Localization and regulation of plasma membrane Ca2+-ATPase in bovine spermatozoa

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European Journal of Cell Biology 86 (2007) 265–273 www.elsevier.de/ejcb

Localization and regulation of plasma membrane Ca2+-ATPase in bovine spermatozoa Jo¨rg Triphan, Gerhard Aumu¨ller, Timo Brandenburger, Beate Wilhelm Department of Anatomy and Cell Biology, Philipps-University, Robert-Koch-Str. 8, D-35037 Marburg, Germany Received 1 June 2006; received in revised form 5 January 2007; accepted 15 February 2007

Abstract Calcium (Ca2+) signals, produced by the opening of plasma membrane entry channels, regulate a number of functions in spermatozoa such as capacitation and motility. The mechanisms of Ca2+ removal from the sperm, required to restore resting [Ca2+]i, include plasma membrane Ca2+-dependent ATPase (PMCA) isoenzymes as well as a plasma membrane Na+–Ca2+ exchanger. We have recently shown that bovine sperm PMCA is stimulated by PDC109, a secretory protein of bovine seminal vesicles. To demonstrate the subcellular localization and regulation of bovine sperm PMCA, we have performed cell fractionation, enzyme activity determination and Western blotting studies of PMCA in spermatozoa removed from the cauda epididymidis of bull. Fractionation of sperm heads and tails resulted in a distinct association of ATPase activity with the tail membrane fraction. In vitro stimulation studies with PDC-109 using intact and fractionated sperm showed an increase in enzyme activity up to 105% in sperm tail membranes. Furthermore, thapsigargin inhibition did not alter the stimulatory effect of PDC-109 on ATPase activity, indicating that no sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), but only PMCA isoenzymes are involved in this effect. Western blotting studies using a polyvalent PMCA antibody showed the exclusive presence of a 135 kDa band in the tail plasma membrane fraction. To elucidate whether or not the stimulatory effect was a direct one or indirectly mediated through PKA and PKC activation, PKA and PKC inhibitors, respectively, were used in the Ca2+ATPase activity assays, which was followed by PDC-109 stimulation. The stimulatory effect of PDC-109 on PMCA was still observed under these conditions, while no phosphotyrosine proteins could be detected by Western blotting in sperm extracts following PDC-109 treatment. Co-immunoprecipitation studies, PDC-109 affinity chromatography as well as overlay blots failed to show a strong association of both PMCA and PDC-109, pointing to an indirect, perhaps phospholipid-mediated effect. r 2007 Elsevier GmbH. All rights reserved. Keywords: Plasma membrane calcium ATPase; PMCA; Seminal vesicle secretion; PDC-109; Spermatozoa

Introduction

Corresponding author. Tel.: +49 6421 286 3838; fax: +49 6421 286 8983. E-mail address: [email protected] (B. Wilhelm).

0171-9335/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2007.02.003

Mammalian spermatozoa, like other eukaryotic cells, use calcium (Ca2+) signals to control physiological responses. Ca2+ influx is considered a prime regulator of sperm motility, a participant in capacitation and an essential element in the initiation of the acrosome

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reaction (Babcock and Pfeiffer, 1987; Darszon et al., 1999; O’Toole et al., 2000; Publicover and Barratt, 1999; Ren et al., 2001). Ca2+ ions entering the sperm cytoplasm from outside or from intracellular stores must be returned to the extracellular milieu or the intracellular storage organelles. The two major systems capable of pumping Ca2+ against the large concentration gradient outside the cell or into the endoplasmic reticulum are the plasma membrane Ca2+-ATPases (PMCAs) and the sarco/endoplasmic reticulum Ca2+ATPases (SERCAs). As has recently been shown in mouse sperm (Wennemuth et al., 2003), the Ca2+ATPase pump of the plasma membrane is essential in the final stages of recovery to achieve a low resting [Ca2+]i, whereas the contribution of the Na+–Ca2+ exchanger, mitochondrial uniporter and the SERCA pump to this effect is low or nil. The PMCAs belong to the family of P-type primary ion transport ATPases, which form a phosphorylated intermediate (an aspartyl phosphate) during the reaction cycle. A conformational change of the phosphorylated enzyme exposes Ca2+ to the extracellular side and promotes Ca2+ release prior to the hydrolytic cleavage of the phosphorylated intermediate, caused by the low Ca2+ affinity of the phosphorylated form of the pump. After its cleavage, the pump returns to its initial conformation (Carafoli, 1994; Carafoli et al., 1996; Guerini et al., 1998). Multigene families code for these Ca2+ pumps, and additional isoform subtypes are generated via alternative splicing (Strehler and Zacharias, 2001). As different PMCA isoforms (1–4) are characterized by different regulatory and kinetic properties, they are obviously optimized for the different functional tasks fulfilled by each pump in setting resting cytosolic or intra-organellar Ca2+ levels and in shaping intracellular Ca2+ signals with spatial and temporal resolution (Strehler and Treiman, 2004). All four basic gene products of PMCA (1200 amino acids) are organized in the plasma membrane with 10 putative transmembrane segments and protrude into the cytosol with four main units that serve different functions. The first intracellular loop contains a stretch of 40 basic amino acids that binds activating acidic phospholipids. The C-terminal domain is particularly important as it carries a calmodulin-binding domain as well as consensus sites for protein kinases A and C and sites for Ca2+ binding (Carafoli and Brini, 2000). Using inhibitors of either SERCA or PMCA, e.g., thapsigargin and quercetin, it was shown in sperm that under such conditions the acrosomal reaction is induced, but sperm motility decreases. This points to the contribution of different ATPase activities in the spatial and temporal patterning of different sperm functions (Williams and Ford, 2003). Schuh et al. (2004) have recently shown in homozygous mice with a targeted gene deletion of isoform 4 of PMCA that

these animals are infertile due to severely impaired sperm motility. They therefore claim a pivotal role of PMCA 4 on the regulation of sperm function and intracellular Ca2+ levels in sperm. This was confirmed by Okunade et al. (2004) in PMCA 4 (/) Black Swiss mice where the male animals were infertile, but had normal spermatogenesis and mating behaviour. Sperm taken from these animals that had not undergone capacitation exhibited normal motility, but could not achieve hyperactivated motility needed to traverse the female genital tract. We have recently shown that PDC-109, the predominant secretory protein from bovine seminal vesicles, in vitro significantly increases sperm motility of epididymal sperm. Enzyme activities of both Mg2+-dependent and Mg2+-independent Ca2+-ATPases were increased in a dose-dependent manner following the addition of PDC109. The effect was dose-, temperature- and pHdependent and organ-specific and seemed to be dependent on a narrow spatial proximity of the enzyme and the bound seminal protein (Sanchez-Luengo et al., 2004). In the current study, we, therefore, tried to identify the localization of the bovine sperm PMCA and its regulation under the influence of PDC-109.

Materials and methods Purification of PDC-109 Native PDC-109 was purified from bovine seminal vesicle fluid following a modified method described by Calvete et al. (1996), using a Heparin Sepharose column (Amersham Pharmacia Biotech, Freiburg, Germany); (for details see (Sanchez-Luengo et al., 2004)). Bound PDC-109 was eluted from the column with 10 mM phosphorylcholine in 50 mM Tris–HCl buffer, pH 7.4, 150 mM NaCl and 5 mM EDTA. To determine protein concentrations the Bio-Rad protein assay was used (BioRad Laboratories, Munich, Germany). In order to check the purity of the fractions, tricine–SDS–PAGE (Schagger and von Jagow, 1987) was performed using 12% separating slab gels. Subsequently, the gels were stained with Coomassie Brilliant Blue R-250.

Fractionation of bovine sperm into sperm head and tail Epididymides were obtained from young mature bulls immediately after slaughtering in the local slaughterhouse. Sperm were released from bovine cauda epididymidis by cutting the duct several times with a sharp razor blade and swirling the tissue in 250 mM sucrose, 5 mM Tris–HCl, pH 7.4, 1 mM EDTA, 10 mM PMSF [phenylmethyl sulfonyl fluoride]. Sperm released were

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centrifuged for 10 min at 2500g. Bovine sperm were immersed in imidazole buffer (50 mM imidazole, 1 mM EDTA, 1 mM DTE [1,4-dithioerythritol], and 250 mM sucrose, pH 8.5) and then treated with ultrasound (sonoplus, Bandelin electronic, Berlin, Germany, cycle 9, 30 s) in order to separate the sperm into heads and tails. The separation was microscopically checked. Subsequently, fragmented sperm were separated into head and tail fractions using differential centrifugation. A sucrose cushion (1.8 M) was overlaid with the solution containing fragmented sperm and was subsequently centrifuged under the following two different conditions, while in both cases three different layers were formed: a plasma membrane-containing supernatant, a layer containing sperm tails and finally a pellet containing sperm heads. (1) In order to achieve pure sperm heads, the sample was centrifuged at 1500g, 4 1C for 30 min, and (2) in order to get a pure sperm tail fraction, the sample was centrifuged at 2500g, 4 1C for 30 min. The layer containing sperm tails contaminated with sperm heads, as obtained using the primary centrifugation conditions and the sperm head fraction contaminated with sperm tails, as obtained after the second conditions, respectively, were discarded. Samples containing pure sperm heads and pure sperm tails were collected separately and washed in imidazole buffer (2500g, 4 1C, 10 min). The pellet was resuspended in a small volume of imidazole buffer. The plasma membrane-containing supernatant (see above) was centrifuged at 12,000g (4 1C, 10 min), and membranes were pelleted for 1 h at 100,000g at 4 1C. Protein concentrations were determined by using the Bio-Rad protein assay. An aliquot of each sample was used for activity assays, Western blot and electron microscopy (see below).

Preparation of sperm for tyrosin phosphorylation studies Cauda epididymal sperm were collected as described above and immersed in a modified Tyrode’s bicarbonate buffered medium (SpTALP) as described (Parrish et al., 1988). Sperm were washed twice in SpTALP medium by centrifugation at 500g for 10 min. Sperm concentration was determined using a hemocytometer and adjusted to 50  106 cells/ml with SpTALP. Then, sperm were incubated under the following conditions for 0, 2 and 4 h at 39 1C in a humidified 5% CO2 environment: (1, control) SpTALP alone, (2) SpTALP with PDC-109 (20 mg/250 ml), (3, positive control) SpTALP with heparin (4 mg/240 ml) plus dbcAMP (1 mM) plus IBMX (100 mM), (4) SpTALP with heparin (4 mg/240 ml) plus dbcAMP (1 mM) plus IBMX (100 mM) plus PDC-109 (20 mg/250 ml), and (5, control) SpTALP with BSA (20 mg/250 ml). To analyse tyrosine phosphorylation,

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sperm were concentrated at 10,000g for 5 min and washed in 1 ml phosphate-buffered saline. The sperm pellet was then resuspended in SDS–gel sample buffer without 2-mercaptoethanol and boiled for 5 min. After centrifugation at 10,000g for 5 min, the supernatant was removed, boiled in the presence of 5% 2-mercaptoethanol for 5 min and was subjected to SDS–PAGE and blotting as described below. Signal intensity was analysed using Scion Image for Windows, beta 4.0.2 (Scion Corporation, Frederick, Maryland).

Enzyme activity measurements Activity assays for Mg2+-dependent and Mg2+independent Ca2+-ATPase were performed according to Sikdar et al. (1991) as previously described (SanchezLuengo et al., 2004). In addition to intact whole sperm taken from the cauda epididymidis, Ca2+-ATPase activity assays were also performed with purified fractions of sperm heads, sperm tails and sperm plasma membranes present in the supernatant. Ten microgram protein was used in the activity assays. To identify the potential participation of protein kinase A (PKA) or protein kinase C (PKC) in the PMCA reaction, a specific inhibitor of PKA, H89 (Santa Cruz, CA) and an inhibitor of PKC (PKC inhibitor, Santa Cruz, CA), were used. The stimulatory effect of PDC-109 on Mg2+-independent and Mg2+-dependent Ca2+-ATPase activities was determined by adding PDC-109 in a concentration of 20 mg/250 ml to the reaction mixture. The SERCA inhibitor thapsigargin (Calbiochem, Darmstadt, Germany) was used to discriminate between SERCA and PMCA activity. Both PDC-109 and the respective inhibitors were added to the reaction mixture prior to the addition of the membrane preparation and the substrate (ATP). Concentrations ranging from 1 to 30 mM for H89 and PKC inhibitor and 0.05–0.2 mM for thapsigargin were used in the experiments.

SDS–PAGE and Western blot SDS–PAGE and Western blot were performed as previously described (Wilhelm et al., 1998). For analysing the presence of PMCA or phosphotyrosine in bovine sperm fractions, sperm proteins were separated on 4–12% Bis-Tris SDS gels with MES running buffer (NuPAGE, Invitrogen, Paisley, UK) according to the manufacturer’s instructions and transferred either onto nitrocellulose (Hybond, Amersham, Freiburg, Germany) or PVDF (Immobilon P, Millipore, Schwalbach, Germany) membranes. Prior to the incubation with antibodies non-specific binding was blocked either with 5% dried non-fat milkpowder (anti-PMCA) or 5% teleostean gelatin from cold water fish skin

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(anti-phosphotyrosine) in Tris-buffered saline. Blots were incubated with primary antibodies against PMCA in dilution of 1:1000 (monoclonal, 5F10, Dianova, Hamburg) or against phosphotyrosine in a dilution of 1:3000 (clone 4G10, Biomol, Hamburg, Germany) at room temperature overnight. Subsequently blots were incubated either with ECL-anti-mouse IgG horseradish peroxidase (POD) antibody (1:2000, Amersham, Freiburg, Germany) or protein A-POD (1:4000, Sigma). To visualize the peroxidase reaction, the enhanced chemiluminescence detection kit (Amersham) was used according to the manufacturer’s instructions.

Transmission electron microscopy To check the purity of the sperm fractions, aliquots were fixed in 1.25% glutaraldehyde, 2.5% paraformaldehyde and 0.05% aqueous picric acid, 0.1 M sodium cacodylate, pH 7.3, for 6 h at room temperature. Fixed samples were collected and washed by repeated centrifugation and resuspension in 0.1 M sodium cacodylate buffer. An aliquot of each sample was postfixed for 1 h at room temperature using 1% aqueous osmic acid solution. Washed samples were dehydrated in a graded series of ethanol, followed by propylene oxide and soaked overnight in a mixture of equal parts of propylene oxide and Epon. Samples were then transferred into fresh pure Epon in gelatin capsules and polymerized by 60 1C for 24 h. Ultrathin sections were cut on a Reichert ultramicrotome, stained with uranyl acetate and lead citrate and examined in a Zeiss EM 10 electron microscope.

Results and discussion Localization of Ca2+-ATPase in bovine cauda epididymal sperm In a previous study, we demonstrated that sperm motility and the activity of the plasma membrane Ca2+ATPase of sperm taken from the cauda epididymidis are significantly stimulated by the addition of PDC-109, the major bovine seminal vesicle protein (Sanchez-Luengo et al., 2004). Interestingly, bound PDC-109 is located underneath the plasma membrane of the midpiece of bovine sperm once these get in contact with seminal vesicle secretion (Aumu¨ller et al., 1988). In the current study, we tried to identify the localization of the bovine sperm PMCA. Therefore, sperm from the epididymal cauda were fractionated into heads and tails using ultrasound treatment and differential centrifugation (Fig. 1(A)). Three fractions were obtained. As shown by electron microscopic analysis, the pellet contained sperm heads with plasma membranes (Fig. 1(B)). A thin

layer on top of the sucrose cushion included membranefree sperm tails (Fig. 1(C)), while the supernatant exclusively contained plasma membranes (Fig. 1(D)). Less than 5% of the sperm tails still retain the plasma membrane. As plasma membranes are still adhered to the sperm heads, the membranes identified in the supernatant must belong to the sperm tails. The presence of Ca2+-ATPase was identified in the different sperm fractions by activity assays and Western blotting experiments. Our experiments clearly demonstrated that Mg2+-dependent and Mg2+-independent Ca2+-ATPase activity is restricted exclusively to the supernatant containing the (tail) plasma membranes. ATPase activity was stimulated by PDC-109 in tail plasma membranes as has previously been shown for total plasma membranes from bovine sperm (SanchezLuengo et al., 2004). Following the addition of 20 mg PDC-109 to the activity assay Mg2+-dependent Ca2+ATPase activity was increased by 104% in sperm membranes whereas Mg2+-independent Ca2+-ATPase activity was increased by 103% in sperm membranes. No or less activity was measured in the sperm head and plasma membrane-free sperm tail fraction (Fig. 2). The SERCA inhibitor thapsigargin (Guerini, 1998) did not alter the Ca2+-ATPase activity nor the stimulatory effect of PDC-109 in the plasma membrane fraction, indicating that no SERCA, but only PMCA isoenzymes are present in the fraction (Fig. 3). Performing Western-blotting studies using the pan anti-PMCA antibody 5F10 we detected immunoreactive bands of about 135 kDa in size in total sperm and sperm tail membranes. In addition, a band of 180 kDa was present in sperm tail membranes. No immunoreactive material was found in protein extracts from sperm heads and demembranated tails (Fig. 4). Unfortunately, the 5F10 antibody did not work on bovine sperm smears, perhaps due to species incompatibility. Up to now, four isoforms of PMCA are described, ranging from 133 to 139 kDa in size (Carafoli, 1992). The antibody (clone 5F10) used recognizes an epitope located between amino acids 724–783 in human erythrocyte PMCA. This epitope is located in the highly conserved hinge region of the intracellular loop, which is identical in all four PMCA isoforms (Borke et al., 1989). The immunoreactive band of approximately 180 kDa, which was visible in the sperm tail membrane extracts on SDS gels, could result from the aggregation of one PMCA isoform as described by Borke et al. (1989). This band has also been observed in our studies in rat coagulating gland plasma membranes (Post et al., 2002). In summary, both the activity assays and the Western blot gave clear evidence that PMCA is exclusively restricted to the plasma membrane of bovine sperm tails. The presence of PMCA in mouse epididymal sperm tails was previously shown by Wennemuth et al. (2003) using the same antibody. Furthermore, in this species the

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isoform 4 was detected in the principal piece of the sperm tail, whereas PMCA 4 knockout mice showed no immunoreactivity for PMCA 4. In addition, the knock-

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Fig. 2. Mg2+-dependent and -independent Ca2+-ATPase activity in bovine sperm from the epidiymal cauda and sperm fragments obtained after ultrasonic treatment and differential centrifugation. Ca2+-ATPase activities were measured in the absence (white columns) or presence of PDC-109 (black columns). (A) Total sperm, (B) sperm heads, (C) sperm tails, (D) membranes. (*) denotes statistical significant stimulatory effect of PDC-109 (Po0.05). Vertical bars indicate SEM.

out studies clearly pointed out that PMCA 4 is the isoform that is important for fertility, as the knockout mice are infertile. Sperm taken from these animals exhibited normal motility, but having not undergone capacitation, could not achieve hyperactivated motility, which is needed to traverse the female genital tract. (Okunade et al., 2004; Schuh et al., 2004). We, therefore,

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Fig. 1. Separation of bovine sperm from epididymal cauda into sperm heads, sperm tails and membranes using ultrasonic treatment and differential centrifugation. (A) Cartoon showing the 3 different fractions obtained after differential centrifugation at 1500 or 2500g. To obtain pure sperm head fractions, 1500g was used, and 2500g was used to achieve pure sperm tail fractions. Contaminated fractions containing both sperm heads and tails were discarded (indicated by X). (B–D) Transmission electron micrographs of the fractions obtained. In the pellet fraction sperm heads with plasma membranes were present (B), in the fraction on the top of the sucrose cushion demembranated sperm tails were detected (C) and in the supernatant exclusively membranes were visible (D). Bars: 1 mm (B), 0.5 mm (C, D).

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Fig. 3. Activity of Mg2+-dependent and -independent Ca2+ATPase of membranes obtained from cauda epididymal bovine sperm (fraction D). Activities were assayed in the presence (black column) or absence (white columns) of PDC109 and SERCA inhibitor thapsigargin in concentrations from 0.05 to 0.2 mM. Thapsigargin had no influence on Ca2+ATPase activity, indicating that only plasma membranes but no membranes of the ER are present in the membrane fraction. Vertical bars indicate SEM.

presume that PMCA 4 might be the isoform of the enzyme, which is activated by PDC-109.

Influence of protein kinase A and protein kinase C on PMCA activity It is well known that the C-terminal domain of PMCA harbours consensus sites for protein kinases A and C and sites for Ca2+ binding (Carafoli and Brini, 2000). Therefore, in a second set of experiments, we analysed whether or not the stimulatory effect of PDC109 on Ca2+-ATPase is mediated though PKA or PKC, respectively. It is known that PKA regulates tyrosine phosphorylation in sperm by direct or indirect mechanisms on tyrosine kinase or phosphatase (Visconti et al., 2002). We, therefore, analysed if the PKA inhibitor H89 or a PKC inhibitor decrease the stimulatory effect of PDC-109 on Ca2+-ATPase activity. Also, the potential

Fig. 4. SDS–PAGE and Western blot analysis of bovine sperm and sperm fractions. Proteins (15 mg protein per lane) were stained with Coomassie Brilliant Blue or probed with an antibody against Ca2+-ATPase (clone 5F10). Lane A, total sperm; lane B, sperm heads; lane C, sperm tails; lane D, sperm (tail) membranes; lane M, protein marker (peqGOLD prestained protein-marker IV, Peqlab, Erlangen, Germany). A pronounced immunoblot reaction was observed for total sperm at 135 kDa and for sperm (tail) membranes at 135 and 180 kDa.

protein tyrosine phosphorylation being mediated through PDC-109 in bovine sperm was checked. Mg2+-independent Ca2+-ATPase activity was not at all influenced, neither by H89 nor by PKC inhibitor. ATPase activities and the stimulatory effect of PDC-109 on PMCA were still observed under these conditions. Even though a light stimulation of Mg2+-dependent Ca2+-ATPase activity was observed after adding the PKC inhibitor (1–30 mM), the differences measured where not significant. In addition, no significant change in Mg2+-dependent Ca2+-ATPase activity was detected when using the PKA inhibitor H89 (1–30 mM) (Figs. 5 and 6). Furthermore, there was no increase in the intensity of tyrosine phosphorylation after addition of PDC-109 (Fig. 7). In contrast, an increased phosphorylation pattern could be detected in control experiments inducing capacitation in vitro using heparin (4 mg/ 250 ml), dbcAMP (1 mM) and IBMX (100 mM). In addition, no further increase in phosphorylation intensity was visible after addition of PDC-109 to the capacitating media. No effect was detected, even if PDC-109 was present over a period of 4 h (Fig. 7). In contrast, it was demonstrated by other groups that PKA plays a central role in several sperm processes, such as capacitation, motility and the acrosome reaction (Bielfeld et al., 1994; Harrison et al., 2000; Skalhegg et al.,

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Fig. 5. Activity of Mg -dependent and -independent Ca ATPase of membranes obtained from bovine epididymal cauda sperm, assayed in the presence (black column) or absence (white columns) of PDC-109 and PKA inhibitor H98 in concentrations from 1 to 30 mM. No significant inhibition of Ca2+-ATPase activity was observed after addition of H89. Vertical bars indicate SEM.

2002; Visconti et al., 1995). It is established that PKA inhibitors prevent tyrosine phosphorylation as well as capacitation (Galantino-Homer et al., 1997; Leclerc et al., 1996; Osheroff et al., 1999; Visconti et al., 1995). Furthermore, it has been reported that PKC present in sperm may be activated during the acrosomal reaction (Naor and Breitbart, 1997). Beside activating Ca2+-ATPase, PDC-109 has been shown to increase sperm motility (Sanchez-Luengo et al., 2004). In addition, PDC-109 was shown to provoke a cholesterol and phospholipid efflux from membranes (Wah et al., 2002). Cholesterol efflux is a particularly important event in sperm capacitation (Moreau et al., 1999; Moreau and Manjunath, 2000; Muller et al., 1998). Therefore, PDC-109 has been suggested to play an important role in capacitation (Manjunath and Therien, 2002). In summary, our data support that the accelerating effect of PDC-109 on Ca2+-ATPase activity is not mediated through PKA or PKC. We, therefore, favour the assumption that activation of bovine sperm Ca2+-ATPase and the effects of PDC-109 on sperm motility and capacitation are independently regulated.

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Fig. 6. Activity of Mg2+-dependent and -independent Ca2+ATPase of membranes obtained from bovine epididymal cauda sperm, assayed in the presence (black column) or absence (white columns) of PDC-109 and PKC inhibitor in concentrations from 5 to 30 mM. Incubation in the presence of the PKC inhibitor had no significant effect on Ca2+-ATPase activity. Vertical bars indicate SEM.

Conclusion and outlook Our data give evidence that PMCA is exclusively restricted to bovine sperm tail plasma membrane and, therefore, is located in direct vicinity to incorporated PDC-109 from seminal vesicle secretion. Furthermore, we could show that the stimulating effect of PDC-109 on Ca2+-ATPase activity is not mediated through PKA and PKC. Nevertheless, we have not been able to show that PDC-109 and PMCA might interact in a direct manner. Experiments such as overlay blots, co-immunoprecipitation and PDC-109 affinity chromatography (data not shown) could not prove a direct interaction between PDC-109 and PMCA. It was demonstrated by other groups that PDC-109 binds to phosphatidylcholine (Muller et al., 1998). We, therefore, favour the possibility that the stimulating effect of PDC-109 on PMCA is indirectly mediated through the binding of PDC-109 to phospholipids. Further studies using PDC109 deletions mutants lacking the phospholipidbinding site will show whether the stimulating effect

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Fig. 7. Influence of PDC-109 on protein tyrosine phosphorylation over a period of 0–4 h. 1. SpTALP buffer, negative control; 2. SpTALP buffer+PDC-109 (80 mg/ml); 3. SpTALP buffer with heparin (4 mg/250 ml)/dbcAMP (1 mM)/IBMX(100 mM), positive control; 4. SpTALP buffer with heparin/dbcAMP/IBMX+PDC-109 (80 mg/ml); 5. SpTALP buffer+BSA (80 mg/ml), control. PDC109 had no effect on tyrosine phosphorylation.

on Ca2+-ATPase activity is due the binding of PDC-109 to phospholipids.

Acknowledgements The authors gratefully acknowledge the excellent technical assistances of Anne Henkeler and Gudrun Hoffbauer. This study was supported by the Deutsche Forschungsgemeinschaft (Graduiertenkolleg 533 Giessen-Marburg, Project A1).

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