| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the * Department of Anatomy and Cell Biology,
Philipps-Universität, Marburg, Germany;
Department of Biochemistry,
Philipps-Universität, Marburg, Germany; and
Department of Chemistry, Bose-Institute,
Calcutta, India.
| Correspondence to: Prof Dr Gerhard Aumüller, Department of Anatomy and Cell Biology, Robert-Koch-Str 8, D-35033 Marburg, Germany (e-mail: aumuelle{at}mailer.uni-marburg.de). |
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Key words: Seminal vesicles, bovine sperm, plasma membrane Ca2+-ATPase
Electrophoretic studies have detected a number of basic proteins in the secretion of the bovine seminal vesicles, including a "basic acrosin inhibitor" (BUSI [Tamblyn, 1983]) and 5'-nucleotidase (Fini and Cannistrado, 1990).
Esch et al (1983) have purified three different major proteins from bovine seminal plasma named BSP I, BSP II, and BSP III. BSP II and III were later found to be identical and were designated PDC-109, referring to their composition of 109 amino acids. Calvete et al (1994) demonstrated that BSP I is the glycosylated form of PDC-109. The bull seminal proteins BSP-A1 and BSP-A2 described by Seidah et al (1987) and Major Protein (Scheit et al, 1988) were also identified as PDC-109. Structure analysis revealed that the peptide chain of PDC-109 is arranged in 2 antiparallel ß-sheets, which lie perpendicular to each other, and 2 irregular loops supporting a large hydrophobic site (Constantine et al, 1992). The protein belongs to a group of fibronectin type II module proteins, present in the seminal secretions of several mammals (Saalmann et al, 2001) which display a particular protein-lipid binding ability.
PDC-109 binds to the choline group of phosphorylcholine-containing phospholipids in a very rapid process (Muller et al, 1998). Interaction of PDC-109 with artificial phosphorylcholine vesicles provoked permeability changes and partial disruption of the vesicles (Gasset et al, 2000). Protein crystallography revealed that the 2 fibronectin domains are separated by a shorter linker polypeptide that supports the clustering of the 2 domains (Wah et al, 2002). Thereby, PDC-109 stimulates cholesterol efflux after binding to the sperm membrane and phospholipid efflux in the early stages of capacitation (Gasset et al, 2000; Manjunath and Therien, 2002). Furthermore, it binds to choline lipids, thereby increasing the heparin-docking sites on the sperm surface (Therien et al, 1999). Greube et al (2001) have demonstrated that PDC-109 alters the membrane structure of lipid vesicles and likewise of bovine spermatozoa.
A comparable membrane effect has previously been suggested by our group (Aumüller et al, 1988), based on immunoelectron microscopic findings of the protein's being located underneath the plasma membrane of the sperm midpiece of epididymal sperm, once these were incubated with seminal vesicle secretion.
In the present study, we describe a novel biochemical feature of PDC-109 during its interaction with bovine epididymal sperm. Studying the activity of plasma membrane–bound calcium ATPases in bovine sperm, we found a strong stimulatory effect of PDC-109. This was analyzed in detail using conventional biochemical assays. Our results point to dose-dependent, saturable, and potentially irreversible cooperative effect of PDC-109 on sperm plasma membrane ATPases, resulting in increased motility of the spermatozoa.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Purification of PDC-109
PDC-109 was purified from bovine seminal vesicles secretion, using a
modified method described by Calvete et al
(1996). To isolate the
secretion, seminal vesicles were minced in tissue pieces (1 cm x 1 cm)
and subsequently immersed in a solution containing 50 mM Tris-HCl buffer, pH
7.4, 150 mM NaCl, and 5 mM EDTA (ethylenediamine tetra-acetic acid). After the
aqueous secretion extract was centrifuged at 12,000 x g for 15
minutes, the supernatant was applied to a Heparin Sepharose column (Amersham
Pharmacia Biotech, Freiburg, Germany) equilibrated with 50 mM Tris-HCl buffer
pH 7.4, containing 150 mM NaCl and 5 mM EDTA. Bound PDC-109 was eluted with 10
mM phosphorylcholine in 50 mM Tris-HCl buffer pH 7.4, 150 mM NaCl, and 5 mM
EDTA. The Bio-Rad protein assay was used to determine protein concentrations
(Bio-Rad Laboratories, Richmond, Calif). To check the purity of the fractions,
tricine-SDS-PAGE was performed according to the method of Schagger and von
Jagow (1987), using 12%
separating slab gels. Subsequently, the gels were stained with Coomassie
brilliant blue R-250.
Amino Acid Sequence Analysis
Purified PDC-109 (80 µg) was run on a preparative tricine-SDS-PAGE (12%
separating slab gels) and transferred onto a PVDF membrane (Immobilon-P,
Millipore) according to Kyhse-Andersen
(1984). The blotted protein
bands were stained with Coomassie brilliant blue for 1 minute. After
destaining the membranes in 50% methanol and washing thoroughly in water, the
13 kDa and 14 kDa bands, respectively, were excised and cut into small pieces.
N-terminal sequence analysis of both samples was carried out by Edman
degradation, as described by Linder et al
(1994).
Western Blotting Experiments
Isolated rat PDC-109 protein in native and in denatured form (SDS-PAGE
strips) was used for immunization of rabbits. The immunization was carried out
by Seramun (Berlin, Germany) using the standard immunization protocol.
Polyclonal antisera raised in rabbits against native PDC-109 and denaturated
PDC-109, respectively, were subsequently used for Western blotting. Western
blotting was performed as previously described by Wilhelm et al
(1998). Seminal vesicle
secretion (15 µg) and purified PDC-109 (3 µg) were transferred from
SDS-PAGE onto nitrocellulose membranes (Hybond; Amersham Pharmacia Biotech,
Freiburg, Germany). The strips were incubated with Roti-block (Bio-Rad,
Munich, Germany) to block nonspecific binding sites and were then incubated
overnight at room temperature either with the antibody against native PDC-109
(1:20,000) or with the antibody against denaturated PDC-109 (1:1000).
Detection of the immunoreaction with a peroxidase-labelled antibody against
rabbit IgG (1:100,000; Pierce, Bonn, Germany) was performed using the
SuperSignal system (Pierce, Bonn, Germany) according to the manufacturer's
instructions.
Preparation of Microsomal Membranes
Microsomal membranes were prepared from epididymal sperm from bovine and
rat organs as well as from different bovine tissues (liver, heart, kidney).
Sperm were released from bovine and rat epididymides, respectively, by cutting
the different epididymal segments several times with a sharp razor blade and
swirling the tissue in 250 mM sucrose solution containing 5 mM Tris-HCl buffer
pH 7.4, 1 mM EDTA, and 10 µm PMSF (phenylmethane sulfonyl fluoride). Sperm
were isolated from the following epididymal regions: 1) distal corpus to
proximal cauda, briefly termed epididymal sperm, 2) caput, 3) corpus, 4)
cauda, and 5) ampulla of the vas deferens. The sperm were centrifuged for 10
minutes at 2500 x g. To obtain a higher amount of membrane
protein, sperm were incubated in hyperosmotic swelling or HOS-buffer (65 mM
saccharose, 65 mM sodium citrate, 1 mM EDTA, 10 µm PMSF) at 37°C for 45
minutes (Bohring and Krause,
1999). Samples were then centrifuged again at 2500 x
g for 10 minutes and sperm were resuspended in homogenization buffer
(500 mM imidazole, 1 mM EDTA, 1 mM DTE [1,4-dithioerythritol], and 250 mM
sucrose, pH 8.5). The sperm, as well as the control tissues, were homogenized
in homogenization buffer by 8 strokes in a glass potter. The homogenate was
centrifuged for 10 minutes at 2500 x g at 4°C, the
supernatant was collected, and the pellet was resuspended in homogenization
buffer and centrifuged again at 2500 x g. This step was
repeated and the pooled supernatants were spun at 15 000 x g at
4°C for 15 minutes. The microsomal membranes were sedimented for 1 hour at
100 000 x g at 4°C. The pellet was resuspended in a small
volume of homogenization buffer and the protein concentration was determined
by using the Bio-Rad protein assay.
Ca2+-ATPase Activity Assay
Mg2+-dependent and Mg2+-independent
Ca2+-ATPase activity assays, respectively, were performed according
to Sikdar et al (1991). To
assay the activity of Mg2+-dependent Ca2+-ATPase, 5
µg of the microsomal membrane preparation and 3 mM ATP were added to the
reaction buffer containing 54.5 mM histidine hydrochloride, pH 7.5, 2 mM EGTA
(ethylene glycol-bis[2-aminoethylether]-N,N,N',N'-tetra-acetic
acid), 0.2 mM DTE, 50 mM sucrose, 1 mM MgCl2, and 4 mM
CaCl2 in a total volume of 250 µL. After incubation for 30
minutes at 37°C, the reaction was stopped by adding 70 µL of 30%
ice-cold trichloroacetic acid (TCA); the sample was subsequently diluted with
550 µL of distilled water. To determine the amount of enzymatically
released inorganic phosphate (Pi), a solution containing 100 µL
of 1.75% ammonium molybdate and 100 µL of 2% ascorbic acid was added. After
10 minutes of incubation at room temperature, the complexed Pi was
quantified using a spectrophotometer at 820 nm. Enzyme activity was expressed
µM Pi/mg protein/h. The activity of the
Mg2+-independent Ca2+-ATPase was measured in a buffer
system containing 50 mM imidazole, pH 8.5, 2 mM EGTA, 0.2 mM DTE, 50 mM
sucrose, and 4 mM CaCl2, as described above. In both cases,
Ca2+-ATPase activity was defined as the difference of activity in
the presence and absence of Ca2+-ions.
To identify the specificity of enzyme activities, Ca2+-antagonists such as nifedipine (0.2 mM), verapamil (0.2 mM), and trifluoperazine (TFP, 0.2 µM) were used. The stimulatory effect of PDC-109 on Mg2+-independent and Mg2+-dependent Ca2+-ATPase activities, respectively, was determined by adding different amounts of PDC-109 (range: 5 µg to 80 µg) to the reaction mixture. Both PDC-109 and the respective antagonists were added to the reaction mixture prior to the addition of the microsomal enzyme preparation and the substrate (ATP).
Temperature and pH Sensitivity of PDC-109
Ca2+-ATPase activities were measured as described above in the
presence of 20 µg PDC-109. PDC-109 was preincubated at different
temperatures (25°C, 33°C, 37°C, 41°C) or different pHs (6.6,
7.0, 7.5, 8.0, 8.5, 9.0) for 30 minutes. Activity determinations with and
without addition of 20 µM PDC-109 were then performed as described
above.
Mechanism of the Stimulatory Effect
In order to identify the substrate optimum for the stimulatory effect of
PDC-109 on enzyme kinetics, ATPase activity was measured as a function of ATP
concentrations at a ratio of 5 µM to 20 µg PDC-109. The dependence of
the enzyme reaction velocity on substrate (ATP) concentration was analyzed
using nonlinear regression to estimate [S] 0.5 (the substrate concentration at
half maximal velocity) and Vmax (describing the maximum velocity of
the reaction at saturating substrate concentration).
In order to prove the reversibility/irreversibility of the stimulating effect of PDC-109 on Ca2+-ATPase-activity, the microsomal fraction was incubated with PDC-109 for 30 minutes in a relation of 5 µg membranes: 20 µg PDC-109, and the Ca2+-ATPase activity was estimated within an aliquot. In order to remove unbound PDC-109, a fraction of the incubated enzyme was centrifuged for 1 hour at 100 000 x g at 4°C, and the supernatant containing the unbound PDC-109 was removed. The pellet was resuspended and the ATPase activity in the resultant pellet was measured. The procedure of washing was repeated 6 times. The total experiment was repeated 3 times. To check the amount of membrane loss during the washing steps, a control sample was treated in the same way but without adding PDC-109. Furthermore, an aliquot (6 µg) of the pellets obtained was analyzed by SDS-PAGE (12% tricine-gel) as described above.
CASA-Method
To record the grade of motility, the computer-assisted, computer-aided
software system (CASA type SM-CMA; Mika Medical, Montreal, Canada) method was
used. CASA technologies calculate a number of parameters characterizing sperm
motion. Sperm released from epididymis were washed by centrifugation in
HBS-puffer at 300 x g during 10 minutes and the pellet was
resuspended in HBS-buffer. Sperm concentration was adjusted to approximately
106 and spread in a Makler counting chamber with 10 µM volume.
The chamber was maintained at 37°C temperature. The following parameters
were measured by the CASA system: VCL = curvilinear velocity (µm/s), VSL =
straight-line velocity (µm/s), and VAP = average path velocity (µm/s).
For obtaining statistically significant results, 250 spermatozoa were counted
in every experiment.
Statistical Evaluation
For the statistical evaluation, Statgraphics® plus software
(Manugistics, Inc, Rockville, Md) was used. The data presented here were
confirmed in at least 4 independent experiments. Statistical analyses
performed were the one-way analysis of variance (ANOVA) and Wilcoxon's test
for correlated data. In all the cases, a P-value < .05 was
considered to have statistical significance.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
Ca2+-ATPase Activity in Bovine Epididymal Sperm
Both Mg2+-dependent and Mg2+-independent
Ca2+-ATPase activities were present in epididymal sperm
(Figure 2, left column;
Table 1). Specificity was
proven as ATPase activities were inhibited by the specific
Ca2+-antagonists verapamil, TFP and nifedipine, respectively.
Twenty-five µm of TFP inhibited the activity of Mg2+-independent
Ca2+-ATPase by 56% and of Mg2+-dependent
Ca2+-ATPase by 65%, while 0.2 mM verapamil decreased the
Mg2+-independent Ca2+-ATPase by 65% and
Mg2+-dependent Ca2+-ATPase by 76%. Addition of 0.2 mM
nifedipine inhibits 80% of the Mg2+-independent
Ca2+-ATPase activity, and 77% of the Mg2+-dependent
Ca2+-ATPase activity, respectively.
|
|
Influence of PDC-109 on Ca2+-ATPase Activity in Epididymal Sperm
To prove the hypothesis of a stimulatory effect of PDC-109 on sperm
membrane Ca2+-ATPases, the influence of different concentrations of
PDC-109 on Mg2+-independent and Mg2+-dependent
Ca2+-ATPases, respectively, in microsomal membranes of epididymal
sperm was measured. A concentration-dependent activity increase was observed
for both Mg2+-independent and Mg2+-dependent
Ca2+-ATPase following the addition of PDC-109. A plateau of enzyme
stimulation was reached at a concentration of about 20 µg PDC-109
(Figure 2), that is, addition
of amounts of PDC-109 above 20 µg did not further increase enzyme activity.
Following the addition of 20 µg PDC-109, Mg2+-independent
Ca2+-ATPase was increased by 154%, whereas
Mg2+-dependent Ca2+-ATPase activity was increased by
149% (Table 1). Effects were
statistically significant (P < .05). As deduced from these
experiments, the PDC-109 concentration for the following experiments was
chosen at 20 µg.
Temperature effects on the activation of Ca2+-ATPase by PDC-109 were studied by preincubation of PDC-109 at 25°C, 33°C, 37°C, and 41°C for 30 minutes prior to adding the protein to the ATPase assay. Incubation at 37°C revealed no change in the stimulatory effect of PDC-109 on ATPase activity. Preincubation above 37°C led to a decrease of the stimulatory effect. It was completely lost after heating the protein up to 40°C.
Effects of the pH on the stimulation of Ca2+-ATPase activity by PDC-109 were studied by preincubation of PDC-109 at different pH values ranging from 6.5 to 9. Incubation at pH ranging from 7.5 to 8.5 revealed no change of ATPase activity stimulation. Incubation at pH values below pH 6.5 and above pH 8.5 led to the decrease of the stimulatory effect.
Mechanism of Stimulation
The kinetic parameters [S] 0.5 (substrate concentration at half maximal
velocity) and Vmax (maximal velocity) for the substrate ATP were
determined by varying substrate concentrations (range 5 µM to 4 mM, final
concentration) in the ATPase assays. The experiments were performed with and
without 20 µg PDC-109. The results are depicted in
Figure 3. The addition of
PDC-109 to the assay did not influence the affinity of the enzyme to the
substrate (ATP). The [S] 0.5 values were nearly identical in both assays, that
is, with and without PDC-109; however, different values were shown for
Mg2+-independent (0.9 mM) and Mg2+-dependent
Ca2+-ATPase (0.35 mM) activities, respectively. In contrast,
Vmax was increased double by addition of PDC-109 to the
Mg2+-dependent and -independent activity assays.
|
To check the reversibility/irreversibility of the interaction between PDC-109 and Ca2+-ATPase, microsomal membranes from epididymal sperm were incubated for 30 minutes with PDC-109. Following the incubation time, Ca2+-ATPase activity was determined showing enzyme activation up to 150%. To separate the presumed Ca2+-ATPase/PDC-109-complex, a fraction of the reaction mixture was diluted in buffer and then centrifuged at 100 000 x g. This washing procedure was performed several times. Enzyme activity of the pellets formed was measured again and showed the same stimulating effect, compared to the respective control (without PDC-109), even after 6 washing steps. Furthermore, SDS-PAGE of the resulted pellets clearly demonstrated that PDC-109 was still present in the membrane samples (Figure 4), suggesting that binding of PDC-109 to the enzyme is irreversible.
|
Organ and Species Specificity of the Stimulatory Effect of PDC-109 on Ca2+-ATPase
In order to measure the potential activating effect of PDC-109 (20 µg)
on Ca2+-ATPase of sperm membranes from different parts of the
epididymis (caput, corpus, cauda) and the ampulla of the vas deferens,
microsomal membrane fractions were prepared from the respective sperm samples.
In membranes prepared from caput sperm, the activity of
Mg2+-independent Ca2+-ATPase was stimulated by PDC-109
by 66%, in those from corpus sperm by 177%, from cauda sperm by 292%, and from
ampulla sperm by 137%. Mg2+-dependent Ca2+-ATPase
activity was increased in membranes obtained from caput sperm by 70%, from the
corpus sperm by 196%, from cauda sperm by 111%, and from ampulla sperm by 78%.
The highest stimulatory effect was hence observed in sperm membrane ATPase of
sperm obtained from the cauda and corpus region of the epididymis, both in
Mg2+-dependent and Mg2+-independent ATPases
(Table 2 and
Figure 5).
|
|
In order to determine whether the enzyme activating effect of PDC-109 is restricted to sperm Ca2+-ATPase, activity measurements were performed with membrane preparations from different bovine organs. The respective membrane fractions were isolated from heart, kidney, and liver. A stimulatory effect on membrane-bound Mg2+-independent Ca2+-ATPase activity was lower or insignificant for all three organs, while in the case of Mg2+-dependent Ca2+-ATPase activity, a stimulatory effect of PDC-109 was detected in the case of liver plasma membranes (50%), while a minor effect was observed in case of heart and kidney membrane enzymes (Table 3).
|
In order to investigate the species specificity of the stimulatory effect of PDC-109 on sperm ATPases, rat epididymal sperm were treated in exactly the same way as bovine sperm, and enzyme activity was measured in these samples. Again, following the addition of PDC-109 to the reaction mixture, enzyme activity was significantly increased in both Mg2+-independent (131%) and -dependent (96%) Ca2+-ATPase activities (Figure 6).
|
Effect of PDC-109 on Sperm Motility
The CASA method detects curvilinear velocity (VCL, µm/s), straight-line
velocity (VSL, µm/s) and average path velocity (VAP, µm/s) in freshly
prepared native epididymal sperm. VSL values increased from a basal value of
9.6 ± 0.14 to 42.4 ± 0.10 following the addition of 2 µM of
PDC-109. VAP values increased from a basal value of 19.2 ± 0.07 to 52.2
± 0.11 after adding 2 µM protein. VCL values ranged from 44.5
± 0.13 without protein to 100.9 ± 0.23 after adding 2 µM of
PDC-109. The velocity was not further enhanced by increasing the amount of
PDC-109 to 4 µm (Figure 6).
The relative number of spermatozoa with enhanced motility was not increased
after addition of PDC-109.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
First, we analyzed whether plasma membranes of epididymal sperm show Ca2+-ATPase activity. We could identify both Mg2+-dependent and -independent Ca2+-ATPase activities in epididymal sperm. Both activities were also shown for goat spermatozoa (Sikdar et al, 1991; Sikdar et al, 1993; Sikdar et al, 1999). Sperm Ca2+-ATPase has been identified in microsomal membranes in some other species revealing the influence of this sort of pumps in calcium transport (Sikdar et al, 1991; Bhattacharyya and Sen, 1998; Sikdar et al, 1999). The plasma membrane Ca2+-ATPase is an essential membrane protein controlling the intracellular calcium concentration in cells and tissues (Carafoli, 1992; Carafoli and Stauffer, 1994). In most organs Ca2+-ATPase requires Mg2+ for its activation (Robinson, 1976; Niggli et al, 1979). This Mg2+-dependent Ca2+-ATPase is called plasma membrane Ca2+-ATPase (PMCA). Four distinct isoforms and several splice variants of the PMCA are described (PMCA1–4) so far (Carafoli, 1992; Carafoli and Stauffer, 1994; Guerini, 1998).
To analyze the potential effect of PDC-109 on the Ca2+-ATPase activity, we purified PDC-109 from the secretion of bovine seminal vesicles to homogeneity, using a novel, highly efficient purification procedure developed by Calvete et al (1996). The purified protein showed a double band at 13/14 kD on SDS-PAGE. Upon sequencing, the N-termini of both protein bands were found to be identical with the N-terminus of mature PDC-109 (Esch et al, 1983; Kemme and Scheit, 1988). This is in agreement with investigations performed by Calvete and coworkers (1996). They could show that the PDC-109 preparation contained a mixture of 2 species with molecular weights of 12.789 and 13.44 kDa, respectively. Both proteins had the identical N-terminus of PDC-109, the same amino acid composition, and the same tryptic fingerprint (Calvete et al, 1996). The difference in the molecular weight corresponds to the nonglycosylated and O-glycosylated isoform (Calvete et al, 1994).
After incubation of microsomal sperm membranes with bovine PDC-109, we observed an increase on Ca2+-ATPase activity, dependent on the concentration of the seminal protein, until it reached a level of about 20 µg/250 µL where activity remained constant. This stimulatory effect of about 150% was shown for both Mg2+-dependent and Mg2+-independent ATPase activities in epididymal sperm membranes. Interestingly, Sen and coworkers described another modulator protein from rat brain that stimulated the Mg2+-dependent Ca2+-ATPase activity in goat sperm but inhibited Mg2+-independent Ca2+-ATPase (Bhattacharyya and Sen, 1998).
To analyze the dependence of the velocity and the substrate concentration,
detailed kinetic experiments were performed. The results show that the
affinity of the enzyme to the substrate ATP is higher in case of
Mg2+-dependent Ca2+-ATPase activity rather than in case
of Mg2+-independent activity in microsomal membranes of epididymal
sperm, as the resulting [S] 0.5 value of Mg2+-dependent
Ca2+-ATPase activity is lower. The affinity of the enzyme to the
substrate was the same after adding of PDC-109, while the maximal velocity was
increasing. PDC-109 and the enzyme substrate obviously do not compete for the
same binding site; conversely, there is a positive cooperativity. Therefore,
it can be concluded that the binding site of PDC-109 and the binding site for
the substrate ATP on the enzyme are different. Furthermore, the effect on the
ATPase activity was not reduced after dilution, repeated centrifugation, and
washing of the stimulated sample. This indicates that the binding of PDC-109
and ATPases is irreversible. Different binding sites for substrate and
modulator, yet reversibility of the modulator binding, was shown for the
brain-modulator protein that stimulates Ca2+-ATPase activity in
goat spermatozoa (Bhattacharyya and Sen,
1998). The stimulatory effect of PDC-109 is quite sensitive to
temperature and to pH. Preincubation at 41°C and at pH between 6.5 to 7.5
and at pH above 8.5 completely abolishes the Ca2+-ATPase activation
ability. This effect might be due to disturbing its secondary structure by
unfolding monomers or by dissociation of oligomers. Heat-induced denaturation
of PDC-109 oligomers has been reported to lead to an irreversible melting
transition at 36°C (Gasset et al,
1997).
|
Both Ca2+-ATPases were inhibited by Ca2+-antagonists such as nifedipine, TFP, and verapamil (Scharff and Foder, 1984; Kim and Raess, 1988; Sikdar et al, 1991; Olorunsogo and Bababunmi, 1992; Kanwar et al, 1993; Bhattacharyya and Sen, 1998). Actually, there are no reports whether Mg2+-dependent and Mg2+-independent ATPases are 2 different enzymes or just 1 with 2 different catalytic sites. Since their behavior in terms of inhibition with Ca2+-antagonists and stimulation with PDC-109 is the same, it is conceivable that they resemble just 1 enzyme with 2 different binding sites. Sen and coworkers favored the model of Ca2+-ATPase being only 1 enzyme molecule with different catalytic sites (Sikdar et al, 1993).
We analyzed whether the Ca2+-ATPase is stimulated to the same degree in sperm membranes present in the different epididymal regions. Our results clearly demonstrate that Ca2+-ATPase activity grows from caput sperm to cauda sperm and the stimulatory effect of PDC-109 increases, too. Spermatozoa undergo a maturation process during their transport from the testis to the cauda epididymidis (Jones, 1989; Cooper, 1995). Therefore, changes in ATPase activity as well in the response to the stimulatory protein are conceivable.
In order to determine whether the activation of PDC-109 is a species-specific event, ATPase activities in micosomal membranes of rat epididymal sperm were analyzed. Our results clearly indicate that the Ca2+-ATPase activity of rat as well as human and goat (unpublished observation) epididymal sperm is also activated by bovine PDC-109. This result indicates that the Ca2+-ATPase in the different species exhibit a high homology resulting in a similar functional response. Ca2+-ATPase activity in micosomal membrane preparations of different organs was checked to determine whether enzyme activation exists in extra genital tissues or only in sperm cells. A stimulatory effect was shown for Mg2+-dependent ATPase activity only in the case of bovine liver microsomes but was less pronounced compared with sperm preparations. In the case of heart and kidney microsomes, no ATPase stimulation by PDC-109 was observed.
To date, it is not known which PMCA isoform is present in sperm. Wennemuth et al (2003), using an antibody against all 4 isoforms, demonstrated in mouse sperm that PMCA is located in the sperm tail. Different commercially available antibodies against PMCA isoforms showed ambiguous results in the bovine samples, maybe due deficient cross-reactivity with the respective bovine antigen(s). Performing RT-PCR using total RNA from human testis and specific primers for the PMCA isoforms 1 through 4, we demonstrated that the isoforms PMCA1 and PMCA4 are expressed in human testis, while PMCA2 and PMCA3 are not expressed (unpublished data). PMCA1 and PMCA4 are known to be ubiquitously transcribed, while PMCA2 and 3 are expressed in a more restricted manner, predominantly in brain and striated muscle; PMCA 2 is present in addition in kidney and liver (Greeb and Shull, 1989; Stahl et al, 1992; Stauffer et al, 1993; Carafoli and Stauffer, 1994; Howard et al, 1994; Keeton and Shull, 1995). As PDC-109 acts predominantly on ATPase in sperm, it is likely that a particular splice variant of PMCA1 or 4 is present in sperm membranes.
Besides activating Ca2+-ATPases, we showed that PDC-109 accelerates sperm motility in a dose-dependent manner. Furthermore, it was shown from different groups that PDC-109 binds to the choline group of phospholipids and provokes cholesterol- and phospholipid-efflux from the membranes. The loss of membrane cholesterol is an important event in sperm capacitation (Muller et al, 1998; Moreau et al, 1999; Moreau and Manjunath, 2000). Therefore, Majunath and coworkers propose that PDC-109 may play an important role in the process of capacitation too (Manjunath and Therien, 2002). Therefore, PDC-109 seems to be a multifunctional protein. Further experiments are necessary to clarify whether the 2 effects of PDC-109 shown in our study and the effects shown by other groups are joint or independent events. Motility and Ca2+-ATPase activity being interdependent events was already predicted by studies of Breitbart et al (1985; cf. Kanwar et al, 1993; Khanduja et al, 2001). They have suggested from inhibition experiments that Ca2+-ATPase has a functional role in the regulation of sperm motility. In contrast, studies performed in the group around Suarez (Ho et al, 2002; Ho and Suarez, 2003; Suarez and Ho, 2003) indicated that increased free intracellular Ca2+ plays a major role in regulating hyperactivated motility. They further showed that although hyperactivation and capacitation occur simultaneously, both events are regulated by different pathways (Ho and Suarez, 2001).
Our data show that PDC-109 increases sperm motility and the pumping efficiency of plasma membrane Ca2+-ATPases in an irreversible, cooperative manner. The effect is tissue specific but not species specific.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Aumüller G, Riva A. Morphology and functions of the human seminal vesicle. Andrologia. 1992; 24: 183 –196.[Medline]
Aumüller G, Vesper M, Seitz J, Kemme M, Scheit KH. Binding of a major secretory protein from bull seminal vesicles to bovine spermatozoa. Cell Tissue Res. 1988; 252: 377 –384.[Medline]
Babcock DF, Singh JP, Lardy HA. Alteration of membrane permeability to calcium ions during maturation of bovine spermatozoa. Dev Biol. 1979;69: 85 –93.[Medline]
Bhattacharyya D, Sen PC. Purification and functional characterization of a low-molecular-mass Ca2+, Mg2+- and Ca2+-ATPase modulator protein from rat brain cytosol. Biochem J. 1998; 330(Pt 1): 95 –101.
Bohring C, Krause W. The characterization of human spermatozoa membrane proteins–surface antigens and immunological infertility. Electrophoresis. 1999; 20: 971 –976.[Medline]
Breitbart H, Darshan R, Rubinstein S. Evidence for the presence of ATP-dependent calcium pump and ATPase activities in bull sperm head membranes. Biochem Biophys Res Commun. 1984; 122: 479 –484.[Medline]
Breitbart H, Rubinstein S, Nass-Arden L. The role of calcium and
Ca2+-ATPase in maintaining motility in ram spermatozoa.
J Biol Chem. 1985; 260: 11548
–1153.
Calvete JJ, Raida M, Sanz L, Wempe F, Scheit KH, Romero A, Topfer-Petersen E. Localization and structural characterization of an oligosaccharide O-linked to bovine PDC-109. Quantitation of the glycoprotein in seminal plasma and on the surface of ejaculated and capacitated spermatozoa. FEBS Lett. 1994; 350: 203 –206.[Medline]
Calvete JJ, Varela PF, Sanz L, Romero A, Mann K, Topfer-Petersen E. A procedure for the large-scale isolation of major bovine seminal plasma proteins. Protein Expr Purif. 1996; 8: 48 –56.[Medline]
Carafoli E. The Ca2+ pump of the plasma membrane.
J Biol Chem. 1992; 267
: 2115
–2118.
Carafoli E, Stauffer T. The plasma membrane calcium pump: functional domains, regulation of the activity, and tissue specificity of isoform expression. J Neurobiol. 1994; 25: 312 –324.[Medline]
Constantine KL, Madrid M, Banyai L, Trexler M, Patthy L, Llinas M. Refined solution structure and ligand-binding properties of PDC-109 domain b. A collagen-binding type II domain. J Mol Biol. 1992; 223: 281 –298.[Medline]
Cooper TG. Role of the epididymis in mediating changes in the male gamete during maturation. Adv Exp Med Biol. 1995; 377: 87 –101.[Medline]
Esch FS, Ling NC, Bohlen P, Ying SY, Guillemin R. Primary structure of PDC-109, a major protein constituent of bovine seminal plasma. Biochem Biophys Res Commun. 1983; 113: 861 –867.[Medline]
Fini C, Cannistrado S. 5'-Nucleotidase from bull seminal plasma. Biochemical and biophysical aspects. Andrologia. 1990; 22(suppl 1): 33 –43.
Gasset M, Magdaleno L, Calvete JJ. Biophysical study of the perturbation of model membrane structure caused by seminal plasma protein PDC-109. Arch Biochem Biophys. 2000; 374: 241 –247.[Medline]
Gasset M, Saiz JL, Laynez J, Sanz L, Gentzel M, Töpfer-Petersen E, Calvete JJ. Conformational features and thermal stability of bovine seminal plasma protein PDC-109 oligomers and phosphorylcholine-bound complexes. Eur J Biochem. 1997; 250: 735 –744.[Medline]
Greeb J, Shull GE. Molecular cloning of a third isoform of the
calmodulin-sensitive plasma membrane Ca2+-transporting ATPase that
is expressed predominantly in brain and skeletal muscle. J Biol
Chem. 1989;264: 18569
–18576.
Greube A, Muller K, Topfer-Petersen E, Herrmann A, Muller P. Influence of the bovine seminal plasma protein PDC-109 on the physical state of membranes. Biochemistry. 2001; 40: 8326 –8334.[Medline]
Guerini D. The significance of the isoforms of plasma membrane calcium ATPase. Cell Tissue Res. 1998; 292: 191 –197.[Medline]
Ho HC, Granish KA, Suarez SS. Hyperactivated motility of bull sperm is triggered at the axoneme by Ca2+ and not cAMP. Dev Biol. 2002; 250: 208 –217.[Medline]
Ho HC, Suarez SS. Hyperactivation of mammalian spermatozoa: function and regulation. Reproduction. 2001; 122: 519 –526.[Abstract]
Ho HC, Suarez SS. Characterization of the intracellular calcium
store at the base of the sperm flagellum that regulates hyperactivated
motility. Biol Reprod. 2003; 68: 1590
–1596.
Howard A, Barley NF, Legon S, Walters JR. Plasma-membrane calcium-pump isoforms in human and rat liver. Biochem J. 1994;303(pt 1): 275 –279.
Jones R. Membrane remodelling during sperm maturation in the epididymis. Oxf Rev Reprod Biol. 1989; 11: 285 –337.[Medline]
Kanwar U, Anand RJ, Sanyal SN. The effect of nifedipine, a calcium channel blocker, on human spermatozoal functions. Contraception. 1993; 48: 453 –470.[Medline]
Keeton TP, Shull GE. Primary structure of rat plasma membrane Ca2+-ATPase isoform 4 and analysis of alternative splicing patterns at splice site A. Biochem J. 1995; 306(pt 3): 779 –785.
Kemme M, Scheit KH. Cloning and sequence analysis of a cDNA from seminal vesicle tissue encoding the precursor of the major protein of bull semen. DNA. 1988; 7: 595 –599.[Medline]
Khanduja KL, Verma A, Bhardwaj A. Impairment of human sperm motility and viability by quercetin is independent of lipid peroxidation. Andrologia. 2001; 33: 277 –781.[Medline]
Kim HC, Raess BU. Verapamil, diltiazem and nifedipine interactions with calmodulin stimulated (Ca2+ + Mg2+)-ATPase. Biochem Pharmacol. 1988; 37: 917 –920.[Medline]
Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984;10: 203 –209.[Medline]
Lilja H, Oldbring J, Rannevik G, Laurell CB. Seminal vesicle-secreted proteins and their reactions during gelation and liquefaction of human semen. J Clin Invest. 1987; 80: 281 –285.
Linder M, Wenzel V, Linder D, Stirm S. Structural elements in
glycoprotein 70 from polytropic Friend mink cell focus-inducing virus and
glycoprotein 71 from ecotropic Friend murine leukemia virus, as defined by
disulfide-bonding pattern and limited proteolysis. J
Virol. 1994;68: 5133
–5141.
Manjunath P, Therien I. Role of seminal plasma phospholipid-binding proteins in sperm membrane lipid modification that occurs during capacitation. J Reprod Immunol. 2002; 53: 109 –119.[Medline]
Moreau R, Frank PG, Perreault C, Marcel YL, Manjunath P. Seminal plasma choline phospholipid-binding proteins stimulate cellular cholesterol and phospholipid efflux. Biochim Biophys Acta. 1999; 1438: 38 –46.[Medline]
Moreau R, Manjunath P. Characteristics of the cholesterol efflux induced by novel seminal phospholipid-binding proteins. Biochim Biophys Acta. 2000;1487: 24 –32.[Medline]
Muller P, Erlemann KR, Muller K, Calvete JJ, Topfer-Petersen E, Marienfeld K, Herrmann A. Biophysical characterization of the interaction of bovine seminal plasma protein PDC-109 with phospholipid vesicles. Eur Biophys J. 1998; 27: 33 –41.[Medline]
Niggli V, Penniston JT, Carafoli E. Purification of the
(Ca2+-Mg2+)-ATPase from human erythrocyte membranes
using a calmodulin affinity column. J Biol Chem. 1979; 254: 9955
–9958.
Olorunsogo OO, Bababunmi EA. Calmodulin antagonism and inhibition of erythrocyte plasma membrane Ca2+-pump by nifedipine, a calcium channel blocker. Afr J Med Med Sci. 1992; 21: 17 –21.[Medline]
Robinson JD. (Ca + Mg)-stimulated ATPase activity of a rat brain microsomal preparation. Arch Biochem Biophys. 1976; 176: 366 –374.[Medline]
Saalmann A, Munz S, Ellerbrock K, Ivell R, Kirchhoff C. Novel sperm-binding proteins of epididymal origin contain four fibronectin type II-modules. Mol Reprod Dev. 2001; 58: 88 –100.[Medline]
Schagger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987; 166: 368 –379.[Medline]
Scharff O, Foder B. Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on the rate of calmodulin binding to erythrocyte Ca2+-ATPase. Biochim Biophys Acta. 1984; 772: 29 –36.[Medline]
Scheit KH, Kemme M, Aumuller G, Seitz J, Hagendorff G, Zimmer M. The major protein of bull seminal plasma: biosynthesis and biological function. Biosci Rep. 1988; 8: 589 –608.[Medline]
Seidah NG, Manjunath P, Rochemont J, Sairam MR, Chretien M. Complete amino acid sequence of BSP-A3 from bovine seminal plasma. Homology to PDC-109 and to the collagen-binding domain of fibronectin. Biochem J. 1987;243: 195 –203.[Medline]
Sikdar R, Ganguly U, Chandra GA, Sen PC. Calcium uptake and Ca2+-ATPase activity in goat spermatozoa membrane vesicles do not require Mg2+. J Biosci. 1993; 18: 73 –82.
Sikdar R, Ganguly U, Pal P, Mazumder B, Sen PC. Biochemical characterization of a calcium ion stimulated-ATPase from goat spermatozoa. Mol Cell Biochem. 1991; 103: 121 –130.[Medline]
Sikdar R, Roy K, Mandal AK, Sen PC. Phosphorylation and dephosphorylation of Mg2+-independent Ca2+-ATPase from goat spermatozoa. J Biosci. 1999; 24: 317 –321.
Stahl WL, Eakin TJ, Owens JW, Jr, Breininger JF, Filuk PE, Anderson WR. Plasma membrane Ca2+-ATPase isoforms: distribution of mRNAs in rat brain by in situ hybridization. Brain Res Mol Brain Res. 1992;16: 223 –231.[Medline]
Stauffer TP, Hilfiker H, Carafoli E, Strehler EE. Quantitative
analysis of alternative splicing options of human plasma membrane calcium pump
genes. J Biol Chem. 1993; 268: 25993
–26003.
Suarez SS, Ho HC. Hyperactivated motility in sperm. Reprod Domest Anim. 2003; 38: 119 –124.[Medline]
Tamblyn TM. A bovine seminal plasma inhibitor of actin-stimulated myosin adenosine triphosphatase. Biol Reprod. 1983; 29: 725 –732.[Abstract]
Therien I, Moreau R, Manjunath P. Bovine seminal plasma
phospholipid-binding proteins stimulate phospholipid efflux from epididymal
sperm. Biol Reprod. 1999; 61: 590
–598.
Vijayasarathy S, Shivaji S, Balaram P. Plasma membrane bound Ca2+-ATPase activity in bull sperm. FEBS Lett. 1980;114: 45 –47.[Medline]
Wah DA, Fernandez-Tornero C, Sanz L, Romero A, Calvete JJ. Sperm coating mechanism from the 1.8 A crystal structure of PDC-109-phosphorylcholine complex. Structure (Camb). 2002; 10: 505 –514.
Wennemuth G, Babcock DF, Hille B. Calcium clearance mechanisms of
mouse sperm. J Gen Physiol. 2003; 122: 115
–128.
Wilhelm B, Keppler C, Hoffbauer G, Lottspeich F, Linder D,
Meinhardt A, Aumuller G, Seitz J. Cytoplasmic carbonic anhydrase II of rat
coagulating gland is secreted via the apocrine export mode. J
Histochem Cytochem. 1998;46: 505
–511.
Yanagimachi R, Usui N. Calcium dependence of the acrosome reaction and activation of guinea pig spermatozoa. Exp Cell Res. 1974;89: 161 –174.[Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
