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Regular Article |
a Department of Cell Biology and Human Anatomy, School of Medicine, University of California at Davis, Davis, California 95616-8643
b Zonagen, Inc., The Woodlands, Texas 77380
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 subunit of the human spinal cord GlyR or with 50 nM strychnine caused significant inhibition in the rhZP3-initated AR. Finally, studies using fura-2-loaded human sperm showed that 50 nM strychnine was also able to inhibit the Ca2+ influx associated with addition of rhZP3. These results further support the view that rhZP3 and the ZP work through the same mechanisms, show that the GlyR is involved in rhZP3-initiated AR, and suggest that the GlyR may also play a role in the early signal transduction cascades associated with ZP-initiated AR in vivo.
acrosome reaction, calcium, fertilization, glycine receptor, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A common element of the ZP-initiated AR mechanism in eutherian mammals is involvement of the Gi protein, and pertussis toxin, which is an inhibitor of the Gi protein function, inhibits the ZP-initiated AR in mammalian sperm [4]. Interestingly, pertussis toxin only inhibits the ZP-initiated AR, not the progesterone-initiated AR, of human and mouse sperm [5–9]. It has also been shown that a glycine receptor/Cl- channel (GlyR) is involved in the ZP-initiated AR, but not in the progesterone-initiated AR, of pig sperm [10]. Therefore, partially different signal transduction pathways appear to be involved in the AR initiated by these two different ligands.
Several reports suggest that the sperm GlyR is important to sperm function. Immunochemical studies have demonstrated that the GlyR is present in the periacrosomal region of pig and mouse sperm and have detected both the and ß GlyR subunits in pig sperm [11, 12]. Strychnine-binding studies have shown that the GlyR is also present in hamster at approximately 17 x 103 sites per sperm [13]. Importantly, strychnine inhibited the solubilized ZP-initiated AR of pig and mouse sperm at the same 50 nM concentration that inhibited the glycine-initiated AR of pig, human, hamster, and mouse sperm [10, 13, 14]. Furthermore, recent studies using mice with defects in their neuronal GlyR or ß subunits (spasmodic or spastic) have demonstrated that the sperm of these mice are deficient in their ability to undergo the glycine-initiated and solubilized ZP-initiated AR in vitro. Finally, a monoclonal antibody directed against the spinal cord GlyR completely blocked the ZP-initiated AR in normal mouse sperm. In view of all these findings demonstrating the importance of the sperm GlyR to the mouse and pig ZP-initiated AR, we investigated whether this is also true for human sperm.
The limited quantity and questionable quality of human ZP available for research is a significant obstacle in any study of human sperm/ZP interactions. In recent years, recombinant human ZP3 (rhZP3) has been produced from various protein expression systems as a potential substitute for solubilized ZP. Preparations of rhZP3 expressed in Chinese hamster ovary (CHO) cells stimulate both the AR and the associated essential Ca2+ influx [15, 16]. In the present study, we have used a purified rhZP3 obtained from CHO cells transfected with and expressing a modified form of the human ZP3 protein [17]. We report on the biological activity of this rhZP3, confirm that its mechanism of action is via signal transduction pathways similar to those activated by solubilized ZP, and show that the human sperm GlyR appears to be required for initiation of the AR by that recombinant molecule.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Salts and metabolites used for incubation and wash media were of reagent grade and purchased from Fisher Scientific (Pittsburgh, PA), Irvine Scientific (Irvine, CA), Mallinckrodt (Paris, KY), or Sigma (St. Louis, MO). Percoll was purchased from Sigma, concanavalin A-fluorescein isothiocyanate (ConA-FITC) from EY Laboratories (San Mateo, CA), Fura-2-AM and cell impermeant BAPTA (tetra sodium salt) from Molecular Probes, Inc. (Eugene, OR), pertussis toxin from List Biological Laboratories (Campbell, CA), pertussis toxin B oligomer from Calbiochem-Novabiochem Corp. (La Jolla, CA), fluoromount mountant Gurr from Gallard-Schlesinger Industries (Garden City, NY), and fraction V bovine albumin (Pentax no. 81-066, lot 59) from Miles (Kankakee, IL). A polyclonal antibody against a keyhole limpet hemocyanin-conjugated synthetic peptide based on the first 19 amino acids of the N-terminus of the human neuronal GlyR 1 subunit (antibody G0666) and strychnine hydrochloride were purchased from Sigma. Western blotting molecular weight standards, donkey anti-rabbit antibody horse radish peroxidase (HRP), and enhanced chemiluminescence (ECL) reagents were purchased from Amersham-Pharmacia (Piscataway, NJ). Frozen rat spinal cords were purchased from Harlan Bioproducts for Science (Indianapolis, IN). Deionized water used in these experiments was purified to 18 M
-cm with a NANO-pure ion-exchange system (Barnstead/Thermolyne, Dubuque, IA). The following materials were also purchased: 15-ml conical polypropylene centrifuge tubes (Grenier Labortechnik) from Applied Scientific (South San Francisco, CA), and 1.5-ml siliconized polypropylene microcentrifuge tubes from United Scientific Products (San Leandro, CA)
Preparation and Capacitation of Human Sperm
Protocols for human sperm studies were approved by the Human Subjects Committee at the University of California, Davis. Semen samples were obtained by masturbation from a pool of seven healthy donors. For any single study, three or more experiments were carried out, and different donors were used for each experiment. A population of >95% motile sperm was obtained by centrifugation of semen samples through a discontinuous Percoll gradient and subsequent washing in human sperm medium (HSM) as previously described [18]. For sperm to respond to AR initiators, they must first undergo a series of molecular changes collectively termed capacitation [1, 19, 20]. The final sperm suspensions were prepared by diluting the sperm to 6 x 106 sperm/ml in an HSM containing 26 mg/ml of BSA (HSM-26B) and capacitated by incubation of 0.5-ml aliquots in loosely capped, 15-ml polypropylene centrifuge tubes or by incubation of 0.2-ml aliquots in loosely capped, 1.5-ml polypropylene microcentrifuge tubes for 24 h at 37°C and 5% (v/v) CO2 [21].
Immunoprecipitation and Western Blot Analysis of the GlyR
The sperm preparations were obtained by Percoll-gradient centrifugation and washed (all solutions without BSA) as previously described [11]. The final pellet was resuspended in 500 µl (6 x 106 sperm/ml) of medium A (capacitation medium: 0.5 mM EDTA, 5 mM EGTA, 1 mM dithiothreitol, protease inhibitors [1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mM benzamidine HCl, 20 µM leupeptin, 1 µM pepstatin A, and 1 µg/ml of aprotinin], and phosphatase inhibitors [1 mM sodium fluoride, 10 mM ß-glycerophosphate, and 0.1 mM sodium orthovanadate]). The GlyR was then solubilized according to the method described by Pfeiffer et al. [22]. Basically, the sperm suspension was incubated with 2% (v/v) Triton X-100 on ice for 1 h and centrifuged at 10 000 x g for 20 min. To the supernatant was added 1 µg of affinity-purified rabbit polyclonal antibody against the 1 subunit of the human GlyR (catalog no. G0666; Sigma), and the mixture was incubated on rotation for 4 h at 4°C. To this preparation, 20 µl of 50% (v/v) protein A slurry pre-equilibrated in medium A were added, and the mixture was incubated for 1 h on rotation at 4°C. The immunocomplex was then centrifuged at 10 000 x g for 10 sec, and the pellet was washed four times with medium A by a 10-min incubation on rotation at 4°C for each wash and finally resuspended in 25 µl of 1x SDS-sample buffer. A 20-µl aliquot of the resuspended immunocomplex was loaded onto a 4–15% (v/v) polyacrylamide gel (Ready Gel; Bio-Rad, Hercules, CA), and the separated proteins were transferred onto a polyvinylidene fluoride membrane. The membrane was blocked for 2 h with 5% (w/v) dried milk powder in TBST (10 mM Tris [pH 7.4], 150 mM NaCl, and 0.05% Tween 20) and then incubated overnight at 4°C with a 1:1000 dilution of antibody G0666 (1 µg/µl). The membrane was washed three times with TBST and incubated with a 1:50 000 dilution of the donkey anti-rabbit antibody conjugated to HRP for 1 h at room temperature. The membrane was subsequently washed four times with TBST and developed using the Amersham-Pharmacia ECL kit.
Rat spinal cord extracts were prepared as a positive control. Briefly, three frozen spinal cords were thawed and homogenized using a Potter-Elvehjem tissue grinder in 500 µl of buffer A (25 mM KPO4 [pH 7.4], 280 mM sucrose, plus protease and phosphatase inhibitors as described above for sperm extracts). The spinal cord extracts were spun at 10 000 x g for 30 min, and the pellet was resuspended in buffer A. Triton X-100 extraction, immunoprecipitation, and Western blot analysis were performed as described above for sperm.
Production of rhZP3
The procedures used to express and to isolate the secreted rhZP3 have been described previously [17]. Briefly, human ZP3 cDNA was transfected and expressed in CHO cells; the expressed protein rhZP3 was engineered with an additional C-terminal, six-histidine (His6) segment (to aid purification of the protein by metal-affinity chromatography) and lacked the C-terminal transmembrane-like domain. The secreted rhZP3 was purified from the collected CHO protein-free medium by cation-exchange chromatography, followed by immobilized metal-ion chromatography on a nickel-binding column (utilizing the His6 tag). The eluted ZP3-containing fraction was then subjected to concentration and dialysis. Following determination of the concentration of the collected rhZP3 by protein microassay, aliquots (40 µl) of rhZP3 in 150 mM NaCl and 20 mM NaH2PO4, pH 7.2, were stored at -70°C until use.
AR Assay
Aliquots (50 µl) of capacitated human sperm suspensions in HSM-26B were placed in microcentrifuge tubes for AR determination. Additions of rhZP3 (final concentration, 100 µg/ml) or ZP3 buffer were made to sperm suspensions, and the treated suspensions were incubated at 37°C in 5% CO2/95% (v/v) air for 20 min. Time-course studies regarding the effect of the rhZP3 had indicated that, following this time interval, a maximum number of sperm in a sample underwent the AR (see Results). At the end of the incubation period, 5-µl aliquots were removed from each treatment group for evaluation of motility. The remaining sperm were immediately fixed in 4% (v/v) formaldehyde in PBS. The acrosomal status of the fixed sperm was then assessed using the ConA-FITC AR assay [23]. Following staining, sperm were mounted in fluoromount, and a minimum of 200 sperm from each treatment were scored in a blind fashion.
To test the effects of various inhibitors on the rhZP3-initiated AR, selected aliquots were pretreated with pertussis toxin or, as a negative control, with pertussis toxin B oligomer (100 ng/ml, added 3 h before rhZP3 addition), strychnine (50 nM, added 10 min before rhZP3), or the GlyR antibody (1 µg/ml, added 30 min before rhZP3). Appropriate solvent controls were performed in parallel in all experiments. For experiments in which extracellular Ca2+ was removed from the medium, the chelator BAPTA (7.5 mM) was added to the aliquots 2 min before the addition of rhZP3.
Motility Determinations
Aliquots of sperm (5 µl) to be used for AR or Ca2+ studies were removed immediately before and following treatment, and the motility of the sample was determined. The percentage of motile sperm was determined using phase-contrast microscopy (100–200 sperm/treatment at 400x magnification), and a subjective score was assigned to the quality of sperm motility using a scale from 1 (twitching, nonprogressive motion) to 4 (vigorous forward motility) [21].
Measurement of [Ca2+]i
Aliquots (1 ml) of capacitated sperm suspensions in HSM-26B were prepared for [Ca2+]i studies by loading the acetoxy-methyl ester of fura-2 as previously described [21]. Briefly, fura-2-AM (final concentration, 1 µM; dissolved in dimethyl sulfoxide 0.01% final concentration) was incubated with the cells for 30 min at 37°C. Excess fura-2-AM was removed from the sample by washing twice (15 ml of Hepes-buffered fura-2 medium containing 3 mg/ml of FM-3B [21]) and centrifugation at 300 x g for 10 min). Following washing, the pelleted sperm were diluted to a final concentration of 6 x 106 sperm/ml in FM-3B medium.
Measurement of [Ca2+]i was performed using an Hitachi F-2000 spectrofluorometer (Hitachi Instruments, San Jose, CA) with an excitation wavelength of 364/385 nm, an emission wavelength of 510 nm, and an excitation/emission bandpass of 5 nm as previously described [24]. Measurements were performed on 200-µl aliquots of fura-2-loaded sperm placed in quartz microcuvettes. Throughout all experiments, the samples were stirred magnetically, and the temperature was maintained at 37°C. A constant flow of 5% CO2 was passed over the top of the sample to maintain pH throughout the experiment.
Following an initial 300-sec time period to allow equilibration and establishment of a stable baseline, rhZP3 (100 µg/ml) was added. In experiments concerned with the effect of strychnine on the action of rhZP3, strychnine (50 nM) was added 5 min before the beginning of the experiment. The fluorescence emission intensity of fura-2-loaded sperm was not influenced by the addition of strychnine. Additionally, fura-2-loaded and nonloaded sperm showed no change in autofluorescence on the addition of strychnine.
Toward the end of each experiment, a calibration step was performed by the sequential addition of 20 µM digitonin and 25 mM Tris-EGTA. Calculation of [Ca2+]i was performed as described previously [25] using a dissociation constant of 285 nm for fura-2 at 37°C
Statistical Analysis
All AR percentage data were transformed to the arcsine of their square roots [26]. The Duncan new multiple-range test [27] was used for the comparison of group mean differences. When appropriate, differences were determined by Student t-test for paired data. In all studies, statistical significance was determined at P 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two bands of similar apparent molecular mass (48 kDa) were detected by immunoprecipitation of the human sperm or rat spinal cord extracts followed by Western blot analysis (Fig. 1) using an antibody against the 1 subunit of the human neuronal GlyR.
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Initiation of AR by rhZP3 but Not by rhZP1 and rhZP2
Incubation with rhZP3 significantly stimulated AR in capacitated human sperm (Fig. 2). Time-course experiments indicated that rhZP3 was able to significantly initiate the AR as early as 5 min after addition, but it was not until 20 min that a maximal level of stimulation was reached. As negative controls, we also tested recombinant versions of the human ZP glycoproteins ZPB (rhZP1) and ZPA (rhZP2) expressed by CHO cells and purified as previously described [17]. The recombinant human proteins (rhZP1 and rhZP2) did not significantly initiate the AR when assayed 20 min after the addition at 100 µg/ml to capacitated human sperm. The following results were obtained (n = 3, %AR ± SEM, P > 0.05 for rhZP1 and rhZP2 and P 0.05 for rhZP3): rhZP3, 27.5% ± 4.7%; rhZP1, 9.9% ± 0.8%; rhZP2, 12.7% ± 1.3%; and recombinant protein buffer (PBS), 9.3% ± 1.1%.
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Additionally, rhZP3 was not able to significantly initiate the AR in uncapacitated sperm at 20 min after addition. The following results were obtained (n = 3, %AR ± SEM, P > 0.05 for uncapacitated sperm and P 0.05 for capacitated sperm): rhZP3 added to uncapacitated sperm, 4.1% ± 0.9%; rhZP3 buffer added to uncapacitated sperm, 5.0% ± 0.8%; rhZP3 added to those sperm after capacitation, 19.6% ± 1.1%; and rhZP3 buffer added to those sperm after capacitation, 8.1% ± 1.4%.
The removal of extracellular Ca2+ from the medium by the addition of BAPTA significantly inhibited (P 0.05) any stimulation of the AR by rhZP3 (n = 3, %AR ± SEM): rhZP3 plus BAPTA solvent, 21.4% ± 1.4%; rhZP3 plus BAPTA, 8.1% ±1.1%; and ZP3 buffer plus BAPTA, 9.4% ± 0.4%.
The percentage of motile sperm in all samples to which recombinant proteins were added was the same as that of the solvent controls (range, 70–80%). No differences were found between the quality of motility in experimental and control tubes in any single experiment.
Effect of Pertussis Toxin
Preincubation of sperm with pertussis toxin (a classical Gi protein inhibitor, 100 ng/ml) completely inhibited the effect of rhZP3 (Fig. 3). The motility of the sperm was not altered by the addition of pertussis toxin. As a negative control, we also tested the effect of preincubating capacitated sperm for 3 h with 100 ng/ml of pertussis toxin B oligomer (enzymatically inactive component of pertussis toxin) dissolved in PBS. Pertussis toxin B oligomer did not inhibit the AR initiated by rhZP3. The following results were obtained (n = 3, %AR ± SEM): rhZP3 after 3-h preincubation with PBS, 33.2% ± 1.8%; rhZP3 after 3-h preincubation with pertussis toxin B oligomer, 31.9% ± 1.5%; and rhZP3 buffer [PBS] after 3-h preincubation with PBS, 11.7% ± 1.1%. No significant difference was found between the percentages of rhZP3-initiated AR obtained after preincubation with pertussis toxin B oligomer or after preincubation with PBS (P > 0.05).
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Inhibition of rhZP3-Initiated AR by Strychnine and GlyR Antibody
A potential role for the GlyR in the rhZP3-initiated AR was also examined. Preincubation of sperm with the classic inhibitor of the neuronal GlyR, strychnine (50 nM), significantly inhibited AR initiation by rhZP3 (Fig. 4). Additionally, the rhZP3-initiated AR was abolished by the preincubation of capacitated human sperm with the polyclonal GlyR antibody G0666 (Fig. 5). The motility of the sperm was not altered by the addition of strychnine or the GlyR antibody.
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Inhibition of rhZP3-Stimulated Ca2+ Influx by Strychnine
Figure 6 shows the change in intracellular Ca2+ levels seen in a typical experiment (n = 3) after addition of rhZP3 to capacitated human sperm. The addition of rhZP3 caused a sudden rise in intracellular Ca2+ levels (peak), followed by a sustained elevation in intracellular Ca2+ levels (plateau). The addition of strychnine before rhZP3 addition significantly reduced both the peak and the sustained response.
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The values for the basal and plateau levels of [Ca2+]i in each experiment (n = 3) were determined by calculating the mean [Ca2+]i level at 100 sec before and after the addition of rhZP3. No difference was found in the basal [Ca2+]i levels in untreated sperm (252 ± 6.6 nM) when compared to strychnine-treated sperm (251 ± 19.9 nM), suggesting that preincubation with strychnine alone had no effect on the [Ca2+]i levels (n = 3).
A Ca2+ response was elicited in the untreated sperm by the addition of 100 µg/ml of rhZP3 (peak, 370 ± 20.3 nM; plateau, 336 ± 13.1 nM). However, in samples preincubated with 50 nM strychnine, on the addition of rhZP3 both the peak value for [Ca2+]i (308 ± 16.7 nM) and the plateau value (271 ± 10.9 nM) were reduced. The relative changes in Ca2+ levels (
[Ca2+]i) from basal to the peak and from basal to the plateau were significantly lower (P 0.05) with strychnine plus rhZP3 ([Ca2+]i peak, 56.7 ± 3.1 nM; [Ca2+]i plateau, 20.2 ± 12.1 nM) compared to rhZP3 alone ([Ca2+]i peak, 117 nM ± 18.9 nM; [Ca2+]i plateau, 83.3 ± 15.5 nM). Overall, the reduction in Ca2+ influx by the presence of strychnine represents a 51% inhibition of the mean peak value and a 75% inhibition of the mean plateau value compared to untreated sperm.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Historically, the limited availability of human ZP has been a major obstacle in the study of human sperm/egg interactions. In recent years, rhZP3 has been produced from various protein expression systems as a potential substitute for solubilized ZP [15, 16, 28, 29]. Biological activity has been demonstrated for rhZP3 protein expressed in CHO cells, with rhZP3 produced using this system being shown to initiate the AR [15, 16] and to mediate the associated calcium influx [16].
In the present study, we have used rhZP3 obtained from CHO cells transfected with and expressing a modified form of the human ZP3 protein. The details of the construction of this expression system and subsequent purification of the secreted rhZP3 have been published previously [17]. To confirm the validity of using this purified rhZP3 preparation as a tool for study of the ZP-initiated AR in human sperm, a series of characterization experiments have been undertaken. Time-course experiments have shown that, whereas a significant response (i.e., AR increase) could be seen after just 5 min of incubation of capacitated sperm with rhZP3, 20 min of incubation were sufficient to initiate the maximal percentage of AR in responsive sperm (Fig. 2). The percentage AR initiated by rhZP3 at 20 min in the present study was higher than that reported at 6 h for another purified rhZP3 preparation produced in CHO cells [15], but it was lower than that detected at 30 min for an unpurified CHO cell rhZP3 preparation [16]. Our 20-min AR results with rhZP3 are comparable to those reported for longer incubations (30 min to 2 h) of human sperm with solubilized human ZP [5, 7, 8] and to those reported for human sperm bound to intact human ZP for 15 min [30]. Additionally, we have demonstrated that only capacitated sperm respond to rhZP3 by undergoing the AR. Such a requirement is an established characteristic of the mammalian AR initiated by the intact or solubilized ZP [1]. Moreover, the influx of calcium into the sperm cytosol is an obligatory event in the mammalian AR initiated by the ZP [1]. Thus, our demonstration that the rhZP3-initated AR requires extracellular calcium and that the recombinant protein mediates an influx of sperm calcium (Fig. 6) supports the likelihood that rhZP3 is initiating the AR by a mechanism similar to that of the ZP. Brewis et al. [16] previously reported that an unpurified rhZP3 preparation from CHO cells could mediate human sperm Ca2+ influx.
Pertussis toxin, a classical Gi protein inhibitor, is known to inhibit the mammalian AR initiated by intact or solubilized ZP [4]. Thus, it is important that pertussis toxin completely inhibited the effect of rhZP3 (Fig. 3), with a concentration and time comparable to those required for inhibition of the human AR initiated by solubilized human ZP [5–9]. As a control, we used a component of pertussis toxin, the pertussis toxin B oligomer, which cannot inactivate Gi protein but can have other cellular effects [8]. Pertussis B toxin oligomer did not inhibit the human sperm AR initiated by solubilized human ZP in the study by Tesarik et al. [8] and, in the present study, did not inhibit the rhZP3-initiated AR. Our results strongly suggest that the rhZP3 was acting through the same Gi protein-mediated mechanism as solubilized native human ZP. Recently, pertussis toxin was also shown to inhibit the AR initiated by rhZP3 produced from human ovarian teratocarcinoma cells [29].
Sperm-ZP binding that leads to the AR is dependent on specific carbohydrate components of ZP3, but the protein backbone of ZP3 is also required for the AR [1]. The ZP is generally believed to be synthesized solely by the oocyte [1]. Thus, the carbohydrate composition of rhZP3, expressed and glycosylated in CHO cells, is unlikely to be identical to that of native human ZP3. Moreover, the protein backbone of rhZP3 is not identical to that of human ZP3 synthesized by the human oocyte, because rhZP3 was engineered with a C-terminal, His6 segment (to aid purification of the protein by metal-affinity chromatography) and lacks the C-terminal transmembrane-like domain. Nevertheless, rhZP3 and the ZP appear to initiate the AR by a similar mechanism, suggesting that components of ZP3 important to AR initiation, or highly related components, are still present in rhZP3. It should be noted that neither rhZP1 nor rhZP2 initiated the human sperm AR even though both proteins were produced and glycosylated in CHO cells as was rhZP3.
Detection of GlyR in Human Sperm
Immunochemical studies have demonstrated the presence of the GlyR in pig and mouse sperm [11, 14], and strychnine-binding studies have indicated its presence in golden hamster sperm [13]. Here, we have confirmed its presence in human sperm. Using immunoprecipitation and Western blot analysis in conjunction with an antibody directed at the 1 subunit of the human neuronal GlyR (G0666), we have detected a band from sperm that runs at the same apparent molecular mass (48 kDa) as the rat spinal cord 1 subunit of the GlyR. The 1 subunit of the mammalian (including human) neuronal GlyR has been previously reported to be 48 kDa [31].
Evidence for Involvement of the GlyR in the Human AR Initiated by rhZP3
Previous studies have suggested the GlyR plays an important role in the ZP-initiated AR of several different species [10, 12–14]. In the present study, we found that strychnine inhibited the rhZP3-initiated AR at 50 nM (Fig. 4), a concentration that inhibits the neuronal GlyR and, also, the pig AR initiated by solubilized pig ZP [10]. Recently, it was shown that nanomolar levels of strychnine inhibit the neuronal 9 nicotinic acetylcholine receptor [32]. This receptor is also inhibited by (+)-bicuculline, with a median inhibitory dose of 0.8 µM [32]. Because we have found that 1 µM (+)-bicuculline does not inhibit the rhZP3-initiated human AR (data not shown), the effect of strychnine on the rhZP3-inititiated AR is not due to inhibition of an 9 nicotinic acetylcholine receptor. Moreover, 1 µM (+)-bicuculline does not inhibit the pig sperm AR initiated by solubilized pig ZP [10]. We have also shown that an antibody against the 1 subunit of the human neuronal GlyR inhibits the AR initiated by rhZP3 (Fig. 5). All these results provide further evidence that, like the native ZP-initiated AR of other species, the rhZP3-initiated human AR involves the GlyR.
Putative Mechanism of Action for GlyR in the ZP-Initiated AR
The neuronal GlyR is also apparently present in some other somatic cells, including GK-P3 cells derived from pancreatic islet cells [33], neutrophils [34], and adrenal chromaffin cells [35]. Usually, in the central nervous system, the GlyR is involved in the inhibition of neurotransmission, because glycine inhibits neuronal activity by increasing Cl- influx through the GlyR, leading to hyperpolarization [36]. However, in some neurons, in neutrophils, and in the GK-P3 cells, glycine appears to be able to cause depolarization by activating the GlyR (presumably stimulating Cl- efflux), resulting in increased [Ca2+]i and secretion [33, 37, 38].
Depolarization of the mouse and bovine sperm occurs due to interaction with ZP3 during the ZP-initiated AR [39, 40]. This depolarization involves ion flux through a pertussis toxin-insensitive, poorly selective cation channel and leads to activation of voltage-operated Ca2+ channels [40, 41]. Several cations, including Na+ and Ca2+, appeared to be important for maximal ZP-induced depolarization of mouse and bovine sperm, but "robust" ZP-induced depolarization still occurred after replacement of Cl- by any of several anions, including gluconate and Br- [39]. However, the initial rate of depolarization observed in those previous studies appeared to be greater in the presence of Cl- than when gluconate was substituted for Cl-.
The presence of Cl- in the extracellular medium is essential to the hamster and pig sperm AR initiated by the ZP [10, 42] and to the human and pig sperm AR initiated by progesterone [10, 43]. Moreover, Cl- channel blockers inhibit the mouse, pig, and hamster AR initiated by the ZP [10, 12, 44], and mutations in the sperm GlyR inhibit the ZP-initiated mouse AR [12]. Voltage-operated Ca2+ channels seem to be present in human sperm, because antagonists of such channels can inhibit progesterone-mediated Ca2+ influx [24]. The Cl- efflux through mammalian sperm amino acid neurotransmitter receptor/Cl- channels could contribute to membrane depolarization leading to activation of voltage-operated Ca2+ channels during ZP or progesterone initiation of the AR [13, 45]. We suggest that the human sperm GlyR is involved in such a mechanism during the ZP-initiated AR. Our data showing that the GlyR antagonist strychnine inhibits the rhZP3-mediated increase in sperm intracellular Ca2+ supports this suggestion. We have no data, at present, regarding the direction of Cl- flux during the ZP-initiated AR, but Cl- efflux does occur during the progesterone-initiated AR via another amino acid receptor/Cl- channel, the GABAA receptor/Cl-channel [46]. Based on the intracellular Cl- concentration in sperm and the membrane potential of capacitated mammalian sperm, such an efflux, presumably via the GlyR, would occur during the ZP-initiated AR and would contribute to depolarization [47].
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FOOTNOTES |
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1 Supported by NIH grant HD-33368 to S.M. 
2 Correspondence: Stanley Meizel, Department of Cell Biology and Human Anatomy, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616-8643. FAX: 530 752 8520; smeizel{at}ucdavis.edu
Accepted: August 15, 2001.
Received: June 27, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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9 nicotinic acetylcholine receptor shares pharmacological properties with type A 
-aminobutyric acid, glycine, and type 3 serotonin receptors. Mol Pharmacol 1999; 55:248-254This article has been cited by other articles:
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Di Zhang and M. Gopalakrishnan Sperm Ion Channels: Molecular Targets for the Next Generation of Contraceptive Medicines? J Androl, November 1, 2005; 26(6): 643 - 653. [Full Text] [PDF]
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A.M. Petrunkina, R.A.P. Harrison, M. Ekhlasi-Hundrieser, and E. Topfer-Petersen Role of volume-stimulated osmolyte and anion channels in volume regulation by mammalian sperm Mol. Hum. Reprod., November 1, 2004; 10(11): 815 - 823. [Abstract] [Full Text] [PDF]
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 J. W. Lynch Molecular Structure and Function of the Glycine Receptor Chloride Channel Physiol Rev, October 1, 2004; 84(4): 1051 - 1095. [Abstract] [Full Text] [PDF]
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A. M. Guzman-Grenfell and M. T. Gonzalez-Martinez Lack of Voltage-Dependent Calcium Channel Opening During the Calcium Influx Induced by Progesterone in Human Sperm. Effect of Calcium Channel Deactivation and Inactivation J Androl, January 1, 2004; 25(1): 117 - 122. [Abstract] [Full Text] [PDF]
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 P. Redecker, M. R. Kreutz, J. Bockmann, E. D. Gundelfinger, and T. M. Boeckers Brain Synaptic Junctional Proteins at the Acrosome of Rat Testicular Germ Cells J. Histochem. Cytochem., June 1, 2003; 51(6): 809 - 819. [Abstract] [Full Text] [PDF]
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J.-H. Son and S. Meizel Evidence Suggesting That the Mouse Sperm Acrosome Reaction Initiated by the Zona Pellucida Involves an {alpha}7 Nicotinic Acetylcholine Receptor Biol Reprod, April 1, 2003; 68(4): 1348 - 1353. [Abstract] [Full Text] [PDF]
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Y.Y. Yuan, W.Y. Chen, Q.X. Shi, L.Z. Mao, S.Q. Yu, X. Fang, and E.R.S. Roldan Zona Pellucida Induces Activation of Phospholipase A2 During Acrosomal Exocytosis in Guinea Pig Spermatozoa Biol Reprod, March 1, 2003; 68(3): 904 - 913. [Abstract] [Full Text] [PDF]
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C. Bray, J.-H. Son, and S. Meizel A Nicotinic Acetylcholine Receptor Is Involved in the Acrosome Reaction of Human Sperm Initiated by Recombinant Human ZP3 Biol Reprod, September 1, 2002; 67(3): 782 - 788. [Abstract] [Full Text] [PDF]
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A. A. Schuffner, H. S. Bastiaan, H. E. Duran, Z.-Y. Lin, M. Morshedi, D. R. Franken, and S. Oehninger Zona pellucida-induced acrosome reaction in human sperm: dependency on activation of pertussis toxin-sensitive Gi protein and extracellular calcium, and priming effect of progesterone and follicular fluid Mol. Hum. Reprod., August 1, 2002; 8(8): 722 - 727. [Abstract] [Full Text] [PDF]
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