at 4 °C *

Affinity chromatography on Chelating Sepharose Fast Flow Gel-Zn(2+) was used for fractionation of boar seminal plasma proteins. Approximately 30% of total boar seminal plasma proteins showed affinity for zinc ions (ZnBP fraction). Native electrophoresis (PAGE) of ZnBP revealed six protein fractions which separated into 27 bands under denaturing conditions (SDS/PAGE). Two-dimensional electrophoresis (2D PAGE) showed 148 polypeptides with isoelectric points mostly in the basic and neutral pH range. The zinc-binding proteins comprise mainly 10-20 kDa polypeptides which are probably members of the spermadhesin family. ZnBP present in the incubation mixture of spermatozoa stored for 1 or 24 h at 4 °C allowed preservation of a higher percentage of cells exhibiting linear motility in comparison to a control sample stored in PBS. Presented results indicate that proteins binding Zn(2+) ions have a shielding effect on the sperm plasma membrane and acrosome of spermatozoa, protecting these structures against consequences of cold shock.


INTRODUCTION
Numerous functions carried out by seminal plasma proteins result from their ability to bind low-molecular weight ligands, such as Zn 2+ ions.They protect chromatin of spermatozoa and regulate their motility.Additionally, these protein ligands exert immunomodulating, antibacterial and antioxidant functions in the reproductive tract (Strzezek et al., 1987;Al-Somai et al., 1994;Vivacqua et al., 2004;Caballero et al., 2006, Edstrom et al., 2008).
Proteins that originate in seminal vesicles are the major components of human semen coagulum, collectively named semenogelins (Sg) (Robert & Gagnon, 1999).Semenogelins and their fragments have a high binding capacity for zinc ions secreted by the prostate gland and they shuttle Zn 2+ to the sperm nucleus where they regu-late DNA stability (Robert & Gagnon, 1999;de Lamirande et al., 2007).
During the epididymal sperm transit zinc elimination is a mandatory step in sperm maturation to obtain motility.Research of Henkel et al. (1999;2003) showed that zinc is located also in the flagella, where it plays an important function in sperm motility regulation in humans.During spermatogenesis, when outer dense fibers (ODF) are formed, zinc ions bind cysteinyl residues in ODF and act as an antioxidant factor.
Zinc is present in human, bull, boar, rat and hamster spermatozoa and its removal by chelating agents causes an increase in sperm motility (Silverstroni et al., 1989;Andrews et al., 1994).
A zinc-binding protein secreted by boar seminal vesicle glands has been isolated from the seminal plasma (Strzeżek et al., 1987;Strzeżek & Hopfer, 1987).It possesses antibacterial properties and regulates sperm motility.However the molecular structure and polypeptide composition of zinc-binding proteins and their influence on the functions of spermatozoa subjected to different storage conditions have not been elucidated as yet.
In this study we isolated and characterized biochemically a zinc-binding protein fraction from boar seminal plasma and determined the effect of the isolated fraction on some quality parameters of sperm after its storage under cold shock conditions (4 °C).

MATERIALS AND METHODS
Preparation of seminal plasma.Boars used in this study were fed a commercial feed and kept in individual pens under standard environmental conditions.Approval of a local ethics committee was obtained for this study (No. 23/2003).Whole ejaculates were collected from ten sexually mature Polish Large White boars using the gloved-hand technique.The gel fraction was removed from the ejaculate using sterile gauze.Filtered semen samples were centrifuged for 15 min at room temperature at 10 000 × g.The supernatant (seminal plasma) was stored at -20 °C until required.Before chromatographic separations seminal plasma was dialyzed for 24 h against deionized H 2 O and centrifuged (15 min, 10 000 × g).Obtained supernatant was used for isolation of zinc-binding proteins (ZnBP).
Protein measurement.The protein content was measured according to Lowry et al. (1951) using bovine serum albumin (BSA; Serum and Vaccine Production, Cracow, Poland) as a standard.
Separation of Zn 2+ -binding proteins.Separation of Zn 2+ -binding proteins was conducted on a BioPilot chromatography system with 500 ml of Chelating Sepharose Fast Flow gel packed in an XK50 column (Amersham Biosciences).The column was equilibrated in 0.5 M Tris/HCl, 0.5 M NaCl buffer (pH 8.0) and saturated with an aqueous solution of ZnCl 2 (Sigma) until a concentration of 30 µM of Zn 2+ ions/ml of gel was obtained (Hoóody & Strzezek, 1999).After the column had been washed with equilibration buffer, 100 ml of dialyzed seminal plasma (20-50 mg proteins/ml) was loaded.Zn 2+ -binding proteins were eluted from the column with 0.5 M Tris/HCl buffer (pH 7.5) with 0.1 M imidazole and collected as 10-ml fractions.The column was regenerated with 0.5 M Tris/acetate, 0.5 M NaCl and 0.5 M EDTA (pH 7.0).The ZnBP fractions were dialyzed against deionized H 2 O and lyophilized.
Determination of molecular mass of Zn 2+ -binding proteins using gel filtration.A Superose 12 column and an FPLC (Fast Protein Liquid Chromatography) system (Amersham Biosciences) were used for molecular mass determination of isolated proteins under nondenaturing conditions.For calibration, the following molecular mass standards were used: ribonuclease (15.6 kDa), chymotrypsinogen (22.8 kDa), ovalbumin (48.9 kDa), albumin (65.4 kDa), aldolase (146 kDa) and catalase (226 kDa).Calibration and separation of protein fractions were conducted at a flow rate of 0.5 ml/min using 0.5 M Tris/HCl, 0.3 M NaCl and 0.02 % (w/v) sodium azide (pH 7.5) buffer.The molecular mass of proteins was determined according to Andrews (1964).(Davis 1964).Glass tubes (0.5 cm × 8.7 cm) were filled with a 7.5 % resolving gel solution to obtain 7.5 cm long gel.After polymerization a 0.5 cm layer of 4 % stacking gel was added on the surface of the resolving gel.Tubes with polymerized gel were fitted in an electrophoresis chamber filled with electrophoresis buffer (0.5 M Tris, 0.25 M glycine, pH 8.3).Samples (200 µl, 8 mg of total protein) were loaded on the gels and electrophoretic separation was performed at a constant current of 5 mA/gel.Gels were stained with 0.5 % (w/v) amido black in 7 % (v/v) acetic acid and destained with several changes of 7 % acetic acid.

Electrophoretic characterization of isolated proteins. Non-denaturing electrophoresis -PAGE
Denaturing electrophoresis -SDS/PAGE (Laemmli, 1970).Protein samples were diluted at a 1:1 ratio with 2 x concentrated lysis buffer (0.34 M Tris/HCl, 10 % SDS (w/v), 20 % glycerol (v/v), 2 % β-mercaptoethanol (v/v), 2 % bromophenol blue (w/v), pH 6.8) and heated for 5 min at 95 ºC.Before loading, samples were centrifuged at 10 000 × g for 5 min at room temp.Electrophoresis was performed in 12 % polyacrylamide gels, 6.5 cm long, at a constant voltage (120 V) in a buffer containing 0.5 M Tris, 0.25 M glycine and 0.5 % SDS (w/v), pH 8.3.Different amounts of protein were loaded in gel wells to obtain optimal resolution of protein bands occurring at various concentrations in the isolated ZnBP fraction.After electrophoresis, the gel was silver-stained according to Heukeshoven and Dernick (1985).Low molecular mass standards (Amersham Biosciences) and the Multi-Analyst software (BioRad) were used for polypeptide molecular mass determinations.
Electrophoresis and staining of the gels were performed using procedures described for SDS/PAGE.Gel Zinc-binding proteins from boar seminal plasma analysis was conducted using PDQUEST (BioRad) software.
The effect of ZnBP proteins on boar spermatozoa stored at 4 °C.Sample preparation.Characteristics of plasma membrane integrity, sperm morphology and linear movement were determined in the following incubation variants: -Control (without ZnBP) -spermatozoa washed with PBS and suspended in PBS at a final concentration 3 × 10 7 cells/ml.
-Treatment (with ZnBP) -spermatozoa rinsed with PBS and suspended in PBS supplemented with 3 mg/ml of the ZnBP fraction at a final concentration of 3 × 10 7 cells/ml.
Measurements were made after semen dilution (time 0) and after 1 and 24 h of incubation at 4 °C (277.2K).
Sperm motility (CASA).Motility of spermatozoa was measured using a CASA (Computer Assisted Semen Analysis) system (CMA-Mika System, Strömberg-Mika, Germany).Total motility of spermatozoa was assessed visually by the same technician throughout the study.For the assessments, aliquots (6 μl) of diluted semen samples were placed on a pre-warmed slide, covered with a glass cover slide and examined under a light microscope (200× magnification) equipped with an attached heated stage (37 °C).Ejaculates used in the experiment showed an initial motility above 80 %.
The percentage of total motile sperm cells was calculated.Among the motile cells percentage of spermatozoa showing linear movement was determined using a CASA system.
Sperm morphology.Spermatozoa were Giemsa stained according to Watson's (1975) method.After smears were prepared on slides they were analyzed under a light microscope (Olympus CO 11) at a 100× magnification using an immersion lens.The percentage of cells with damaged acrosome was recorded.
Sperm plasma membrane status -fluorescence measurements.Sperm plasma membrane integrity was assessed using a combination of two fluorochromes: carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) (both from Sigma, St. Louis, MO, USA), as described in a previous study (Fraser et al., 2002).Spermatozoa were examined under an epifluorescence microscope (Olympus CH 30, Tokyo, Japan).Spermatozoa showing PI staining were assumed to have damaged sperm plasma membrane.Two slides were assessed per sample and 200 spermatozoa were evaluated per slide.
Statistical analysis.Data were subjected to analysis of variance (ANOVA) using the General Linear Model (GLM) procedure from Statistica software package, version 8 (StatSoft Incorporation, Tulsa, OK, USA).All results are expressed as mean ± standard error of the mean (S.E.M.), and were considered significant at P < 0.05.

RESULTS
After separations on Chelating Sepharose Fast Flow gel, two protein fractions were obtained from boar seminal plasma.The first one contained zinc non-binding proteins eluted from the resin with equilibration buffer.The second fraction comprised zinc-binding proteins (ZnBP) eluted with imidazole buffer.Planimetric analysis of chromatogram showed that the ZnBP constituted approx.30 % of the total amount of seminal plasma proteins.
Electrophoresis of the ZnBP fraction under non-denaturing conditions (PAGE) showed the presence of six protein bands (Fig. 1).The molecular messes of two of them were determined at over 226 kDa and the others were approx.150, 85, 40 and below 15 kDa (Fig. 2).The action of reducing and denaturing factors (SDS/PAGE) on native forms of ZnBP resulted in an increased heterogeneity of the analyzed sample.Electrophoregrams showed the presence of 27 ZnBP bands from below 14 kDa to over 94 kDa with those between 14 and 20 kDa predominating (Fig. 3).
The polypeptide map (2D PAGE) (Fig. 4) showed that the ZnBP of boar seminal plasma consisted of 148 polypeptides, of which most had the isoelectric point (pI) within the pH 6 to 10 range.Only few of them were acidic (pI = 3 to 6).The molecular msses of the analyzed polypeptides ranged from below 14 to above 94 kDa.
Significant differences (P < 0.05) were found between the percentage of spermatozoa with linear motility after 1 h incubation in 4 °C between the control without ZnBP and treated samples with ZnBP.The proportion of spermatozoa with damaged acrosome were markedly lower (P < 0.05) in the treated samples (Table 1).
Even though there were no motile spermatozoa in the treated samples the proportions of spermatozoa with damaged acrosome were lower (P ≤ 0.05) after 24 h incubation in 4 °C in treated samples with ZnBP (Table 1).
After 1 and 24 h incubation in 4 °C the percentage of spermatozoa with intact membranes (CFDA/PI stained) was higher (P < 0.05) in treated samples compared with control (Table 2).

DISCUSSION
We isolated zinc-binding proteins from boar seminal plasma (ZnBP).Our results indicate that ZnBP in the native state form high-molecular mass aggregates (150 kDa and over 226 kDa) which dissociate under the influence of denaturing and reducing conditions.These findings are in agreement with other authors' research, which showed that the processes of association and dissociation of native forms of seminal plasma proteins occur under the influence of different factors, including the presence of specific ligands and changes in the pH of the solution (Jelinkova et al., 2004;Jonakova & Ticha, 2004).Siciliano et al. (2008) found in boar seminal plasma high molecular mass prostasome-like vesicles which stimulated acrosome reaction.These structures under denaturating conditions showed molecular masses of 60 and 90 kDa.
Proteins of bull seminal plasma in their native state have a wide range of molecular mass from 5 to 500 kDa (Shannon et al., 1987;Hameed et al., 1991).These are mainly high-molecular aggregates, which under the influence of low pH and in the presence of citrate are dissociated to low-molecular mass components (Al-Somai et al., 1994).In the human seminal plasma protein complexes with mass over 660 kDa and approx.250 kDa were identified (Vivacqua et al., 2004).
We used two-dimensional electrophoresis (2D PAGE) to generate a peptide map of ZnBP isolated from boar seminal plasma.This method allows detailed identification of seminal plasma proteins participating in the fertilization process.This system has been used before to characterize seminal plasma proteins from different   mammalian species, involved in the regulation of fertility and suitability of semen for cryopreservation (Brandon et al., 1999;Flowers et al., 2001;Killian et al., 1993;Jobim et al., 2004).
In this study all polypeptides of analyzed fractions under denaturing conditions (SDS/PAGE; 2D PAGE) mass from below 14 kDa to over 94 kDa.The highest percentage of polypeptides was found within the range of molecular weights from 10 to 20 kDa and a basic pI, which corresponded with molecular mass of zinc-binding peptides identified in human seminal fluid (Rehault et al., 2002;Fung et al., 2004).Analysis by 2D revealed their molecular mass < 30 kDa and basic pI.These peptides were identified as truncated forms of semenogelins I and II.In physiological state they occur after prostate-specific antigen (PSA) action.This process is regulated by zinc ions (Malm et al., 2000;Jonsson et al., 2005).It is interesting that these peptides displayed antibacterial activity, while the holoproteins did not (Edstrom et al., 2008).Additionally, a direct MALDI-TOF-MS analysis of unfractionated human seminal fluid revealed the presence of 0.8-7.3-kDapeptides, the majority of which were found to be derived from SgI (Fung et al., 2004).The same method was used to detect seminal basic protein, also a fragment of Sg I, that is belived to inhibit sperm motility (Robert & Gagnon, 1999).A 19-kDa protein, probably derived from Sg processing, is present at the periphery of detergent-treated and sonication-resistant human sperm nuclei (Zalensky et al., 1993).
The low molecular mass polypeptides found on the 2D gels suggest the presence of spermadhesins in the analyzed fraction.Spermadhesins are synthesized by accessory sex glands of the male reproductive system and are deposited on the sperm surface during ejaculation (Sanz et al., 1992).According to Calvete et al. (1997) spermadhesins account for approx.90 % of the total protein in seminal plasma.In turn, two main spermadhesins in boar, i.e.PSP-I and PSP-II, represent over 50 % of the total protein content of the seminal plasma (Rutherfurd et al., 1992).
The structure of spermadhesins is based on a combination of several modules, of which each contains a CUB fragment.It is a conserved domain containing in its structure four cysteine residues, between which two disulfide bridges are formed (Bork & Beckman, 1993).It is a commonly known fact that bonds are formed between zinc ions and cysteine residues (Bjorndahl et al., 1990).Presumably this type of interaction may be the way of binding of zinc ions with spermadhesins containing CUB fragments.
Apart from the above-mentioned binding mechanism of zinc ions with proteins, Zn 2+ affinity for phospholipids and lipoproteins also should be taken into account.Interactions of Zn 2+ ions with phosphate groups and double bonds of unsaturated fatty acids are also possible (Bettger & O'Dell, 1981).Moreover, zinc ions and proteins binding them may also bind heparin.This type of interaction is well known for the spermadhesin family (Björnadhl 1986;Hołody & Strzeżek, 1999).
In boar spermatozoa, coating of the sperm membranes by plasma proteins may stabilize its structure (de Leeuw et al., 1990).Seminal plasma from good freezer boars has a positive effect on cryopreserved sperm function, among other things by enhancing their resistance to cold shock (Hernandez et al., 2007).
Heparin-binding proteins (HBP) and the PSP-I/PSP-II heterodimer have an antagonistic effect on the viability, motility and mitochondrial activity of diluted spermatozoa incubated at 38 °C for 5 h.The HBP have a negative effect while PSP-I/PSP-II a positive one (Centurion et al., 2003).
It has been found that an addition of PSP-I/PSP-II to highly diluted (1 × 10 6 cells/ml) spermatozoa preserves their viability, motility and mitochondrial activity for at least 10 h (Caballero et al., 2006).It is of interest that Zn 2+ ions participate in the stabilization of the PSP-I/ PSP-II structure, while low pH destabilizes this heterodimer (Campanero-Rhodes et al., 2005;Rodriguez-Martinez et al., 2005).The positive effect on some spermatozoa parameters achieved in our study may be partly caused by the PSPI/PSPII heterodimer present among the zinc-binding proteins.
Our investigations made it possible to state that the analyzed fraction has an advantageous effect on the percentage of cells exhibiting linear movement important for sperm quality.Fluorescence studies showed a stabilizing effect of the zinc-binding proteins on the plasma membranes of spermatozoa incubated at a cold shock temperature (4 °C), and morphological analyses confirmed a positive effect of these proteins on acrosome.
It is possible that the zinc-binding fraction isolated from boar seminal plasma contains some albumin (known as a zinc-binding protein), and a part of the positive results obtained in our experiment could be due to its action.Moreover, albumin has been shown to elimi- nate free radicals generated by oxidative stress, enhance sperm motility and protect sperm plasma membrane integrity against cold shock during freezing-thawing of semen (Uysal et al., 2005;Matsuoka et al., 2006).
Our results indicate that proteins binding Zn 2+ ions in boar seminal plasma can presumably protect the sperm plasma membrane against cold shock and stabilize spermatozoal acrosome, which may be used in semen preservation for this species.This pertains particularly to the protection of spermatozoa at stages of high semen dilution, which may be used in modern biotechnological methods such as cell sorting and freezing.

Table 1 . Percentage of spermatozoa exhibiting linear movement and spermatozoa with damaged acrosome
Time 0, after semen dilution and after incubation at 4 °C for 1 and 24 h.Control, spermatozoa incubated with PBS; Treatment -spermatozoa incubated with zinc-binding proteins (ZnBP) from boar seminal plasma.n = 10.Values denoted with letters a, b differ statistically significantly at P ≤ 0.05.

Table 2 . Percentage of spermatozoa with intact plasmalemma (CFDA/PI staining).
Time 0, after semen dilution and after incubation at 4 °C for 1 and 24 h.Control, spermatozoa stored after dilution in PBS; Treatment, spermatozoa stored with zinc-binding proteins (ZnBP) isolated from boar seminal plasma.n = 10.Values denoted with letters a, b differ statistically significantly at P ≤ 0.05.