD
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHOD
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

The following reagents were purchased from Sigma Chemical Co. (St. Louis, MO): {alpha}-bungarotoxin (BTX), methyllycaconitine citrate (MLA), fertilization-promoting peptide (P-Glu-Glu-Pro-amide), polyvinylpyrrolidone (PVP, embryo tested, average molecular weight 40 000), and Coomassie brilliant blue G. The {alpha}-conotoxin IMI trifluoroacetate (CTX IMI) and pertussis toxin (PTX) were from Calbiochem-Novabiochem (La Jolla, CA), and the Pentax fraction V bovine albumin (81-066) was from Bayer (Kankakee, IL). Paraformaldehyde and salts and metabolites used for incubation and washing media were reagent grade and were purchased from Fisher Scientific (Pittsburgh, PA), Irvine Scientific (Irvine, CA), Mallinckrodt (Paris, KY), or Sigma. Frozen ovaries from 21-day-old Swiss-Webster mice were purchased from Harlan Bioproducts for Science (Indianapolis, IN). The following materials were also purchased: 15-ml conical polypropylene centrifuge tubes (Greiner Labortechnik) from Applied Scientific (San Francisco, CA), 1.5-ml siliconized polypropylene microfuge tubes from United Scientific Product (San Leandro, CA), 35- x 10-mm polystyrene cell culture dishes from Falcon (Becton Dickinson Labware, Lincoln Park, NJ), and eight-well (10 mm) microscope slides from Erie Scientific (Portsmouth, NH). For all experiments, deionized water was further purified to 18M{Omega}-cm with a NANO-pure ion exchange system (Barnstead/Thermolyne, Dubuque, IA).

Sperm Preparation and Capacitation

Mature 8- to 10-wk-old male Swiss-Webster mice were purchased from Charles River Laboratory (Wilmington, MA) and cared for in accordance with U.S. Public Health Service guidelines for animal use. All procedures were approved in advance by the Institutional Animal Care and Use Committee of the University of California, Davis.

Mice were killed by exposure to CO2 for about 30 sec followed by cervical dislocation. The cauda epididymides and vas deferentia were removed, rinsed in PBS (pH 7.4), and equilibrated in a tissue culture dish in 2 ml of Whittingham medium containing 30 mg/ml of BSA for 3 min at 37°C in an incubator with a moist 5% CO2/95% air atmosphere. Following equilibration, the tissues were minced and further incubated under the same conditions for 15 min to allow release of sperm into the medium. The tissue was then removed from the sperm-rich medium, and the sperm were incubated for 2 h to permit capacitation. All sperm samples were treated with 250 nM of fertilization-promoting peptide at the beginning of the 2-h capacitation period to increase capacitation and decrease spontaneous AR [27]. After completion of 2-h capacitation, sperm were washed twice with 12 ml of wash medium (Whittingham medium containing 3 mg/ml BSA) at 300 x g for 5 min, diluted to 4 x 106/ml, and aliquoted (100 µl) into 1.5-ml siliconized microcentrifuge tubes. Usually, 20–40 x 106 sperm/ml with 75–85% motility were obtained from one mouse.

Mass Isolation of ZP from Mouse Ovaries

ZPs were prepared from frozen ovaries of 21-day-old female Swiss-Webster mice as previously described [28] using the methods of Bleil and Wassarman [29] and Leyton et al. [30]. ZPs were separated by Percoll gradient centrifugation after homogenization of 33 pairs of frozen ovaries. After recovery from the Percoll gradient, ZPs were solubilized by heating at 60°C for 60 min in PBS-PVP. Yield of heat-solubilized ZPs was quantitated by a Micro BCA protein assay kit (Pierce, Rockford, IL), and the purity of the ZP preparations was determined by SDS-PAGE followed by silver staining. Normally, three bands (ZP1, ZP2, and ZP3) were detected by silver staining, and AR assays with capacitated sperm were performed thereafter to determine whether prepared ZPs were able to initiate AR.

AR Initiation and Assay

All pretreatments of capacitated mouse sperm with antagonists and treatments with AR initiators were carried out at 37°C in a moist 5% CO2/95% air atmosphere. All additions were made to sperm suspensions aliquots (100 µl final volumes) in 1.5-ml microcentrifuge tubes.

The effects of different concentrations of ACh (2 µM, 10 µM, 50 µM, 250 µM, and 1 mM) as AR initiators were tested on capacitated mouse sperm. After additions of ACh (2 µl) or control solvent (PBS), sperm were incubated for 10 min prior to fixation for AR assay.

To test the effects of various nAChR antagonists on the initiation of the AR by ACh, heat-solubilized ZP, or calcium ionophore A23187, capacitated sperm were pretreated with BTX (100 nM), CTX IMI (100 nM), or MLA (100 nM) for 10 min. Appropriate controls were used in parallel in all experiments: PBS for ACh and BTX, sodium citrate for MLA, sodium trifluoroacetate for CTX, PBS-PVP for ZP, and dimethyl sulfoxide (DMSO, 0.01% final concentration) for A23187. After additions of ACh (250 µM), ZP (12 µg/ml), or calcium ionophore A23187 (20 µM) to pretreated sperm, incubations were continued for 10 min except for calcium ionophore A23187 (5 min). Based on a protein content of 4.8 ng/mouse ZP [31], 12 µg/ml of ZP is the equivalent of 2.5 ZP/µl of sperm suspension.

In another study, sperm were treated with PTX (100 ng/ml) for the last hour of the 2-h capacitation period, followed by ZP (12 µg/ml) or 250 µM ACh for 10 min (still in the presence of PTX) prior to fixation. Samples were coded and identified only after AR assay.

Immediately after incubations of sperm with AR initiators, 10-µl aliquots were removed from each treatment group for evaluation of motility. The percentage of motile sperm was determined by examination using phase contrast microscopy (at least 100 sperm/treatment at 400x magnification). Additionally, a subjective score was recorded for sperm motility [32] using a scale of 1 (twitching, no progressive motion) to 4 (vigorous forward motility).

The remaining 90 µl of sperm was fixed with an equal volume of 4% paraformaldehyde in PBS for 10 min at room temperature. A 10-µl aliquot of fixed sperm was placed in a slide well and dried at 37°C, and the acrosomal status of these sperm was then determined by Coomassie blue staining [33, 34]. After staining, 2 µl of Vectashield (Vector Laboratories, Burlingame, CA) was applied to prevent photobleaching. At least 100 sperm were counted at 1250x magnification for each sample.

Statistical Analysis

All AR percentage data were transformed to the arcsine of their square roots [35]. The Duncan new multiple-range test (SAS Institute, Cary, NC) was used for the comparison of group mean differences.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHOD
 RESULTS
DISCUSSION
 REFERENCES
 
Studies with ACh and AChR Antagonists

To determine whether ACh would initiate the mouse sperm AR, capacitated sperm were treated for 10 min with various concentrations of ACh (2 µM to 1 mM). There was no significant initiation of the AR at concentrations up to 50 µM, but 250 µM and 1 mM ACh significantly initiated the AR compared with a solvent control (Fig. 1). Because AR initiation at 1 mM was not significantly greater than that at 250 µM (P > 0.05), the lower concentration was used in antagonist studies. The addition of ACh to uncapacitated mouse sperm did not result in significant AR initiation (n = 3; PBS: 4.73 ± 1.04; ACh: 6.53 ± 0.50; P > 0.141). A similar lack of response was obtained previously when ACh was incubated with uncapacitated human sperm [26].



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  		FIG. 1. Effect of ACh on the mouse sperm AR. Capacitated sperm (100-µl aliquots of 4 x 106 sperm/ml) were incubated with various concentrations of ACh (2 µM to 1 mM) for 10 min in PBS or with PBS alone. Each group value represents the mean ± SEM percentage AR of three experiments. Means with different superscripts among treatments are significantly different (P < 0.001)

To determine whether AR initiation by ACh involved an nAChR, we tested the effects of several nAChR antagonists: BTX, an antagonist of several nAChRs including {alpha}7nAChRs [12]; CTX IMI, a highly selective antagonist of {alpha}7nAChRs [36]; and MLA, a relatively selective antagonist of {alpha}7nAChRs [37, 38]. Preincubation of capacitated sperm with BTX, CTX IMI, and MLA for 10 min significantly blocked the AR initiated by ACh (Fig. 2).



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  		FIG. 2. Effect of nAChR antagonists on the ACh-initiated AR. A) Effect of BTX on the mouse sperm AR initiated by ACh. Capacitated sperm (100-µl aliquots of 4 x 106 sperm/ml) were preincubated with BTX (100 nM) for 10 min followed by a further 10-min incubation with ACh (250 µM) in PBS or with PBS alone. Bars represent the mean ± SEM percentage AR of four experiments. Means with different superscripts among treatments are significantly different (P < 0.002). B) Effect of CTX IMI on the mouse sperm AR initiated by ACh. Capacitated sperm (100-µl aliquots of 4 x 106 sperm/ml) were preincubated with CTX IMI (100 nM) as a trifluoroacetate (TFA) salt in PBS or with Na TFA in PBS or with PBS alone for 10 min followed by a further 10-min incubation with ACh (250 µM) in PBS or with PBS alone. Bars represent the mean ± SEM percentage AR of three experiments. Means with different superscripts among treatments are significantly different (P < 0.011). C) Effect of MLA on the mouse sperm AR initiated by ACh. Capacitated spermatozoa (100-µl aliquots of 4 x 106 sperm/ml) were preincubated with MLA (100 nM) as a citrate salt in PBS or with sodium citrate (Na Cit) in PBS or with PBS alone for 10 min followed by a further 10-min incubation with ACh (250 µM) in PBS or with PBS alone. Bars represent the mean ± SEM percentage AR of four experiments. Means with different superscripts among treatments are significantly different (P < 0.001)

Studies with Isolated Heat-Solubilized ZP and nAChR Antagonists

To determine whether AR initiation by mouse ZP involved an nAChR, we tested the effects of BTX, CTX IMI, and MLA. Preincubation of capacitated mouse sperm with BTX, CTX IMI, or MLA at 100 nM for 10 min significantly blocked the AR initiated by isolated heat-solubilized ZP (Fig. 3).



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  		FIG. 3. Effect of nAChR antagonists on the ZP-initiated AR. A) Effect of BTX on the mouse sperm AR initiated by isolated heat-solubilized ZP. Capacitated spermatozoa (100-µl aliquots of 4 x 106 sperm/ml) were preincubated with BTX (100 nM) in PBS or with PBS alone for 10 min followed by a further 10-min incubation with ZP (12 µg/1 ml) in PBS-PVP or with PBS-PVP alone. Bars represent the mean ± SEM percentage AR of five experiments. Means with different superscripts among treatments are significantly different (P < 0.001). B) Effect of CTX IMI on the mouse sperm AR initiated by isolated heat-solubilized ZP. Capacitated spermatozoa (100-µl aliquots of 4 x 106 sperm/ml) were preincubated with CTX IMI (100 nM) as a trifluoroacetate (TFA) salt in PBS or with Na TFA in PBS for 10 min followed by a further 10-min incubation with ZP (12 µg/ml) in PBS-PVP or with PBS-PVP alone. Bars represent the mean ± SEM percentage AR of four experiments. Means with different superscripts among treatments are significantly different (P < 0.001). C) Effect of MLA on the mouse sperm AR initiated by isolated heat-solubilized ZP. Capacitated spermatozoa (100-µl aliquots of 4 x 106 sperm/ml) were preincubated with MLA (100 nM) as a citrate salt in PBS or with Na citrate (Na Cit) in PBS or with PBS alone for 10 min followed by a further 10-min incubation of ZP (12 µg/ml) in PBS-PVP or with PBS-PVP alone. Bars represent the mean ± SEM percentage AR of four experiments. Means with different superscripts among treatments are significantly different (P < 0.001)

Other AR Studies

To test whether inhibition of the mouse AR by nAChR antagonists is specific to initiation by ACh or ZP and whether events downstream from Ca2+ entry are inhibited, we examined the effects of BTX, CTX IMI, and MLA on the AR initiated by calcium ionophore A23187. Preincubation of capacitated mouse sperm with BTX, CTX IMI, or MLA at 100 nM for 10 min did not significantly inhibit the calcium ionophore-initiated AR (Fig. 4).



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  		FIG. 4. Effect of nAChR antagonists on the AR initiated by calcium ionophore A23187. Capacitated spermatozoa (100-µl aliquots of 4 x 106 sperm/ml) were preincubated with 100 nM MLA, BTX, or CTX IMI in PBS for 10 min followed by a further 5-min incubation with calcium ionophore A23187 (20 µM) in DMSO or with DMSO (0.01% final concentration) alone. Bars represent the mean ± SEM % AR of three experiments. Means with different superscripts among treatments are significantly different (P < 0.001)

Because a PTX-insensitive, poorly selective cation channel plays a role in the ZP-initiated AR and because PTX inhibits the ZP-initiated AR [6], the effect of PTX on the ACh-initiated AR was tested. Preincubation of mouse sperm with PTX during the last hour of capacitation and during AR initiation (100 ng/ml) did not inhibit the ACh-initiated AR (Fig. 5). As a control in the same experiments, we tested the effect of PTX on the ZP-initiated AR and found that the PTX did inhibit the ZP-initiated AR (Fig. 5).



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  		FIG. 5. Effect of PTX on mouse AR initiated by isolated heat-solubilized ZP. Capacitated spermatozoa (100-µl aliquots of 4 x 106 sperm/ml) were preincubated with PTX (100 ng/ml) in PBS for 1 h followed by a further 10-min incubation of ACh (250 µM) in PBS or ZP (12 µg/ml) in PBS-PVP or PBS-PVP alone. Bars represent the mean ± SEM percentage AR of three experiments. Means with different superscripts among treatments are significantly different (P < 0.001)

Motility

Motility ranged from 71% to 83% in different experiments and was the same in each tube in any one experiment, except that A23187-treated sperm displayed a weaker flagellar beat. The nAChR antagonists, PTX, and their controls had no significant effect on the percentage of motile sperm nor any obvious effect on the intensity of motility (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHOD
 RESULTS
 DISCUSSION
REFERENCES
 
We previously reported that ACh initiated the human sperm AR [26]. In those human sperm studies, the optimal AR was significantly initiated by 10, 50, and 250 µM ACh. In the present studies, ACh concentrations <250 µM did not significantly initiate the mouse sperm AR. Putative differences in the affinities of mouse and human sperm nAChRs for ACh may account for the differences in concentration effects. Another possible explanation might be the postcapacitation presence in the two media of different amounts of acetylcholinesterase, an enzyme that destroys ACh. This enzyme is found in mammalian sperm [39, 40] and could leak from dead sperm during capacitation.

BTX, CTX IMI, and MLA are competitive nAChR antagonists, although inhibition by BTX is almost irreversible [12, 41, 42]. These three antagonists at 100 nM (and in some cases even at lower concentrations) significantly inhibited the human sperm AR initiated by ACh or by a purified recombinant human ZP3 [26]. In the present studies, each antagonist at 100 nM (the only concentration tested) inhibited the mouse AR initiated by ACh or by mouse ovarian ZP. Such concentrations also antagonize neuronal {alpha}7nAChRs [37, 41, 43–45]. BTX binds to the nAChR {alpha}1, {alpha}7, {alpha}8, and {alpha}9 subunits [12, 36]. MLA is considered to be a selective {alpha}7nAChR antagonist [18], but this antagonist might also bind to an nAChR containing {alpha}3 and {alpha}6 subunits [37]. CTX IMI is a highly selective antagonist of {alpha}7nAChRs [36]. The specificity of each of the antagonists that inhibited the ACh- and ZP-initiated mouse AR suggests that they act on {alpha}7nAChR (i.e., only the {alpha}7 subunit binds all three antagonists). Similar antagonist studies have suggested that {alpha}7nAChR plays a role in exocytosis by chromaffin cells [18]. The {alpha}7 subunits of rat brain nAChRs can be present as functional nAChR homomers [15], but we cannot rule out the presence of additional nAChR {alpha} subunit types in the sperm {alpha}7nAChR.

Micromolar concentrations of BTX can inhibit human sperm motility [46]. However, neither BTX, CTX IMI, nor MLA at 100 nM (an effective concentration for antagonism of neuronal nAChRs and for inhibition of the ACh- or ZP-initiated mouse AR) inhibited the percentage or intensity of mouse sperm motility. Therefore, mouse sperm nAChR containing an {alpha}7 subunit probably is not involved in those aspects of mouse sperm motility. Similar results were obtained in our studies with human sperm [26]. However, we have not examined the effects of the antagonists on hyperactivated motility in mouse or human sperm [47].

None of the three nAChR antagonists were able to significantly inhibit the AR initiated by calcium ionophore A23187, a molecule that increases Ca2+ influx without the involvement of ion channels [48]. Those results suggest that the mouse sperm AR events involving the sperm {alpha}7nAChR inhibited by the three antagonists during AR initiation by ACh or ZP were upstream from ion fluxes mediated by that receptor/channel. Activation of the sperm {alpha}7nAChR involved in the AR, like activation of neuronal {alpha}7nAChRs, probably results in an influx of Na+ and Ca2+ [12]. Homomeric {alpha}7nAChRs may exhibit more Ca2+ influx than other types of nAChRs [20, 21], but it is not yet known whether the mouse sperm {alpha}7nAChR is a homomer or a heteromer. Entry of Na+ and Ca2+ contributes to membrane depolarization and subsequent activation of voltage-gated Ca2+ channels and activation of phospholipase C, early events in the mechanism of AR initiation by the ZP [7, 8]. Cl- efflux via the glycine receptor/Cl- channel, another neurotransmitter receptor in sperm, may also contribute to membrane depolarization and resulting Ca2+ influx during the ZP-initiated AR [49]. In the present studies, A23187, by allowing a large amount of Ca2+ influx that stimulates various steps of the Ca2+-requiring AR, would bypass the need for sperm {alpha}7nAChR and thus the nAChR antagonists would have no effect. Moreover, the failure of the antagonists to inhibit the A23187-initiated AR demonstrates that their inhibition of the mouse AR initiated by ZP and ACh was not due to nonspecific membrane effects.

PTX did not inhibit the ACh-initiated AR but, as has been previously discussed [6, 7], did inhibit the ZP-initiated AR (used in the same experiments as a positive control). The inhibition of the mouse ZP-initiated AR by PTX is due to inhibition of Gi protein function, resulting in inhibition of a transient increase in intracellular pH that is needed, along with depolarization, to activate sperm voltage-gated calcium channels [6, 7, 10]. Thus, the insensitivity of mouse sperm AR initiation by ACh to PTX suggests that the intracellular pH increase was not required for that event. Direct activation of the sperm {alpha}7nAChR by added ACh may provide sufficient cation influx, and thereby depolarization, to activate voltage-gated calcium channels even without the pH effect required by the ZP.

A PTX-insensitive, poorly selective cation channel, permeable to Ca2+ [11, 50] and probably to Na+ [7], is involved in membrane depolarization and voltage-gated Ca2+ channel activation early during ZP initiation of the bovine and murine AR. As is the case for other nAChRs, the sperm {alpha}7nAChR should be permeable to Na+ and Ca2+. The fact that PTX does not inhibit the ACh-initiated AR lends some support to the possibility that sperm {alpha}7nAChR is the PTX-insensitive, poorly selective cation channel involved in these early steps of the ZP-initiated AR.

Whether or not the sperm {alpha}7nAChR and the poorly selective cation channel are identical, the present {alpha}7nAChR antagonist studies with ZP obtained from ovary-derived mouse oocytes and used as AR initiator strongly support conclusions from human sperm studies with recombinant human ZP3 [26], i.e., that an {alpha}7nAChR is important to the mammalian AR initiated by the ZP. Activation of {alpha}7nAChR during ZP initiation of the AR might involve receptor cross-talk occurring after sperm binding to one of the candidate ZP receptors for the sperm [6] and even putative sperm synthesis and release of ACh [26]. Studies are being undertaken to further characterize the structure of the sperm {alpha}7nAChR, to determine its role in sperm depolarization and cation influx, and to investigate the manner of its activation. Elucidation of the role of sperm nAChR will be important to our understanding of the initiation of the AR, an essential fertilization event.


   FOOTNOTES
 
1 This work was supported by NIH grant HD-33368 to S.M. Back

2 Correspondence: S. Meizel, Department of Cell Biology and Human Anatomy, School of Medicine, University of California, One Shields Ave., Davis, CA 95616-8643. FAX: 530 752 8520; smeizel{at}ucdavis.edu Back

Received: 9 September 2002.

First decision: 30 September 2002.

Accepted: 28 October 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHOD
 RESULTS
 DISCUSSION
 REFERENCES
 

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