Sperm membrane protein profiles of fertile and infertile men: identification and characterization of fertility-associated sperm antigen

S.K. Rajeev1 and K.V.R. Reddy1,2

1 Division of Immunology, National Institute for Research in Reproductive Health, Indian Council of Medical Research, Mumbai, India

2 To whom correspondence should be addressed at: Division of Immunology, National Institute for Research in Reproductive Health, Indian Council of Medical Research, J.M. Street, Parel, Mumbai 400012, India. e-mail: shrichi{at}rediffmail.com


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Male infertility is the major cause of conception failure in about 25% of all infertile couples. Understanding the causes of male infertility depends to a certain extent on the proteins present on the spermatozoa. The study aim was to investigate first, whether there is any difference in the expression of sperm membrane proteins between fertile and infertile males; and second, whether there is any functional significance of these proteins in the spermatozoa. METHODS: Six different protocols were employed to extract sperm membrane proteins. A 57 kDa protein was identified and purified using different chromatographic techniques. The homogeneity and isoelectric point of the protein was confirmed by 2D-electrophoresis. The protein was characterized by immunofluorescence, ELISA, flow cytometry, SDS–PAGE and Western blot analysis. The role of 57 kDa protein in sperm–oocyte binding was studied in vitro. RESULTS: All six sperm extracts of normozoospermic and infertile subjects showed 16–18 major and 12–15 minor protein bands. However, in one of the methods, the lysis buffer containing N-octyl-{beta}-D-glycopyranoside (NOG) resulted in an additional protein band at the 57 kDa region in 95% of normozoospermic samples. The protein was either absent (~80%) or negligible (~20%) in infertile subjects. The protein was localized to the head of non-acrosome-reacted spermatozoa (NAR), and shifted to the equatorial segment in acrosome-reacted (AR) spermatozoa. The antibody directed against purified 57 kDa protein inhibited binding of human sperm to zona-free hamster oocytes in a dose-dependent manner. CONCLUSIONS: The results suggest that lack and/or low expression of 57 kDa protein may be one of the reasons for infertility in men. Therefore, the protein could be used as a marker for sperm quality in men.

Key words: acrosome/fertile/infertile/in-vitro sperm–oocyte binding/spermatozoa


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although infertility or subfertility (the first term is used to cover both conditions) is difficult to define and affects men and women worldwide, estimates of its prevalence are not very accurate and vary from region to region. It is estimated that 15% of couples are infertile, and that infertility is distributed evenly between men and women (Dallapiccola and Novelli, 2000Go; Nieschlag and Leifke, 2000Go). In 30% of cases the problem is attributed to the male partner, and of these approximately 50% are due to abnormal spermatogenesis which results in oligospermia and asthenospermia (Diekman and Goldberg, 1994Go; De Kretser and Baker, 1999Go). The sperm plasma membrane plays a critical role in sperm–oocyte recognition, adhesion and fertilization (O’Rand et al., 1979Go). It has been reported that loss of integrity of the sperm plasma membrane is frequently associated with infertility in men, despite normal semen parameters (Wassarman, 1990Go; Batova et al., 1998Go). Although many investigations have been carried out on fertilization, the sperm membrane proteins which are directly or indirectly involved in this process are poorly understood. A detailed knowledge on the sperm surface molecules would be useful in understanding some of the factors associated with male infertility.

Several studies have reported on the identification of sperm membrane proteins as potential receptors for zona binding. The known molecules include tyrosine kinase receptor (Burks et al., 1995Go), protein kinases (Roten et al., 1990Go), galactosyl transferase (Cardullo and Wolf, 1992Go), adhesins (Sanz et al., 1992Go), integrins (Klentzeris et al., 1995Go; Glander et al., 1996Go; Reddy et al., 1998Go; Rajeev and Reddy, 2000Go), extracellular matrix proteins such as vitronectin, fibronectin and laminin (Glander et al., 1996Go; Wennemuth et al., 2001Go), PH-20, PH-30 (Primakoff et al., 1987Go; Blobel et al., 1992Go), G proteins (Kopf, 1989Go) and proto-oncogenes such as c-kit, c-fos, c-myc and c-ros (Wolfes et al., 1989Go; Naz et al., 1991Go, 1992Go; Kodaira et al., 1996Go). The clinical relevance of many of these proteins remains either elusive or at least controversial (Wolf et al., 1992Go; Diekman et al., 2000Go). Therefore, the objectives of the present study were to: (i) compare differences in the sperm membrane proteins between fertile and infertile males; and (ii) identify, purify and characterize novel sperm membrane proteins (if any) and to evaluate their role in male infertility.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
The study was approved by the Institutional Ethics Committee of National Institute for Research in Reproductive Health, Mumbai. Handling of animals (hamsters, mice and rabbits) and experimental procedures were performed according to the guidelines for care and use of laboratory animals.

Extraction of sperm membrane proteins
Semen samples from men [normozoospermic, in-vitro fertilization failure (IVF-F), oligozoospermic, asthenozoospermic and oligoasthenozoospermic] undergoing evaluation for infertility at King Edward Memorial Hospital, Wadia Infertility Clinic and Fertility and IVF Centre, Mumbai, were collected for the study. Ejaculates were obtained by masturbation after a 3-day period of sexual abstinence. On liquefaction (37°C, 30–45 min), sperm count, motility (progressive and forward progressive), morphology and viability were evaluated as per World Health Organization guidelines (WHO, 1992). Those samples with a ratio of spermatozoa to leukocytes >100:1, viscous ejaculates and samples with a positive immunobead test (IBT) test (>10% adherent spermatozoa) were excluded.

Preparation of spermatozoa
Spermatozoa were separated from seminal plasma using a discontinuous Percoll gradient (McClure et al., 1989Go). Briefly, 1.5 ml 40% Percoll solution was layered over 1.5 ml 80% Percoll solution in a conical falcon tube. An aliquot (1 ml) of seminal fluid was layered onto the gradient and centrifuged at 600 g for 20 min. The recovery of spermatozoa was >85%. Spermatozoa were washed in physiological saline (0.9% NaCl) by centrifugation at 600 g for 10 min and used for further studies.

Induction of the acrosome reaction
Capacitation was induced by incubating motile sperm suspensions for 18 h at room temperature in Biggers–Whitten–Whittingham (BWW) medium supplemented with 1% bovine serum albumin (BSA) (Auer et al., 2000Go). The acrosome reaction was induced by incubating with the calcium ionophore A23187 (CaI-A23187; Sigma, St Louis, MO, USA). CaI-A23187 was prepared as 5 mmol/l stock in dimethyl sulphoxide (DMSO). Aliquots (100 µl) were frozen at –80°C; subsequently, 5 µl of this was used to challenge 245 µl of sperm suspension (108 spermatozoa), giving a final CaI concentration of 10 µmol/l. Following the addition of CaI-A23187, the sperm suspension was incubated at 37°C under 5% CO2 for exactly 30 min to derive the degree of spontaneous acrosome loss. Under these conditions, more than 90% of spermatozoa showed complete acrosome reaction. After washing twice, spermatozoa (104) were coated onto a clean glass slide, air-dried and fixed with cold methanol for 10–15 min. Slides were incubated at 37°C for 30 min in the presence of fluoroscein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (FITC-PSA; Sigma). A sample (245 µl) of sperm suspension (108 spermatozoa) without CaI-A23187 was subjected to identical incubation conditions. The same batch of CaI-A23187 was used throughout the study. The acrosome status was monitored using an epifluorescence microscope (Olympus, BX 51). Spermatozoa that showed staining of the head region were classified as non-acrosome-reacted (NAR), while those with complete loss of PSA staining were classified as acrosome-reacted (AR) (Troup et al., 1994Go)

Extraction and purification of sperm membrane proteins
Sperm pellet suspensions were subjected to membrane lysis using buffers containing six different detergents: (i) 0.5% Nonidet P-40 (NP-40); (ii) 8 mol/l urea; (iii) 0.1% Tween 20; (iv) 30 mmol/l N-octyl-{beta}-D-glycopyranoside (NOG); (v) 0.5% Triton X-100; and (vi) 1% sodium dodecyl sulphate (SDS; Sigma). For each extraction, approximately 108 spermatozoa were subjected to sonication (eight bursts, 15 s each) using buffers containing respective detergents. Before being subjected to gel electrophoresis, the sperm extracts were centrifuged at 5000 g for 10 min at 4°C, the supernatants collected, and the protein concentrations determined (Lowry et al., 1951Go).

Purification of 57 kDa protein
For the purification of 57 kDa protein, a total of 784 normal semen samples was obtained from couples undergoing evaluation for primary infertility (median age 30 years; range 25–47 years). Typically, the samples showed a mean (± SD) sperm count of 87.76 ± 29.21x106, motility of 57.55 ± 20.43%, morphology of 46.11 ± 19.06%, and viability of 66.10 ± 26.66%. The methods of sample collection, processing and acrosome induction were as referred to above. Spermatozoa were frozen and stored at –80°C in the presence of protease inhibitors [1 mmol/l benzamidine, 5 mmol/l EDTA, 1 mmol/l pepstatin-A, 2 mmol/l leupeptin and 2 mmol/l phenylmethylsulphonyl fluoride (PMSF; Sigma)].

Of the six procedures employed, that using NOG resulted in an additional protein band of 57 kDa specific to the normozoospermic samples. Attempts were made to purify this protein using a published method (Yoshida and Aketa, 1987Go) where the authors have shown the purification of 58 kDa sperm binding protein from the eggs of sea urchin, Hemicentrotus pulcherrimus. Briefly, the NOG sperm extract was treated with 1 mol/l HCl (pH 3.0) and centrifuged at 5000 g for 10 min. To the precipitate 1 mol/l urea was added and dialized. To this 50 µmol/l calcium acetate was added to precipitate carbohydrate and lipid contents. The supernatant was treated overnight at 4°C with saturated ammonium sulphate. The precipitate was dissolved in 0.01 mol/l PBS (pH 7.2) and dialysed extensively overnight at 4°C against the same buffer using a 10 kDa cut-off dialysis membrane.

Gel filtration chromatography
Sepharose CL-4B chromatography was carried out on a 48x1.8 cm glass column using standard proteins for calibration. The void volume was determined using blue dextran (Sigma). Lyophilized sample (~1.38 mg protein) was dissolved in 0.01 mol/l PBS (pH 7.2) containing NaN3 and loaded onto the column. Fractions (1 ml) were collected and their absorbance was monitored at 280 nm using a spectrophotometer (UV-160A; Shimadzu, Japan). Based on the protein content, fractions were pooled separately, concentrated, dialysed overnight at 4°C, and lyophilized.

Ion-exchange chromatography
DEAE–Sephadex A-25 chromatography was carried out on a 25x1.2 cm glass column using standard proteins for calibration. Previous attempts to purify 57 kDa protein using anion-exchange chromatography on DEAE–Sephadex-A25 were unsuccessful because of strong adsorption of the protein to column material. Therefore, 0.01% Triton X-100 was added to the sample buffer to elute the adsorbed proteins. Peak-I fractions (~0.837 mg protein) obtained on gel filtration chromatography were dissolved in 0.05 mol/l NaCl and loaded onto the DEAE-column. Protein was eluted by changing the molarity of NaCl from 0.05 to 2 mol/l, and finally to 4 mol/l. The molarity of buffer was increased so that the protein that had affinity for DEAE at lower ionic strength would be eluted at a higher ionic strength of buffer. Fractions (1 ml) were collected and their absorbance was monitored at 280 nm. Fractions were pooled according to the peaks, dialysed overnight at 4°C, and lyophilized.

Affinity chromatography
Affinity chromatography on calcium hydroxyapatite matrix was performed using 12x0.9 cm glass column. Peak-I fractions (~0.382 mg protein) obtained using ion-exchange chromatography were dissolved in 0.01 mol/l PBS (pH 7.2) and applied onto the column. At lower molarity of phosphate buffer (0.01 mol/l), the protein was bound to the matrix and hence buffer gradient was increased from 0.2 mol/l to 1 mol/l. Fractions (1 ml) were collected and their absorbance was monitored at 280 nm. Fractions were pooled according to the peaks, dialysed overnight at 4°C, and lyophilized. In order to obtain substantial amounts of protein in pure form, the purification was carried out five times.

Characterization of 57 kDa protein
Semen samples were obtained from 20 fertile subjects (mean age 28 ± 5 years) (sperm count 76.94 ± 8.30x106; motility 59.18 ± 5.64%; morphology 51.76 ± 4.09%; viability 65.66 ± 7.81%) in whom fertility had been demonstrated in the previous year, and from 61 patients (mean age 33 ± 6 years) who were consulting for infertility. None of these infertile subjects had fathered a child. Of these patients, nine were classified as IVF-F (sperm count 58.99 ± 5.32x106; motility 50.20 ± 4.63%; morphology 40.26 ± 4.97%; viability 63.26 ± 8.06%), 20 as oligozoospermic (sperm count 9.34 ± 3.26x106; motility 39.76 ± 3.50; morphology 23.36 ± 2.70%; viability 44.51 ± 6.13%), eight as asthenozoospermic (sperm count 43.14 ± 2.02x106; motility 11.27 ± 1.10 %; morphology 10.28 ± 0.91%; viability 18.09 ± 1.02%), and 24 as oligoasthenozoospermic men (sperm count 13.26 ± 3.91x106; motility 6.26 ± 2.83%; morphology 10.16 ± 2.23%; viability 8.86 ± 2.04%). The sample processing and acrosome induction were performed as described earlier for the extraction of sperm membrane proteins.

SDS–Polyacrylamide gel electrophoresis (SDS–PAGE)
One-dimensional SDS–PAGE was performed using vertical slab gel apparatus (Gibco-BRL, Life Technologies) with the stacking gel containing 4% polyacrylamide and the resolving gel 12% polyacrylamide (Laemmli, 1970Go). Briefly, NOG sperm membrane extracts of normozoospermic and infertile subjects were diluted 1:1 with reducing sample buffer (20% glycerol, 4% SDS, 0.125 mol/l Tris–HCl, pH 6.5, 10% {beta}-mercaptoethanol and 0.5% bromophenol blue), heated for 15 min at 100°C and centrifuged at 10 000 g for 5 min. The supernatant (10 µl corresponding to 20 µg isolated protein) was loaded into each well and subjected to electrophoresis at 200 V for 15 min, followed by 150 V for 45 min. The gel was stained with Coomassie blue (Coomassie Brilliant blue R-250; Sigma) and calibrated using molecular weight markers (Gibco-BRL).

Two-dimensional electrophoresis
Purified 57 kDa protein was solubilized in 0.01 mol/l PBS, and isoelectric focusing (IEF) performed under equilibrated conditions (Berube and Sullivan, 1994Go). Briefly, IEF as a first dimension was performed on slab gels using glass tubes (14 mm length, 1.5 mm thickness) filled with 4% polyacrylamide, 8 mol/l urea, 2.5% carrier ampholyte with a pH range 2–10 and 2% Nonidet P-40. The gels were allowed to polymerize for 2 h by dispersing 20 µl 8 mol/l urea onto the slab top. The anode reservoir was replenished with 25 mmol/l H3PO4 and the cathode reservoir with 0.1 mol/l NaOH. The 20 µl 8 mol/l urea laid on the slab top was changed for 20 µl denaturing IEF lysis buffer (8 mol/l urea, 2% Nonidet P-40 and 1% {beta}-mercaptoethanol), and a pre-equilibration step was run gradually at 200 V for 15 min followed by 300 V for 30 min and later at 400 V for 30 min. The purified 57 kDa protein corresponding to 5 µg was loaded, and electrophoresis performed at 400 V for 16 h. For the second dimension (SDS–PAGE), the slab gels were positioned across the top of a 12% acrylamide gel (1 mm thickness, 15 cm length) and electrophoresis continued at 15 mA for 1 h and 20–22 mA for 4 h. After electrophoresis, the protein was visualized by silver staining and the molecular weight determined using SDS–PAGE standards (Gibco-BRL).

Development of polyclonal antibody against affinity-purified 57 kDa protein
Antibody against the eluted protein (peak-II of affinity chromatography) were raised in two adult female New Zealand White rabbits by intradermal injection of 20 µg protein in Freund’s complete adjuvant, and one week later with Freund’s incomplete adjuvant. On completion of five weekly injections, animals were given a booster dose without adjuvant in PBS. At 1 week after the booster dose, blood samples (10 ml) were obtained from the marginal ear vein and tested for their reactivity with corresponding 57 kDa protein by ELISA. The immunoglobulin fraction was separated from serum by precipitation with 40% ammonium sulphate. Pre-immune rabbit serum was used as a negative control.

Western blot analysis-purified protein fractions
Protein concentrations were determined for all peaks obtained during each step of purification. For SDS–PAGE, 5 µg protein was loaded per well. Sperm proteins were electrotransferred from gels onto 0.45 µm pore size nitrocellulose membranes (Pharmacia, Sweden) using a mini trans-blot Cell apparatus (Gibco-BRL) under reducing conditions (Towbin et al., 1979Go). Ponceau S staining was used to evaluate the transfer efficiency of proteins. After transfer, the membranes were soaked in transfer buffer (192 mmol/l glycine, 25 mmol/l Tris, 20% methanol, pH 8.3) and blocked for 30 min with 5% (w/v) skimmed milk powder to saturate unoccupied protein binding sites. The blots were incubated for 1 h at 37°C with 57 kDa antibody at a dilution of 1/1000 in Tris buffer saline containing 1% BSA (T-BSA). After washing three times in T-BSA, the blots were incubated with goat anti-rabbit-horseradish peroxidase (HRP) conjugated antibody (Sigma) at a dilution of 1/1000. The blots were washed and the bound peroxidase activity was visualized with substrate, 3-3'-diaminobenzidine (Sigma) (2 mg/ml) in the presence of 0.02% H2O2. Pre-immune rabbit serum and secondary antibody alone were included as negative controls.

Immunolocalization of 57 kDa protein on spermatozoa
The presence and distribution of 57 kDa protein on human spermatozoa were analysed by indirect immunofluorescence (IF) with methanol-fixed and viable, non-capacitated, capacitated and AR spermatozoa. Motile spermatozoa were collected from a discontinuous Percoll gradient, washed and resuspended in Tyrode buffer containing 1% BSA. For IF on methanol-fixed sperm, 100 µl of the sperm suspension diluted to 5x106 cells/ml was centrifuged at 300 g for 5 min. The smears were fixed for 10 min in methanol and blocked for 30 min in Tyrode buffer containing 5% BSA (Tyr-BSA) before incubation for 1 h at room temperature with pre-immune rabbit serum at a dilution of 1/1000 in Tyr-BSA. For IF on viable sperm cells, 50 µl of sperm suspension containing 20x106cells was incubated for 1 h at 37°C with 100 µl pre-immune rabbit serum at a 1/1000 dilution. After two centrifugations in Tyr-BSA, the motile spermatozoa were resuspended at a concentration of 5x106 cells/ml and centrifuged at 300 g for 5 min as described above. The live sperm suspensions and smears of methanol-fixed spermatozoa were incubated with 57 kDa antibody at a dilution of 1/1000 for 1 h at room temperature. After washing, samples were incubated with goat-anti rabbit-FITC conjugated secondary antibody (Gibco-BRL), washed twice and mounted in a glycerol:PBS solution (1:1 v/v) containing 0.1% paraphenylenediamine. Pre-immune sera and secondary antibody alone were included as negative controls. Sperm smears were examined for fluorescence pattern under epifluorescence microscope (BX51, Olympus). The pictures were obtained using a digital camera (PM-10SP, Olympus).

Enzyme-linked immunosorbent assay (ELISA)
Quantification of 57 kDa protein in spermatozoa of fertile and infertile subjects was carried out using a cell-ELISA (Reddy et al., 1998Go). To measure the differences in the expression of the protein, both NAR and AR spermatozoa were used. Spermatozoa at a concentration of 1x106/ml were coated onto polystyrene microtitre plates and incubated overnight at 4°C. The sperm were fixed using 0.25% glutaraldehyde (Jonakova et al., 1998Go). The non-specific sites were blocked with normal goat serum at a dilution of 1/1000. Endogenous peroxidase was quenched with 0.5% H2O2 in PBS. Primary antibody at a dilution of 1:1000 was added into the wells and incubated at 37°C for 1 h, followed by goat-anti rabbit-HRP secondary antibody at a dilution of 1/1000. In between each step the plates were washed with PBS–Tween 20. Bound peroxidase activity was visualized after adding substrate, o-phenylenediamine (OPD; Sigma) in 0.03% (v/v) H2O2 in citrate phosphate buffer (pH 5.5). After incubation in the dark for 20 min, the reaction was terminated by adding 100 µl 2 mol/l H2SO4 to each well. Absorbance was measured at 492 nm using an ELISA reader (ELX-800; BIO-TEK Instruments, Germany). Pre-immune sera and secondary antibody alone were included as negative controls.

Flow cytometric analysis
Single colour flow cytometry (FACScan; Beckman Coulter) was used to evaluate the surface expression of 57 kDa protein on human spermatozoa (Reddy et al., 2003Go). Briefly, capacitated and ionophore-treated sperm suspensions (105 spermatozoa) of fertile and IVF-F subjects were incubated with anti-57 kDa antibody (10 µg/ml) for 1 h. After washing in PBS–BSA, the cells were incubated with FITC-conjugated goat anti-rabbit secondary antibody at a dilution of 1/1000. Fluorescence signals (FLI -FITC) of each stained cell were detected as it passes through the focus of a 520 nm argon-ion laser. The green pulse width (the time taken for a cell to pass through the laser beam) was also recorded and used to distinguish single cells from debris, if any. The percentage positive cells and mean fluorescence were calculated. Data for a minimum of 10 000 cells were collected for each sample and presented in log scale on the x-axis, and number of cells showing fluorescence on the y-axis, in flow cytograms. Channels MI and M2 represented low- and high-intensity staining respectively. Pre-immune rabbit sera and secondary antibody alone were included as negative controls.

Zona-free hamster oocyte binding assay
Motile spermatozoa were collected from a discontinuous Percoll gradient and capacitated and acrosome-reacted as described above. Spermatozoa (5x106) were incubated at 37°C for 30 min with different concentrations of 57 kDa antibody (10, 5, 2.5, 1.25, 0.625, 0.3125 µg /ml). The CaI-A23187 was added at a final concentration of 10 µmol/l for the last 15 min of incubation with the antibody.

Female golden hamsters (aged 6–8 weeks) were induced to superovulate by administering an intraperitoneal injection (25 IU) of pregnant mare’s serum gonadotrophin (PMSG) followed 48 h later by 25 IU hCG. The hamsters were killed 16 h later and the cumulus–oocyte masses collected from the ampulla. Cumulus cells were dispersed with 0.1% bovine testicular hyaluronidase in BWW–BSA (Sigma) After washing in BWW, the eggs were placed in 0.1% bovine pancreatic trypsin (type XII; Sigma) in BWW–BSA to remove the zona pellucida (ZP). Finally, zona-free oocytes were washed in three changes of fresh BWW medium and kept at 37°C in 5% CO2 in air before use. After washing, the sperm suspension was incubated at 37°C for 1 h with zona-free oocytes in Petri plates containing mineral oil. Oocytes were washed three times by aspiration through a narrow pipette in order to remove any loosely bound spermatozoa, mounted onto slides, and then examined using phase-contrast optics (100x objective). Between 15 and 20 oocytes were examined in each experiment; the results were expressed as number of sperm bound per oocyte.

Statistical analysis
In most instances the results were expressed as mean ± SD. Where appropriate, Student’s t-test was applied for comparison of mean values, and differences were considered significant at P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Comparison of sperm antigens between fertile and subfertile men
The characteristics of semen samples (normozoospermic and infertile) and the protocol used for the extraction of sperm membrane proteins by different detergents are provided in Table I and Figure 1 respectively. SDS–PAGE analysis of sperm extracts of normal and infertile subjects showed approximately 16–18 major and 12–15 minor protein bands, with relative molecular masses ranging from 5 to 100 kDa. Different sperm extraction methods showed almost identical protein profiles, though with some minor quantitative differences. The sperm from normozoospermic males, when extracted with NOG, showed an additional protein band with a molecular weight of 57 kDa. This protein was present in ~95% of normozoospermic males, but was found to be low in the remaining ~5%. In 80% of infertile males the protein expression was found to be absent, and was marginal in the remaining 20% (Figure 2).


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Table I. Results of semen analyses of samplesa used for the extraction of membrane proteins
 


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Figure 1. Extraction and purification of 57 kDa protein.

 


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Figure 2. SDS–PAGE analysis of human spermatozoa from normal and infertile men. Sperm membrane proteins were extracted and 20 µg loaded into each well. Gels were stained with Coomassie Brilliant blue. Extracts showed similar protein profiles, but with some quantitative inter-individual variations. One representative sample from each group is shown. Molecular weight markers: lane 1, 57 kDa protein (<-) was present in extracts of fertile (lane 2) and normozoospermic (lane 3) samples, but absent from oligo zoospermic (lane 4), IVF-failure (lane 5) and oligoastheno zoospermic (lane 6) samples.

 
Purification of 57 kDa protein
Partially purified NOG sperm extract (~1.38 mg protein) was loaded onto the Sepharose CL-4B column, and three protein peaks were obtained. SDS–PAGE analysis of peak-I showed the presence of a protein in the 57 kDa region as well as several other proteins at various molecular regions (Figure 3, lane 1). None of the other two peaks showed the protein band in the 57 kDa region (data not shown).



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Figure 3. SDS–PAGE (lanes 1–3) and Western blot analysis (lanes 4–6) of protein peaks obtained during purification. Protein (5 µg) was loaded into each well; gels were stained with Coomassie Brilliant blue. Lane 1 = peak-I proteins after gel filtration chromatography; lane 2 = peak-I proteins after ion-exchange chromatography; lane 3 = peak-II proteins after affinity chromatography. The purified protein appeared as a single band of molecular weight 57 kDa (<-). Lanes 4–6 were corresponding protein peaks on Western blotting. The 57 kDa protein was recognized in all three peaks by anti-57 kDa antibody. All experiments were performed in triplicate. For details of SDS–PAGE and Western blotting, see Materials and methods.

 
The fractions of peak-I (~0.873 mg protein) were further purified using DEAE–Sephadex chromatography. Protein elution varied with the ionic strength of NaCl, but three peaks were obtained, with peak-I being eluted in 2 mol/l NaCl. SDS–PAGE of the peak showed the presence of 57 kDa protein in addition to a few other proteins at various molecular regions (Figure 3, lane2).

The fractions of peak-I (~0.382 mg protein) were loaded onto a hydroxyapatite affinity chromatography column. Protein elution varied with changes in molarity of the phosphate buffer (pH 7.2); three peaks were obtained, and peak-II (~0.185 mg protein) was eluted with 0.2 mol/l PBS. SDS–PAGE of the peak showed a single protein band at the 57 kDa region (Figure 3, lane 3).

Western blot analysis of rabbit polyclonal antibody raised against the purified 57kDa protein
Antibody obtained from rabbits immunized with 57 kDa protein was tested for reactivity against the immunized 57 kDa protein and protein fractions of ion-exchange (Figure 3, lane 4) and gel filtration (Figure 3, lane 5) chromatography and separated by one-dimensional SDS–PAGE (Figure 3, lanes 1–3). Anti-57 kDa serum at 1/40 000 dilution recognized a single 57 kDa protein band (Figure 3, lane 6). Pre-immunized serum at the same dilution did not react with any of the protein bands. The immunoreactivity was not seen after prior incubation of immune serum with 57 kDa protein (data not shown).

Two-dimensional PAGE analysis of 57 kDa protein
Two-dimensional (2D) electrophoresis offers resolution and separation of proteins according to the molecular weight and isoelectric point (pI). The homogeneity of 57 kDa protein and its electrophoretic mobility was analysed by 2D electrophoresis. The purified protein appeared as a single spot corresponding to the 57 kDa region with pI of 5.2 (Figure 4).



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Figure 4. Two-dimensional electrophoresis of purified 57 kDa protein (5 µg) separated by isoelectric focusing in the first dimension, followed by SDS–PAGE (12% resolving gel) in the second dimension. The isoelectric point range is shown horizontally at the top of the gel; molecular weight markers (kDa) are shown vertically at the left. Proteins were detected by silver staining.

 
Indirect immunofluorescence
The pattern of binding of rabbit anti-57 kDa antibody to methanol-fixed human spermatozoa is shown in Figure 5A–F. NAR spermatozoa of fertile men showed localization of 57 kDa protein on the acrosome cap (Figure 5A). After the acrosome reaction, it was mainly found on the equatorial segment. A similar staining pattern was observed in live NAR and AR spermatozoa. The expression of protein was found to be low in infertile subjects compared with the fertile. Omission of the primary antibody resulted in an absence of fluorescence in the acrosome and equatorial region of the sperm (Figure 5C and F).



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Figure 5. Immunofluorescence localization of 57 kDa protein on spermatozoa of fertile and infertile (IVF-F) subjects. Protein was localized on the acrosome of non-acrosome-reacted (NAR) (A) and the equatorial region in acrosome-reacted (AR) spermatozoa (D). The expression was significantly lower in NAR (B) and AR (E) spermatozoa of IVF-F subjects. Pre-immune rabbit serum did not stain on NAR (C) and AR (F) spermatozoa.

 
Flow cytometric analysis of 57 kDa protein in fertile and infertile subjects
Flow cytometry was used to compare the percentage of AR spermatozoa labelled by anti-57 kDa antibody between fertile and infertile subjects. The data revealed that the percentage of spermatozoa reacting with 57 kDa antibody increased significantly in fertile samples when compared to infertile (Figure 6). The AR spermatozoa of fertile males showed higher mean fluorescence intensity (43 ± 4.6%) when compared with infertile subjects (IVF-F, 6.89 ± 2.16%). In controls, only 2–3% of spermatozoa weakly reacted with pre-immune rabbit serum.



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Figure 6. Flow cytometric analysis of 57 kDa protein on human spermatozoa. (A) Fertile; (B) IVF-F; (C) negative control. Comparison of 57 kDa positivity for both groups was made by comparing fluorescence in the M2 channel. LFI = log fluorescence.

 
Quantitative analysis of 57 kDa protein by ELISA
ELISA was used to measure the levels of 57 kDa protein in spermatozoa obtained from fertile and infertile subjects, and the results obtained were in agreement with IF and flow cytometry data. The expression of protein was ~50% higher in fertile subjects compared with infertile. After the acrosome reaction, expression was increased from 0.987 ± 0.06 to 1.348 ± 0.107 (P < 0.05) (Figure 7). No difference was observed among the infertile subjects. An absorbance value of 1 detected after the acrosome reaction was considered to be a cut-off point in order to differentiate fertile from infertile. Some 90% of the fertile samples (18/20) showed an absorbance value >1, whereas 93% of infertile samples (46/49) showed a value <1 (Figure 8). The sensitivity and specificity of the test was >90%.



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Figure 7. Quantitative analysis of 57 kDa protein in normal spermatozoa before (A) and after (B) the acrosome reaction. Values are mean ± SD of six samples. The expression of 57 kDa protein was significantly increased (P < 0.05) after the acrosome reaction.

 


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Figure 8. Quantitative analysis of 57 kDa protein in human spermatozoa. (A) Fertile; (B) oligozoospermic; (C) oligoastheno zoospermic; (D) IVF failure. Levels of 57 kDa protein were significantly lower (P < 0.05) in infertile subjects than in fertile subjects. The absorbance value of 1, detected after the acrosome reaction, was considered to be a cut-off point to differentiate fertile from infertile males.

 
Effect of rabbit anti-57 kDa antibody on binding of human spermatozoa to zona-free hamster oocytes
Several experiments were conducted with human spermatozoa from various fertile donors, after overnight capacitation and treatment with 10 µmol/l CaI-A23187A in the presence or absence of rabbit anti-57 kDa antibody. A significant association was observed between the expression of 57 kDa and number of sperm bound to the oocyte (Figure 9). The effect of the 57 kDa antibody on sperm motility before binding to oocyte and the induction of premature acrosome reaction were evaluated. No change in the percentage of progressively motile and hyperactivated spermatozoa was observed in the presence of antibody. The forward progressive motility with 10 µg/ml antibody was 72.%, and 71% with pre-immune sera. Mean data acquired from three experiments are illustrated in Figure 9. In the presence of anti-57 kDa antibody, the proportion of spermatozoa adhered to the oocyte was significantly decreased (P < 0.05), and the effect was found to be dose-dependent. An optimum inhibitory effect was observed at a concentration of 10 µg/ml. In controls, pre-immune serum did not inhibit the binding.



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Figure 9. The effect of rabbit anti-57 kDa antibody (10 µg–0.312 µg) on the capacity of human spermatozoa treated with ionophore A23187 to bind zona-free hamster oocytes. Numbers of sperm bound per oocyte varied with antibody concentration. Inhibition was >90% with 10 µg antibody (square). In controls (diamond), 57 kDa antibody was replaced with pre-immune rabbit sera, and no effect was observed on sperm binding to hamster zone-free oocytes.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Worldwide, the problem of infertility is increasing, and the quality of sperm is reported to be decreasing over the years, with about one-third of patients who attend infertility clinics being diagnosed as idiopathic. Analyses of male fertility have revealed that several sperm surface proteins are associated with sperm function (Wassarman, 1990Go; Kerr, 1993Go; Batova et al., 1998Go; Dallapiccola and Novelli, 2000Go). Moreover, specific sperm membrane proteins have been identified which might be important for the diagnosis of certain cases of infertility in humans (Liu et al., 1996Go; Naz and Leshie, 1999Go).

The recent development of a two-dimensional protein database of human sperm proteins has aided the identification of sperm surface molecules. By using immunoblot and immunoprecipitation techniques, a number of sperm antigens have been identified, although characterization did not proceed further in most cases (Primakoff et al., 1987Go; Mathur et al., 1988Go; Ash et al., 1995Go; Paradesi et al., 1996Go; Batova et al., 1998Go; Naz and Leshie, 1999Go). In other studies, sera from infertile patients and vasectomized men were used to identify antigens expressed in testis cDNA libraries, and many of the detected molecules have been investigated, though without much success (Hjort and Griffin 1985Go; Diekman and Goldberg, 1994Go; Auer et al., 2000Go). Vectorial labelling of the sperm surface by biotinylation and iodination resulted in the identification of 98 dual-labelled sperm surface proteins (Naaby-Hansen et al., 1997Go), and by using 2D-electrophoresis of infertile sera, six immunodominant sperm surface antigens have been identified (Shetty et al., 1999Go). In the present study, a close relationship was demonstrated between the expression of 57 kDa protein and sperm function.

The results of the present study indicated that 57 kDa protein was present in the spermatozoa of ~95% of normozoospermic males when extracted with NOG buffer, but in the remaining ~5% of samples it was relatively weaker in intensity. In all four infertile groups examined—namely IVF-F, oligozoospermic, asthenozoospermic and oligoasthenozoospermic—the 57 kDa protein was absent in ~80% of men and only marginally expressed in the remaining ~20%. The absence of this protein in the electrophoretic profiles of infertile subjects raises the possibility that it might be associated with sperm function. The absence and/or low expression of the protein in the spermatozoa of infertile males might be due either to the lack of expression during early stages of spermatogenesis or to the masking of antigenic sites by antisperm antibodies (a positive IBT result). In order to avoid the latter situation, semen samples were excluded that were positive for anti sperm antibodies. The experiment was repeated three times with different semen samples, and similar results were obtained.

The 57 kDa protein was further characterized by raising antibodies against it. The protein was purified by using different chromatographic techniques, and in the blots obtained with the purified protein fractions of sperm membrane the anti-57 kDa antibody reacted specifically with the protein corresponding to the immunizing protein. The purified protein was homogeneous, with a molecular weight of 57 kDa and an isoelectric point of 5.2, which suggested that it might contain acidic amino acids. The ELISA results indicated that levels of the 57 kDa protein were higher in fertile than in infertile subjects. Following the acrosome reaction, the levels were increased significantly, suggesting that the epitopes recognized by the specific rabbit 57 kDa antibody were less available on the surface of acrosome-intact (NAR) spermatozoa. Transmission electron microscope studies using immunogold labelling are currently in progress to investigate the precise location of 57 kDa protein in relation to the acrosome reaction.

Spermatozoa are highly polarized in the distribution of their surface components (Batova et al., 1998Go). In the present study, the NAR and AR spermatozoa were stained after they had been fixed with methanol, and subsequent immunofluorescence studies revealed that the 57 kDa protein was localized on the acrosome of NAR spermatozoa. After induction of the acrosome reaction, the main staining pattern was shifted to the equatorial segment, a situation which might be attributed to the incorporation of intracellular proteins into the plasma membrane. A similar distribution pattern has been reported for the ram sperm protein, ESA152 (McKinnon et al., 1991Go). Although factors responsible for the translocation of the protein are not known, one group (Allen and Green, 1995Go) showed that novel antigens or epitopes are expressed on the post-acrosome region after the acrosome reaction through the exposure of a previously masked protein. A similar localization pattern was observed in live NAR and AR spermatozoa. The localization of this 57 kDa protein suggests that it may be involved in the process of initial sperm–oocyte recognition and binding.

Flow cytometry was used to measure the number of sperm expressing 57 kDa protein in both fertile and infertile subjects, and the results obtained mirrored the changes observed by using ELISA and IF. It is tempting to speculate that a lack of and/or low expression of this protein may be one of the possible reasons for the low binding of sperm to hamster zona-free oocytes. The AR spermatozoa, when treated with anti-57 kDa antibody, inhibited the binding in dose-dependent manner; that is, as the concentration increased from 0.31 µg to 10 µg, the numbers of sperm bound to oocytes was decreased by >90%. This inhibition was not the result of an effect of the antibody on sperm motility; neither was it due to premature induction of the acrosome reaction. As the antibody had no effect on sperm motility, the inhibition of sperm binding to the hamster zona-free oocytes by 57 kDa antibody may primarily be due to neutralization of the antigen. When considered in conjunction with the data for ESA152, the present data reinforce the idea that the functional and physical properties of sperm plasma membranes are inseparable. Both the distribution and redistribution of sperm surface components appear to be critical for the union of male and female gametes. Thus, anti-57 kDa antibody may inhibit both sperm–oolemma adhesion and fusion. To date, no fusion proteins have been reported in human spermatozoa, but several have been proposed for other mammalian spermatozoa. Such proteins are recognized by monoclonal antibodies that bind to the equatorial segment of AR spermatozoa and inhibit sperm–oocyte binding in mice (Toshimori et al., 1998Go), rats (Cohen et al., 1996Go) and guinea-pigs (Allen and Green, 1995Go). Some of these monoclonal antibodies have been shown to react with human spermatozoa, but the involvement of the corresponding antigens in human infertility has not been demonstrated (Auer et al., 2000Go).

In conclusion, in the present study several biochemical and immunological properties of a 57 kDa sperm surface protein have been characterized, and the results have suggested that this protein might vary significantly between normal and infertile subjects. The positive relationship between expression of the protein and sperm quality strengthens the hypothesis that this protein will be useful in identifying and explaining some cases of infertility. Consequently, in order to evaluate the physiological role of the protein its sequence is the subject of ongoing studies. Likewise, cDNA cloning and the availability of recombinant protein will greatly facilitate investigations into the molecular identity of the 57 kDa protein.


    Acknowledgements
 
The authors thank Dr Chander P.Puri, Director, for his consistent encouragement throughout these studies. The investigations were supported by the Department of Biotechnology, Government of India (Grant No. BT/PRO587/Med/09/115/97 to K.V.R.R). The assistance of Drs M.Hansotia, V.Mangoli, M.Bhattacharya and K.N.Ganla for providing semen samples for the study is gratefully acknowledged. The technical assistance of Prasanna Chavan is also greatly appreciated.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Allen CA and Green DPL (1995) Monoclonal antibodies which recognize equatorial segment epitopes presented de novo following the A23187-induced acrosome reaction of guinea-pig sperm. J Cell Sci 108,767–777.[Abstract/Free Full Text]

Auer J, Senechal H, Desvaux FX et al. (2000) Isolation and characterization of two sperm membrane proteins recognised by sperm associated antibodies in infertile men. Mol Reprod Dev 57,393–405.[CrossRef][ISI][Medline]

Ash K, Berger T, Horned CM and Culvert CC (1995) Identification of porcine sperm plasma membrane proteins that may play a role in sperm-egg fusion. Zygote 3,163–170.[ISI][Medline]

Batova IN, Ivanova MD, Mollova S et al. (1998) Human sperm surface glycoprotein involved in sperm-zona pellucida interaction. Int J Androl 21,141–153.[CrossRef][ISI][Medline]

Berube B and Sullivan B (1994) Inhibition of in vitro fertilization by active immunization of male hamsters against a 26 kDa sperm glycoprotein. Biol Reprod 16,1255–1263.

Blobel CP, Wolfsberg TG, Turck CW et al. (1992) A potential fusion peptide and integrin ligand domain is a protein in sperm-egg fusion. Nature 35,248–252.

Burks DJ, Carballada R, Moore HDM et al. (1995) Interaction of tyrosine kinase from human sperm with the zona pellucida at fertilization. Science 269,83–86.[ISI][Medline]

Cardullo RA and Wolf DE (1992) Cross linking mouse surface galactosyl transferase results in its redistribution but does not complete the acrosome reaction. J Cell Biol 110,1137–1145.

Cohen DJ, Munuce MJ and Causnicu PS (1996) Mammalian sperm-egg fusion: the development of rat oolemma fusibility during oogenesis involves the appearance of binding sites for sperm protein ‘DE’. Biol Reprod 55,200–206.[Abstract]

Dallapiccola B and Novelli G (2000) Male infertility pleiotropic genes and increased risk of diseases in future generations. J Endocrinol Invest 23,557–559.[ISI][Medline]

De Kretser DM and Baker HWG (1999) Infertility in men: recent advances and continuing controversies. J Clin Endocrinol Metab 84,3443–3450.[Free Full Text]

Diekman AB and Goldberg E (1994) Characterization of a human antigens with sera from infertile patients. Biol Reprod 50,1070–1087.

Diekman AB, Norton EJ, Westbrook VA et al. (2000) Antisperm antibodies from infertile patients and their cognate sperm antigens: a review – Identity between SAGA-1, the H6-3C4 antigen and CD52. Am J Reprod Immunol 43,134–143.[CrossRef][ISI][Medline]

Glander HJ, Schaller J, Weber WB et al. (1996) In vitro fertilization: increased VLA (very late antigen and fibronectin after acrosome reaction. Arch Androl 36,177–185.[ISI][Medline]

Hjort T and Griffin PD (1985) The identification of candidate antigens for the development of birth control vaccine. J Reprod Immunol 8,271–278.[CrossRef][ISI][Medline]

Jonakova J, Kraus M and Veselsky L (1998) Sperm adhesins of the AQN and AWN families, DQH sperm surface protein and HNK protein in the heparin binding fraction of boar seminal plasma. J Reprod Fertil 114,25–34.[Abstract]

Kerr LE (1993) Sperm antigens and immunocontraception. Reprod Fertil Dev 7,825–830.

Klentzeris LD, Fishel S, McDermott H et al. (1995) A positive correlation between expression of {alpha}1-integrin cell adhesion molecules and fertilizing ability of human spermatozoa in vitro. Mol Hum Reprod 10,728–733.

Kodaira K, Takahashi R, Hirabayashi M et al. (1996) Over expression of c-myc induces apoptosis at the prophase of meiosis of rat primary spermatocytes. Mol Reprod Dev 45,403–410.[CrossRef][ISI][Medline]

Kopf G (1989) Mechanisms of signal transduction in mouse sperm. Ann N Y Acad Sci 564,289–302.[Abstract]

Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227,680–685.[ISI][Medline]

Liu QY, Wang LF, Miao SY et al. (1996) Expression and characterization of a novel human sperm membrane protein. Biol Reprod 54,323–330.[Abstract]

Lowry OH, Rosebrough NJ, Farr AL et al. (1951) Protein measurement with Folin phenol reagent. J Biol Chem 193,265–275.[Free Full Text]

Mathur S, Chao L, Goust JM et al. (1988) Special antigens on sperm from autoimmune infertile men. Am J Reprod Immunol 17,5–13.[ISI]

McClure RD, Nunez L and Tom R (1989) Semen manipulation: improved sperm recovery and function with a two layer Percoll gradient. Fertil Steril 51,874–877.[ISI][Medline]

McKinnon CA, Weaver FE, Yoder JA et al. (1991) Cross linking and maturation dependent ram sperm plasma membrane antigen induces the acrosome reaction. Mol Reprod Dev 29,200–207.[ISI][Medline]

Naaby-Hansen S, Flickinger CJ and Herr JC (1997) Two dimensional gel electrophoretic analysis of vectorizoa. Biol Reprod 56,771–787.[Abstract]

Naz RK and Leshie MH (1999) Sperm surface protein profiles of fertile and infertile men: search for a diagnostic molecular marker. Arch Androl 43,173–181.[CrossRef][ISI][Medline]

Naz RK, Ahmad K and Kumar G (1991) Presence and role of c-myc proto-oncogene in product in mammalian sperm cell function. Biol Reprod 44,842–850.[Abstract]

Naz RK, Ahmad K and Kaplan P (1992) Expression and function of ras-proto-oncogene proteins in human sperm cells. J Cell Sci 102,487–494.[Abstract]

Nieschlag E and Leifke E (2000) Empirical therapies for idiopathic male infertility. In Nieschlag E and Behre MB (eds), Andrology, Male Reproductive Health and Dysfunction. Springer-Verlag, Berlin, p. 313.

O’Rand MG, Widgren EE and Fisher SJ (1979) Changes of sperm surface properties correlated with capacitation. In Fawcett DW and Bedford JM (eds), The Spermatozoon. Urban and Schwarzenberg, Baltimore, USA.

Paradesi R, Bellavia E, Pession A et al. (1996) Characterization of human sperm antigens reacting with sperm antibodies from homologous serum and seminal plasma in an infertile population. Biol Reprod 55,54–61.[Abstract]

Primakoff P, Hyatt H and Tredick-Kline J (1987) Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion. J Cell Biol 104,141–149.[Abstract]

Rajeev SK and Reddy KVR (2000) Integrins and disintegrins: the candidate molecular players in sperm-egg interaction. Ind J Exp Biol 38,1217–1221.[Medline]

Reddy KVR, Meherji PK and Shahani SK (1998) Integrin cell adhesion molecules on human spermatozoa. Ind J Exp Biol 36,450–463.

Reddy KVR, Rajeev SK and Vijayalaxmi G (2003) {alpha}6{beta}1 integrin is a clinical marker to evaluate the sperm quality in men. Fertil Steril 79,1590–1596.[CrossRef][ISI][Medline]

Roten R, Paz GF, Hamonnai ZT et al. (1990) Protein kinase C is present in human sperm: possible role of flagellar motility. Proc Natl Acad Sci USA 87,7305–7308.[Abstract]

Sanz L, Cavete JJ, Man K et al. (1992) The complete primary structure of the sperm adhesion AWN, a zona pellucida-binding protein isolated from boar spermatozoa. FEBS Lett 300,213–218.[CrossRef][ISI][Medline]

Shetty J, Naaby-Hansen S, Shibahara H et al. (1999) Human sperm proteome: Immunodominant sperm surface antigens identified with sera from infertile men and women. Biol Reprod 61,61–69.[Abstract/Free Full Text]

Toshimori KM, Sexena DK, Tanii I et al. (1998) An MN9 antigenic molecule, equatorin, is required for successful sperm-oocyte fusion in mice. Biol Reprod 59,22–29.[Abstract/Free Full Text]

Towbin H, Staehelin T and Gordon TJ (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76,4350–4354.[Abstract]

Troup SA, Lieberman BA and Matson PL (1994) The acrosome reaction to ionophore challenge test: assay reproducibility, effect of sexual abstinence and results for fertile men. Hum Reprod 9,2079–2083.[Abstract]

Wassarman PM (1990) Profile of a mammalian sperm receptor. Development 108,1–17.[Abstract]

Wennemuth G, Meinhardt A, Mallidis C et al. (2001) Assessment of fibronectin as a potential new clinical tool in andrology. Andrologia 33,43–46.[CrossRef][ISI][Medline]

Wolf DE, McKinnon CA, Leyton L et al. (1992) Protein dynamics in sperm function membranes: implication for sperm function during gamete interaction. Mol Rep Fertil 33,228–234.

Wolfes H, Kogawa K, Millette CF et al. (1989) Specific expression of nuclear proto-oncogenes before entry into meiotic prophase of spermatogenesis. Science 24,740–744.

World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen–Cervical Mucus Interactions. 3rd edition. Cambridge University Press, London, UK.

Yoshida M and Aketa K (1987) Purification of the sperm binding factor and identification of a sperm attack molecule from the egg of the sea urchin, Hemicentrotus pulcherrimus. Gamete Res 18,1–16.[ISI][Medline]

Submitted on June 25, 2003 resubmitted on July 25, 20 ; accepted on October 7, 2003



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