Comparative Studies on the Aggregation Behavior ...

Protein & Peptide Letters, 2008, 15, 000-000

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Comparative Studies on the Aggregation Behavior of HBPs from Human Seminal Plasma by Dynamic Light Scattering Vijay Kumar1, Md. Imtaiyaz Hassan1,2, Tej P. Singh1 and Savita Yadav1,* 1

Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India, 2Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India Abstract: Heparin binding proteins (HBPs) from human seminal plasma (HSP) were obtained by heparin affinity and size exclusion chromatography. The aggregation/disaggregation of HBPs was followed by dynamic light scattering (DLS) in presence of various physiological ligands such as CaCl2, NaCl, EDTA, cholesterol, adenosine, D-glucose and D-fructose. The aggregation pattern of lactoferrin was also analyzed and compared. The study of interactions of HBPs of HSP may contribute to an understanding mechanisms underlying the fertilization process.

Keywords: protein-protein aggregation, chromatography, dynamic light scattering, HBPs, HSP, sperm capacitation INTRODUCTION HSP is a complex mixture of secretions originating from male accessory sex glands. Seminal plasma proteins (SPPs) are bound to sperm surface during ejaculation and forming a protein coat around spermatozoa, which is essential for fertilization [1-3]. SPPs interact among themselves as well as with different types of glycoconjugates and membrane phospholipids to form aggregates, which play an important role in individual steps of the fertilization processes. These protein aggregates are of different molecular mass, composition and binding properties. The existence of aggregated forms of proteins in seminal plasma is known only in some species [4,5]. Interestingly, most of these proteins tend to show aggregations in boar are heparin binding proteins (HBPs) [6]. Many studies have been documented for the potential physiological role of HBPs [7]. Sperm capacitation is a multi-step process and involves several biochemical and ultra structural changes in the sperm membrane, resulting in the modification of membrane lipid composition and increased permeability of ions and efflux of the membrane cholesterol leads to the sperm capacitation [8]. Sperm capacitation can be inhibited or the sperm even decapacitated by adding seminal plasma or lipid vesicles composed of synthetic phospholipids liposomes containing cholesterol to the sperm, as these prevents the loss of cholesterol from the membrane [9]. Interestingly, when cholesterol was added to the incubation medium, sperm from several species were inhibited from undergoing the acrosome reaction [10,11]. Albumin, highdensity lipoproteins (HDLs), follicular and oviductal fluids are effectors of capacitation in human and bovine spermatozoa [12]. The other agent for sperm capacitation is adenosine, which is present in micro molar concentration and stimulate capacitation and fertilizing ability in uncapacitated mouse sperm while in capacitated cells they inhibit *Address correspondence to this author at the Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, Índia; Tel: +91 11 26593201; Fax: +91 11 26588663; E-mail: [email protected] 0929-8665/08 $55.00+.00

spontaneous acrosome loss [13]. The effect of D-glucose on capacitation varies between species [14,15]. D-fructose produced mainly by the seminal vesicles, which is essential for spermatozoal metabolism and motility as an energy source. A little correlation between seminal D-fructose concentrations and seminal activity was reported by Matschalut et al. [16]. Biswas et al. [17] reported that a decrease in D-fructose concentrations leads to the increase in sperm motility. Although, many proteins from HSP have been studied separately in detail, however a little attention has been paid to their physiological forms in seminal fluid. In the present article, we have studied the response of various constituents of seminal plasma like D-fructose, D-glucose, cholesterol and adenosine in the context of aggregation and disaggregation of HBPs. These proteins have been isolated by aid of affinity chromatography on Heparin-Sepharose followed by size-exclusion chromatography. The hydrodynamic radii and approximate relative molecular mass (Mr) of these proteins in solution were measured by DLS to observe aggregation/dis-aggregation. The present studies were undertaken to understand the aggregation process of HBPs, which might shed some light on capacitation and acrosome reaction and thus fertilization. MATERIALS AND METHODS Sample and Reagents Human semen samples were obtained from Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi. Cocktail of protease inhibitor (Sigma, USA) was added to samples immediately after the collection. The normal ejaculates were pooled and centrifuged at 1500g for 30 min at 4 °C to remove spermatozoa. The seminal plasma was further clarified by centrifugation at 10,000g for 15 minutes and supernatant was stored at -20° C until next use. All reagents used were of analytical grade. D-glucose, D-fructose, adenosine and cholesterol were purchased from Sisco Research Laboratories Pvt. Ltd., Mumbai, India.

© 2008 Bentham Science Publishers Ltd.

2 Protein & Peptide Letters, 2008, Vol. 15, No. 6

Affinity Chromatography on Heparin-Sepharose Heparin-sepharose CL-6B (GE-Healthcare, Uppsala, Sweden) was packed in a column (2.620cm), which was pre-equilibrated with phosphate buffered saline (PBS: 150mM NaCl in 20mM phosphate, pH 7.4 buffer). About 50mg of lyophilized seminal plasma powder was dissolved in 50mL of PBS and loaded on heparin-sepharose column. The column was washed with PBS to remove unbound fraction completely. HBPs were obtained by eluting the proteins with 0.5 M NaCl and measured at 280 nm to analyze the protein content of eluent. The protein containing fractions were pooled, concentrated and desalted using ultra-filtration (Millipore, USA), lyophilized and used for all the further analysis. Size Exclusion Chromatography Lyophilized HBP fraction (20mg) was dissolved in 50mM Tris pH 8.0 (1.0 mL) and loaded on Sephadex G-100 column (Sigma, USA) (1.6125cm), pre-equilibrated with 150 mM NaCl in 50mM Tris pH 8.0. Fractions eluted at the flow rate of 6 mL/hour were measured at 280 nm. Protein peaks were pooled and concentrated by ultra-filtration using 3 kDa membrane (Millipore, USA). The experiment was performed under the same conditions in presence of physiological concentrations of D-glucose (1mM) and Dfructose (0.04mM) also. Gel Electrophoresis Samples were prepared under reducing conditions according to Lamelli [18], separated on 10% Sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The Mr of the proteins separated was estimated by using protein standards run in parallel. Reference molecular markers: Phosphorylase b (M 97, 000 Da), Bovine serum albumin (M 66,000 Da), Ovalbumin (M 45,000 Da), Carbonic anhydrase (M 29,500 Da), Soybean trypsin inhibitor (M 21,500 Da), Hen egg white lysozyme (M 14,400 Da) and Aprotinin (M 6,500 Da). Dynamic Light Scattering Studies (DLS) HBPs under various pH, salt, sugars and ligands were analyzed by DLS measurements at 20°C. All reagents were filtered using 0.22 filters (Millipore). A protein concentration of 3 mg/mL was used. Equal volumes of samples were adjusted to a range of pH from 1.5 to 11.0 by mixing it with buffer (20mM phosphate buffer). Effects of various agents were also analyzed: (a) NaCl (0.2M, 0.3M and 0.5M), (b) EDTA (1mM, 2mM and 5mM) and (c) CaCl2 (1mM, 2mM and 5mM). Effect of major seminal plasma components under physiological conditions and beyond, like D-fructose (2%, 4%, and 6%), D-glucose (2%, 4% and 6%), Adenosine (1mM, 2mM and 5mM) and Cholesterol (1mM, 2mM and 5mM) were also studied. A series of measurements with a sampling time of 30 s and a wait time of 1 s was conducted using a Spectroscatter 201 (RiNA). A diode laser of wavelength 659 nm was used as the source. The scattered light was collected at a fixed angle of 90°. The autocorrelation functions were analyzed with the program CONTIN to obtain hydrodynamic radius (RH) distributions. The RH is related to the diffusion coefficient by the Einstein-Stokes equa-

Kumar et al.

tion. The data were analyzed using PMgr v3.01p17 software supplied with the instrument. All the DLS experiments were repeated ten times and average value were calculated with standard error (Table 1). We have used the theory of DLS as given by Murphy in 1997 [19]. RESULTS Affinity Chromatography on Heparin -Sepharose Affinity chromatography was used to separate HBPs and non-HBPs of HSP. All adsorbed proteins were eluted with 1M NaCl (w/v). Gel Filtration Chromatography Size exclusion chromatography was used to show the tendency of HBPs of HSP to associate specifically in higher molecular mass aggregates. Fractions containing HBPs when separated on Sephadex G-100, eluted in void volume of the column, suggesting that these proteins forms a high molecular mass aggregate (Fig. 1A, peak 1). When HBPs incubated with 5mM D-glucose, it aggregates but to a less extent as compared to HBPs alone which is clearly indicated by the peak shift towards elution volume (Fig. 1A, peak 2). In presence of 5mM D-fructose, HBPs separates into two fractions as (i) eluted in the void volume corresponds to higher molecular mass aggregate (Fig. 1A, peak 3A) and (ii). eluted at 100mL of elution volume which correspond to low molecular mass aggregate of 50 kDa (Fig. 1A, peak 3B). Gel Electrophoresis SDS PAGE analysis showed that HBPs fractions are heterogeneous and comprised of a broad range of polypeptides in the range of 97-10 kDa (Fig. 1B, lane I). Similarly, the gel filtration eluent in presence of D-glucose showed a broad range of polypeptides of molecular mass of 97-10 kDa, but has been greatly resolved (Fig. 1B. lane II). Gel filtration in presence of D-fructose yields two peaks. Peak I fraction comprised of high molecular mass proteins in the range of 97-30 kDa (Fig. 1B, lane IV) while peak II comprised of low molecular weight proteins in the range of 3010 kDa (Fig. 1B, lane III). Dynamic Light Scattering Analysis In addition to SEC experiments, DLS was also performed. Under physiological conditions, aggregated forms of HBPs strongly predominated over the monomeric forms in HSP. Table 1 lists the value of hydrodynamic radii of HBPs under various conditions with respective standard error. Dynamic light scattering analysis was carried out by inserting the modified values for refractive index and viscosity in the instrument software in case of various salts and cholesterol. Effect of pH To determine the behavior of HBPs in solution, we performed DLS experiments at a wide range of pH from 1.5 to 11.0. The hydrodynamic radii, R H increases from 18.8 nm to 23.6nm as the pH changes from 1.5 to 7.0. While after 7.0, it starts decreasing from 23.6 nm to 21.5

Aggregation Studies of HBPs

Table 1.

Protein & Peptide Letters, 2008, Vol. 15, No. 6

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Hydrodynamic Radii of HBPs in Different Conditions Condition

Range

Hydrodynamic radii (nm)

Standard error

pH

1.5

18.18

(0.10)

3.0

20.01

(0.12)

5.0

20.36

(0.11)

7.0

23.59

(0.15)

9.0

22.01

(0.14)

11.0

21.50

(0.11)

100

10.77

(0.11)

200

10.28

(0.11)

500

7.58

(0.15)

1.0

8.95

(0.11)

2.0

8.71

(0.10)

5.0

6.11

(0.14)

1.0

5.56

(0.15)

2.0

4.08

(0.11)

5.0

3.90

(0.20)

0.0

23.59

(0.12)

1.0

20.56

(0.14)

2.0

18.51

(0.15)

5.0

14.70

(0.17)

0.0

23.59

(0.11)

0.02

18.72

(0.16)

0.04

14.63

(0.12)

0.06

11.03

(0.11)

0.0

23.59

(0.11)

0.02

27.96

(0.10)

0.04

31.23

(0.15)

0.06

35.03

(0.16)

0.0

23.59

(0.20)

0.02

18.55

(0.12)

0.04

14.35

(0.14)

0.06

8.12

(0.12)

NaCl (mM)

CaCl2 (mM)

EDTA (mM)

D-glucose (mM)

D-fructose (mM)

Cholesterol (mM)

Adenosine (mM)

nm as pH increases to 11.0. However, the increase or decrease in hydrodynamic radii is not significant (Fig. 2). Effect of NaCl, CaCl2 and EDTA We selected a wide range of concentrations of NaCl, CaCl2 and EDTA to see the effect on hydrodynamic radii (R H) of HBPs. These reagents showed dissociating effect

on HBPs (Fig. 3). The measured diffusion coefficient (D) is higher in presence of NaCl, corresponding to smaller hydrodynamic radii (R H). The hydrodynamic radii decreases 3 fold as we increase the concentration to 500mM NaCl. Similarly, in case of CaCl2, the R H decreases 4 fold as the concentration is increased to 5mM. In presence of 5mM EDTA there is a major 6 fold decrease in R H value.


Kumar et al.

Figure 3: Effect of Salts on HBPs. The Hydrodynamic radii was measured in presence of increasing concentration of NaCl (•), CaCl2 () and EDTA () by DLS. The Hydrodynamic radii (Y axis) plotted as a function of pH (X axis).

The molecular mass, as estimated from the hydrodynamic radius, is an average value, and influenced by the heterogeneity of the sample. Figure 1: (A). HBPs were concentrated to 50 mg/ml. One ml of protein was loaded on sephadex G-100 that was previously equilibrated with 50 mM Tris-HCl buffer (pH 8.0) and 0.15 M NaCl. Loaded protein was eluted at a flow rate of 6ml/hr. 2.0 ml of fraction was collected and analyzed by SDS-PAGE. Peak 1: HBPs alone; Peak 2: HBPs in the presence of 1mM D-glucose; Peak 3A and Peak 3B: HBPs in the presence of 0.04 mM D-fructose. (B). SDSPAGE: M. Marker [97 kDa Phosphorylase b, 66 kDa Bovine serum albumin, 45 kDa Ovalbumin, 30 kDa Carbonic anhydrase, 20 kDa Soyabean trypsin inhibitor, 14 kDa Hen egg white lysozyme and 6 kDa Aprotonin from top to bottom]. Lane I: 1.0 M NaCl elution from Heparin Sepharose; Lane II: Gel filtration eluent in presence of D-glucose; Lane III: Peak 3B of gel filtration eluent in presence of D-fructose and Lane IV: Peak 3A of gel filtration eluent in presence of D-fructose.

Effect of Cholesterol, Adenosine, D-Glucose and DFructose We investigated the effect of major seminal plasma constituents on oligomeric status of HBPs. HBPs respond differently to some extent to both the saccharides. In presence of D-glucose, R H decreases to 14.7 nm (1.7 fold) as we increase the concentration to 5mM. However, Dfructose at 0.06 mM causes dissociation of HBPs to a large extent than D-glucose as shown by a decrease in R H to 11.0 nm (2 fold). Similarly, in the presence of adenosine the hydrodynamic radii of HBPs decrease to 8.1 nm (3 fold) at 0.06 mM. On the contrary, in the absence of cholesterol, the RH is 23.6 nm that increases to 35.0 nm (1.5 fold) as the concentration increased to 0.06 mM (Fig. 4). Effect of pH, Cholesterol, Adenosine, D-Glucose and DFructose on Lactoferrin

Figure 2: Effect of pH on HBPs. The Hydrodynamic radii were measured at different pH by DLS. The Hydrodynamic radii (Y axis) plotted as a function of pH (X axis).

Seminal lactoferrin is one of the heparin binding proteins which were also studied to know the effect of these agents on the individual HBP. It was purified as described [20] which (Mw 80 kDa) in solution under physiological condition was shown to exist in pentameric to octameric state (400 kDa 600 kDa) as evident by its hydrodynamic radii, and did not show any significant change in hydrodynamic radii from pH 4.0 to 9.0 (Fig. 5A). It is interesting to note that, hydrodynamic radii of isolated lactoferrin did not change to a large extent with addition of adenosine (Fig. 5B), D-glucose (Fig. 5C) and D-fructose (Fig. 5D). However, in presence of cholesterol, hydrodynamic radii increase to 3 fold than native isolated lactoferrin (Fig. 5E) and aggregate to a larger extent (900kDa-1.0MDa). Autocorelation function of purified lactoferrin sample with a homogeneous molecular mass


distribution is illustrated in Fig. 6A. The intensity distribution of purified lactoferrin under physiological conditions in solution is shown in Fig. 6B.

Figure 4: Effect of different agents on aggregation of HBPs. The Hydrodynamic radii was measured in presence of increasing concentration of cholesterol (•), D-glucose (), D-fructose () and adenosine () by DLS. The Hydrodynamic radii (Y axis) plotted as a function of pH (X axis).

DISCUSSION SPPs are known to interact with various types of ligands, such as saccharide moiety of glycoproteins, polysaccharides of glycosaminoglycan, phospholipids, lipoproteins, collagen,


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protease inhibitors, sperm surface, and zona pellucida [12,21-24]. Specific interactions between SPPs lead to the formation of higher-molecular-mass aggregates. To study the mutual interactions between HBPs of HSP, we have used affinity-chromatography on Heparin-Sepharose, sizeexclusion chromatography and DLS. Affinity chromatography on Heparin-Sepharose column was used to separate human SPPs into heparin-binding fraction (H+) and nonheparin binding fraction (H-). Our result showed that human HBPs eluted at 0.5 M NaCl elution are in aggregated form which was confirmed by size-exclusion chromatography and DLS. Seminal HBPs was eluted as a high molecular mass fraction in gel-filtration, which could be due to their mutual interactions to form aggregates. Heparin and high-density lipoproteins stimulates sperm capacitation in the female reproductive tract, although very little information is available which deals with the exact mechanism of sperm capacitation. This is the process by which a spermatozoon becomes capable of fertilizing an ovum after it reaches the ampullary portion of the uterine tube [21,25]. Light scattering studies was used to determine the protein aggregation, polydispersity/heterodispersity, which is the inherent property of proteins in the solution. HBPs showed wide intensity distribution, which suggested the presence of aggregates with many different molecular masses. For the interpretation of DLS data, it is always necessary to consider that the larger molecules produce a stronger signal than smaller ones. The intensity distribution function must be further transformed in order to get the distribution based on mass. The transformation was performed by the following equation: Mw = kRH

Figure 5: Effect of pH (A), adenosine (B), D-glucose (C), D-fructose (D), and cholesterol (E) on aggregation of purified lactoferrin through the use of Cumulant analysis by DLS. The Hydrodynamic radii (Y axis) plotted as a function of % relative width(X axis).


Figure 6: Autocorrelation function (a) and intensity distribution (b) of purified lactoferrin under physiological conditions.

Where Mw is the molecular mass, k is empirical factor, RH is the hydrodynamic radius and is an exponent dependent on the molecular type: here value of is considered as 3 for globular proteins. The correlation function and intensity distribution of HBPs are shown in Fig. 7, which is a heterogeneous sample containing a population of molecules with a large molecular mass distribution. Due to heterogeneity of analyzed samples we could barely determine the perfect correlation function for calculating absolute RH values. For this reason, the analyzed samples were only qualitatively compared for the aggregation and disaggregation from the several size distributions.

Figure 7: Autocorrelation function (a) and intensity distribution (b) of purified HBPs under physiological conditions.

The studies on dissociation of aggregates dependent on a varied pH range from acidic (1.5) to alkaline (11.0), suggest that the protein-protein interaction is very strong leading to stable size of protein aggregates. The aggregates of HBPs in HSP shows dissociation in presence of salts like NaCl, CaCl2, and EDTA. Increasing concentration of salt causes rapid dissolution of aggregates as evident by the decrease in hydrodynamic radii, which can be explained by change in repulsive electrostatic forces by the salt [26,27]. The sugar components of seminal plasma like Dglucose and D-fructose influences the aggregation state of HBPs. The capacitation of mammalian spermatozoa

Kumar et al.

requires the consumption of significant amount of energy, which is species-specific [28]. D-glucose is the major energy source needed in maintaining in vitro capacitation in mice and human spermatozoa because this sugar induces much penetration-rates and capacitation-like changes than other monosaccharide like D-fructose and mannose [29]. However, D-fructose causes the dissociation of high molecular mass protein aggregates giving rise to formation of lower molecular mass protein aggregates. In our case, Dglucose decreased the aggregation of HBPs, but to a less extent than D-fructose, as evident by less decrease in hydrodynamic radii. From these results it can be suggested that D-glucose under physiological concentration (1mM to 5mM) supports sperm capacitation that is essential for fertilization. On the other hand D-fructose (0.05mM) is required for sperm motility and sperm-oocyte fusion [30,31]. The decrease in aggregation of HBPs in presence of D-fructose may cause increase in sperm motility. Interestingly, these sugars therefore have dissimilar effect on aggregation of HBPs in human, indicating human spermatozoa must have a highly developed metabolic system that allows them to perform specific functionality of sperm. We observed the changes in hydrodynamic radii of aggregates by DLS to investigate further the role played by adenosine and cholesterol on HBPs. It is evident from earlier reports which entails the capacitation and increased motility are related to an increase of protein tyrosine phosphorylation [32,33], which is modulated by adenosine [34]. Our in-vitro results shows that adenosine decreases the hydrodynamic radii of HBPs as their concentration increases resulting in disaggregation of HBPs and this decrease might have some effects on signal transduction pathways leading to tyrosine phosphorylation and thus capacitation. The rate at which a particular sperm initiates capacitation and the acrosome reaction depends largely on the cell membrane status and in particular on the amount of cholesterol present in the plasma membrane [35-37]. During capacitation, cholesterol is lost from the sperm plasma membrane, and when sufficient cholesterol is removed, the membrane becomes unstable, enhancing its ability to fuse with the outer acrosomal membrane, resulting in acrosomal reaction. Many studies have shown that albumin [38,39] and highdensity lipoprotein (HDL) in the female genital tract facilitate the efflux of cholesterol that occurs during the early step of capacitation [40,41]. The sperm coated HBPs in bovine seminal plasma interact with HDL in the female genital tract and mediate the exchange of cholesterol and phospholipids between the sperm membrane and HDL. This may result in decrease in cholesterol/phospholipids ratio, leading to capacitation [42]. We have observed that the presence of cholesterol in increasing concentrations associates the HBPs of HSP, as evident by increase in hydrodynamic radii of HBPs. This aggregation may be responsible for weakening the interaction of HBPs with HDL and may decrease the efflux of cholesterol from the membrane and thus inhibit capacitation. The ability of the aggregates of HBPs to interact with cholesterol might be significant in the process of capacitation of human sperm. These aggregates may become acceptors of cholesterol molecules released from the sperm membrane during the process of cholesterol efflux. Recently, the presence of hydrophobic cavities in the aggregates from bovine SPPs, in



which cholesterol could be trapped, has been described [43]. Thus cholesterol could be considered as a stabilizing agent for membranes, its mere presence in the medium inhibits capacitation and acrosome reaction. Lactoferrin is a major heparin-binding protein in HSP [44]. Under physiological conditions the purified Lactoferrin that is also a HBP form aggregates from 400 kDa to 600 kDa. The composition and size of purified homogenous preparation of lactoferrin were stable and not influenced by change in pH from 4.0 to 9.0 or by increasing concentration of D-glucose, D-fructose and adenosine. Interestingly, the components of HBPs alone (lactoferrin) do not show any significant changes upon subsequent addition of physiological ligands like D-glucose, D-fructose and adenosine. From these results it may be suggested that aggregation of HBPs in HSP accompanied by its molecular environment, could be a possible mechanism for the capacitation, mediated by SPPs. In conclusion, our results show that HSP HBPs are characterized by a great tendency to form differently aggregated forms. Mutual specific interaction and hydrophobic nature of these proteins participates in this process. Various compounds, including low molecular mass substances present in the environment of seminal plasma influence the aggregation state of these proteins and thus play an important role in the fertilization process. Aggregation of HSP HBPs is probably an important phenomenon in the fertilization process. In vivo studies on the basis of these in-vitro results will help us to clarify some of the still enigmatic aspects of human fertilization. Further in vivo studies are required to know the role played by aggregation of HBPs on capacitation and hence in fertilization. Altogether, these data help to understand the complex behavior of HBPs in solution and may correlate with the diversity of biological properties of these proteins.

We greatly appreciate Dr. Sarman Singh for arranging to provide human seminal sample. This work is supported by grants from Department of Biotechnology (DBT), Government of India. Kumar V and Hassan MI are thankful to Council of Scientific and Industrial Research (CSIR) for fellowship. ABBREVIATIONS Heparin Binding Protein

DLS =

Dynamic Light Scattering

HSP =

Human seminal Plasma

SPP =

Seminal plasma proteins

[3] [4]

[8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29]

[31] [32] [33] [34] [35] [36]

[39] [40] [41]

Evans, J. P. and Kopf, G. S. (1998), Andrologia, 30, 297-307. Topfer-Petersen, E., Calvete, J. J., Sanz, L. and Sinowatz, F. (1995), Andrologia, 27, 303-24. Topfer-Petersen, E. (1999), J Exp Zool, 285, 259-66. Manjunath, P. and Therien, I. (2002), J Reprod Immunol, 53, 10919.

Received: November 13, 2007

[7]

[37] [38]

REFERENCES [1] [2]

[6]

[30]

ACKNOWLEDGEMENT

HBP =

[5]

Revised: February 24, 2008

Accepted: February 26, 2008

[42] [43] [44]

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