Changes in calmodulin immunocytochemical localization associated with capacitation and acrosomal exocytosis of ram spermatozoa

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Theriogenology 71 (2009) 789–800 www.theriojournal.com

Changes in calmodulin immunocytochemical localization associated with capacitation and acrosomal exocytosis of ram spermatozoa C. Cola´s a, P. Grasa a,2, A. Casao a, M. Gallego b, J.A. Abecia c, F. Forcada c, J.A. Cebria´n-Pe´rez a,1, T. Muin˜o-Blanco a,1,* a

Department of Biochemistry and Molecular and Cell Biology, University of Zaragoza, Miguel Servet, 177, 50013 Zaragoza, Spain b Department of Animal Pathology and Anatomy, University of Zaragoza, 50013 Zaragoza, Spain c Department of Animal Production, School of Veterinary Medicine, University of Zaragoza, 50013 Zaragoza, Spain Received 9 June 2008; received in revised form 6 October 2008; accepted 14 October 2008

Abstract The aim of this study was to determine the localization of calmodulin (CaM) in ram sperm and the possible changes during in vitro capacitation (CA) and the ionophore-induced acrosome reaction (AR). Likewise, changes in intracellular calcium levels ([Ca2+]i) were also analysed by using flow cytometry. CA was induced in vitro in a medium containing BSA, CaCl2, NaHCO3, and AR by the addition of the calcium ionophore A23187. The acrosomal status was assessed by the chlortetracycline-fluorescence (CTC) assay. Flow cytometry (FC) analyses were performed by loading samples with Fluo-3 AM, that emits fluorescence at a high [Ca2+]i, combined with propidium iodide (PI) that allowed us to discriminate sperm with/without an integral plasma membrane both with high/low [Ca2+]i. Immunocytochemistry localized CaM to the flagellum, and some sperm also contained CaM in the head (equatorial and post-acrosomal regions). CA and AR resulted in a slight increase in the post-acrosomal labelling. The treatment of sperm with increasing concentrations of two CaM antagonists, W7 and calmidazolium (CZ), accounted for an increase in capacitated and acrosome-reacted CTC-sperm patterns. CZ induced a significant reduction in the content of three protein tyrosinephosphorylated bands of approximately of 30, 40 and 45 kDa. However, W7 showed no significant effect at any of the studied concentrations. Neither of them significantly influenced protein serine and threonine phosphorylation. FC analysis revealed that the main subpopulation in the control samples contained 70% of the total sperm with integral plasma membrane and a medium [Ca2+]i. After CA, 67.1% of the sperm preserved an integral membrane with a higher [Ca2+]i. After AR, only 7.2% of the total sperm preserved intact membranes with a very high [Ca2+]i. These results imply that CaM appears to be involved in ram sperm capacitation, and both treatments increased its localization in the post-acrosomal region. # 2009 Elsevier Inc. All rights reserved. Keywords: Fluo-3 AM; Capacitation; CTC; Calmodulin antagonists

1. Introduction

* Corresponding author. Tel.: +34 976761639; fax: +34 976761612. E-mail address: [email protected] (T. Muin˜o-Blanco). 1 These authors contributed equally to this article and share senior co-authorship. 2 Current address: Department of Pharmacology, University of Oxford, UK. 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.10.003

Although fertilization is essential for species survival, the molecular events underlying gamete interaction remain poorly understood. In mammals, freshly ejaculated spermatozoa have to undergo the process of capacitation in the female genital tract to acquire the ability to fertilize mature oocytes [1].

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Calcium has been shown to play a key role in sperm function [2,3]. Although the influence of extracellular calcium in sperm capacitation in mammals has been widely demonstrated [4–7], controversial results about the effect of extracellular calcium on in vitro sperm capacitation and protein tyrosine phosphorylation have been found, this depending on the species and/or laboratories [4,6,8,9]. Capacitation has been associated with Ca2+ uptake [10], which produces increased cyclic adenosine monophosphate concentration (cAMP [11–13]), activation of cAMP-dependent protein kinase A (PKA [14]), resulting in phosphorylation of certain proteins [4– 6,8,15–18], and decreased binding of proteins to calmodulin (CaM) [19–21]. Calcium can act through binding to calmodulin, a 17-kDa Ca2+-binding protein, putative Ca2+ sensor, that plays a central role in many cell signalling pathways by modulating the activity of key regulatory enzymes, ion pumps and proteins associated with calcium-regulated exocytosis [22,23]. The Ca2+/CaM complex regulates the activity of multiple enzymes, including adenylyl cyclases [24,25], phosphatases [26,27], phosphodiesterases (PDEs) [28] and protein kinases [29,30]. CaM appeared to accelerate capacitation in mouse sperm [2,31,32] and, as demonstrated in several studies, CaM antagonists inhibit different aspects of sperm function, including the onset of sperm hyperactivation [3] and the agonist-induced acrosome reaction (AR) in mouse sperm [32]. It was reported that the activation of sperm motility requires extracellular Ca2+ [33–35] and that sperm binding to zona pellucida causes further activation of PKA [36]. The role of PKA in the acrosome reaction may be to release Ca2+ from the acrosome leading to an increase in cytosolic calcium. The depletion of calcium in the acrosome will activate a store-operated calcium entry mechanism in the plasma membrane, which leads to a higher increase in cytosolic calcium and allows the subsequent membrane fusion and acrosome reaction [37,38]. One goal of our laboratories is to understand the molecular regulation mechanism of capacitation in ram sperm. In this study, we have attempted to examine the potential changes in intracellular calcium levels in ram sperm during capacitation and the ionophore-induced acrosomal exocytosis by using flow cytometry analysis. Since Ca2+/CaM plays a significant role in several cell signalling pathways and membrane fusion events, we have used a pharmacological approach to examine the role of CaM in in vitro ram sperm capacitation.

2. Materials and methods 2.1. Sperm preparation All experiments were carried out with fresh semen taken from eight mature Rasa Aragonesa rams using an artificial vagina. The rams, which belonged to the National Association of Rasa Aragonesa Breeding (ANGRA), ranged from 2 to 4 years of age. They were kept at the Veterinary School under uniform nutritional conditions. Experiments were carried out over time, maintaining sires with an abstinence period of 2 days according to previous results [39]. In order to eliminate individual differences, second ejaculates were pooled and used for all assays. The experimental protocol was reviewed and approved by the Ethical Committee of the University of Zaragoza, according to the Guide for Care and Use of Laboratory Animals as adopted by the Society for the Study of Reproduction. A seminal plasma-free sperm population was obtained by a dextran/swim-up procedure [40] performed by using a medium consisted of 200 mM sucrose, 50 mM NaCl, 18.6 mM sodium lactate, 21 mM HEPES, 10 mM KCl, 4 mM NaHCO3, 3 mM CaCl2, 2.8 mM glucose, 0.4 mM MgSO4, 0.3 mM sodium pyruvate, 0.3 mM K2HPO4, 5 mg/ml of BSA 1.5 IU/ml penicillin, and 1.5 mg/ml streptomycin, pH 7.2 (adjusted by NaOH addition). 2.2. Immunocytochemistry Because of the high sensitivity of ram spermatozoa, centrifugation was avoided by working with cells adhered to poly-L-lysine-coated slides. Therefore, after treatments, aliquots of 8  106 cells (500 ml) were fixed with 3.7% formaldehyde [(v/v in PBS), 100 mM sodium phosphate and 50 mM NaCl, pH 7.2] for 20 min at room temperature. Then, the cells were centrifuged and the pellet was resuspended in PBS. After fixation, 20 ml of cell suspension were smeared onto the slide, and maintained at room temperature during 30 min to ensure good adhesion onto the slide. For permeabilization, slides were immersed in methanol for 15 min at room temperature. Endogenous peroxidase was inactivated with 1.7% hydrogen peroxide in 100% ethanol for 30 min, and then non-specific antibody binding sites were blocked by incubation with 5% BSA (w/v in PBS) for 1 h at 37 8C. Thereafter, spermatozoa were incubated with a mouse anti-calmodulin antibody (clones 2D1+1F11+6D4, C7055, Sigma Chemical Co., Madrid, Spain) at a 1:15 dilution in PBS with 1% BSA (overnight/

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4 8C). A Vectastain ABC kit (Vector Laboratories, Burlingame, CA 94010) was used to detect the binding of the primary antibody to the calmodulin. The sections were incubated with a biotinylated anti-mouse antiserum for 40 min. An avidin–biotin–peroxidase complex (Vector Laboratories, Burlingame, CA 94010) was subsequently applied for 45 min. The peroxidase reaction was developed with diaminobenzidine (DAB) and hydrogen peroxide solution (20 mg DAB in 100 ml of 0.05 M Tris–HCl buffer containing 0.005% H2O2) for 5 min. To eliminate the excess of reagents after every incubation step, slides were rinsed with PBS. As negative controls, either the primary or the secondary antibody was omitted. 2.3. In vitro treatments In vitro capacitation was performed by incubation of swim-up obtained samples (1  108 sperm) during 4 h at 39 8C in a humidified incubator with 5% CO2 in air. For the ionophore-induced acrosomal exocytosis, samples obtained by the swim-up procedure were diluted until a 2  107 cells/ml concentration with HEPES glucose buffer (149 mM NaCl, 2.5 mM KCl, 10 mM glucose, 20 mM Hepes and 3 mM CaCl2), pH 7.4 [41]. After sperm dilution, 3 mM calcium ionophore A23187 (Sigma Chemical Co., Madrid, Spain) in 0.3% dimethyl sulphoxide (DMSO, Sigma Chemical Co., Madrid, Spain) was added. Control tubes had DMSO added but no ionophore, which was found to be without effect (data not shown). The samples were incubated at 39 8C for 1 h, and the acrosomal status and cell viability were determined. All assays were performed comparing control (immediately after the selection procedure), capacitated and ionophore-treated samples. 2.4. Evaluation of sperm samples Capacitation status was assessed by the chlortetracycline-fluorescence (CTC) assay that we previously validated for the evaluation of capacitation and acrosome reaction-like changes in ram sperm [17], using a slightly modified method of that described by Ward and Storey [42]. CTC solution (750 mM, Sigma Chemical Co., Madrid, Spain) was prepared daily in a buffer containing 20 mM Tris, 130 mM NaCl and 5 mM cysteine, pH 7.8, and passed through a 0.22 mm filter (Millipore Ibe´rica, Madrid, Spain). To 18 ml of sperm sample, 2 ml of ethidium homodimer-1 (EthD-1, 23.3 mM) were added and then incubated at 37 8C in the dark for 10 min.

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Thereafter, 20 ml of CTC-solution and 5 ml of 12.2% (w/v) paraformaldehyde in 0.5 M Tris–HCl, pH 7.8, were added. An aliquot of 4 ml of stained sample was placed onto a glass slide at room temperature, and mixed with 2 ml 0.22 M Triethylenediamine (an antifade-reagent, Sigma Chemical Co., Madrid, Spain) in glycerol:phosphate buffered saline (PBS, 9:1). The samples were covered with 24 mm  48 mm coverslips, sealed with colorless enamel, and stored at 4 8C in the dark. For the evaluation of CTC patterns, the samples were observed within 12 h under a Nikon Eclipse E-400 microscope under epifluorescence illumination using a V-2A and G-2A filters. All samples were processed in duplicates, and at least 200 spermatozoa per slide were scored. No fluorescence was observed when CTC was omitted from the preparation. Three sperm types were estimated [43]: not capacitated (NC, even distribution of fluorescence on the head, with or without a bright equatorial band), capacitated (C, with fluorescence in the anterior portion of the head) and acrosomereacted cells (AR, showing no fluorescence on the head). 2.5. Effect of calmodulin antagonists on in vitro ram sperm capacitation To determine whether Ca2+/CaM plays a significant role in ram sperm capacitation, we assessed the capacitation state of ram spermatozoa in the presence of two antagonist of calmodulin, calmidazolium (CZ), a strong inhibitor of Ca2+/CaM-dependent phosphodiesterase (type I PDE [52]) and W7, inhibitor of Ca2+dependent phosphodiesterases [53]. The samples were incubated in capacitating conditions (described above) in a medium with/without 5, 10, 20, 50, 75, 100, 200 mM CZ or 50, 100, 150, 200, 500, 750, 1000 mM W7, and the capacitated status was determined by the CTC assay. 2.6. Extraction of proteins For calmodulin identification, sperm proteins were extracted by mixing 1 ml of the swim-up obtained sample with 1 ml of extraction buffer (2% Triton X-100, 19.25 mM Tris–HCl pH 8, 115.5 mM NaCl, 0.77 mM EDTA, 0.77 mM Na3VO4, 0.77 mM NaF), supplemented with a protease inhibitor cocktail (Sigma Chemical Co., Madrid, Spain, P 2714). After 10 min of incubation in ice, the sample was sonicated in ice by four pulses of 10 s, 30% power (Cell disrupter VCX-130, Sonics & Materials, Inc., Newtown, CT, USA), and then, the cells

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were maintained in ice and shaken in an orbital shaker (SO3 Stuart Sc., Stone, Staffordshire, SA) for 1 h. After that, the sample was centrifuged to separate pellet (membrane-bound fraction) and supernatant (cytosolic fraction). For protein phosphorylation analysis, the procedure was already described [17]. Aliquots of 0.5 ml of control or capacitated samples diluted 1:10 (107 cells) were centrifuged at 7500  g in a microfuge for 5 min at room temperature and the supernatant discarded. The resulting sperm pellet was resuspended in 100 ml of extraction medium (2% SDS, 28% sucrose, 12.4 mM TEMED, 185 mM Tris–HCl, pH 6.8) and immediately incubated for 5 min at 100 8C. After centrifugation at 7500  g for 5 min, the supernatant was recovered and the protein concentration was determined [44]. After adding 10% of a protease and phosphatase inhibitor cocktail (Sigma Chemical Co., St. Louis, MO) it was stored at 20 8C. 2.7. SDS-PAGE and immunoblotting To detect calmodulin, proteins extracted with Triton X-100 were supplemented with 5% b-mercaptoethanol and 1% glycerol (final concentrations), heated at 100 8C for 5 min and loaded onto 12% (w/v) SDSPAGE gels, following the Laemmli method [45]. Separated proteins were blotted onto Immobilon-P membrane (Millipore, Bedford, MA, USA) with a Mini-PROTEAN 3 Cell and Mini Trans-Blot1 module (Bio Rad 165-3317). Non-specific sites on the membranes were blocked for 1 h with 5% BSA in blocking buffer (Tris–HCl 25 mM pH 7.5, NaCl 150 mM and 0.1% Tween). The blots were incubated with a monoclonal anti-calmodulin antibody (clones 2D1+1F11+6D4, C7055, Sigma Chemical Co., Madrid, Spain) diluted 1:200 for 3 h at 25 8C. After washing (with blocking buffer, 1 of 15 min followed by 3 of 5 min each, in the orbital shaker described above), the membranes were incubated for 1 h with a secondary sheep anti-mouse peroxidase-conjugated IgG (NA931VS Amersham, Madrid, Spain) diluted 1:5000 in blocking buffer. After washing (as indicated above) the proteins that bound the antibody were visualized by enhanced chemiluminescence. As negative controls, either the anti-calmodulin or the secondary antibody was omitted, to rule out nonspecific binding. To analyse phosphoproteins, protein samples were separated by electrophoresis on 4–22.5% (w/v) SDSPAGE gels [45] and electrotransferred onto ImmobilonP (Millipore, Bedford, MA) as previously described

[46]. Non-specific binding sites on membranes were blocked with 5% BSA (w/v) in Tris-buffered saline (Tris–HCl 10 mM, pH 8, 120 mM NaCl and 0.05% Tween 20) for 1 h. The phosphoproteins were immunodetected by incubating with the primary mouse monoclonal anti-phosphotyrosine (1:2000), phosphoserine (1:4000) or phosphothreonine (1:4000) antibody (PT-66, PSR-45 and PTR-8, respectively, Sigma Chemical Co., Madrid, Spain) for 3 h at room temperature. After washing (as indicated above) in Tris-buffered saline, the membranes were incubated for 1 h with a secondary goat anti-mouse alkaline phosphatase-conjugated IgG (1:4000, Sigma Chemical Co., Madrid, Spain). Thereafter, phosphorylated proteins were visualized as described above. Negative and positive controls for both antibodies anti-phosphoserine and threonine have already been shown [47]. Likewise, negative controls in which either the primary or the secondary antibody was omitted were carried out. To determine whether Ca2+/CaM influences protein phosphorylation, proteins were extracted from samples incubated in capacitated conditions (described above) in a medium with/without 5–200 mM CZ or 50– 1000 mM W7 (as previously indicated), and phosphorylated proteins were visualized as described above. The Gel Doc System with Molecular Analyst software (Bio Rad, Hercules, CA) was used to quantify changes in the phosphorylated proteins. The phosphorylation signal was evaluated as volume (area  intensity), and results are presented as changes relative to control sample. 2.8. Flow cytometry analysis The protocol used for these assays was based on the one already described [48]. Control (0 h), capacitated (4 h) and ionophore-treated samples were loaded with Fluo-3 acetoxymethyl (AM, Molecular Probes, Eugene, OR) that emits fluorescence in the presence of Ca2+. Aliquots of 2  107 cells/ml were mixed with 10 mM Fluo-3 AM and incubated for 20 min at room temperature in the dark. The use of Fluo-3 AM with its large fluorescence response may be a sensitive means for detecting destabilizing changes in sperm membrane [48]. Moreover, this fluorochrome was coupled with propidium iodide (PI) to discriminate different subpopulations according to calcium concentration and membrane integrity. Thus, after incubation with Fluo-3 AM, sperm suspensions were diluted 1:10 with the adequate medium, 5 mg/ml PI were added and samples were kept in the dark until flow cytometry analysis.

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Flow cytometry was performed on a Coulter Epics Elite flow cytometer equipped with a 20-mW aircooled 488 nm argon-ion laser at a power output of 100 mW. Fluorescence of Fluo-3 was measured using a band-pass filter of 525  20 nm, while fluorescence of PI was detected using a 675  20 nm band-pass filter. Sperm cells were analysed at a rate of 500– 800 events/s using Isoton (Acid free balanced electrolyte solution, Beckman Coulter) as sheath fluid, and for each sample, 10,000 events were stored in the computer for further analysis. Fluorescence data were collected in logarithmic mode while lightscatter data were collected in linear mode. Through inspection of forward light-scatter data (indicative of size), only sperm cell-specific events that appeared in a typical L-shaped scatter profile were selected for further analysis. Then, ‘‘windows’’ were set up to delineate the different categories of cells observable in the experiment. These subpopulations include spermatozoa unstained with PI, showing either a low Fluo-3 fluorescence signal (live cells with low Ca2+), or with more than the minimal Fluo-3 signal (live cells with high Ca2+). The software computed for each sample the percentage of cells in each category. To reduce background in the flow cytometry analysis, all media were passed through a 0.2-mm filter (Millipore Ibe´rica, Madrid, Spain). 2.9. Statistical analysis Results are shown as mean (standard error) of the number of different samples indicated in each case. Means were compared by analyses of variance tests to determine whether there were any significant differences between samples using INSTAT for Windows (version 3.01, license GTA-31516-136). P < 0.05 was considered to be statistically significant.

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Fig. 1. Immunocytochemical localization of calmodulin in ram spermatozoa. (a) Aliquots of 8  106 sperm were fixed with formaldehyde, permeabilized with methanol and incubated with a rabbit anticalmodulin antibody. All tails are labelled. Magnification 1000. (b) Spermatozoa with staining at the post-acrosomal area, at the equatorial region, and with no reactivity in the head are shown.

immuno-patterns, the main sperm type in control samples showed no reactivity in the head, which represented 66% of total sperm. Approximately 19% showed staining at the equatorial region and 15% at the post-acrosomal area. Replacement of the anti-serum with preimmune serum was used as a negative control to rule out non-specific binding, and no labelling was found (data not shown). The capacitating status of control, in vitro capacitated and ionophore-induced acrosome-reacted sperm samples was determined by the CTC staining (Fig. 2)

3. Results 3.1. Immunolocalization of calmodulin in ram spermatozoa We firstly evidenced calmodulin in ram sperm using an immunocytochemical technique. As ram sperm cells are very sensitive to centrifugation, sperm fixation was performed in suspension with no previous centrifugation in order to avoid any possible artefactual result. Immunocytochemical analysis demonstrated the presence of calmodulin in ram sperm (Fig. 1), and also that all tails were labelled. According to the

Fig. 2. Assessment of the capacitation state of ram spermatozoa by the CTC-EtD1 staining of the swim-up obtained sample (control, 0 h), after induced in vitro capacitation and acrosome reaction. Percentage (mean values  S.E.M., n = 5) of cells: not capacitated; total capacitated; acrosome-reacted. Significant differences related to control samples: ***P < 0.001. Significant differences related to capacitated samples:  P < 0.05.

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that we previously validated for ram semen [17]. As expected, an increase (P < 0.001) in total capacitated and acrosome-reacted sperm was found after 4 h of incubation in capacitating conditions, related to the control samples (0 h). Likewise, a significant increase (P < 0.01) in the acrosome-reacted CTC-pattern was found in samples stimulated with the calcium ionophore. This increase corresponded to a decreased proportion of non-capacitated sperm compared to control (P < 0.0001) and capacitated (P < 0.05) samples, respectively (Fig. 2). However, in vitro capacitation scarcely influenced the incidence of the CaMstaining patterns and resulted in a slight decrease in the proportion of sperm without head labelling (52%), and an increase in the percentage of sperm with reactivity at the post-acrosome (35%). The onset of the acrosomal exocytosis by ionophore A23187 produced a further loss of the head labelling (43%) and an increase (44%) with labelling at the post-acrosome.

3.2. Effect of calmodulin antagonists on in vitro ram sperm capacitation To determine whether Ca2+/CaM plays a significant role in ram sperm capacitation, we assessed the capacitation state of ram spermatozoa incubated in a medium with/without different concentrations of two antagonist of calmodulin, CZ, a strong inhibitor of Ca2+/ CaM-dependent phosphodiesterase (type I) PDE [52] and W7, inhibitor of Ca2+-dependent phosphodiesterases [53]. As shown in Fig. 3, the addition of increasing concentrations of both compounds during incubation in capacitating conditions accounted for a gradual increase in the proportion of capacitated and acrosome-reacted sperm patterns, according to CTC staining. A significant (P < 0.05) reduction in the proportion of non-capacitated sperm pattern, related to control samples, was found in the presence of 20 mM of CZ (Fig. 3a) and 500 mM of W7 (Fig. 3b).

Fig. 3. Effect of different calmidazolium (a) and W7 (b) concentrations on capacitation state (CTC staining) of ram sperm selected by swim-up and incubated 4 h in capacitating conditions. Percentage of & not capacitated, capacitated, acrosome-reacted sperm. Mean values  S.E.M. (n = 5). Significant differences relative to control (0 mM) at 0 h: ***P < 0.001. Significant differences relative to control (0 mM) at 4 h:  P < 0.001;  P < 0.01;  P < 0.05.

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Fig. 4. Western-blot analysis of the presence of calmodulin in ram sperm protein extracts: molecular weight markers (lane 1), supernatant (cytosolic fraction, lane 2) and pellet (non-soluble fraction, lane 3). Proteins extracted from about 108 sperm obtained by swim-up were analysed by western-blotting, as described under M&M. The experiment was performed two times, and a representative image is shown.

3.3. Identification of calmodulin in ram sperm protein extracts by western-blotting We also corroborated the immunocytochemistry results by western-blot analysis of protein extracts obtained after sperm lyses with Triton X-100. As shown in Fig. 4, we identified one band of about 17 kDa in the pellet sample (non-soluble fraction) while no reactivity was found in the supernatant (cytosolic fraction). 3.4. Effect of calmodulin antagonists on ram sperm membrane protein phosphorylation The tested calmodulin antagonists affected protein tyrosine phosphorylation in a different way. Densitometry quantification of the content of protein bands from sperm incubated in capacitating conditions with increasing concentrations of CZ (Fig. 5) or W7 (Fig. 6) revealed that CZ accounted for a significant reduction in the content of three bands of approximately of 30, 40 and 45 kDa (Table 1). However, W7 showed no significant effect at any of the studied concentrations (data not shown). Both calmodulin antagonists hardly affected protein serine and threonine phosphorylation as no densitometric differences were found in the presence of increasing concentrations of CZ (Fig. 5) or W7 (Fig. 6) related to control sperm.

Fig. 5. Western-blot analysis of ram sperm protein phosphorylated in tyrosine (a), serine (b) and threonine (c) residues and the effect of increasing concentration of calmidazolium (CZ, 5–75 mM). Sperm were obtained by swim-up (control samples, lane 1) and incubated in capacitation conditions for 4 h at 39 8C under 100% humidity and 5% CO2 (capacitated samples, lane 2). Proteins extracted from 107 spermatozoa were analysed by western-blotting, as described under M&M. The experiment was performed three times, and representative images are shown.

3.5. Changes in intracellular calcium concentration Changes in the concentration of intracellular calcium during in vitro capacitation and acrosome reaction were determined by flow cytometry. As shown in Fig. 7a, control samples already showed different sperm subpopulations according to their calcium content. The main subpopulation contained about 70% of the total cells with an integral plasma membrane and a medium level of calcium. Approximately 29.5% of cells were also stained with PI (non-intact membrane cells).

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concentration (quadrant 2, Fig. 7c). Only 7.2% of the total cells preserved integral membranes containing a very high [Ca2+]i (quadrant 1). 4. Discussion

Fig. 6. Western-blot analysis of ram sperm protein phosphorylated in tyrosine (a), serine (b) and threonine (c) residues and the effect of increasing concentration of W7 (0.05–1 mM). Sperm were obtained by swim-up (control samples, lane 1) and incubated in capacitation conditions for 4 h at 39 8C under 100% humidity and 5% CO2 (capacitated samples, lane 2). Proteins extracted from 107 spermatozoa were analysed by western-blotting, as described under M&M. The experiment was performed three times, and representative images are shown.

And only 7% of the total sperm showed an integral plasma membrane with a low calcium level. The concentration of intracellular calcium was progressively increased during in vitro capacitation (Fig. 7b) and the acrosome reaction (Fig. 7c) according to equivalent increases in the Fluo-3 AM intensity. After capacitation, discrimination of sperm subpopulations was improved due to a high increase in calcium concentration in integral plasma membrane cells. A calcium-enriched subpopulation containing 67.1% of cells with intact plasma membrane (quadrant 1) was well separated from another containing about 30% of damaged-membrane sperm (quadrant 2, Fig. 7b). After the acrosome reaction, a drastic increase in the intracellular calcium level was shown. The main subpopulation (92.1%) was PI-stained (damagedmembrane cells) and contained a high calcium

At least some of the effects of Ca2+ are likely to be achieved through CaM, since it was shown that inhibition of CaM decreases sperm motility [3,13,54,55]. Using both immunocytochemistry and immunoblotting techniques, we showed that CaM is present in ram sperm with a complex compartmentalization pattern. Immunocytochemical localized CaM to the flagellum of all sperm in control samples. In addition, we showed that a certain proportion of sperm contains CaM also in the head (equatorial and postacrosomal regions). The localization appears to be specific, based on the lack of staining in two different control experiments in which the primary antibody was substituted for normal mouse serum. Consistent with these results, the protein extraction studies indicated the presence of CaM in insoluble structures. Coherent with this broad distribution of CaM in sperm, there is convincing evidence that Ca2+/CaM is involved in multiple functions in sperm, including motility, capacitation and the acrosome reaction [2,3,30,49– 51,55–57]. Furthermore, our findings showed that in vitro capacitation and the ionophore-induced acrosome reaction produced an increase in CaM localization in the post-acrosomal region. Similar CaM localization has been shown by immunoelectron microscopy in other species supporting the idea that CaM is in an appropriate location for regulating sperm membrane changes which are important for fertilization [58,59]. It has been reported that CaM antagonists inhibit capacitation and protein tyrosine phosphorylation [2,3,32,51]. Thus, we expected that CaM antagonists would inhibit these processes. However, treatment of sperm with increasing concentrations of CZ, a highly specific antagonist of CaM, and with W7, another CaM antagonist structurally unrelated to CZ, accounted for an increase in capacitated and acrosome-reacted sperm patterns, according to CTC staining that was used as a probe for monitoring capacitation-dependent sperm membrane changes, since it is correlated with the sperm ability of to undergo the zona pellucida [60] and lysophosphatidyl choline [17]-induced AR. Since CZ is associated with an increase in intracellular Ca2+ [13] while no such increase was observed in W7-treated sperm [61], both antagonists probably influence sperm capacitation by different pathways. The apparent contra-

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Table 1 Densitometry quantification of the content of certain phosphotyrosine protein bands from sperm incubated in capacitating conditions with increasing concentrations of CZ. The experiment was performed three times, and presented data are means  standard error of relative intensity units. CZ (mM)

0 (swim-up) 0 (control 4 h) 5 10 20 50 75 100 200

Molecular weight (kDa) 30

40

45

65,862  1,554 82,930  3,010 82,884  2,898 84,654  3,405 85,218  2,814 97,896  10,802 83,794  12,444 61,080  6,559* 71,540  4,756

133,471  5,324 140,137  12,288 136,437  7,945 128,080  10,226 139,023  26,045 126,702  10,977 100,827  33,867 100,426  43,071* 56,276  4,000*

225,127  13,133 233,425  5,500 228,655  3,410 216,033  12,515 201,068  24,502 164,690  7,341** 131,794  49,766 94,012  58,143* 51,294  2,465***

Significant differences related to control (without CZ incubated 4 h) samples. * P < 0.05 ** P < 0.005 *** P < 0.001.

Fig. 7. Fluorescence intensities of individual sperm in Fluo-3 AM-loaded samples stained with propidium iodide and analysed by flow cytometry. (a) Control, (b) capacitated, (c) acrosome-reacted. Up-left quadrant (1): intact-membrane cells with high Fluo-3 fluorescence. Up-right quadrant (2): dead cells with high Fluo-3 fluorescence. Down-left quadrant (3): intact-membrane cells with low Fluo-3 fluorescence. Down-right quadrant (4): dead cells with low Fluo-3 fluorescence. The log scales at the bottom and left-hand side of each plot indicate true relative fluorescence intensities. Data are representative of three replicates.

diction could be explained because the stimulating effect shown in this study was found at concentrations much higher than those used in previous reports which described an effective inhibition of capacitation and protein tyrosine phosphorylation [2,3,32], sperm motility and hyperactivation [62] with very low CaM antagonist concentrations. However, the stimulation of ionophoreinduced acrosomal exocytosis in human spermatozoa by very low concentration of CaM inhibitors has already been reported [63], which indicates that CaM modulates acrosomal exocytosis negatively. Since cAMP is known to be important for sperm membrane changes during capacitation [6], enzymes involved in cAMP metabolism might likely be targets of CaM. Thus, it has been suggested that CaM might act by

direct stimulation of the plasma membrane adenylyl cyclase, increasing cAMP levels within the sperm [3]. In addition, a mammalian testis-specific cytosolic adenylyl cyclase has been found [64] and provides another possible route by which CaM might directly influence cAMP levels. Although the molecular mechanisms of cAMP action in sperm are not completely understood, it is assumed to involve the phosphorylation of proteins through PKA, resulting in membrane lipid remodelling and the regulation of ion channels [65]. The cyclic nucleotide PDE modulated by CaM was found in rat, bovine and mouse sperm [3,28,66] and agents that are known to inhibit PDEs, and are thereby likely to increase intracellular cyclic nucleotides, have been shown to increase sperm

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motility [67,68]. The interdependence of Ca2+ and cAMP is being recognized as a new paradigm for signal transduction [69] and a Ca2+/CaM-regulated PDE is likely to be a major regulator of the process [70]. Moreover, a new variant of Ca2+/CaM-dependent PDE (PDE1A [70]) appears to be activated by CaM and alters depolarization-evoked calcium increases [68]. Given that CZ is a strong inhibitor of Ca2+/CaM-dependent PDE [52], it seems likely that the rise in the proportion of capacitated-sperm pattern might occur due to the increase in the intracellular cAMP concentration produced by the inhibition of Ca2+/CaM-dependent PDE activity. Tyrosine phosphorylation of specific sperm proteins has been shown to be correlated with capacitation and to be dependent on cAMP [6]. However, our findings showed that CZ and W7 probably influence protein tyrosine phosphorylation in a different way as concentrations of CZ that increased the sperm capacitated-CTC pattern accounted for a reduction in certain protein bands while W7 did not affect significantly the level of protein phosphorylation. These results would indicate that at least some protein phosphorylation in sperm can be uncoupled from capacitation-dependent membrane changes, and therefore that these processes operate at least by partially separated pathways, as already suggested [3]. Similarly, changes in the capacitated-CTC pattern, the LPCinduced AR, and IVF induced by CaM inhibitors were not correlated to changes in protein tyrosine phosphorylation in mouse sperm [3]. The results of flow cytometry indicate that the use of the calcium-sensitive fluorescent probe Fluo-3 AM allows for the detection of changes in the [Ca2+]i in ram sperm under different experimental conditions. Capacitation induced a large increase in [Ca2+]i in a live sperm subpopulation (propidium iodide negative). The ionophore-induced acrosome reaction accounted for a much higher increase in [Ca2+]i in a small subpopulation of live cells. The great increase in the percentage of damaged-membrane cells (PI+) might probably be a consequence of the fusion between the outer acrosomal membrane and the overlying plasma membrane occurred during the ionophore-induced acrosomal exocytosis. Our results suggest that ram sperm capacitation is associated to increases in [Ca2+]i, despite the apparent irrelevance of the addition of extracellular calcium in the capacitation process that we described previously [17]. However, in that study, a significantly higher value of acrosome-reacted sperm was found in response to the addition of 4 mM Ca2+, suggesting the participation of

calcium as an inducer of membrane changes that are needed for the subsequent acrosome reaction [17]. The higher increase in [Ca2+]i found in capacitatedsperm might explain the enhancement in CaM localization in the post-acrosomal region, as the redistribution of CaM is known to occur in other cell types in response to changes in intracellular calcium levels [71]. High levels of CaM are associated with the post-acrosomal portion of the sperm, and suggest a potential role for CaM in the events of cell fusion during fertilization [50]. The findings of the present study implicate CaM in ram sperm capacitation, and might suggest interplay between cAMP and Ca2+/CaM-dependent PDE activity. Our results also evidence that the increase in intracellular calcium level starts during capacitation, and that this increase may take place by uptake as well as by mobilization from the intracellular calcium stores, suggesting the implication of this cation in the molecular events that lead to capacitation and acrosome reaction in ram spermatozoa. However, further prospective studies are essential to define the significance of CaM in ram sperm capacitation and its molecular role. Acknowledgments Supported by grants CICYT-FEDER AGL 200502614, CICYT-FEDER AGL 2007-61229 and DGA A26/2005. The authors thank ANGRA for supplying the sires, M. Cebria´n for the artwork and S. Morales for the collection of semen samples. References [1] Yanagimachi R. Mammalian Fertilization. In: Knobil E, Neill JD, editors. The physiology of reproduction. New York: Raven Press Ltd.; 1994. p. 189–317. [2] Bendahmane M, Lynch II C, Tulsiani DR. Calmodulin signals capacitation and triggers the agonist-induced acrosome reaction in mouse spermatozoa. Arch Biochem Biophys 2001;390:1–8. [3] Si Y, Olds-Clarke P. Evidence for the involvement of calmodulin in mouse sperm capacitation. Biol Reprod 2000;62:1231–9. [4] Luconi M, Krausz C, Forti G, Baldi E. Extracellular calcium negatively modulates tyrosine phosphorylation and tyrosine kinase activity during capacitation of human spermatozoa. Biol Reprod 1996;55:207–16. [5] Tardif S, Dube C, Chevalier S, Bailey JL. Capacitation is associated with tyrosine phosphorylation and tyrosine kinaselike activity of pig sperm proteins. Biol Reprod 2001;65:784–92. [6] Visconti PE, Bailey JL, Moore GD, Pan D, Olds-Clarke P, Kopf GS. Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. Development 1995;121:1129–37. [7] Visconti PE, Kopf GS. Regulation of protein phosphorylation during sperm capacitation. Biol Reprod 1998;59:1–6.

C. Cola´s et al. / Theriogenology 71 (2009) 789–800 [8] Baker MA, Hetherington L, Ecroyd H, Roman SD, Aitken RJ. Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitation. J Cell Sci 2004;117:211–22. [9] Tardif S, Dube C, Bailey JL. Porcine sperm capacitation and tyrosine kinase activity are dependent on bicarbonate and calcium but protein tyrosine phosphorylation is only associated with calcium. Biol Reprod 2003;68:207–13. [10] Handrow RR, First NL, Parrish JJ. Calcium requirement and increased association with bovine sperm during capacitation by heparin. J Exp Zool 1989;252:174–82. [11] Parinaud J, Milhet P. Progesterone induces Ca2+-dependent 30 ,50 -cyclic adenosine monophosphate increase in human sperm. J Clin Endocrinol Metab 1996;81:1357–60. [12] Parrish JJ, Susko-Parrish JL, Uguz C, First NL. Differences in the role of cyclic adenosine 30 ,50 -monophosphate during capacitation of bovine sperm by heparin or oviduct fluid. Biol Reprod 1994;51:1099–108. [13] White DR, Aitken RJ. Relationship between calcium, cyclic AMP, ATP, and intracellular pH and the capacity of hamster spermatozoa to express hyperactivated motility. Gamete Res 1989;22:163–77. [14] Visconti PE, Johnson LR, Oyaski M, Fornes M, Moss SB, Gerton GL, et al. Regulation, localization, and anchoring of protein kinase A subunits during mouse sperm capacitation. Dev Biol 1997;192:351–63. [15] Aitken RJ, Harkiss D, Knox W, Paterson M, Irvine DS. A novel signal transduction cascade in capacitating human spermatozoa characterised by a redox-regulated, cAMPmediated induction of tyrosine phosphorylation. J Cell Sci 1998;111(Pt 5):645–56. [16] Galantino-Homer HL, Visconti PE, Kopf GS. Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cyclic adenosine 30 50 -monophosphate-dependent pathway. Biol Reprod 1997;56:707–19. [17] Grasa P, Cebrian-Perez JA, Muino-Blanco T. Signal transduction mechanisms involved in in vitro ram sperm capacitation. Reproduction 2006;132:721–32. [18] Leclerc P, de Lamirande E, Gagnon C. Cyclic adenosine 30 ,50 monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. Biol Reprod 1996;55:684–92. [19] Leclerc P, Langlais J, Lambert RD, Sirard MA, Chafouleas JG. Effect of heparin on the expression of calmodulin-binding proteins in bull spermatozoa. J Reprod Fertil 1989;85:615–22. [20] Leclerc P, Sirard MA, Chafouleas JG, Lambert RD. Decreased binding of calmodulin to bull sperm proteins during heparininduced capacitation. Biol Reprod 1990;42:483–9. [21] Leclerc P, Sirard MA, Chafouleas JG, Lambert RD. Decrease in calmodulin concentrations during heparin-induced capacitation in bovine spermatozoa. J Reprod Fertil 1992;94:23–32. [22] Cheung WY. Calmodulin plays a pivotal role in cellular regulation. Science 1980;207:19–27. [23] Means AR, Lagace L, Guerriero Jr V, Chafouleas JG. Calmodulin as a mediator of hormone action and cell regulation. J Cell Biochem 1982;20:317–30. [24] Gross MK, Toscano DG, Toscano Jr WA. Calmodulin-mediated adenylate cyclase from mammalian sperm. J Biol Chem 1987; 262:8672–6. [25] Mons N, Guillou JL, Jaffard R. The role of Ca2+/calmodulinstimulable adenylyl cyclases as molecular coincidence detectors in memory formation. Cell Mol Life Sci 1999;55:525–33.

799

[26] Rusnak F, Mertz P. Calcineurin: form and function. Physiol Rev 2000;80:1483–521. [27] Tash JS, Krinks M, Patel J, Means RL, Klee CB, Means AR. Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm. J Cell Biol 1988;106:1625–33. [28] Wasco WM, Orr GA. Function of calmodulin in mammalian sperm: presence of a calmodulin-dependent cyclic nucleotide phosphodiesterase associated with demembranated rat caudal epididymal sperm. Biochem Biophys Res Commun 1984;118: 636–42. [29] Hook SS, Means AR. Ca(2+)/CaM-dependent kinases: from activation to function. Annu Rev Pharmacol Toxicol 2001;41: 471–505. [30] Marin-Briggiler CI, Jha KN, Chertihin O, Buffone MG, Herr JC, Vazquez-Levin MH, et al. Evidence of the presence of calcium/calmodulin-dependent protein kinase IV in human sperm and its involvement in motility regulation. J Cell Sci 2005;118:2013–22. [31] Adeoya-Osiguwa SA, Fraser LR. Evidence for Ca(2+)-dependent ATPase activity, stimulated by decapacitation factor and calmodulin, in mouse sperm. Mol Reprod Dev 1996;44:111–20. [32] Zeng HT, Tulsiani DR. Calmodulin antagonists differentially affect capacitation-associated protein tyrosine phosphorylation of mouse sperm components. J Cell Sci 2003;116:1981–9. [33] Carlson AE, Westenbroek RE, Quill T, Ren D, Clapham DE, Hille B, et al. CatSper1 required for evoked Ca2+ entry and control of flagellar function in sperm. Proc Natl Acad Sci USA 2003;100:14864–8. [34] Marquez B, Suarez SS. Different signaling pathways in bovine sperm regulate capacitation and hyperactivation. Biol Reprod 2004;70:1626–33. [35] Schuh SM, Carlson AE, McKnight GS, Conti M, Hille B, Babcock DF. Signaling pathways for modulation of mouse sperm motility by adenosine and catecholamine agonists. Biol Reprod 2006;74:492–500. [36] Breitbart H, Spungin B. The biochemistry of the acrosome reaction. Mol Hum Reprod 1997;3:195–202. [37] Breitbart H. Intracellular calcium regulation in sperm capacitation and acrosomal reaction. Mol Cell Endocrinol 2002;187: 139–44. [38] Spungin B, Margalit I, Breitbart H. Sperm exocytosis reconstructed in a cell-free system: evidence for the involvement of phospholipase C and actin filaments in membrane fusion. J Cell Sci 1995;108(Pt 6):2525–35. [39] Ollero M, Muino-Blanco T, Lopez-Perez MJ, Cebrian-Perez JA. Viability of ram spermatozoa in relation to the abstinence period and successive ejaculations. Int J Androl 1996;19:287–92. [40] Garcı´a-Lo´pez N, Ollero M, Muin˜o-Blanco T, Cebria´n-Pe´rez JA. A dextran swim-up procedure for separation of highly motile and viable ram spermatozoa from seminal plasma. Theriogenology 1996;46:141–51. [41] Shams-Borhan G, Harrison RA. Production, characterization, and use of ionophore-induced, calcium-dependent acrosome reaction in ram spermatozoa. Gamete Res 1981;4:407–32. [42] Ward CR, Storey BT. Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay. Dev Biol 1984;104:287–96. [43] Perez LJ, Valcarcel A, Heras MAD, Moses DF, Baldassarre H. In vitro capacitation and induction of acrosomal exocytosis in ram spermatozoa as assessed by the chlortetracycline assay. Theriogenology 1996;45:1037–46.

800

C. Cola´s et al. / Theriogenology 71 (2009) 789–800

[44] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–54. [45] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. [46] Perez-Pe R, Grasa P, Fernandez-Juan M, Peleato ML, CebrianPerez JA, Muino-Blanco T. Seminal plasma proteins reduce protein tyrosine phosphorylation in the plasma membrane of cold-shocked ram spermatozoa. Mol Reprod Dev 2002;61: 226–33. [47] Barrios B, Fernandez-Juan M, Muino-Blanco T, Cebrian-Perez JA. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins that protect ram spermatozoa against cold shock. J Androl 2005;26:539–49. [48] Harrison RA, Mairet B, Miller NG. Flow cytometric studies of bicarbonate-mediated Ca2+ influx in boar sperm populations. Mol Reprod Dev 1993;35:197–208. [49] Carrera A, Moos J, Ning XP, Gerton GL, Tesarik J, Kopf GS, et al. Regulation of protein tyrosine phosphorylation in human sperm by a calcium/calmodulin-dependent mechanism: identification of A kinase anchor proteins as major substrates for tyrosine phosphorylation. Dev Biol 1996;180:284–96. [50] Jones HP, Lenz RW, Palevitz BA, Cormier MJ. Localization of calmodulin in mammalian spermatozoa by immunofluorescence methods. Ann N Y Acad Sci 1980;356:393–4. [51] Tulsiani DR, Zeng HT, Abou-Haila A. Biology of sperm capacitation: evidence for multiple signalling pathways. Soc Reprod Fertil Suppl 2007;63:257–72. [52] Sunagawa M, Yokoshiki H, Seki T, Sperelakis N. Intracellular application of calmidazolium increases Ca2+ current through activation of protein kinase A in cultured vascular smooth muscle cells. J Vasc Res 1998;35:303–9. [53] Leung PC, Minegishi T, Wang J. Inhibition of follicle-stimulating hormone- and adenosine-30 ,50 -cyclic monophosphateinduced progesterone production by calcium and protein kinase C in the rat ovary. Am J Obstet Gynecol 1988;158:350–6. [54] Ahmad K, Bracho GE, Wolf DP, Tash JS. Regulation of human sperm motility and hyperactivation components by calcium, calmodulin, and protein phosphatases. Arch Androl 1995;35: 187–208. [55] Schlingmann K, Michaut MA, McElwee JL, Wolff CA, Travis AJ, Turner RM. Calmodulin and CaMKII in the sperm principal piece: evidence for a motility-related calcium/calmodulin pathway. J Androl 2007;28:706–16. [56] Ignotz GG, Suarez SS. Calcium/calmodulin and calmodulin kinase II stimulate hyperactivation in demembranated bovine sperm. Biol Reprod 2005;73:519–26. [57] Lopez-Gonzalez I, De La Vega-Beltran JL, Santi CM, Florman HM, Felix R, Darszon A. Calmodulin antagonists inhibit T-type

[58]

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67] [68]

[69] [70]

[71]

Ca(2+) currents in mouse spermatogenic cells and the zona pellucida-induced sperm acrosome reaction. Dev Biol 2001; 236:210–9. Kann ML, Feinberg J, Rainteau D, Dadoune JP, Weinman S, Fouquet JP. Localization of calmodulin in perinuclear structures of spermatids and spermatozoa: a comparison of six mammalian species. Anat Rec 1991;230:481–8. Moriya M, Katagiri C, Yagi K. Immuno-electron microscopic localization of calmodulin and calmodulin-binding proteins in the mouse germ cells during spermatogenesis and maturation. Cell Tissue Res 1993;271:441–51. Ward CR, Storey BT. Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescent assay. Dev Biol 1984;104:287–96. Iwasa F, Hasegawa Y, Ishijima S, et al. Effects of calmodulin antagonists on motility and acrosome reaction of sea urchin sperm. Zoolog Sci 1987;4:61–72. Ahmadi A, Ng SC, Liow SL, Ali J, Bongso A, Ratnam SS. Intracytoplasmic sperm injection of mouse oocytes with 5 mM Ca2+ at different intervals. Hum Reprod 1995;10:431–5. Yunes R, Tomes C, Michaut M, De Blas G, Rodriguez F, Regazzi R, et al. Rab3A and calmodulin regulate acrosomal exocytosis by mechanisms that do not require a direct interaction. FEBS Lett 2002;525:126–30. Buck J, Sinclair ML, Schapal L, Cann MJ, Levin LR. Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals. Proc Natl Acad Sci USA 1999;96:79–84. Harrison RA. Cyclic AMP signalling during mammalian sperm capacitation—still largely terra incognita. Reprod Domest Anim 2003;38:102–10. Chaudhry PS, Casillas ER. Calmodulin-stimulated cyclic nucleotide phosphodiesterases in plasma membranes of bovine epididymal spermatozoa. Arch Biochem Biophys 1988;262: 439–44. Henkel RR, Schill WB. Sperm preparation for ART. Reprod Biol Endocrinol 2003;1:108. Wennemuth G, Carlson AE, Harper AJ, Babcock DF. Bicarbonate actions on flagellar and Ca2+-channel responses: initial events in sperm activation. Development 2003;130:1317–26. Zaccolo M, Pozzan T. CAMP and Ca2+ interplay: a matter of oscillation patterns. Trends Neurosci 2003;26:53–5. Vasta V, Sonnenburg WK, Yan C, Soderling SH, ShimizuAlbergine M, Beavo JA. Identification of a new variant of PDE1A calmodulin-stimulated cyclic nucleotide phosphodiesterase expressed in mouse sperm. Biol Reprod 2005;73:598–609. Teshima Y, Kakiuchi S. Membrane-bound forms of Ca2+-dependent protein modulator: Ca2+-dependent and independent binding of modulator protein to the particulate fraction from brain. J Cyclic Nucleotide Res 1978;4:219–31.