A 14 kDa cytosolic protein purified from bovine brain homogenate has been recently reported as a stimulator of goat spermatozoa Mg2+-independent Ca2+-ATPase. In the present study, we demonstrate the formation of the [γ-32P]ATP-labelled phosphoenzyme as the 110 kDa phosphoprotein and its rapid decomposition in presence of the stimulator protein. Together with the cross-reactivity of this 110 kDa protein with an anti-SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 2a antibody, the ATPase can now be conclusively said to belong to the SERCA family, which is activated by the stimulator. The ability of the stimulator to enhance the Ca2+ transport has been elucidated from 45Ca2+ uptake studies and was found to be sensitive to Ca2+ channel blockers. CD revealed an α-helical structure of the stimulator. The amino acid analysis suggests that it is composed primarily of hydrophobic and some acidic amino acid residues. The pI of 5.1 has been re-confirmed from two-dimensional electrophoresis. Immuno-cross-reactivity studies indicate that the stimulator or similar proteins are present in cytosolic fractions of liver, kidney or testes in different species, but brain is the richest source. Proteomic analyses of its trypsinized fragments suggest its similarity with bovine THRP (thyroid hormone-responsive protein). The physiological significance of the stimulator has been suggested from its ability to activate sperm-cell motility.

INTRODUCTION

The Mg2+-independent Ca2+-ATPase is an important enzyme in goat spermatozoa [1], playing a vital role in cellular calcium homoeostasis. Like most other Ca2+-ATPases, it is involved in ATP-dependent Ca2+-transport in sperm, and undergoes phosphorylation/dephosphorylation in the transport cycle [2,3]; with transient phosphorylation of the enzyme by ATP on an active aspartate group being the key step in Ca2+ translocation. The enzyme has a molecular mass of approx. 97–110 kDa and has been shown to cross react with a monoclonal (mouse) anti-SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 2a antibody [4]. Its activity is inhibited by vanadate and lanthanum chloride, but it is insensitive to calmodulin stimulation [1], suggesting its similarity with the SERCA family of Ca2+-ATPases. A recent study has identified SERCA 2 isoforms in mammalian sperm of similar molecular masses [5].

Ca2+-ATPase activity has been shown to be affected by a wide range of substances such as proteins and peptides, drugs, hormones and growth factors etc. under different conditions. They serve to activate or inhibit Ca2+-ATPase activity in different cells and tissues and can be collectively referred to as modulators of Ca2+-ATPases [6]. An interesting aspect of modulation of Ca2+-ATPase activity in the SR (sarcoplasmic reticulum) in various cells and tissues [712] is that modulation of enzymatic activity is directly reflected in the calcium-uptake process [13,14]. This is because active transport, which is required to import calcium into the lumen of the SR, is driven by ATP hydrolysis by the Ca2+-transporting ATPase of the SR, i.e. SERCA, which is usually a 100–110 kDa membrane-bound protein [7] which forms the obligatory phosphoenzyme in the presence of Ca2+ and ATP in the reaction cycle.

Different laboratories have reported direct regulation of this calcium transport by affecting the ATP hydrolysing activity of the SR pump by various modulators of proteinous origin. Colca et al. [15] reported unidentified factors in the cytosol of islet cells which served to increase the rate of active Ca2+ uptake. They suggested that the activator was either highly charged or had a molecular mass >1000 Da as it was retained by membrane dialysis and was different from calmodulin. They speculated that the activation resulted either from phosphorylation of ER (endoplasmic reticulum) components or from the involvement of active transport of cytosolic activators into the ER. Overexpression of Sorcin, a 21.6 kDa Ca2+-binding protein of the penta-EF hand family, activates Ca2+-ATPase-mediated Ca2+ uptake in the SR of rat cardiomyocytes in a dose-dependent manner and has been thought to play critical roles in both Ca2+ homoeostasis and cardiac dysfunction in failing hearts [16]. Prolactin directly enhances Ca2+-ATPase activity in the duodenum of female rats while stimulating calcium uptake in duodenal epithelial cells [17], thus playing the key role in duodenal calcium absorption. Regucalcin, a Ca2+-binding protein which does not contain an EF hand motif, differing from calmodulin, has also been reported to activate Ca2+, Mg2+-ATPase activity as well as ATP-dependent Ca2+ transport [18]. Previous work in our laboratory has identified a 12 kDa cytosolic protein from rat brain which acts as an endogenous inhibitor of this Mg2+-independent form of Ca2+-ATPase [4], inhibiting the phosphorylation reaction of the enzyme [19].

The Mg2+-independent Ca2+-ATPase activity has been recently reported to be stimulated by a 14 kDa protein purified from bovine brain cytosol [20]. In the present study, we consolidate our previous findings by demonstrating formation of the acylphosphate intermediate as the [γ-32P]ATP-labelled phosphoprotein band observed after analysis by gel electrophoresis under acidic conditions, followed by autoradiography. Since the stimulator protein activates ATP hydrolysis, an investigation of its probable role in active Ca2+-transport coupled with the Mg2+-independent Ca2+-ATPase enzymatic activity was undertaken. Ca2+-ATPase has long been suggested to play a vital role in regulation of sperm-cell motility [21,22]. The physiological significance of the stimulator has been demonstrated from the results showing the ability of the stimulator to act as a sperm motility-enhancing factor. Along with elucidation of the role of the stimulator on Ca2+-ATPase activity and active Ca2+ transport, parallel characterization studies of the stimulator were also performed.

MATERIALS AND METHODS

Materials

EDTA, EGTA, PMSF, Tos-Lys-CH2Cl (‘TLCK’, tosyl-lysylchloromethane), Tos-Phe-CH2Cl (‘TPCK’, tosylphenyl-alanylchloromethane), Mops (sodium salt), SDS, DTT (dithiothreitol), 2-mercaptoethanol, β-alanine, TFP (trifluoroperazine) hydrochloride, verapamil hydrochloride, thapsigargin, C12E8 [octa(ethylene glycol) dodecyl ether], A23187 (calcium ionophore), iodoacetate and Ficoll 400 were from Sigma–Aldrich. Trypsin was purchased from Promega. All other reagents were of analytical grade, purchased from SRL, Mumbai, India. Nitrocellulose filter membranes (0.45 μm, type HA) were from Millipore. [γ-32P]ATP (specific activity 4000 Ci/mmol) was from Jonaki Laboratories (BRIT), Hyderabad, India. 45CaCl2 in HCl (specific activity 40.7 MBq/g) was from BARC, Mumbai, India. X-ray films, Kodak Developer D-19 (containing hydroquinone and sodium carbonate) and Kodak Industrex Fixing salt with hardener were from Kodak. MilliQ water was used throughout the study. Complete and Incomplete Freund's adjuvant were from Bangalore Genei, Bangalore, India. Anti-rabbit IgG antibody (whole molecule) was from Sigma–Aldrich.

Isolation and purification of Mg2+-independent Ca2+-ATPase-enriched microsomal membranes from goat spermatozoa

Goat testes were collected from the local slaughterhouse immediately after the animals were killed and brought to the laboratory on ice. This procedure was performed according to the guidelines of the animal ethics committee of the Institute. The microsomal membranes were prepared following a method described previously [1], followed by purification of the ATPase with the non-ionic detergent octylglucoside (C12E8) [1]. The purified membrane fraction was found to comprise two major proteins with molecular masses of approx. 97 and 110 kDa when analysed by SDS/PAGE (7.5% gels) and was used as a source of Mg2+-independent Ca2+-ATPase.

Purification of the low-molecular-mass stimulator protein

The stimulator protein was purified from the cytosolic fraction of bovine brain by ammonium sulfate precipitation (50–65%) and Sephadex G100 chromatography, followed by DEAE-cellulose anion-exchange chromatography as described previously [20]. The purified stimulator protein collected from flow-through and buffer wash fractions of the DEAE-cellulose anion exchanger was used for subsequent experiments.

Protein content estimation

Protein content was estimated according to the method described by Bradford [23], using BSA as a protein standard.

Analysis of the acylphosphate intermediate

The acid-stable acylphosphate intermediate for the Mg2+-independent Ca2+-ATPase was analysed following a method described previously [24] with slight variations. Typically, for each tube, 250 μg of microsomal proteins were added to 50 mM Tris/HCl (pH 6.8) buffer containing 1 μM calcium-EGTA and 5 mM KCl in the absence and presence of a fixed concentration of the stimulator protein and were incubated in an ice-cold water bath (0°C) for 10 min. Phosphorylation was initiated by the addition of 20 μCi of [γ-32P]ATP in 50 μM unlabelled ATP, and subsequently terminated after 20 s with the addition of 250 μl of ice-cold TCAP (trichloroacetic acid, phosphoric acid and ATP; 10% trichloroacetic acid, 50 mM phosphoric acid and 1 mM ATP). For dephosphorylation experiments, 0.5 mM CaCl2 was added to the assay mixture after phosphorylation for 20 s, followed by a further incubation in the ice-cold water bath for different time intervals (20, 30, 60, 100 and 120 s) and subsequent termination of the reaction in the same way as described above. The samples were vortexed, incubated on ice for 5 min, centrifuged at 5000 g for 5 min at 0–2°C, washed twice with 1.5 ml of TCAP and then resuspended in 40 μl of freshly prepared sample buffer containing 10 mM Mops (sodium salt, pH 5.5), 1 mM EDTA, 3% (w/v) SDS, 10% (w/v) sucrose, 2 mM DTT and 0.1% Methylene Green as the tracking dye. The samples were further vortexed vigorously for 15–20 min and then electrophoresed immediately by SDS/PAGE (10% gels) at pH 4 according to a method described previously [25] at 4°C at 90 V (constant voltage) until the dye front reached the bottom of the gel. The gel was stained with Coomassie Blue, destained, dried and autoradiographed by exposure to X-ray film (Kodak) for 96–120 h at −70°C, followed by developing of the film.

Effect of the stimulator protein on 45Ca2+ uptake in microsomal membranes of goat spermatozoa

Calcium uptake by Mg2+-independent Ca2+-ATPase was performed according to methods described previously [2], with slight modifications. Radioactivity was measured using a Wallac 1409 Liquid Scintillation Counter (PerkinElmer). The effect of the calcium ionophore A23187 and calcium-channel blockers on 45Ca2+ uptake was studied using 7.8 μCi of CaCl2 in each sample, followed by incubation at 37 °C for various time intervals. The uptake studies were also carried out in the presence of inhibitors at different concentrations, as described in the Figure and Table legends.

Sperm motility study

Spermatozoa were obtained from goat cauda epididymes following a standard procedure described previously [26]. Freshly extracted sperm preparations contained (1–2)×108 cells/ml and were used within 15 min of extraction for motility assays. To eliminate the possibility of sperm adhesion to glass, motility assays were carried out in the presence of an anti-sticking factor, EP (epididymal plasma), prepared by a method described previously [26].

Microscopic procedure

An aliquot of the freshly extracted sperm preparation (0.8×106–1×106 cells) containing EP (0.3 mg) was incubated in a total volume of 0.5 ml of RPS (modified Ringer's solution) in the absence and presence of increasing concentrations (0.21–0.98 μM) of the stimulator protein for 10 min at room temperature (25 °C) before assessing sperm motility in a haemocytometer. In the microscopic method to assay sperm motility, all cells which showed some degree of motility, forwardly motile and total motile sperm were counted along with the total count of sperm cells [26].

Spectrophotometric assay of sperm motility

The preparation of freshly extracted cauda epidiymal spermatozoa at 2×108 cells/ml (200 μl) was mixed with 50 μl of 10% (w/v) Ficoll 400 in a total volume of 500 μl of RPS in the absence or presence of increasing concentrations of the stimulator protein (0.57–2.0 μM), according to a method described previously [27].

Development of antiserum and immunoblotting

Antiserum against the stimulator protein was raised in a male albino rabbit (approx. 1 kg body weight) following six successive immunizations with the purified protein according to standard protocols as described previously [28]. For the first intramuscular injection, 200 μg of pure protein was emulsified in complete Freund's adjuvant at a 1:1 ratio. The same amount of protein emulsified in incomplete Freund's adjuvant (1:1 ratio) was used for the second injections. The animal was then boosted with 250 μg of protein a further four times. The first four injections were given at weekly intervals, followed by the last two injections at fortnightly intervals. The rabbit was bled one week after the last injection and the antiserum was collected. This procedure was performed according to the guidelines of the animal ethics committee of the Institute.

An immuno-cross-reactivity study using different concentrations of the stimulator protein against the polyclonal antibody raised against it was performed according to standard procedures described previously [29]. Immunodetection was performed using the rabbit antisera (1:1000 dilution) as the primary antibody. ALP (alkaline phosphatase)-conjugated goat anti-rabbit IgG (1:30000 dilution) used as the secondary antibody, followed by development of the membrane with NBT (Nitro Blue Tetrazolium)/BCIP (5-bromo-4-chloroindol-3-yl phosphate) solution.

Organ and species specificity of the protein were tested by cross-reaction studies of cytosolic fractions prepared from brain, liver, kidney and testes of mice, rat and goat with the antibody raised against the stimulator protein, with 20 μg of the cytosolic protein used as the antigen in each case.

Since the protein purified from bovine brain cytosol exhibited stimulatory effects on Mg2+-independent Ca2+-ATPase activity, the antibody raised against the protein was tested for immunoinhibitory effects. For this purpose, 5 μg of the stimulator protein was incubated with increasing amounts of the antisera (10–800 ng) for 30 min at 37°C and the effect on the Mg2+-independent Ca2+-ATPase activity was examined according to standard assay protocols described previously [1,20]. The purified enzyme was used for the assay (5 μg) and 5 μg of the stimulator protein without the antisera was used as a control.

Amino acid analysis of the stimulator protein

Amino acid analysis was performed in a PicoTag® system according to the manufacturer's instructions (Waters). The number of residues were determined on the basis of a molecular mass of 14000 Da.

Far UV-CD spectroscopic measurement

Far UV-CD spectra of the stimulator protein (0.25 mg/ml) in 5 mM Tris/HCl buffer (pH 7.5) were recorded at 25°C in a Jasco J-600 spectropolarimeter flashed with dry nitrogen. Spectra were collected from 250 nm to 200 nm in a quartz cell of 1 mm path-length using a slit width of 1 nm and a scan speed of 20 nm/min as the average of five scans. Analysis of the spectra was done using the CDPro software package (http://lamar.colostate.edu/~sreeram/CDPro) [30]. Using a reference set of soluble proteins (SP 22x), the results have been represented for evaluation by the CONTINLL curve-fitting program, which always gives the best agreement of the experimental spectrum with the fitted spectrum and has the lowest normalized RMSD (root mean square deviation).

Two-dimensional electrophoresis of the stimulator protein

For two-dimensional PAGE, the first dimension IEF (isoelectric focusing) electrophoresis was performed according to a method described previously [31] with a non-linear immobilized pH gradient (pI 4–7, 7 cm) in a Protean IEF tray (Bio-Rad) according to the manufacturer's instructions. The second dimension was resolved by SDS/PAGE (15% gels) in a Mini-Protean electophoresis cell (Bio-Rad) at 200 V (constant voltage) for 40 min. The gel was Coomassie Blue stained and imaged in a gel documentation instrument (Gel Doc XR 1708170, Bio-Rad). The molecular mass of the stimulator protein was obtained by comparison with the position of the standard protein molecular-mass markers on the gel. The approximate pI of the relevant spot was calculated from a standard plot of pH as a function of distance traversed by the protein on the gel (percentage gel length) at 20°C and 8 M urea as provided by the manufacturer of the IPG strips (immobilized pH gradient strips).

Peptide mass fingerprinting

In-gel digestion of the stimulator protein with MALDI (matrix-assisted laser-desorption ionization) grade trypsin (Promega) was performed as described previously [32] with slight modifications. HCCA (α-cyano hydroxyl cinnamic acid) was used as the matrix and the trypsinized peptides were subjected to MALDI–TOF (time-of-flight)-MS in an Autoflex II TOF/TOF mass spectrometer (Bruker Daltonics, Germany). Data acquisition was performed using Flex Control software and Flex Analysis software (Bruker Daltonics) was used for data analysis. Peak detection was performed after calibration of the mass spectrum with internal standards. Biotools (Mascot Search at http://www.matrixscience.com) was used for a peptide mass fingerprinting search of the trypsin-digested fragments by transferring the list of peptide masses into the peptide mass fingerprinting search program. The enzyme used for the proteolytic cleavage was specified as trypsin with carbamidomethyl as a fixed modification and deamidation and dioxidation as variable modifications.

RESULTS

A transient phosphoenzyme was formed using a goat spermatozoal microsomal membrane fraction phosphorylated with 20 μCi of [γ-32P]ATP in the presence of 50 mM Tris/HCl (pH 6.8), 1 μM calcium-EGTA and 5 mM KCl after incubation for 20 s without the requirement of Mg2+, which was abolished on dephosphorylation with excess calcium at a time interval of 120 s (Figure 1). To examine the degradation of the phosphorylated intermediate, the dephosphorylation reaction was carried out at different time intervals (20, 30, 60 and 100 s) when a time-dependent decrease of the phosphoenzyme was observed (Figure 2A).

A representative autoradiograph showing the acylphosphate intermediate of Mg2+-independent Ca2+-ATPase

Figure 1
A representative autoradiograph showing the acylphosphate intermediate of Mg2+-independent Ca2+-ATPase

Microsomal membranes (250 μg) were phosphorylated with 20 μCi of [γ-32P]ATP in 50 μM unlabelled ATP in medium containing 50 mM Tris/HCl (pH 6.8), 1 μM calcium-EGTA and 5 mM KCl. Dephosphorylation was induced by the introduction of excess unlabelled 0.5 mM CaCl2 into the assay mixture after 120 s. Lane 1, phosphorylated intermediate after 20 s incubation; lane 2, dephosphorylation of this intermediate after 20 s interval (see the Materials and methods section for details).

Figure 1
A representative autoradiograph showing the acylphosphate intermediate of Mg2+-independent Ca2+-ATPase

Microsomal membranes (250 μg) were phosphorylated with 20 μCi of [γ-32P]ATP in 50 μM unlabelled ATP in medium containing 50 mM Tris/HCl (pH 6.8), 1 μM calcium-EGTA and 5 mM KCl. Dephosphorylation was induced by the introduction of excess unlabelled 0.5 mM CaCl2 into the assay mixture after 120 s. Lane 1, phosphorylated intermediate after 20 s incubation; lane 2, dephosphorylation of this intermediate after 20 s interval (see the Materials and methods section for details).

Autoradiograph showing the time course (A) and level (B) of dephosphorylation of the acylphosphate intermediate of Mg2+-independent Ca2+-ATPase

Figure 2
Autoradiograph showing the time course (A) and level (B) of dephosphorylation of the acylphosphate intermediate of Mg2+-independent Ca2+-ATPase

(A) Lane 1, acylphosphate intermediate formed after 20 s incubation. Lanes 2, 3, 4 and 5, dephosphorylation of the intermediate after 20, 30, 60 and 100 s respectively (see the Materials and methods section for details). (B) Lane 1, phosphoenzyme intermediate formed after 20 s of incubationl; Lane 2, level of phosphoryl intermediate after 30 s dephosphorylation in the absence of the stimulator protein (control). Lanes 3 and 4, the levels of dephosphorylation after 30 s in the presence of 1.4 and 2.8 μM stimulator protein respectively (see the Materials and methods section for details).

Figure 2
Autoradiograph showing the time course (A) and level (B) of dephosphorylation of the acylphosphate intermediate of Mg2+-independent Ca2+-ATPase

(A) Lane 1, acylphosphate intermediate formed after 20 s incubation. Lanes 2, 3, 4 and 5, dephosphorylation of the intermediate after 20, 30, 60 and 100 s respectively (see the Materials and methods section for details). (B) Lane 1, phosphoenzyme intermediate formed after 20 s of incubationl; Lane 2, level of phosphoryl intermediate after 30 s dephosphorylation in the absence of the stimulator protein (control). Lanes 3 and 4, the levels of dephosphorylation after 30 s in the presence of 1.4 and 2.8 μM stimulator protein respectively (see the Materials and methods section for details).

Investigation of the effect of the stimulator on the formation of the phosphoenzyme intermediate revealed that the presence of the stimulator had no effect on the amount of phosphoenzyme formed (results not shown) at a concentration of up to 2.8 μM, which corroborated with our findings from previous membrane filtration experiments [20]. To examine its effect on dephosphorylation, two different concentrations of the stimulator were studied at a dephosphorylation time interval of 30 s, by introducing an excess amount of non-radioactive CaCl2 (0.5 mM) into the assay mixture after a standard phosphorylation interval of 20 s. A rapid loss of radiolabelling was observed on addition of the stimulator, suggesting an enhancement of the rate of the dephosphorylation reaction of the enzyme (Figure 2B).

The microsomal vesicles showed an ATP-dependent Ca2+ uptake in the absence of Mg2+, the uptake being maximum at approx. 12 min, beyond which the accumulated calcium was slowly released from the microsomal vesicles. The addition of the calcium ionophore A23187 after 10 min of uptake led to immediate release of accumulated calcium, whereas the presence of the stimulator in the reaction mixture led to a significant rise in 45Ca2+ uptake (Figure 3). At a concentration of 1.4 μM (20 μg/ml), the stimulator protein caused an increase in calcium uptake from 9 to 13.2 nmol of 45Ca2+/mg of microsomal protein. Figure 4 shows the effect of calcium-transport inhibitors on calcium uptake in a time-dependent manner. In a representative set of experiments, 0.5 mM verapamil caused a decrease in calcium uptake from 9 in the control to 4.9 units; 24 nM thapsigargin caused a decrease to 5.9 units and 60 μM TFP to 5.5 units measured at a 12 min interval. The concentrations of the inhibitor used for such time course experiments was optimized by repeated trial experiments.

Time course of 45Ca2+ uptake by Mg2+-independent Ca2+-ATPase in the microsomal membranes of goat spermatozoa

Figure 3
Time course of 45Ca2+ uptake by Mg2+-independent Ca2+-ATPase in the microsomal membranes of goat spermatozoa

Results show 45Ca2+ uptake by incubation in complete (■) and ATP-depleted (▼) medium, and in the presence of 1 μM calcium ionophore (A23187) added after 10 min of uptake (▲) in complete medium and in the presence of 1.4 μM stimulator protein (●). Results are means±S.E.M. (n=5).

Figure 3
Time course of 45Ca2+ uptake by Mg2+-independent Ca2+-ATPase in the microsomal membranes of goat spermatozoa

Results show 45Ca2+ uptake by incubation in complete (■) and ATP-depleted (▼) medium, and in the presence of 1 μM calcium ionophore (A23187) added after 10 min of uptake (▲) in complete medium and in the presence of 1.4 μM stimulator protein (●). Results are means±S.E.M. (n=5).

Effect of inhibitors on 45Ca2+ uptake by Mg2+-independent Ca2+-ATPase

Figure 4
Effect of inhibitors on 45Ca2+ uptake by Mg2+-independent Ca2+-ATPase

Results show 45Ca2+ uptake by incubation in complete medium (control, ■), and in the presence of 0.5 mM verapamil (●), 60 μM TFP (▲) and 24 nM thapsigargin (▼). Results are means±S.E.M. (n=5).

Figure 4
Effect of inhibitors on 45Ca2+ uptake by Mg2+-independent Ca2+-ATPase

Results show 45Ca2+ uptake by incubation in complete medium (control, ■), and in the presence of 0.5 mM verapamil (●), 60 μM TFP (▲) and 24 nM thapsigargin (▼). Results are means±S.E.M. (n=5).

Next, experiments were designed to investigate whether stimulation of calcium uptake by the protein could be perceived even in the presence of such inhibitors. Table 1 shows that the presence of increasing concentrations of verapamil, thapsigargin or TFP progressively countered such enhanced calcium uptake.

Table 1
Effect of the stimulator on 45Ca2+-uptake by Mg2+-independent Ca2+-ATPase incubated with different concentrations of Ca2+- uptake inhibitors

Control represents the percentage uptake of 45Ca2+ in the presence of enzyme alone; 1, in the presence of 1.4 μM stimulator; 2,3 and 4, in the presence of the stimulator plus 0.3, 0.5 and 0.75 mM verapamil respectively; 5, 6 and 7, in the presence of the stimulator plus 12, 24 and 36 nM thapsigargin respectively; 8, 9 and 10, in the presence of stimulator plus 40, 60 and 80 μM TFP respectively. Results are the means±S.D. (n=5).

Sample45Ca2+ uptake activity (%)
Control 100 
158.7±5.1 
89.8±2.4 
85.3±1.4 
78.1±2.8 
140.8±2.7 
94.6±3.1 
59.2±3.6 
131.5±2.6 
93.1±2.9 
10 57.2±1.3 
Sample45Ca2+ uptake activity (%)
Control 100 
158.7±5.1 
89.8±2.4 
85.3±1.4 
78.1±2.8 
140.8±2.7 
94.6±3.1 
59.2±3.6 
131.5±2.6 
93.1±2.9 
10 57.2±1.3 

In the absence of the exogenous stimulator protein, nearly 20–30% of spermatozoa extracted from goat cauda sperm preparations showed forward progression, and nearly 45% of the sperm exhibited an overall motility following microscopic analysis. In the presence of as low as 0.7 μM stimulator, approx. 53% of the sperm showed forward progression compared with 28% of control sets, resulting in a stimulation of 89%. The overall or total sperm motility stimulation also markedly increased to 91% at this concentration of the stimulator protein (Table 2). The addition of the stimulator thus seemed to induce motility in weakly motile or immotile sperm present in the control. However, further addition of the protein beyond 1 μM led to saturation of the stimulatory effect.

Table 2
Effect of variable concentrations of the stimulator protein on the forward progression of goat caudal sperm assessed microscopically

The assay was performed as described in the Materials and methods section). The percentage stimulation was concentration-dependent for both forward motility (FM) and total motility (TM). Results are the means±S.D. (n>20), with P≤0.05 for TM.

Stimulation (%)
Concentration of stimulator protein (μM)FMTM
0.21 28.6±1.2 20.0±2.7 
0.52 57.1±0.9 40.0±1.9 
0.70 89.3±1.4 91.1±2.4 
0.98 67.8±2.1 66.7±2.8 
Stimulation (%)
Concentration of stimulator protein (μM)FMTM
0.21 28.6±1.2 20.0±2.7 
0.52 57.1±0.9 40.0±1.9 
0.70 89.3±1.4 91.1±2.4 
0.98 67.8±2.1 66.7±2.8 

When analysed spectrophotometrically at a wavelength of 545 nm, a similar enhancement of the vertical motility of goat caudal sperm was observed in the presence of the stimulator, as demonstrated in Figure 5. The vertical motility of the sperm increased with increasing concentrations of the purified protein up to 2 μM, beyond which no further stimulation was observed. Both the optical microscopic method of analysis as well as the spectrophotometric method demonstrated the ability of the protein to function as a stimulator of forward or vertical as well as total motility of goat cauda sperm.

Representative spectrophotometric tracing showing the effect of stimulator protein on the vertical motility of goat caudal sperm

Figure 5
Representative spectrophotometric tracing showing the effect of stimulator protein on the vertical motility of goat caudal sperm

Various concentrations of stimulator protein were tested [0.57 μM (○), 1.14 μM (▲) and 2 μM (Δ)] to examine the effect on sperm motility (200×106 cells/ml) with no stimulation present as a control (●) (see the Materials and methods section for details).

Figure 5
Representative spectrophotometric tracing showing the effect of stimulator protein on the vertical motility of goat caudal sperm

Various concentrations of stimulator protein were tested [0.57 μM (○), 1.14 μM (▲) and 2 μM (Δ)] to examine the effect on sperm motility (200×106 cells/ml) with no stimulation present as a control (●) (see the Materials and methods section for details).

To further characterize the stimulator protein, a rabbit polyclonal antibody was raised against the purified stimulator protein. Western-blot analysis with different concentrations of the stimulator protein against the rabbit antisera (Figure 6) suggested that the antibody recognized the protein in the cytosol, since even 5 μg of the protein generated a strong cross-reactive band. When titrated with different dilutions of rabbit antisera, the antigen produced an intense cross-reactive band even at a 1:4000 dilution (results not shown). This also suggested that the protein is highly antigenic in nature.

Immunodetection of the stimulator protein

Figure 6
Immunodetection of the stimulator protein

Lanes 1, 2 and 3 represent 5, 10 and 15 μg of the stimulator protein probed with the rabbit antisera against the stimulator protein (1:1000 dilution).

Figure 6
Immunodetection of the stimulator protein

Lanes 1, 2 and 3 represent 5, 10 and 15 μg of the stimulator protein probed with the rabbit antisera against the stimulator protein (1:1000 dilution).

Table 3 represents the intensity of the cross-reactive bands with cytosolic fractions from different organs of mouse, rat and goat immunoblotted with the polyclonal antibody raised against the stimulator protein. A strong cross-reactive band was observed with mouse, rat and goat brain at approx. 14 kDa. All other organs from mouse, rat and goat show less intense bands at this region. A faint cross-reactive band also appeared at approx. 20 kDa in the cytosol of rat and goat liver and in goat brain. All of these findings, taken together, suggest that the stimulator or similar proteins might be ubiquitously expressed in different organs; however, the expression level is the highest in the brain.

Table 3
Intensity of cross-reactivity of the antibody with cytosolic fractions of different organs from mouse, rat and goat

Cytosol (20 μg) prepared from different tissues was cross-reacted with the polyclonal antibody raised against the stimulator protein (1:1000 dilution). ++++, strong cross-reactivity; +++, average cross-reactivity; ++, weak cross-reactivity; +, very weak cross-reactivity, N.S., not studied.

Organ
SpeciesBrainLiverKidneyTestes
Mouse ++++ +++ ++ ++ 
Rat ++++ ++ 
Goat ++++ N.S. 
Organ
SpeciesBrainLiverKidneyTestes
Mouse ++++ +++ ++ ++ 
Rat ++++ ++ 
Goat ++++ N.S. 

The specificity of the antibody for the antigen (stimulator protein) was tested by titrating a fixed amount of the purified protein with nanogram quantitites of the antisera, followed by checking the stimulatory effect of the protein on Mg2+-independent Ca2+-ATPase activity. In the presence of the antibody, the stimulatory activity of the protein decreased. With 400 ng of the antisera, approx. 50% of the stimulatory activity of the protein was found to be lost compared with control values (taken as 100%). Beyond this value, no further inhibition was observed (results not shown).

Amino acid analysis of the purified protein (Table 4) showed that it contained relatively high proportions of hydrophobic amino acids along with the individual contributions of hydrophilic amino acids such as serine residues and polar amino acids such as cysteine residues. Acidic amino acids such as aspartic acid and glutamic acid are also present in significant amounts, indicating that the protein has an acidic nature.

Table 4
Amino acid composition of the purified stimulator protein

Stimulator protein (20 μg) free from ions and salts was digested with 6 M HCl in an atmosphere of nitrogen, followed by derivatization with phenyl isothiocyanate and run through a PicoTag® HPLC column. The stimulator protein is composed of 144 animo acid residues.

Amino acid residueResidue per mol of proteinComposition (%)
Aspartic acid/Asparagine 22 10.7 
Glutamic acid/Glutamine 24 12.8 
Serine 11 8.3 
Glycine 11 6.1 
Histidine 4.1 
Arginine 10 4.1 
Threonine 4.1 
Alanine 13 8.2 
Proline 6.0 
Tyrosine 3.1 
Valine 4.5 
Methionine 4.3 
Cysteine 7.9 
Isoleucine 2.8 
Leucine 6.8 
Phenylalanine 1.7 
Lysine 4.6 
Amino acid residueResidue per mol of proteinComposition (%)
Aspartic acid/Asparagine 22 10.7 
Glutamic acid/Glutamine 24 12.8 
Serine 11 8.3 
Glycine 11 6.1 
Histidine 4.1 
Arginine 10 4.1 
Threonine 4.1 
Alanine 13 8.2 
Proline 6.0 
Tyrosine 3.1 
Valine 4.5 
Methionine 4.3 
Cysteine 7.9 
Isoleucine 2.8 
Leucine 6.8 
Phenylalanine 1.7 
Lysine 4.6 

Secondary structure elucidation from far UV-CD spectra of the stimulator protein showed minima at 208 and 222 nm, characteristic of an α-helix, with contributions from a β-sheet shown by the flattened minima at approx. 215–220 nm (results not shown). The percentage of secondary structures present, as analysed with the CONTINLL program from CDPro software (Table 5), indicated that 38.3% of the protein existed as a helix, 16.1% as a β-sheet, 9.1% as a poly-proline structure, whereas the rest of the protein is unordered.

Table 5
Quantitative curve fitting of CD spectra of the stimulator protein using the CONTINLL program (Basis Set SP22X) from CDPro software

PP2, poly-proline structure.

Secondary structure typePercentage of total structure
α-Helix 38.3 
β-Sheet 16.1 
PP2 9.1 
Unordered 36.4 
Secondary structure typePercentage of total structure
α-Helix 38.3 
β-Sheet 16.1 
PP2 9.1 
Unordered 36.4 

A plot of pH against the distance traversed by the spot of interest (expressed as a percentage of gel length) was used in two-dimensional gel electrophoresis to calculate the pI, which was approx. 5.1 (Figure 7), re-confirming our previous results [20].

Two-dimensional PAGE of the purified stimulator protein

Figure 7
Two-dimensional PAGE of the purified stimulator protein

Non-linear immobilized pH gradient [IPG strips (immobilized pH gradient strips), pI 4–7, 7 cm] was used in the first dimension, the direction of the IPG strip in the diagram is pI 4→7 from left to right, starting from the point next to the lane showing molecular-mass markers. SDS/PAGE (15% gel) was used in the second dimension. The position of the spot corresponding to the purified protein is indicated by the arrow, with a calculated pI of 5.1.

Figure 7
Two-dimensional PAGE of the purified stimulator protein

Non-linear immobilized pH gradient [IPG strips (immobilized pH gradient strips), pI 4–7, 7 cm] was used in the first dimension, the direction of the IPG strip in the diagram is pI 4→7 from left to right, starting from the point next to the lane showing molecular-mass markers. SDS/PAGE (15% gel) was used in the second dimension. The position of the spot corresponding to the purified protein is indicated by the arrow, with a calculated pI of 5.1.

Proteomic analysis of the purified protein was carried out by in-gel digestion of the protein spot with trypsin, followed by MALDI–TOF analysis of the trypsinized peptides (Figure 8 and Table 6). Twenty-three peptide fragments were obtained. Matching of the peptide mass fingerprint was done through the Mascot search programs (using the MSDB database). Maximum homology (in terms of peptide fingerprint) was obtained with THRP (Q690M9_BOVIN from Bos taurus). It has an expected mass of 17.19 kDa and a calculated pI of 5.73. Next highest homology was obtained with a protein (Q2TBW8_ BOV IN) from B. taurus and a 60S ribosomal protein L10, the QM protein homologue (RL10_BOVIN from B. taurus) respectively. The protein (Q2TBW8_ BOVIN) has been found to have a molecular mass of 25.026 kDa, and a calculated pI value of 10.21, whereas the 60S ribosomal protein L10, QM protein homologue (RL10_BOVIN) has been reported to be a 24.912 kDa protein with a calculated pI value of 10.16. Different matches with decreased homology compared with those mentioned above were also obtained with other proteins from B. taurus.

MALDI–TOF fragmentation pattern of the tryptic digest of the stimulator protein

Figure 8
MALDI–TOF fragmentation pattern of the tryptic digest of the stimulator protein

Peptides extracted from the tryptic digest of the stimulator protein were analysed in an Autoflex II mass spectrometer and Flex Control software was used for acquisition of the spectra. Intensity (intens.) is measured in absorbance units (a.u.) (y-axis). Peptide masses are in Da (x-axis). The letters A–W correspond to the masses listed in Table 6 (see Table 6 for details).

Figure 8
MALDI–TOF fragmentation pattern of the tryptic digest of the stimulator protein

Peptides extracted from the tryptic digest of the stimulator protein were analysed in an Autoflex II mass spectrometer and Flex Control software was used for acquisition of the spectra. Intensity (intens.) is measured in absorbance units (a.u.) (y-axis). Peptide masses are in Da (x-axis). The letters A–W correspond to the masses listed in Table 6 (see Table 6 for details).

Table 6
MALDI–TOF analysis of the tryptic digest of the purified protein

Monoisotopic peptide masses were obtained using the Autoflex II Mass Spectrometer after in-gel digestion of the protein with trypsin. Flex Analysis software was used to generate the mass list (see Figure 8 for spectra).

PeakPeptide masses (Da)
703.75 
719.80 
724.82 
769.16 
877.16 
893.04 
958.55 
966.62 
972.53 
996.52 
1018.50 
1093.53 
1167.68 
1221.63 
1247.73 
1290.71 
1296.65 
1312.67 
1318.67 
1444.64 
1855.79 
1877.75 
1899.74 
PeakPeptide masses (Da)
703.75 
719.80 
724.82 
769.16 
877.16 
893.04 
958.55 
966.62 
972.53 
996.52 
1018.50 
1093.53 
1167.68 
1221.63 
1247.73 
1290.71 
1296.65 
1312.67 
1318.67 
1444.64 
1855.79 
1877.75 
1899.74 

DISCUSSION

We have demonstrated recently the stimulatory effect of a 14 kDa cytosolic protein from bovine brain on the goat spermatozoa Mg2+-independent Ca2+-ATPase [20]. It has been found that the stimulatory effect of the protein observed on Mg2+-independent Ca2+-ATPase activity is a result of its ability to enhance the rate of dephosphorylation of the phosphoenzyme intermediate [20]. The high-energy acylphosphate intermediary involved in the entire process has now been identified by gel electophoresis. Autoradiographic results show a 110 kDa phosphoprotein band (on an acidic gel) when microsomal vesicles are phosphorylated with [γ-32P]ATP in the presence of micromolar Ca2+ concentrations and in the absence of Mg2+ at 0°C (Figure 1 and Figure 2A). It should be noted that this 110 kDa protein is the only protein in the present study that undergoes phosphorylation under the conditions detailed above. Together with our previous Western-blot analysis of goat sperm microsomes probed with mouse monoclonal anti-SERCA 2a antibody (recognizing a doublet of 97 and 110 kDa) [4], this finding conclusively establishes that the Mg2+-independent Ca2+-ATPase of caprine spermatozoa belongs to the SERCA family of ATPases. Lawson et al. [5] very recently showed the localization of SERCA 2 isoform in mammalian sperm by Western blotting, where a SERCA 2 could be detected by two protein bands of approx. 100 and 110 kDa, comparable with our results [4]. We have found that in the presence of the stimulator, the formation of the phosphoenzyme was not perturbed (results not shown), but the rate of decay of the phosphoprotein band on the gel was markedly increased (Figure 2B), corroborating our previous findings from Millipore membrane-filtration experiments [20]. The optimum time required for maximum radiolabel incorporation and formation of the phosphoprotein band to be analysed by gel electrophoresis under acidic conditions followed by autoradiography is slightly longer (20 s) compared with that obtained when the phosphoenzyme was quantified by a rapid vacuum filtration assay [20] on membranes (10 s). This might be as a result of the lower temperature reaction conditions (0°C) used in the former assay. Although it is difficult to explain why the phosphorylation rate is unaffected, however, similar reports are available in the literature. The stimulation of Ca2+-ATPase activity purified from rabbit skeletal muscle SR by disulfiram has been investigated at partial reaction levels for the enzyme [33] by a rapid kinetic method. Reports have suggested that disulfiram had no effect on the rate of phosphorylation of the ATPase by ATP, but it increased the rates of the Ca2+ transport step (E2PCa2→E2P) and the rate of the dephosphorylation reaction by almost 2-fold. Jasmone, which caused a similar increase in ATPase activity, was also found to increase the rates of these two steps, with no other effects on the ATPase [34]. In both of the above cases, the stimulation of ATPase activity followed from the increase in the dephosphorylation reaction along the exponential decay curve comparable with that observed in the present study.

The Mg2+-independent Ca+2-ATPase of goat spermatozoa microsomal membranes has been known to be involved in calcium uptake without the requirement of Mg2+, suggesting the role of this enzyme in calcium transport across the membrane [2]. However, a detailed study of such calcium transport and sensitivity of the pathway to calcium-transport inhibitors/stimulators was lacking. The interaction of the low-molecular-mass protein with Mg2+-independent Ca2+-ATPase [20], prompted us to elucidate any probable effects of the protein on such calcium-transport phenomenon associated with the enzyme. It is pertinent to mention that under existing isolation and purification conditions, the possibility of having any effect of endogenous calcium is remote. We have not experienced either the presence of any protease and/or phosphatase activity of the 14 kDa protein.

The effect of the stimulator as well as those of three known calcium-transport inhibitors; verapamil, TFP and thapsigargin, and their agonistic or antagonistic effect, if any, on 45Ca2+ uptake have been examined. As expected, the stimulator protein, when present at micromolar concentrations, enhances the time-dependent rate of Ca2+ accumulation in microsomal vesicles. An appreciable increase of 1.5-fold in calcium accumulation has been observed with 1.4 μM of stimulator in 12 min, compared with that in control microsomes (Figure 3). Increasing the concentration of stimulator increases this activation (results not shown). The ATP-dependent 45Ca2+-uptake by spermatozoa microsomal vesicles was inhibited to various degrees by verapamil, TFP and thapsigargin (Figure 4). Inhibition was observed in the range 0.1–1 mM for verapamil, 1–100 nM for thapsigargin and 5–100 μM for TFP. which is comparable with previous reports [3537]. The stimulator-enhanced Ca2+ transport was found to be sensitive to inhibition by verapamil, and more markedly by thapsigargin or TFP (Table 1). Interestingly, we demonstrated previously that the inhibitory effect of verapamil on Mg2+-independent Ca2+-ATPase activity could be reversed by low concentrations of the stimulator, whereas higher concentrations were required for complete restoration of the enzyme activity inhibited by TFP [20]. It may therefore be speculated that the similar nature of effect of the stimulator on ATPase activity and rate of Ca2+ transport in the presence of such inhibitors show the functional coupling of the two processes. It is pertinent to mention here that although the effects of the stimulator protein purified from brain have been tested on Mg2+-independent Ca2+-ATPase of caprine sperm where the ATPase activity is largely enriched, some experiments were also performed with brain ATPase, where comparable effects have been observed (results not shown). Since the level of ATPase is found to be quite low in brain compared with spermatozoa, the latter was used as the source of the enzyme for the present study.

The Ca2+-ATPase has been known to play a vital role in regulating sperm motility through the maintenance of intracellular calcium concentration [21,22]. The calcium-channel blocker verapamil has been shown to retard sperm motility in humans, with a significant decrease in Ca2+-dependent ATPase activity [38]. Nifedipine, another calcium-channel blocker, significantly inhibits Ca2+-uptake and Ca2+-dependent ATPase activity and simultaneously arrests the motility of human spermatozoa [39]. A recent study has localized SERCA 2 isoforms in mammalian sperm [5], especially in the sperm mid piece, suggesting that SERCA could play a role in sperm motility. PDC-109 has been shown to stimulate both Mg2+-dependent as well as Mg2+-independent Ca2+-ATPases in bovine sperm with simultaneous stimulation of sperm motility [40]. These reports linking Ca2+-ATPase activity in sperm and sperm motility led us to investigate any possible effect of the stimulator protein on caprine sperm motility. Evaluation of number of forwardly motile sperm assessed microscopically indicate a marked increment in the presence of increasing micromolar concentration of the stimulator protein (Table 2). To eliminate the possibility of artefact due to sperm adhesion to glass, the motility assay was carried out in the presence of an antisticking factor (EP). The results obtained indicate that the presence of the stimulator protein could initiate forward or overall motility of sperm which were apparently immotile in control sets of experiment. Furthermore, quantitative estimation of forward motile sperm, taking into consideration their vertical velocity, was carried out by spectrophotometric methods. Results revealed a dose-dependent stimulatory action of the purified protein on sperm vertical velocity (Figure 5) or forward motility observed up to a specific concentration. It is to be mentioned here that the concentration of the stimulator (2 μM) causing maximum enhancement of vertical motility measured by spectrophotometric means is greater compared with that (0.7 μM) observed for the light microscopic assessments of forward motility; this lack of correlation between the values obtained by two methods of motility assay has also been noted previously by other researchers [27] who reasoned that while the microscopic method measures the number of forwardly motile cells, the spectrophotometric assay technique takes into consideration the vigorously vertically moving sperm. A 66 kDa sperm-motility promoting glycoprotein FMSF-1 (forward motility-stimulating factor-1) has been reported from buffalo blood serum [41]. From immunoblotting studies the factor has been found to be present in testis and epididymis, although its richest source is reported to be liver. A 78 kDa SMIF (spermatozoa motility-inhibiting factor) has been reported from chicken seminal plasma which probably involves disulfide bonds for its activity [42]. Interestingly, use of anti-SMIF antibodies have shown cross-reactive proteins to be present in chicken blood and liver and also in seminal plasma of cattle and buffalo. A 52 kDa glycoprotein from porcine follicular fluid [43] and a 58 kDa porcine blood serum antithrombin III [44] have also been shown to enhance sperm motility. Although we are presently unable to evaluate the exact mechanism underlying such stimulation of sperm progression by the protein under study, it can be suggested from the above results that activation of goat sperm motility is another physiological function of the stimulator protein which serves to stimulate Mg2+-independent Ca2+-ATPase of goat sperm. Previous results have revealed that the extracellular addition of calmodulin, a well known 17 kDa Ca2+-ATPase activator protein, plays a vital role in sperm capacitation [45], whereas calmodulin antagonists served to block capacitation. Whether the 14 kDa stimulator protein described in the present study plays a similar role needs to be explored further.

In order to further characterize the 14 kDa stimulator protein and the mechanism of stimulation of the ATPase, amino acid analysis of the stimulator was performed (Table 4). The protein is found to contain large amounts of non-polar hydrophobic residues, enabling it to penetrate through the bilayer core of the membrane and bind the enzyme. However, the protein is of an overall acidic nature, as deduced from the two-dimensional gel electrophoresis results, which indicates its pI to be approx. 5.1 (Figure 7). Far UV-CD spectra and analysis of the secondary structural features (Table 5) indicate that the protein exists primarily in an α-helical form, with some β-pleated structural features. In-gel digestion of the purified protein with trypsin, analysis of the peptide fragments by MALDI–TOF (Table 6 and Figure 8) followed by matching of the peptide fingerprint with that of other known proteins using the MSDB search program indicated its maximum similarity with the THRP fragment from B. taurus (17.19 kDa protein, pI 5.73). Both its molecular mass and pI are thus different from the protein in the present study. A literature search revealed that THRP is expressed in rat cerebral tissue and shares similarity with the c-Abl interactor protein Abi-2, which is a substrate for the tyrosine kinase activity of c-Abl [46]. Western blotting has been used to study the localization of the stimulator. Immunoblots with cytosolic fractions from different organs of mouse, rat and goat suggested that the stimulator protein is expressed not only in bovine brain, but similar or homologous proteins are also present in liver, kidney and testes (Table 3). Therefore we might presume that this low-molecular-mass cytosolic stimulator protein is not organ- or species-specific. However, the intensity of the immunoreactive 14 kDa band is highest with brain cytosol fractions from different species, which might suggest that although similar or homologous proteins are present in different organs, the expression level is the highest in brain. The progressive decrease of the stimulatory activity of the purified protein pre-incubated with increasing concentration of its antisera (results not shown) indicate that the antibody raised in rabbit is highly specific for the stimulator protein and hence the immuno-inhibition.

The amino acid sequence of the protein in the present study, which would be useful for elucidating the molecular basis of interaction of the stimulator with the enzyme, is under investigation.

Abbreviations

     
  • C12E8

    octa(ethylene glycol) dodecyl ether

  •  
  • DTT

    dithiothreitol

  •  
  • EP

    epididymal plasma

  •  
  • ER

    endoplasmic reticulum

  •  
  • IEF

    isoelectric focusing

  •  
  • MALDI

    matrix-assisted laser-desorption ionization

  •  
  • RPS

    modified Ringer's solution

  •  
  • SERCA

    sarcoplasmic/endoplasmic reticulum Ca2+-ATPase

  •  
  • SMIF

    spermatozoa motility-inhibiting factor

  •  
  • SR

    sarcoplasmic reticulum

  •  
  • TCAP

    trichloroacetic acid, phosphoric acid and ATP

  •  
  • TFP

    trifluoroperazine

  •  
  • THRP

    thyroid-hormone-responsive protein

  •  
  • TOF

    time-of-flight

Part of this work was funded by the Council of Scientific and Industrial Research by awarding Senior Research Fellowships to S.G. [9/15(261)/2002-EMR-1 and TS9/15(260)/2002-EMR-1] and by funding in part from the Bose Institute, Kolkata, India.

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