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a Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Others have studied effects of CaM antagonists on changes in membranes or motility during capacitation [11–13]. However, it is unlikely that these different aspects of capacitation are always interdependent. For example, hyperactivation, a whiplash-like flagellar movement with large-amplitude, asymmetric bends, occurs during capacitation [1] and is sometimes used as an assay of capacitation [11, 14]. Yet mouse sperm hyperactivation is not always correlated with the ability of those sperm to undergo the zona pellucida-induced acrosome reaction (AR) [15]. In addition, tyrosine phosphorylation of specific sperm proteins occurs during capacitation [16]. Although in the mouse this process is known to be mediated by a cAMP-dependent pathway [17], the relationship of this pathway to capacitation-associated membrane changes leading to the AR is not yet clear. Therefore, in this study we assayed changes in sperm membranes, motility, and tyrosine phosphorylation, as well as the ability of the sperm to fertilize oocytes in vitro. These various assays allowed us to examine the interaction of CaM with different sperm functions associated with capacitation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male mice were produced by crossing 129X1/SvJ-+/+ and C57BL/6-+/+ mice in the colony of Dr. Olds-Clarke. Males used in this study were 3–8 mo old. Female CF1 mice (Charles River Laboratories, Wilmington, MA) were 5–8 wk old at the time of their use.
Chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. The medium used throughout these experiments was a modified Krebs-Ringer-bicarbonate buffer [15] containing 2% BSA, 1.7 mM CaCl2, and 50 mM Hepes (IVF medium), except in some in vitro fertilization (IVF) experiments in which 10 mM TAPSO (3[N-Tris-(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid) was substituted for Hepes. Stock solutions of N-(6-aminohexyl)-1-naphthalenesulfonamide (W5), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), calmidazolium (CZ), and 3-isobutyl-1-methylxanthine (IBMX) were prepared in dimethyl sulfoxide (DMSO) and stored at -20°C. Controls without inhibitors received an equivalent amount of DMSO. The diastereoisomers of the phosphorothioate analogue of cAMP, Sp-adenosine-3',5'-cyclic monophosphothioate (Sp-cAMPS) and Rp-adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS), were purchased from Research Biochemicals International (Natick, MA).
Isolation and Incubation of Sperm
The cauda epididymides were minced and placed in IVF for 10 min to allow the sperm to swim out into the medium; then epididymal tissue was removed, and 50-µl aliquots of the sperm suspension were diluted 1:1 with fresh IVF medium containing CaM antagonists or the equivalent concentration of DMSO. The sperm suspensions were incubated at a concentration of 0.75–1.32 x 108/ml at 37°C in 5% CO2, 95% air for 90 min before use.
Assessment of Viability
The proportions of living and dead sperm were assessed by the newly developed living-cell nucleic acid stain, SYBR-14, in combination with the conventional dead-cell nucleic acid stain, propidium iodide [18]. According to the staining protocol of live/dead sperm viability kit (cat. #L-7011; Molecular Probes, Eugene, OR) with some modifications, 1 µl of 10 µM SYBR-14 working solution was added to 100 µl of sperm suspension (~1 x 107 cells/ml) and incubated at 37°C for 15 min to stain the living cells. Then the dead cells were stained by adding 1 µl of 2.4 mM propidium iodide working solution. After 5 min, 10 µl of the sperm suspension was pipetted onto a glass slide, covered with a coverslip, and observed immediately under a fluorescent microscope equipped with the appropriate filters. The SYBR-14 stained the nucleus of living sperm green, while dead or membrane-damaged sperm were stained red by propidium iodide. At least 100 sperm were counted for each treatment. The mean ± SD percentage of control sperm that were viable by this test was 51 ± 4.5 (n = 7). The percentage of viable sperm exposed to CaM antagonists was calculated as the percentage of viable sperm after treatment divided by the percentage of viable control sperm receiving no added antagonist.
Assessment of Motility
For quantitative sperm motion analysis, the IVOS Sperm Analyzer (Hamilton Thorne Research, Beverly, MA) was used [19]. Sperm were diluted 1:40 in the same medium in which they had been incubated for 90 min or in IVF medium. They were immediately imaged by darkfield illumination at x150 magnification in an 80-µm-deep chamber at 37°C. Eight fields from one sample were pooled to obtain a measure of the entire population. Sperm were analyzed at 60 images/sec for 0.5 sec. Only sperm tracks with 
16 images and a curvilinear velocity >50 µm/sec were used. At least 80 tracks from each treatment in each experiment were analyzed. The percentage of sperm with progressive motility, mean curvilinear velocity (VCL), progressive velocity (VSL), path velocity (VAP), linearity (LIN), and straightness (STR) was determined for each population. VCL is the best estimate of the instantaneous sperm swimming speed; VSL is the ratio of the net length of the track to the time of observation, and VAP is the ratio of the length of a 5-point moving average of the sperm track to the time of observation. LIN (VSL/VCL) and STR (VSL/VAP) are measures of the straightness of the sperm track.
These motility parameters were used to identify hyperactivated sperm as described previously [15, 20, 21]. The rationale was based on the assumption that hyperactivation is normally correlated with capacitation. Sperm from four males were incubated in Ca2+-deficient medium (noncapacitated) or in medium containing 1.7 mM Ca2+ (capacitated) and subjected to motility analysis. The data were then inspected to select limiting values of motility parameters that could identify a large subpopulation of sperm with reasonable velocity but low progressiveness among capacitated populations, but only a small proportion of such sperm among noncapacitated populations. The best such indicators were VCL > 200 µm/sec and STR < 70. Tracks selected by these limiting values appeared hyperactivated upon visual inspection. Therefore hyperactivated sperm were defined as those whose tracks had both an STR of < 70 and a VCL > 200 µm/sec. These parameter values were selected before the beginning of experiments with CaM antagonists.
Assessment of Sperm Membrane Changes
Sperm membrane changes correlated with capacitation were evaluated by two methods. One assay was based on the pattern of sperm head fluorescence after staining with chlortetracycline (CTC): capacitated but acrosome-intact sperm exhibit fluorescence over the anterior two thirds of the head (the B pattern), which is correlated with the ability to undergo a zona pellucida-induced AR [22]. The CTC staining procedure was performed as described previously [22], except that glutaraldehyde was omitted to reduce agglutination, and preparations were examined immediately [23].
The second assay was to incubate sperm with 300 µg/ml L-
-lysophosphatidylcholine (LPC; type I, from egg yolk) for 15 min and then assess the sperm for the AR as determined by the absence of fluorescence over the sperm head after CTC staining [23]. The LPC-induced AR has been correlated with capacitation of bovine, human, and mouse sperm [23–25].
Assessment of Capacitation
Capacitation was determined by IVF [26]. CF-1 females were induced to superovulate and killed 18 h after hCG injection; the cumulus/oocyte clumps (COC) were obtained by puncturing the distended ampulla. One or two COC were transferred with forceps to 2-µl drops of IVF medium containing 0.3% BSA under mineral oil. Sperm incubated for 90 min with 100 µM W7, 100 µM W5, or 1.5% DMSO (control) were diluted to 1 x 106 sperm/ml with IVF medium containing no inhibitors or DMSO. After dilution, the final concentration of W7 in the sperm suspension treated with 100 µM W7 was calculated. This same concentration of W7 (1–2 µM) was then added to the diluted control sperm suspension, so that the concentration of W7 after dilution was the same for both control and W7-treated groups. Ten microliters of the diluted sperm samples was added to 1–2 COC kept in 2 µl of IVF medium and coincubated for 2 h in an atmosphere of 90% N2, 5% O2, 5% CO2.
After a 2-h incubation period, the oocytes were transferred to 0.01% bovine testis hyaluronidase (Calbiochem, San Diego, CA) to disperse cumulus cells. Denuded oocytes were washed four times and placed in 50 µl of IVF medium under mineral oil in an atmosphere of 90% N2, 5% O2, 5% CO2. Twenty-four hours later, the percentage of oocytes that had developed into 2-cell embryos was determined. For each treatment in each experiment, 8–23 oocytes and/or embryos were examined. The effect of CZ treatment on sperm IVF was not examined since sperm motility was severely inhibited; even after dilution in IVF medium, it was not normal (see Table 3).
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SDS-PAGE and Immunoblotting
Sperm proteins were prepared and separated as described previously [27]. Briefly, sperm incubated for 90–120 min were washed by centrifugation and resuspension in PBS containing 1 mM 4-[2-aminoethyl]-benzene-sulfonylfluoride (Calbiochem-Novabiochem, La Jolla, CA). After the third wash, sperm were resuspended in sample buffer [28] without 2-mercaptoethanol and boiled for 4 min; then 2-mercaptoethanol was added to the supernatant at a final concentration of 5%. Samples were frozen at -70°C until use (less than 1 mo).
Proteins from 3.3–4 x 105 sperm were applied to each lane of the gel. CaM standards were from porcine brain ("phosphodiesterase activity"). Proteins were separated by PAGE under reducing conditions in 8% gels, transferred electrophoretically to Immobilon-P (Millipore, Bedford, MA) membranes, and probed with either mouse monoclonal antibodies against CaM (diluted 1:10 000; Upstate Biotechnology, Lake Placid, NY) or phosphotyrosine (4G10; diluted 1:100 000; Upstate Biotechnology). As a control for specificity, the antiphosphotyrosine antibody was first absorbed with 20 mM O-phosphotyrosine. Peroxidase-conjugated goat anti-mouse IgG (Bio-Rad Laboratories, Hercules, CA) at 1:5000 dilution was used as the secondary antibody. Proteins were visualized with chemiluminescence kits (CaM: ECL; Amersham Pharmacia Biotech, Piscataway, NJ; tyrosine-phosphorylated proteins: SuperSignal West Pico Chemiluminescence Substrate, Pierce, Rockford, IL).
Blots probed with phosphotyrosine antibody were scanned and the band intensities quantified with SigmaGel (SPSS, Chicago, IL). For each lane, the intensity of the hexokinase band (116 kDa) was determined separately from that for all other bands. Since tyrosine phosphorylation of hexokinase is not dependent upon capacitation [29], this served as an internal control for equivalent lane loading. The inverse ratio of the intensity of the hexokinase band to the intensity of all other bands was calculated for each treatment and was compared to the inverse ratio of the control (IVF + 1.5% DMSO) in the same experiment. Each treatment was repeated at least three times.
Statistical Analysis
Significant differences among treatments were determined by the ANOVA followed when appropriate by the Newman-Keuls test. Results were considered significantly different when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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While CaM has been shown to be present in cauda epididymal mouse sperm by immunoblot analysis [9], a study of bull sperm suggested that the CaM concentration decreased significantly during capacitation [30]. Therefore, the presence of CaM was examined in capacitated mouse sperm. After incubation of cauda epididymal sperm for 70 min in IVF medium, sperm proteins were extracted, separated by electrophoresis on a 15% SDS-PAGE gel, and subjected to immunoblot analysis. An anti-CaM monoclonal antibody recognized one band in the sperm extract at about 18 kDa, and porcine brain CaM migrated at a similar rate (Fig. 1). The level of intensity of the band from 4 x 105 sperm appeared to be intermediate between the levels of intensity of 10 and 100 ng of porcine brain CaM.
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CaM Antagonists Inhibited the Capacitation-Dependent B Pattern After CTC Staining
Cauda epididymal sperm were isolated in IVF, diluted 1:1 into medium with CaM antagonists or the diluent (DMSO), incubated for 90 min, and assayed by the B pattern of fluorescence after CTC staining. As reported previously, in a capacitated population approximately 70–80% of the sperm exhibit the B pattern, while a noncapacitated population has only about 20–30% with a B pattern [23]. W7 decreased the percentage of B pattern sperm in a concentration-dependent manner, with maximal inhibition at 100–200 µM (Fig. 2). In contrast, W5, a chlorine-deficient W7 derivative, having ~10 times less affinity for CaM relative to W7, did not significantly affect the B pattern even at 200 µM (Fig. 2). Another CaM antagonist, CZ, also showed a concentration-dependent ability to inhibit the B pattern. CZ at 10 µM significantly suppressed the frequency of the B pattern, and inhibition was even greater at higher concentrations (Fig. 2).
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To rule out the possibility that the antagonist-induced inhibition of the B pattern was due to nonspecific toxic effects, the proportion of living and dead sperm after treatment with these drugs was examined. In the concentration range of 100–300 µM for W7 and W5, and 10–30 µM for CZ, none of these CaM antagonists showed significant inhibitory effects on sperm viability (Table 1).
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Cyclic AMP Analogues Could Overcome the Inhibitory Effect of CaM Antagonists on the B Pattern
Several lines of evidence suggest a stimulatory role for cAMP in sperm capacitation [17, 31, 32]. Since the data described above suggest that Ca2+-CaM is involved in mouse sperm capacitation, it was of interest to examine whether the addition of exogenous cAMP analogues could overcome the inhibitory effects of CaM antagonists. When sperm were incubated for 1.5 or 3 h with 100 µM W7, 200 µM IBMX (a nonspecific inhibitor of cAMP and cGMP phosphodiesterases), and 3 mM 8-bromo cAMP (a cell-permeable cAMP agonist), about 60–65% of the sperm displayed the B pattern (Fig. 3). However, when sperm were incubated with 100 µM W7, 200 µM IBMX, and 3 mM 8-bromo AMP (an inactive analogue of 8-bromo cAMP), the level of sperm with the B pattern was not different from that of sperm incubated in W7 alone. Similarly, when Sp-cAMPS, which activates cAMP-dependent protein kinase (PKA) [33], was included with IBMX and W7 in the sperm incubation medium, the percentage of B pattern sperm was more than 55% (Fig. 3). On the other hand, when Sp-cAMPS was replaced by Rp-cAMPS, a membrane-permeable cAMP analogue that antagonizes PKA activity [33], the percentage B pattern sperm remained at levels similar to that of sperm incubated in W7 only. Thus, both an active cAMP analogue and a stimulator of PKA activity could overcome the inhibition of the B pattern among sperm treated with W7.
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When sperm were incubated 1.5 h with 10 µM CZ, 200 µM IBMX, and 3 mM 8-bromo cAMP, 58.1 ± 5.5% of the sperm displayed the B pattern (three experiments). Since this mean is significantly higher than that of sperm incubated in 10 µM CZ, but significantly lower than that of sperm incubated in IVF medium (Fig. 2B), cAMP analogues could at least partially overcome the CZ-induced inhibition of the sperm B pattern after CTC staining.
CaM Antagonists Inhibited the LPC-Induced AR, and cAMP Analogues Could Overcome This Inhibition
It was possible that CaM antagonists could have inhibited the B pattern by artifactual means, or that CaM was necessary only for the pathway leading to expression of the B pattern. Since a previous study had demonstrated that capacitated mouse sperm can be induced to undergo the AR by exposure to the fusogenic lipid LPC [23], this procedure was used as an independent measure of capacitation-associated membrane changes.
When sperm were incubated for 105 min in IVF medium (Fig. 4A, bar labeled IVF, IVF), approximately 12% of the sperm underwent a spontaneous AR. However, if sperm were exposed to 300 µg/ml LPC for the last 15 min of the incubation period (bar labeled IVF, LPC), the percentage of acrosome-reacted sperm significantly increased, to approximately 35%. When sperm were incubated with 100 µM W7 for 90 min, then exposed to LPC for 15 min (bar labeled W7, LPC), only 7% underwent the AR. In another sperm group, W7 was added only for the last 15 min of incubation, at the same time as LPC (bar labeled IVF, LPC+W7). Results of this treatment were similar to the control values (bar labeled IVF, LPC), demonstrating that W7 did not block the LPC-induced AR by interfering with the action of LPC. Sperm incubated for 90 min with either 100 µM W5, or 100 µM W7, 3 mM 8-bromo cAMP, and 200 mM IBMX, and then exposed to LPC (bars labeled W5, LPC and W7+cAMP, LPC), showed a level of AR similar to that of control sperm.
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Equivalent results were also observed in experiments using CZ (Fig. 4B). CZ added only at the time of LPC addition (bar labeled IVF, CZ+LPC) did not block the LPC-induced AR, demonstrating that CZ did not interfere with the action of LPC on sperm. However, sperm incubated 90 min in 10 µM CZ and then exposed to LPC for 15 min displayed significantly fewer AR (bar labeled CZ, LPC). Furthermore, sperm incubated with 10 µM CZ, 3 mM 8-bromo cAMP, and 200 mM IBMX for 90 min and then exposed to LPC for 15 min (bar labeled CZ+cAMP, LPC) had a level of AR similar to that of the control. Thus, the inhibition of the LPC-induced AR by CaM antagonists could be overcome by cAMP analogues.
A CaM Antagonist Inhibited IVF
The original definition of capacitation is "all the events that lead to the development of the capacity of mammalian sperm to penetrate eggs" [34]. Therefore, IVF was used to assay all aspects of capacitation in vitro. Sperm were incubated 90 min in 100 µM W7, then diluted approximately 1:100 in IVF medium without W7, and immediately used to inseminate cumulus-oocyte complexes (COC). After 2 h of coincubation, the COC were removed, divested of remaining cumulus cells and sperm, and incubated in IVF medium for 24 h. As shown in Figure 5, the mean percentage of 2-cell embryos after insemination by sperm treated with 100 µM W7 was approximately 10%. Because the sperm were not washed free of W7 but rather diluted 1:100 with IVF medium, the sperm-COC coincubation medium would be expected to contain 1–2 µM W7. However, it was possible that even this low concentration could affect sperm-oocyte interactions [35]. Therefore, just prior to coincubation with COC, 1–2 µM W7 was added to the control sperm that had been incubated for 90 min in IVF medium. This group was able to fertilize about 80% of the oocytes (Fig. 5). Furthermore, treatment of sperm with 100 µM W5 showed similar results. Thus, W7-treated sperm had a significantly reduced ability to fertilize, relative to that of W5-treated or untreated sperm.
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CaM Antagonists Altered Sperm Motility and Inhibited Hyperactivation
To ascertain whether antagonists of CaM affected sperm motility, sperm were incubated for 90 min and then analyzed for head movements using the IVOS Sperm Analyzer. The mean percentage of motile sperm among sperm populations treated with W7 was significantly less than that of the control, but not significantly less than that for the equivalent concentration of W5 (Table 2). Because the mean percentage of motile sperm after 10 µM CZ treatment was less than 10%, other motility parameters were not examined in this group.
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The mean VCL was not altered by the presence of 100 µM W7; LIN was significantly higher than that of control sperm and sperm incubated in 100 µM W5. The percentage of motile cells that were hyperactivated was significantly less for W7-treated sperm than for W5-treated sperm, and this level was less than that of control sperm. Thus it appears that CaM antagonists alter motility; however, the effect of CZ was more severe than that of W7.
Effects of CaM Antagonists on Motility Were Not Reversed by cAMP Analogues
As described above, cAMP analogues suppressed the inhibitory effect of W7 and CZ on capacitation-dependent sperm membrane changes. Therefore, it was important to test whether the CaM antagonist-induced inhibition of motility could also be overcome by cAMP analogues. When 3 mM 8-bromo cAMP plus 200 µM IBMX was added to the incubation medium containing either 100 µM W7 or 10 µM CZ and incubated for 90 min, the sperm displayed motility similar to that obtained when cAMP analogues were not added (Table 2). Thus, in contrast to the results seen with capacitation-dependent sperm membrane changes, addition of cAMP analogues was unable to reverse the effects of CaM antagonists on motility.
To determine whether inhibition of CaM was irreversible, sperm were incubated as before for 90 min in CaM inhibitors, then diluted into control (IVF) medium and immediately analyzed for motility. Sperm incubated for 90 min in W7 and then diluted with IVF medium exhibited mean percentage motile, VCL, LIN, and hyperactivation levels similar to those of sperm incubated in W5 or IVF medium (Table 3). Sperm incubated 90 min in CZ and then diluted with IVF medium displayed motility parameter values similar to those of sperm incubated in IVF medium, with one exception. The percentage of hyperactivated sperm was significantly less for CZ-treated sperm than for control sperm. Thus, all of the effects of W7 on motility, and most of the effects of CZ on motility, were quickly reversed when the drug concentration in the medium was reduced 40-fold or more.
CaM Antagonists Did Not Specifically Inhibit Tyrosine Phosphorylation of Sperm Proteins
Tyrosine phosphorylation of specific mouse sperm proteins has been shown to be correlated with capacitation [29]. To determine whether CaM antagonists altered this process, sperm were incubated 90–120 min with W5, W7, or CZ; then proteins were extracted and subjected to immunoblot analysis with antibodies to phosphotyrosine as the probe. Tyrosine phosphorylation of hexokinase (Mr = 116 000), which is not dependent on capacitation [29], served as an indication of equivalent lane loading. Treatment of sperm with either 100 µM W7 or W5 reduced most band intensities, relative to values in control sperm, but there was no further decrease in intensity with higher concentrations of W7 (Fig. 6A). Also, W7 did not consistently reduce the intensity of protein tyrosine phosphorylation relative to that of sperm treated with the same concentration of W5 (Fig. 6A).
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Proteins from sperm treated with 10 µM CZ exhibited a level of tyrosine phosphorylation similar to that of control sperm, and band intensity did not appear to decrease consistently with increasing concentrations of CZ (Fig. 6B). Quantitative analysis of all experiments did not demonstrate any significant differences in intensity between sperm treated with the same concentration of W7 and W5, or between sperm treated with 10 µM CZ and IVF medium with 1.5% DMSO (data not shown). Thus, it appears that levels of CaM antagonists that significantly inhibited capacitation-correlated membrane changes as assayed by the CTC B pattern and LPC-induced AR, and capacitation as assayed by IVF, were unable to substantially reduce the tyrosine phosphorylation of sperm proteins that occurs during capacitation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment of sperm with 100 µM W7, a highly specific antagonist of CaM, significantly decreased their fertilization competence, relative to that of control sperm (Fig. 5). However, treatment of sperm with 100 µM W5, an approximately 10-fold less potent W7 analogue, did not affect their ability to fertilize relative to control sperm. This result indicates that specific inhibition of sperm CaM prior to their coincubation with zona pellucida-intact oocytes inhibits fertilization, and thus suggests that CaM plays a role in mouse sperm capacitation. Since IVF requires multiple sperm functions, we wondered which of these might be inhibited by W7.
The B pattern after CTC staining was used as a probe for monitoring capacitation-dependent sperm membrane changes, since it is correlated with the sperm's ability to undergo the zona pellucida-induced AR [22]. Both W7 and CZ, another CaM antagonist structurally unrelated to W7, inhibited the B pattern in a concentration-dependent manner (Fig. 2). Furthermore, both W7 and CZ inhibited the sperm's ability to undergo the LPC-induced AR, an assay for capacitation-dependent membrane changes that is unrelated to the B pattern (Fig. 3). Examination of the percentage viable sperm indicated that the antagonist-induced inhibition was not due to nonspecific toxic effects (Table 1). These results indicate that CaM antagonists inhibit capacitation-dependent membrane changes, and suggest that this pathway involves CaM. Capacitated mouse sperm contain detectable levels of CaM (Fig. 1), and two independent immunoelectron microscopic studies have suggested that CaM is localized to the postacrosomal region of the mouse sperm head [9, 10]—supporting the idea that CaM is in an appropriate location to regulate sperm membrane changes important for fertilization.
The immediate target of CaM remains unclear. Mammalian sperm contain a number of CaM-dependent enzymes, including calcineurin, adenylyl cyclase, and PDE, as well as a CaM-binding protein, calspermin [2–5, 38]. Calcineurin could be involved in flagellar functions (see below). While the function of calspermin is unknown, it could act as a "sink" for CaM [38]. Since cAMP is known to be important for sperm membrane changes during capacitation [17], enzymes involved in cAMP metabolism are likely targets. We observed that the CaM antagonist-induced inhibition of the sperm B pattern (Fig. 3) and LPC-induced AR could be overcome by active cAMP analogues (Fig. 4), suggesting that CaM might act by increasing cAMP levels within the sperm. If so, it is possible that CaM directly stimulates adenylyl cyclase.
There is evidence for CaM regulation of sperm cAMP metabolism in a variety of animals. In abalone and equine sperm, CaM activates adenylyl cyclase to stimulate the synthesis of cAMP. CaM antagonists, e.g., CZ, inhibit the activity of adenylyl cyclase in a concentration-dependent manner, and activity is restored by the addition of CaM [2, 39]. In human beings and hamsters, treatment of sperm with CZ causes a significant decrease in intracellular cAMP concentration [11, 40]. While CaM-dependent PDE is found in rat and bovine sperm [3, 4], it is unlikely that in mouse sperm CaM is acting solely via PDE, since activation of this enzyme would reduce cAMP levels. Although membrane adenylyl cyclase of mouse sperm is stimulated by solubilized zona pellucida proteins even in the presence of EGTA [41], the recent discovery of a mammalian testis-specific cytosolic adenylyl cyclase activity [42] provides another possible route by which CaM might directly influence cAMP levels. Our data are consistent with the hypothesis that CaM is necessary, either directly or indirectly, for an increase in sperm cAMP that in turn causes sperm membrane changes leading to competence in fertilization.
Tyrosine phosphorylation of specific sperm proteins has been shown to be correlated with the ability of sperm to undergo a zona pellucida-induced AR and to be dependent on cAMP [17]. Thus, we expected that CaM antagonists would inhibit this process. However, concentrations of CaM antagonists that significantly inhibited the B pattern, the LPC-induced AR, and IVF were not able to substantially affect the level of protein tyrosine phosphorylation (Fig. 6). Although 100 µM W7 did reduce the level of protein tyrosine phosphorylation relative to that of control sperm, it was not reduced relative to that of sperm treated with a similar concentration of W5. This suggests that the reduction in phosphorylation was a nonspecific effect of naphthalenesulfonamide derivatives, rather than a specific inhibition of CaM-dependent activities. Effects of CZ were more straightforward: at a level that completely inhibited the B pattern and the LPC-induced AR, there was no apparent reduction of protein tyrosine phosphorylation relative to that of control sperm; however, higher concentrations sometimes decreased this process (Fig. 6).
One possible explanation for this result is that tyrosine phosphorylation is stimulated by a lower level of intracellular cAMP than that required for capacitation-related membrane changes. As shown for human and hamster sperm [11, 40], treatment of mouse sperm with CaM antagonists probably results in a decrease in intracellular cAMP, to a level that might not have been sufficient to induce capacitation-dependent membrane changes but could have been enough to activate the cAMP-dependent tyrosine kinase pathway. Regardless of the mechanism, the present data support the conclusions from studies of human and hamster sperm, that protein tyrosine phosphorylation is necessary but not sufficient for capacitation-related membrane changes [32, 43]. Furthermore, these data support a novel conclusion, that protein tyrosine phosphorylation in sperm can be uncoupled from capacitation-dependent membrane changes and thus that these processes operate by at least partially separate pathways.
CaM antagonists also affected sperm motility. At concentrations that inhibited capacitation-dependent membrane changes, both W7 and CZ depressed motility relative to that of untreated sperm (Table 2). In addition, W7 inhibited hyperactivation, as did CZ at lower concentrations, allowing a reasonable percentage of motile sperm (unpublished results). Furthermore, after the resuspension of CZ-treated sperm in drug-free medium, motility was restored but hyperactivation was still inhibited (Table 3). These results are similar to findings from studies of CaM antagonists with human and hamster sperm [11, 40]. A W7 derivative, W13, at the concentration of 200 µM, induced abnormal circular swimming patterns in dog sperm flagella; but its inactive analogue W12 had no effects on sperm motility [44]. Thus, all of these studies suggest that CaM is important for the control of mammalian sperm flagellar motility. Since exogenous CaM converts symmetric flagellar bending to asymmetric flagellar bending of demembranated sea urchin sperm [45], and acquisition of hyperactivation involves the modulation of flagellar asymmetry [1, 46], CaM might directly control the asymmetric bending characteristic of hyperactivated sperm.
Unlike sperm membrane changes, effects of CaM antagonists on motility and hyperactivation were not reversed by exogenous cAMP analogues (Table 2). This finding is in agreement with a study of hamster sperm in which addition of IBMX to CZ-treated sperm increased the intracellular cAMP concentration to a level higher than that in capacitated sperm but did not restore hyperactivated motility [11]. These data suggest that CaM regulation of flagellar movement is independent or downstream of cAMP.
Interestingly, dilution in drug-free IVF medium almost immediately restored the motility of CaM antagonist-treated sperm and hyperactivation among W7-treated sperm (Table 3). This observation has two implications. First, it implies that the W7-induced inhibition of the sperm's ability to fertilize eggs in vitro was not due to the disruption of their motility, and therefore makes it likely that the decrease in IVF by CaM antagonist was due to inhibition of the sperm's ability to undergo membrane changes necessary for fertilization. Second, these data indicate that the CaM antagonist-modulated inhibition of motility is quickly reversible. This suggests that CaM antagonists act differently on flagellar movement and capacitation-dependent membrane changes, since sperm were suspended in drug-free medium for 2 h without completely recovering their ability to fertilize the eggs. Thus, because of the differences in response to cAMP analogue and timing of recovery from the drugs, it appears that the signal transduction pathways involved in regulation of sperm motility and hyperactivation are at least partially separate from those needed for the capacitation-dependent membrane changes.
In summary, our present results provide evidence that CaM, a downstream target for Ca2+, is involved in modulating mouse sperm capacitation as measured by IVF. CaM antagonists inhibit sperm membrane changes as assayed by the B pattern and the LPC-induced AR, and cAMP acts downstream of CaM in this pathway. The failure of CaM antagonists to also block protein tyrosine phosphorylation suggests that this capacitation-related process could be a separate pathway from that leading to sperm membrane changes. Our data also support the hypothesis that CaM is involved in hyperactivation, but cAMP is not acting downstream of CaM in this pathway.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by NIH grant HD 15045. 
2 Correspondence: Patricia Olds-Clarke, Department of Anatomy and Cell Biology, Temple University School of Medicine, 3400 N. Broad St., Philadelphia, PA 19140. FAX: 215 707 2966; polds-cl{at}vm.temple.edu
Accepted: December 28, 1999.
Received: October 25, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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H. Qi, M. M. Moran, B. Navarro, J. A. Chong, G. Krapivinsky, L. Krapivinsky, Y. Kirichok, I. S. Ramsey, T. A. Quill, and D. E. Clapham From the Cover: All four CatSper ion channel proteins are required for male fertility and sperm cell hyperactivated motility PNAS, January 23, 2007; 104(4): 1219 - 1223. [Abstract] [Full Text] [PDF]
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 G. G. Ignotz and S. S. Suarez Calcium/Calmodulin and Calmodulin Kinase II Stimulate Hyperactivation in Demembranated Bovine Sperm Biol Reprod, September 1, 2005; 73(3): 519 - 526. [Abstract] [Full Text] [PDF]
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 C. I. Marin-Briggiler, K. N. Jha, O. Chertihin, M. G. Buffone, J. C. Herr, M. H. Vazquez-Levin, and P. E. Visconti Evidence of the presence of calcium/calmodulin-dependent protein kinase IV in human sperm and its involvement in motility regulation J. Cell Sci., May 1, 2005; 118(9): 2013 - 2022. [Abstract] [Full Text] [PDF]
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 C. Rodeheffer and B. D. Shur Sperm from {beta}1,4-galactosyltransferase I-null mice exhibit precocious capacitation Development, February 1, 2004; 131(3): 491 - 501. [Abstract] [Full Text] [PDF]
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T. A. Quill, S. A. Sugden, K. L. Rossi, L. K. Doolittle, R. E. Hammer, and D. L. Garbers Hyperactivated sperm motility driven by CatSper2 is required for fertilization PNAS, December 9, 2003; 100(25): 14869 - 14874. [Abstract] [Full Text] [PDF]
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H. Ecroyd, R. C. Jones, and R. J. Aitken Tyrosine Phosphorylation of HSP-90 During Mammalian Sperm Capacitation Biol Reprod, December 1, 2003; 69(6): 1801 - 1807. [Abstract] [Full Text] [PDF]
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R. M. Turner Tales From the Tail: What Do We Really Know About Sperm Motility? J Androl, November 1, 2003; 24(6): 790 - 803. [Full Text] [PDF]
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L. Lefievre, E. de Lamirande, and C. Gagnon Presence of Cyclic Nucleotide Phosphodiesterases PDE1A, Existing as a Stable Complex with Calmodulin, and PDE3A in Human Spermatozoa Biol Reprod, August 1, 2002; 67(2): 423 - 430. [Abstract] [Full Text] [PDF]
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F. Urner, G. Leppens-Luisier, and D. Sakkas Protein Tyrosine Phosphorylation in Sperm During Gamete Interaction in the Mouse: The Influence of Glucose Biol Reprod, May 1, 2001; 64(5): 1350 - 1357. [Abstract] [Full Text]
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