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Biology of Reproduction 62, 1875-1881 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Distribution and Localization of Calmodulin-Binding Proteins in Bull Spermatozoa1

Pierre Leclerc2,a, and Serge Goupil

a Endocrinologie de la Reproduction, Centre de Recherche du CHUQ and Centre de Recherche en Biologie de la Reproduction, Quebec, Quebec, Canada G1L 3L5

ABSTRACT

Previous studies from our laboratory have shown that a decrease in the calmodulin binding properties of a few sperm proteins occurs during the capacitation process, an effect associated with a decrease in intracellular calmodulin concentrations. Using biotinylated-calmodulin nitrocellulose overlay assay on protein extracts of subcellular fractions of bull spermatozoa, one of these proteins (p32) is detected in the flagellar-enriched fractions, whereas p30 is found in the fraction enriched with sperm heads. This latter calmodulin binding protein, p30, appears to be associated with the perinuclear theca. None of these binding proteins was solubilized by nonionic detergents. Sodium dodecyl sulfate was effective solubilizing p32, whereas p30 was extracted only in conditions reported to isolate the perinuclear theca. Cellular localization of calmodulin binding proteins was also achieved by incubating spermatozoa fixed on slides with biotinylated calmodulin and revealed in a further step by fluorescein-conjugated streptavidin. Using this procedure, it was found that calmodulin binds to the sub- and postacrosomal areas of the sperm head along with the midpiece in the presence of Ca2+. Only a sharp band of fluorescence at the subacrosomal area was observed when this procedure was performed in the absence of Ca2+ in the presence of EGTA. The pattern of cellular calmodulin binding was highly decreased when spermatozoa were incubated under capacitating conditions, in the presence of heparin, in agreement with the published effect of capacitation on calmodulin binding proteins.

INTRODUCTION

Spermatozoa are highly specialized cells whose major role is to bring its genetic content in the vicinity of the egg. To fertilize the egg successfully, spermatozoa have to express proper mobility and fertilizing ability functions. These two activities are compartmentalized to the flagellum and the head, respectively. Although the fertilization and motility processes are separately controlled, Ca2+ is a key regulator of both functions. Calmodulin (CaM) is the major intracellular Ca2+-binding protein and mediates the Ca2+ regulation over a large number of enzymes [1].

The importance of Ca2+ and CaM in sperm motility has been shown for many years [2]. The role of Ca2+/CaM in the control of the flagellar movement can involve different regulatory mechanisms. Sperm motility has been described for a long time as a process regulated by protein phosphorylation, and different phosphoproteins have been associated with the initiation or maintenance of motility [3–7]. Ca2+/CaM can regulate the phosphorylation status of these proteins through the activation of the CaM-dependent protein phosphatase calcineurin [8]. There is growing evidence supporting the role of protein phosphatases in the control of sperm motility [9–12]. On the other hand, most of these motility-related proteins are protein substrates for the cAMP-dependent phosphorylation pathway, another process that can be affected by CaM. This intracellular modulator can decrease the cellular concentration of cAMP through the activation of a sperm cyclic nucleotide phosphodiesterase [13] or increase the levels of this nucleotide through the activation of sperm adenylyl cyclase [14, 15].

In spermatozoa, the localization of CaM in the acrosomal portion of the head [16–20] suggests a role for this regulatory protein in the capacitation/acrosome reaction processes. It has been proposed that CaM affects sperm capacitation through regulation of Ca2+ intake [21–23]. The modification in CaM compartmentalization during sperm capacitation [19] is also supportive of such an involvement. Numerous CaM-binding proteins have been detected in sperm cells [24–29] and semen [30]. Among those CaM-binding proteins, at least two are known players in the capacitation/acrosome reaction process. Phospholipase A2 is colocalized to CaM in spermatozoa [17], and acrosin is known to interact with CaM in vitro [31]. In addition, a decrease in the CaM binding to specific sperm proteins [27, 28] has been shown to be highly correlated to sperm capacitation as measured by in vitro fertilization. Nevertheless, these proteins remain to be identified. In addition, the localization of these two CaM-binding proteins (CaMBP) has never been described. Therefore, the objective of the present study was to characterize better and to localize these capacitation-modulated CaM-binding proteins in bull spermatozoa.

MATERIALS AND METHODS

Sperm Culture

Either fresh or frozen bull semen, kindly donated by L'Alliance Semex at the Centre d'Insémination Artificielle du Québec (C.I.A.Q. Inc., St-Hyacinthe, PQ), was used throughout this study. The semen was diluted in Sp-TALP [32] supplemented with 6 mg/ml fatty acid–free BSA (Sigma, St. Louis, MO), and washed twice by centrifugation. Resultant spermatozoa were resuspended (10 x 106 cells/ml) in the same medium in the absence or presence of 10 µg/ml of the capacitation inducer heparin (166 IU/mg; Sigma). An aliquot of the suspension was taken before or after a 6-h incubation at 39°C, 5% CO2 in air. At specific times, spermatozoa were centrifuged and washed in Sp-TALP devoid of BSA. Sperm proteins were extracted in solubilization buffer (final concentrations 2% SDS, 62.5 mM Tris-HCl, pH 6.8, 5% ß-mercaptoethanol, 10% glycerol) and heated for 5 min at 100°C. On one occasion, epididymides were collected at a local slaughterhouse and immediately brought to the laboratory on ice. Spermatozoa were obtained by retrograde flushing of the cauda epididymis with PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4)-saturated mineral oil, resuspended in HBS (10 mM HEPES, pH 7.2, 150 mM NaCl), and washed twice by centrifugation as described above.

Identification of CaMBP

Sperm CaMBP were identified using the nitrocellulose CaM overlay procedure [33] modified to use biotinylated bovine brain CaM (Calbiochem, San Diego, CA) instead of iodinated CaM. Sperm proteins were separated by SDS-polyacrylamide gel electrophoresis [34] and electrotransferred to nitrocellulose (0.22 µm pore size; Micron Separations Inc., Westboro, MA) according to the methods described by Towbin et al. [35]. The membranes were incubated for 1 h in TTBS (0.9% w/v NaCl, 20 mM Tris-HCl pH 7.4, 0.05% Tween-20, 1 mg/ml BSA) containing either 1 mM CaCl2 or 1 mM EGTA to prevent nonspecific binding. Biotinylated-CaM was next added (20 ng/ml) and the membranes were incubated for another hour. The blots were next extensively washed in TTBS in the presence of 1 mM CaCl2 or EGTA and further incubated for 1 h with streptavidin conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) in the presence or absence of Ca2+. Again, the blots were extensively washed, and the positive CaMBP bands were detected using an enhanced chemiluminescence kit (Amersham Life Science Inc., Oakville, ON, Canada) and x-ray film exposure (Fuji, Tokyo, Japan).

Detergent Solubilization of Sperm CaMBP

In one experiment, fresh semen was diluted in HBS, and spermatozoa were washed twice by centrifugation and incubated (10 x 106 sp/ml) for 15 min at room temperature in the presence of either nonionic detergents (Triton X-100, NP-40, Tween-20), ionic detergents (SDS, deoxycholate), or zwitterionic detergents (CHAPS). All the detergents were used at the final concentration of 1% (w/v). After treatment, the samples were centrifuged at 4°C (5 min, 10 000 x g) and the presence of CaMBP was investigated in the solubilized material as described above by the nitrocellulose CaM overlay procedure.

Localization of CaMBP

The localization of sperm CaMBP was assessed in two different ways. In the first set of experiments, the subcellular distribution of sperm CaMBP was evaluated using a modified protocol developed to characterize bull sperm perinuclear theca [36] and the nitrocellulose CaM overlay assay. One hundred microliters of fresh semen was diluted in 5 ml HBS and washed twice by centrifugation. The final sperm pellet was suspended in 500 µl HBS containing protease inhibitors (500 µM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin, 50 mM benzamidine) and sonicated twice (45 sec) on ice to dislocate the head from the flagellum. Dislocation was monitored by light microscopy. Under these conditions, more than 85% of spermatozoa were dislocated. The sonicated material was layered onto 500 µl of 90% Percoll-HBS and centrifuged 10 min (725 x g) at 4°C. The supernatant that was presumed to contain membranes and cytosoluble material was kept and further centrifuged at 10 000 x g. The material obtained at the interface, enriched in flagellar fragments, was further centrifuged (725 x g) for 10 min, and the supernatant containing mostly flagellar fragments was kept. The pellet obtained at the bottom of the 90% Percoll layer, enriched in sperm heads, was resuspended with 500 µl HBS and centrifuged again through a 90% Percoll-HBS layer as described above. The resulting pellet consisted mostly in sperm heads. Heads were next processed as previously described to isolate perinuclear theca [36], and CaMBP were determined using the nitrocellulose CaM overlay procedure on proteins from the Triton X-100, NaCl, and NaOH extracts. Protein concentration was determined in each fraction using the BCA assay (Pierce, Rockford, IL), and equivalent amounts of proteins (1.5–2.5 µg) from each fraction were separated by SDS-PAGE.

In a second set of experiments, the localization of CaMBP was investigated on whole sperm cells using a protocol similar to the indirect immunofluorescence assay, with the exception that biotinylated CaM was used instead of immunoglobulins. Permeabilized cells fixed on microscope slides were incubated with biotinylated CaM and fluorescein isothiocyanate-conjugated streptavidin (streptavidin-FITC) was used to detect the localization of biotinylated CaM within the sperm cells. Before or after the 6-h incubation in the absence or presence of heparin, spermatozoa were centrifuged and washed as described above, and the resultant sperm pellet was resuspended in ice-cold ethanol and kept on ice for 30 min. The sperm cells were next smeared on a microscopic slide and allowed to dry. The slides were kept overnight at 4°C until use. The slides were incubated for 1 h in PBS containing 1 mg/ml BSA and either 1 mM CaCl2 or 1 mM EGTA to rehydrate the cells. The spermatozoa were next incubated for another hour in PBS-BSA containing biotinylated CaM (100 ng/ml) in the presence or absence of Ca2+. After three washes in PBS-BSA (±Ca2+), the cells were incubated for 1 h with streptavidin-FITC (Life Technologies Inc., Burlington, ON) in PBS-BSA (±Ca2+). Following several rinses with PBS-BSA, the slides were mounted with coverslips using 90% glycerol containing 1.5% 1,4-diazabicyclo-[2.2.2] octane as an antibleaching agent. Fluorescence was detected by epifluorescence microspcopy with a UV light.

RESULTS

Detection of CaMBP

In our previous studies [27, 28] where we described the effects of sperm capacitation on CaMBP, the experiments were performed using frozen-thawed bull spermatozoa. In the present study, our first aim was to determine whether the freeze-thaw process affects the pattern of sperm CaMBP. In addition, because CaMBP has been detected in bull seminal plasma [30], we investigated whether the CaMBP present on the spermatozoa come from the cells themselves or originate from the seminal plasma and bind to the cells. The presence of CaMBP was thus investigated in epididymal as well as in ejaculated spermatozoa that were used either as fresh or as extended and frozen/thawed semen. As seen in Figure 1, no major difference is detected in the pattern of CaMBP from either epididymal, ejaculated, or frozen-thawed spermatozoa. Using the biotinylated CaM nitrocellulose overlay protocol, three types of CaMBP were detected. Proteins, such as the 202-kDa one, that bind to CaM preferably in the absence of Ca2+ (Ca2+ inhibited), proteins, such as those of 181 and 117 kDa that bind to CaM in the presence of Ca2+ (Ca2+ dependent) and others, such as those of 49, 32, and 30 kDa that bind to CaM in either the presence or absence of Ca2+ (Ca2+ independent) (Fig. 1). To characterize further these last proteins, to which CaM binding is inversely correlated to sperm capacitation [27, 28], biotinylated CaM overlays were performed on protein extracts obtained using different nonionic, ionic, and zwitterionic detergents. Unlike most of the streptavidin-binding proteins detected in spermatozoa, no CaMBP were detected in Triton X-100-, NP-40- (Fig. 2, lanes 2 and 3), Tween-20- (not shown), or CHAPS-solubilized proteins (Fig. 2, lane 6). The CaMBP p49 was extracted by SDS or deoxycholate, whereas p32 was extracted more effectively by SDS than by deoxycholate (Fig. 2). As shown in this figure, the CaMBP p30 was not extracted by any of the abovementioned detergents. This result suggests that this binding protein is associated with or is a component of the sperm cytoskeleton.



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  		FIG. 1. Calmodulin-binding proteins in epididymal and ejaculated spermatozoa. Proteins from 1 x 106 spermatozoa were separated by SDS-PAGE, transferred to nitrocellulose, and processed for biotinylated-CaM blot overlay as described in Materials and Methods. A) Pattern of Coomassie brilliant blue-stained proteins, B) pattern of CaMBP obtained in the presence of Ca2+, and C) pattern of CaMBP obtained in the absence of Ca2+. In each panel, lanes 1, 2, and 3 represent proteins extracted from epididymal, ejaculated, and frozen–thawed spermatozoa, respectively. Molecular weight standards (Mr x 10-3) are shown on the left, arrowheads point to nonspecific bands (134, 76, and 73 kDa) that bind to the peroxidase-conjugated streptavidin used to detect the biotinylated CaM



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  		FIG. 2. Calmodulin-binding proteins in bull sperm detergent extracts. Washed, freshly ejaculated spermatozoa were incubated for 15 min at room temperature in HBS in the presence of 1% (w/v) Triton X-100, NP-40, SDS, deoxycholate, or CHAPS (lanes 2–6, respectively). At the end of the extraction period, the cells were centrifuged as described in Materials and Methods and CaMBP were determined by biotinylated-CaM blot overlay procedure (in the presence of Ca2+) in the solubilized material. The pattern of CaMBP detected in total sperm proteins is shown (lane 1). Molecular weight standards (Mr x 10-3) are shown on the left; arrowheads point to nonspecific bands that bind to the peroxidase-conjugated streptavidin used to detect the biotinylated CaM

Localization of CaMBP

The localization of bull sperm CaMBP was next investigated. As shown in Figure 3, the two CaMBP, p32 and p30, are located differently; p32 is detected in the fraction containing the sperm flagella, whereas p30 is found in the head fraction. On the other hand, in addition to p32, CaMBP of 92, 34 kDa, and a doublet of 19–19.5 kDa were detected in the flagellar fraction, whereas up to 6 CaMBP ranging from 101 to 157 kDa were evident in the sperm heads in addition to p30 (Fig. 3). In the procedure of perinuclear theca isolation, the sperm heads are sequentially extracted with Triton X-100 and 1 M NaCl. Lastly, the perinuclear theca is extracted with 0.1 N NaOH. Subcellular distribution of CaMBP within the sperm head reveals that p30 is detected in the NaOH-solubilized materials, which suggests that this CaMBP resides within the perinuclear theca (Fig. 4, lane 4). However, some p30 remains associated with the heads and is solubilized by 2% SDS treatment (5 min, 100°C; Fig. 4, lane 5), although further p30 solubilization is achieved using reducing agents in addition to SDS (Fig. 4, lane 6).



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  		FIG. 3. Subcellular distribution of bull sperm CaMBP. Washed freshly ejaculated spermatozoa were decapitated by sonication and the various fractions were separated by centrifugation (725 x g) on a discontinuous 45–90% Percoll gradient. The CaMBP were evaluated by nitrocellulose CaM overlay assay in the presence of Ca2+, on proteins from total sonicated spermatozoa (lane 1), in the 10 000 x g supernatant (lane 2) of the initial 725 x g centrifugation, in the flagella- (lane 3) and head-enriched (lane 4) fractions. Molecular weight standards (Mr x 10-3) are shown on the left; arrowheads point to nonspecific bands that bind to the peroxidase-conjugated streptavidin used to detect the biotinylated CaM



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  		FIG. 4. Localization of the CaMBP p30 within the sperm head. Washed, freshly ejaculated spermatozoa were decapitated by sonication and the various fractions were separated by centrifugation (725 x g) on a discontinuous 45–90% Percoll gradient. The head-enriched fraction was further extracted using a protocol known to isolate the perinuclear theca [36]. The CaMBP were evaluated by nitrocellulose CaM overlay assay in the presence of Ca2+, on proteins from isolated heads (lane 1), in the Triton X-100 extract of the heads (lane 2), in the NaCl extract following Triton X-100 treatment (lane 3), in the NaOH-extracted fraction following NaCl treatment (lane 4), in the 2% SDS-extracted materials following NaOH treatment (lane 5), and in the fraction solubilized by 2% SDS in the presence of 2 mM dithiothreitol (lane 6). Molecular weight standards (Mr x 10-3) are shown on the left; arrowheads point to nonspecific bands that bind to the peroxidase-conjugated streptavidin used to detect the biotinylated CaM

Localization of sperm CaMBP was also achieved using biotinylated-CaM on spermatozoa fixed on slides and revealed by FITC-conjugated streptavidin. As shown in Figure 5A, a strong fluorescent signal was observed at the equatorial segment and the postacrosomal portion of the sperm head (Fig. 5A). This signal was specific to the presence of biotinylated-CaM because no signal was observed when the cells were incubated only with FITC-conjugated streptavidin without preincubation with biotinylated-CaM (data not shown). In addition, only a weak signal was detected when the binding of biotinylated-CaM to fixed cells was assessed in the presence of a 100-fold excess concentration of unlabeled CaM (Fig. 5C). On the other hand, preincubation of fixed spermatozoa with unlabeled CaM did not affect the biotinylated-CaM binding pattern (data not shown), suggesting that CaM could be displaced from its binding proteins upon addition of biotinylated-CaM. The Ca2+-dependence of biotinylated-CaM binding to spermatozoa was also assessed. The presence of Ca2+ was necessary for CaM binding to the postacrosomal region since no signal was observed in the sperm postacrosomal region when this cation was replaced by 1 mM EGTA during the entire procedure. Under these conditions, biotinylated-CaM bound only to the sperm equatorial segment (Fig. 5B). The pattern of biotinylated-CaM binding to spermatozoa was unaffected by the freezing/thawing process because similar results were obtained whether fresh or cryopreserved spermatozoa were used (data not shown).



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  		FIG. 5. Calmodulin binding to bull spermatozoa. Washed freshly ejaculated spermatozoa fixed on a microscope slide were incubated with biotinylated CaM and the presence of CaM was next revealed with FITC-conjugated streptavidin. The localization of CaM binding to spermatozoa was evaluated in the presence (A) or absence (B) of Ca2+. The specificity of CaM binding was evaluated in the presence of Ca2+ and unlabeled CaM at a 100-fold concentration over biotinylated-CaM (C). Similar results were obtained using frozen/thawed spermatozoa

In previous studies, we demonstrated that bull spermatozoa incubated in the presence of heparin, to induce capacitation, expressed lower levels of CaMBP [27, 28]. In the present study, we observed a dramatic decrease in the binding of biotinylated-CaM to spermatozoa when these cells were previously incubated for 6 h in the presence of heparin (Fig. 6C) as compared to those incubated for the same length of time under noncapacitating conditions, in the absence of heparin (Fig. 6B). This result is in perfect agreement with our previous results [27, 28]. In fact, the biotinylated-CaM binding pattern in heparin-treated spermatozoa was similar to the one observed when the CaM-binding assay was performed in the absence of Ca2+. Because a long exposure of spermatozoa to heparin might result in the exocytosis of the acrosome [37], a control experiment was performed to ensure that the heparin-induced decrease in CaM binding to spermatozoa was not caused by an increase in sperm acrosomal exocytosis. The evaluation of the acrosomal status of the sperm cells was done by FITC-conjugated Pisum sativum agglutinin binding. Heparin treatment had no effect on sperm acrosomal status (data not shown), suggesting that the decrease in CaM binding to heparin-treated spermatozoa was caused by an alteration in CaMBP properties.



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  		FIG. 6. Effect of capacitation on the binding of calmodulin to bull spermatozoa. Washed freshly ejaculated spermatozoa were fixed on a microscope slide before (A) or after a 6-h incubation period in the absence (B) or presence (C) of heparin to induce capacitation. The localization of CaM binding to spermatozoa was evaluated in the presence of Ca2+. Similar results were obtained using frozen/thawed spermatozoa

DISCUSSION

Previous results from our laboratory showed that specific sperm proteins expressed lower binding activity toward CaM when they were incubated under capacitating conditions [27, 28]. However, these proteins, for which CaM binding was inversely correlated to capacitation, were not further characterized and their localization within the sperm cells had yet to be determined. In the present study, we demonstrate that even though CaMBP are found in the seminal plasma [30], capacitation-affected CaMBP do not originate from the seminal plasma because similar levels of these binding proteins are found in epididymal as well as in ejaculated spermatozoa (Fig. 1).

Our attempts using biotinylated-CaM to determine the localization of sperm CaMBP revealed that these proteins are found mostly in the midpiece, the postacrosomal region, and a bright intense signal is also found in the subacrosomal area. These results are in agreement with a previous study [20] showing a similar localization of CaM in rabbit spermatozoa. In that study, the authors showed that, in the sperm head, CaM is detected in the postacrosomal area and in a subacrosomal region located anterior to the equatorial segment. When we decapitated the spermatozoa by sonication and isolated flagellar and head structures, no CaMBP were detected in the material (cytoplasm and membranes) that was not pulled down by a 10 000 x g centrifugation, whereas both isolated flagellar and head fractions contained CaMBP (Fig. 3). These results suggest that, although CaM is a cytosolic protein found primarily in the acrosomal region of spermatozoa, it remains associated with binding proteins that are resistant to extraction with nonionic detergents (Fig. 2), possibly part of the cytoskeleton. Moreover, our results show that flagella and heads do not share the same CaMBP (Fig. 3). The CaMBP of 92, 34, 32, and a doublet of 19–19.5 kDa are easily detected in the flagellar fraction, whereas a group of at least six CaMBP with a mass ranging from 101 to 157 kDa and a 30-kDa CaMBP are found in the fraction containing the sperm heads.

In the present study, we clearly show that the two CaMBP, p32 and p30, for which binding to the intracellular mediator of Ca2+, CaM, is inversely correlated to sperm capacitation [27, 28], are differently located within the sperm cell. The 32-kDa CaMBP appears associated with the flagellar structures (Fig. 3, lane 3) and is solubilized by SDS, whereas the 30-kDa CaMBP is found in the head (Fig. 3, lane 4). This latter binding protein is poorly solubilized by SDS treatment unless in the presence of reducing agents and is detected in the extract containing the perinuclear theca (Fig. 4). On the other hand, the role of these two CaMBP in sperm function remains to be established. It has been shown recently that, during the process of fertilization, the perinuclear theca is removed from the sperm nucleus upon binding to the oocyte's microvilli and precedes the decondensation of the nucleus and the formation of the male pronucleus [38]. It would be interesting to determine whether the CaMBP p30 is involved in this process and whether sperm capacitation is important not only for the sperm binding to the egg's zona pellucida and the acrosomal exocytosis but also for processes that occur following the acrosome reaction.

The presence of a 32-kDa sperm flagellar CaMBP has been reported for several years and was named calspermin according to its tissue specificity [39, 40]. This protein is encoded by the CaM kinase IV gene [41] and is predominantly expressed in postmeiotic germ cells [39, 40]. However, the exact role of this protein, other than being an intracellular inhibitor of CaM [42] is still undefined. Whether or not the CaMBP p32, described in the present study, is calspermin remains to be established.

In the present study, only a slight CaM binding to spermatozoa was observed when these cells were incubated under capacitating conditions, in the presence of heparin (Fig. 6). This result is in total agreement with our previous reports describing a decrease in the binding of CaM to sperm proteins during the process of capacitation [27, 28]. Nevertheless, the mechanisms involved in this decrease in CaM binding to sperm proteins are not known. Most likely, modifications of sperm CaMBP occur during the process of capacitation so they are unable to bind CaM. This was assessed in an experiment (not shown) where fixed spermatozoa were preincubated with unlabeled CaM, washed to remove unbound CaM, then incubated with biotinylated-CaM and revealed with streptavidin-HRP. Under those conditions, biotinylated-CaM replace bound unlabeled CaM and the final CaM-binding pattern was not affected as compared to samples not preincubated with unlabeled CaM. This experiment suggests that the capacitation (heparin)-induced decrease in CaM binding does not result from CaM-saturated CaMBP, leaving no more sites for biotinylated-CaM, but rather from a loss of CaM binding properties exhibited by these binding proteins.

In spermatozoa, CaM is involved in the regulation of Ca2+ fluxes across membranes [21]. Calmodulin exerts this activity through the activation of Ca2+-ATPase [23], an enzyme localized in the postacrosomal region of the sperm head [23], an area where CaM [20] and CaMBP are found (present study). In the sperm context, the role of this enzyme is to maintain low intracellular levels of Ca2+ by pumping this cation into the acrosome. A decrease in CaM binding to this Ca2+-ATPase would lead to a decrease in the activity of this ATP-dependent Ca2+ extruder that will ultimately result in a net Ca2+ uptake. In fact, the process of sperm capacitation is characterized by an elevation in intracellular Ca2+ concentrations [43–45]. The lower CaM binding to the sub- and postacrosomal regions of the head observed in capacitated spermatozoa (present study) is suggestive of such a mechanism.

On the other hand, the regulation of intracellular Ca2+ concentrations not only occurs through the control of Ca2+-ATPase activity. Inositol triphosphate (IP3) receptor-gated Ca2+ channels also regulate the levels of intracellular Ca2+. However, these receptors are localized over the acrosome and in the postacrosomal region of the mammalian sperm head [46, 47]. Activation of these channels will promote the liberation of Ca2+ stored within the acrosome and induce the acrosomal exocytosis. Although CaM binds to IP3 receptors, its functions on this type of receptor remain controversial and appear to depend on the subtype of receptor. It has been recently shown that CaM binds to type-1 IP3 receptors in a Ca2+-independent manner, inhibits IP3 binding to its receptor, and prevents IP3-mediated Ca2+ release from microsomes [48, 49]. In spermatozoa, binding of CaM to a >200-kDa protein has been observed in the absence of Ca2+ (Fig. 1) and a Ca2+-independent association of CaM with proteins present in the subacrosomal region as well as in the midpiece is observed (Fig. 5). Whether this binding protein is an IP3 receptor is still unknown. In the sperm context, the binding of CaM to the IP3 receptors in uncapacitated cells will prevent precocious release of the stored Ca2+, and the resulting acrosomal exocytosis. Upon capacitation, CaM levels [50], CaM binding to proteins [27, 28] as well as CaM binding to sub- and postacrosomal areas of the sperm head (present study) decrease, leaving IP3 receptors responsive to IP3. During the process of fertilization, capacitated spermatozoa bind to the egg's zona pellucida and undergo the acrosome reaction. During this process, the activation of phospholipase C, the enzyme responsible for IP3 generation, has been demonstrated [51].

The results reported in the present study show that the capacitation-induced decrease in CaM binding to sperm proteins occurs at the sub- and postacrosomal region of the head. Although the mechanism for such a decrease in CaM binding remains elusive, it may be involved in the elevation of intracellular Ca2+ concentrations that occur during sperm capacitation and/or the acrosome reaction. Whether this Ca2+ rise occurs through Ca2+-ATPase inactivation or the inhibition reversal of IP3 receptors upon CaM levels and binding decrease remains to be elucidated.

ACKNOWLEDGMENTS

The authors are thankful to L'Alliance SEMEX for their generous gift of bull semen that was used throughout this study.

FOOTNOTES

First decision: 7 October 1999.

1 Supported by a grant from the Natural Science and Engineering Research Council of Canada to P.L. Back

2 Correspondence: Pierre Leclerc, Endocrinologie de la Reproduction, Pav. St-François d'Assise, 10, de l'Espinay, Québec, PQ, Canada G1L 3L5. FAX: 418 525 4195; pierre.leclerc{at}crsfa.ulaval.ca Back

Accepted: February 1, 2000.

Received: August 2, 1999.

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