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Regular Article |
a Department of Cell Biology and Human Anatomy, School of Medicine, University of California,Davis, California 95616-8643
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-bungarotoxin (-BTX, 100 nM), -conotoxin IMI (-CTX IMI, 250 nM and 25 nM), and methyllycaconitine (MLA, 100 nM and 10 nM), significantly blocked AR initiation by ACh. -BTX is an anatagonist of several nAChRs, including the 7 nAChR, and -CTX IMI and MLA are highly specific antagonists of 7 subunit-containing AChRs. The sperm nAChR plays a role in the AR initiated in vitro by a purified recombinant human ZP protein (rhZP3). Previously, rhZP3 was able to stimulate the AR by mechanisms similar to those seen with native ZP. Preincubation of human sperm with -BTX (from 10 µM to 100 nM), -CTX IMI (250 and 100 nM), or MLA (100 nM and 10 nM) caused a significant inhibition in the rhZP3-initated AR. The inhibition of the ACh-initiated and rhZP3-initiated AR by these nAChR antagonists strongly suggests the involvement of an 7 subunit-containing nAChR in the AR initiated by both ligands. AR initiation by progesterone was not inhibited by MLA or -BTX, suggesting that this particularnAChR is not involved in the AR initiated by that ligand. In vitro results show for the first time that ACh can initiate the human sperm AR and strongly suggest that a human sperm 7 subunit-containing nAChR plays a role in the rhZP3-initiated AR. This nAChR ligand-gated ion channel may be important to the signal transduction events of ZP-initiated AR in vivo.
acrosome reaction, fertilization, neurotransmitters, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Despite a plethora of studies characterizing the initial signaling events and activation of downstream second messenger systems culminating in AR, the exact nature of the primary receptors and channels sequentially activated by AR initiators on the plasma membrane has remained elusive. However, it is generally agreed that following binding of the ZP a capacitated sperm undergoes rapid depolarization, which in turn allows the opening of voltage-operated Ca2+ channels, a step that contributes to the obligatory rise in intracellular Ca2+ levels required for the onset of the AR [2]. An early component of this depolarization in the mouse and bovine sperm AR initiated by ZP involves ion flux through an as yet unidentified, poorly selective cation channel and leads to activation of voltage-operated Ca2+ channels. The cations Na+ and Ca2+ appeared to be important for ZP-induced depolarization and therefore maximal ZP initiation of mouse, hamster, and bovine sperm [3, 4]. Cl- flux via the sperm glycine receptor may also be involved [5, 6]
The nicotinic acetylcholine receptor (nAChR) belongs to the same superfamily of neurotransmitter-gated transmembrane ion channels as the glycine and GABAA receptors [7] In nerve and skeletal muscle, the binding of an activating ligand to an nAChR opens the channel, usually resulting in a large influx of Na+ and a smaller flux of Ca2+ and thereby membrane depolarization [8]. The nAChRs are pentamers composed of various combinations of five different types of proteins (, ß, 
, 
, and 
) that are expressed throughout the central and peripheral nervous systems (neuronal nAChRs are composed of just two types of subunit, and ß) and in skeletal muscle (all five subunit types) [8, 9]. Eight different subunits and four different ß subunits have been identified in mammals. Various nAChRs are also found in other mammalian cells, including several somatic cell types, lymphocytes, endothelial cells, bronchial epithelial cells, and chromaffin cells [10–13]
In addition, the nAChR is present in mammalian sperm. The results of studies utilizing radioactive isotope-labeled ligands and antagonists indicate the presence of nAChR in ram and rabbit sperm [14, 15]. Baccetti and coworkers [16] used -bungarotoxin (-BTX), a nearly irreversible competitive antagonist of the nAChR, to detect the nAChR-like molecules in the postacrosomal and midpiece regions of rabbit and ram sperm and in the acrosomal and midpiece regions of human sperm. Partial transcripts for the 5, 7, and ß4 subunits have been detected in human testis (database of the National Center for Biotechnology Information [NCBI], Bethesda, MD).
In the present study, we investigated whether the nAChR has a role in the AR of human sperm. The evidence strongly suggests that acetylcholine (Ach) is able to initiate the AR by activating an 7 subunit-containing nAChR and that this receptor is essential for the AR initiated by purified recombinant human ZP protein (rhZP3) and thus potentially has a role in the initiation of the AR by intact ZP in vivo.
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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 washing media were of reagent grade and were purchased from Fisher Scientific (Pittsburgh, PA), Irvine Scientific (Irvine, CA) Mallinckrodt (Paris, KY), or Sigma (St. Louis, MO). Percoll, progesterone, ACh, -BTX, and methyllycaconitine (MLA) were purchased from Sigma, -conotoxin IMI trifluoroacetate (-CTX IMI) and (-)-nicotine-L-ditartrate were from Calbiochem-Novabiochem (La Jolla, CA), ConA-fluorescein isothiocynate (FITC) was from EY Laboratories (San Mateo, CA), fluoromount mountant Gurr was from Gallard-Schlesinger Industries (New York, NY), and fraction V bovine albumin (Pentax 81-066, lot 59) was from Miles (Kankakee, IL). 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 microfuge 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 healthy donors. 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 [17]. For sperm to respond to AR initiators, they must first undergo a series of molecular changes collectivity called capacitation [1, 18, 19]. The final sperm suspensions were prepared by diluting the sperm to 6 x 106 sperm/ml in an HSM containing 26 mg/ml BSA (HSM-26B) and capacitated by incubation of 500-µl aliquots in loosely capped 15-ml polypropylene centrifuge tubes or incubation of 200-µl aliquots in loosely capped 1.5-ml polypropylene microcentrifuge tubes for 24 h at 37°C in 5% CO2 [20].
AR Initiation and Assay
Capacitated human sperm suspensions in HSM-26B were placed in microcentrifuge tubes for AR determination; 200-µl aliquots were used for experiments involving ACh, nicotine, and progesterone, and 50-µl aliquots were used for experiments involving rhZP3. The production and purification of rhZP3 (lacking the C-terminal transmembrane-like domain and possessing a C-terminal His 6-tag) from Chinese hamster ovary cells have been described previously [5, 21].
After additions of AR initiators or solvents, i.e., ACh or nicotine or their solvent PBS, rhZP3 or its solvent PBS, and progesterone or its solvent dimethyl sulfoxide (DMSO) at a final concentration of 0.05%, the treated suspensions were incubated at 37°C in 5% CO2/95% air for 20 min. 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% formaldehyde in PBS. The acrosomal status of the fixed sperm was then assessed using the ConA-FITC AR assay [22]. 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 nAChR anatagonists on the initiation of the AR by ACh and rhZP3, selected aliquots were pretreated with -BTX (10 µM, 1 µM, 100 nM, 10 nM, and 1 nM for 1 h), -CTX IMI (250 nM, 100 nM, 25 nM, 5 nM, and 0.5 nM, added 10 min prior), or MLA (1 nM, 10 nM, and 100 nM 30 min prior). Appropriate solvent controls (sodium citrate for MLA, sodium trifluoroacetate [TFA] for -CTX IMI, and PBS for -BTX) were processed in parallel in all experiments.
The percentage of motile sperm was determined by examination using phase contrast microscopy (100–200 sperm/treatment at 400x magnification). A subjective score was assigned to the quality of sperm motility using a scale of 1 (twitching, nonprogressive motion) to 4 (vigorous forward motility) [20].
Statistical Analysis
All AR percentage data were transformed to the arcsine of their square roots [23]. The Duncan new multiple range test [24] was used for the comparison of group mean differences. Where appropriate, differences were determined with a 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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Incubation of capacitated human sperm with 10 µM, 50 µM, and 250 µM ACh significantly initiated the AR (Fig. 1). The incubation of uncapacitated sperm with ACh did not cause initiation of the AR (n = 3; P > 0.05); PBS produced 6.6% ± 1.7% AR, and 250 µM ACh produced 6.3% ± 1.6% AR.
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Nicotine concentrations of 10 nM, 50 nM, and 250 nM significantly initiated the AR (Fig. 2). Because nicotine was prepared as a ditartrate salt, control experiments using the maximum concentration (250 nM) of sodium ditartrate were also performed. These experiments indicated that sodium ditartrate did not significantly initiate the AR.
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To test whether AR initiation by ACh involved annAChR, aliquots of sperm were incubated with several different specific anatagonists of the nAChR. Because -BTX has been used to detect nAChR in human sperm and transcripts for the 7 subunit have been detected in human testis (NCBI database), -BTX and two specific antagonists of the 7 nAChR, -CTX IMI and MLA [25], were tested.
Preincubation of sperm with 100 nM -BTX significantly inhibited the effect of ACh (Fig. 3). Preincubation with lower concentrations of -BTX (10 nM and 1 nM) did not significantly inhibit the AR.
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-CTX IMI also caused inhibition of the AR initiated by ACh. Preincubation of sperm with concentrations of-CTX IMI ranging from 250 to 25 nM significantly inhibited the effect of ACh (Fig. 4). Preincubation with-CTX IMI at lower concentrations (5 nM and 0.5 nM) did not cause significant inhibition.
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A third anatagonist of the nAChR, the plant toxin MLA, was also used to investigate the nature of AR initiation by ACh. Following 30 min of preincubation with the antagonist, concentrations of 100 nM and 10 nM MLA caused significant inhibition of the ACh-initiated AR (Fig. 5).
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Because -CTX IMI and MLA were prepared as TFA and citrate salts, respectively, control experiments were undertaken to determine whether preincubation with sodium TFA and sodium citrate at concentrations equivalent to the highest antagonist concentrations influenced the AR. Neither salt influenced the levels of AR initiation by ACh (Figs. 4 and 5).
In the various experiments, the percentage of control motility in any single experiment was not altered by the addition of the different concentrations of ACh, nicotine, -BTX, -CTX IMI, MLA, ditartrate, TFA, or citrate when compared with the control tubes (range of motility in different experiments was 75–85%), and differences in the quality of motility were not observed.
Effect of AChR Anatagonists on the Initiationof the AR by rhZP3
Recombinant human ZP3 is able to significantly stimulate the AR in a manner equivalent to that of native solubilized zona [5]. A potential role for nAChRs in the rhZP3-initiated AR was examined by studying the effects of the three nAChR antagonists that had inhibited the ACh-initiated AR.
Preincubation of sperm with -BTX inhibited the rhZP3-initiated AR. Concentrations of -BTX ranging from 10 µM to 100 nM caused significant inhibition of the rhZP3-initiated AR (Fig. 6). A slight inhibition seen at 10 nM was not significant (P > 0.05).
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A similar inhibition of the rhZP3-initiated AR was obtained when sperm were preincubated with -CTX IMI. Concentrations of -CTX IMI of 250 and 100 nM caused significant inhibition of the rhZP3-initiated AR (Fig. 7). MLA also caused a concentration-dependent inhibition of the rhZP3-initiated AR. Concentrations of MLA of 100 nM and 10 nM caused significant inhibition of the rhZP3-initiated AR (Fig. 8).
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Because the preparations of -CTX IMI and MLA contained TFA and citrate salts, respectively, control experiments were undertaken to determine whether preincubation with sodium TFA and sodium citrate at concentrations equivalent to the highest antagonist concentrations influenced the AR. Neither salt influenced the rhZP3-initiated AR (Figs. 7 and 8).
The percentage of motility in any single rhZP3 experiment was not altered by the addition of the different concentrations of -BTX, -CTX IMI, MLA, TFA, or citrate when compared with the control tubes (range of motility in different experiments was 75%–85%), and differences in the quality of motility were not observed.
Effect of AChR Antagonists on the Initiation of ARby Progesterone
Progesterone also initiates the human AR in vitro [26, 27]. To investigate whether nAChRs are involved in the signaling pathways activated during progesterone-initiated human AR, capacitated sperm were preincubated with the anatagonists -BTX or MLA prior to the addition of progesterone (3.17 µM or DMSO solvent). Both anatagonists caused small but nonsignificant (P > 0.05) differences in the level of AR initiation. The following results were obtained for -BTX (n = 4): PBS + DMSO, 13.9% ± 0.8% AR; PBS + progesterone, 30.7% ± 2.0% AR; -BTX (10 µM) + progesterone, 25.8% ± 2.8% AR. The following results were obtained for MLA (n =3): sodium citrate (100 nM) + DMSO, 27.7% ± 1.2% AR; sodium citrate (100 nM) + progesterone, 46.5% ± 1.1% AR; MLA (100 nM) + progesterone, 45.8% ± 1.0% AR.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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7 Subunit-Containing nAChR Is Involvedin the Human AR Initiated by ACh and rhZP3
The present results are the first to show that a sperm nAChR can play a role in the AR. We found for the first time that ACh is able to initiate the AR in mammalian (capacitated human) sperm. However, the ligand did not initiate the AR in uncapacitated human sperm, a result indicating that the cellular events of capacitation are a prerequisite for the ACh effect. The fact that the nAChR agonist nicotine also initiated the human sperm AR and that three nAChR antagonists, -BTX, -CTX IMI, and MLA, inhibited the ACh-initiated and rhZP3-initiated AR indicate the involvement of a sperm nAChR. Transcripts for the 5, ß4, and 7 nAChR subunits have been detected in human testis (NCBI database, GenBank accessions AA382668, AA431307, and AA889613, respectively). Although-BTX antagonizes nAChRs by binding to 1, 7, 8, and 9 subunits [8, 28], -CTX IMI and MLA are more specific antagonists of the 7 nAChR [25, 29–31].
The mammalian neuronal 7 nAChR subunit can form a functional nAChR homomer in both expression systems and the brain [32], but it has been suggested that heteromers containing the 7 subunit are present in the embryonic chick neurons [33]. It is not yet known whether the human sperm nAChR important to the ACh-initiated and rhZP3-initated AR is an 7 subunit homomer or a heteromer including that subunit. However -BTX, -CTX IMI, and MLA all inhibited the ACh-initiated and rhZP3-initiated AR at concentrations similar to those that have been shown to antagonize the mammalian neuronal homomeric 7 nAChR [25, 29, 30]. Those results strongly suggest that the sperm nAChR that plays a role in the AR contains an 7 subunit either as a homomer or as a heteromer.
Progesterone is another possible physiological AR initiator [34]. We also tested the nAChR anatagonists to see whether the sperm nAChR was important to the progesterone-initiated AR. However, neither -BTX nor MLA at concentrations shown to block the ACh-initiated and rhZP3-initiated AR caused a significant decrease in the level of the progesterone-initiated AR, indicating that the signaling pathways activated are distinct from those utilized during the ACh-initiated and rhZP3-initiated AR. Other studies support the view that ZP and progesterone have at least partially different signal pathways [35, 36]. In a study of 7 knockout mutant mice, homozygous male and female mice were fertile, although the authors suggested there may be some reduction in fertility [37]. If the 7-containing nAChR is important for the ZP-initiated AR, how would any fertilization occur in such mutant mice? As we have suggested previously, the redundancy and activation of distinct AR signaling pathways by ZP and progesterone may account for fertility in a situation where the ZP-initiated AR does not occur [38].
Nelson [39] suggested that nAChRs are involved in sea urchin sperm motility. In another study, micromolar levels of -BTX inhibited uncapacitated human sperm motility [40], but even basal levels of motility in that study were quite low. In the present study, -BTX, -CTX IMI, and MLA had no obvious effect on sperm motility, suggesting that a human sperm nAChR containing an 7 subunit does not play a role in motility.
Putative Mechanism for nAChR in the ZP-Initiated AR
The nAChRs are cation channels, primarily selective for Na+ but also conducting Ca2+ [8]. However, not all isotypes of the neuronal nAChR are equally permissive to both cations. Studies of mammalian homomeric 7 nAChRs expressed in Xenopus oocytes have indicated 20 times greater permeability for Ca2+ than for monovalent cations [41, 42]. Another study provides evidence that cation influx through the homomeric human 7 nAChR expressed in human embryonic kidney cells is sufficient to cause a significant, transient increase in intracellular Ca2+ [43].
In neurons, flux of Na+ via nAChR can depolarize the membrane [8]. Stimulation by ACh causes membrane depolarization, leading to calcium-dependent exocytosis from some somatic cell types [44, 45]. Depolarization of the plasma membrane elicited by the flux of ions through a number of poorly characterized channels is a critical event in the onset of the mammalian sperm exocytotic event, the AR. In both mouse and bovine sperm, this depolarization is triggered by interaction with ZP3, involves ion flux through a poorly selective cation channel, and leads to activation of voltage-operated Ca2+ channels [2, 46]. Several cations, including Na+ and Ca2+, appear to be important for maximal ZP-induced depolarization and/or AR [3, 47]. The human sperm nAChR described here may be similar to the poorly selective cation channel important in the mouse and bovine sperm ZP-initiated AR. Activation of that sperm nAChR might then contribute to Na+ or Ca2+ flux needed for that depolarization. Although nicotine and ACh at 100 µM were reported to cause hyperpolarization, not depolarization, of human sperm in low K+ and high K+ media, those studies at such high agonist concentrations were performed with uncapacitated sperm [48]. Based on the high Ca2+ permeability observed in heterologously expressed homomeric 7 nAChRs [41–43], it is also possible that the sperm nAChR might contribute to the rise in intracellular Ca2+ important for some other step of the ZP-initiated AR mechanism.
Although exogenous ACh can directly activate the sperm nAChR, how might rhZP3 activate this receptor? Some as yet unidentified cross-talk step via a ZP receptor or direct binding of the rhZP3 to the nAChR are both potential mechanisms. Another possible mechanism would involve sperm synthesis and release of ACh during AR initiation. Ram and human sperm contain the enzyme required for ACh synthesis, choline acetyltransferase [49, 50], which is localized to the human sperm postacrosomal region. Further studies are planned to determine which of those putative mechanisms is responsible for the activation of the 7 subunit-containing nAChR by rhZP3 and to elucidate the role of the subsequent receptor-mediated ion flux.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH grants HD-33368 and HD-23098 to S.M. C.B. is a Lalor Foundation Fellow. 
2 Correspondence: Stanley 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
Accepted: April 8, 2002.
Received: February 14, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxyprogesterone: novel stimulators of calcium influx in human sperm. J Biol Chem 1990 265:1376-1380This article has been cited by other articles:
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