Mol. Hum. Reprod. Advance Access originally published online on August 17, 2007
Molecular Human Reproduction 2007 13(9):633-639; doi:10.1093/molehr/gam049
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ERp57 is a potential biomarker for human fertilization capability
Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, Jiangsu, P.R. China
1 Correspondence address. Tel: +86-25-86862908; Fax: +86-25-86862908; E-mail: bbhxy{at}njmu.edu.cn
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Abstract |
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Human infertility is a growing concern and while many assisted reproductive technologies exist, their success rates are low. Thus, developing tests, possibly by assessing proteins involved in fertilization, that could predict the outcome of these technologies is of great significance. To identify candidate proteins, we used two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF techniques and detected the ERp57 protein from human testis protein profile. Immunohistochemistry showed that ERp57 was mostly located in spermatogenic cell cytoplasm from spermatocytes to the spermatozoa phases and in Leydig cells of human testes; it was also present at low levels in Sertoli cells. ERp57 was evident in human spermatozoa, primarily in the acrosome and tail; moreover, it appeared to translocate to the equatorial segment after the acrosome reaction. During sperm capacitation, the ERp57 protein underwent post-translational modification. Blocking ERp57 with antibodies significantly inhibited human sperm from penetrating zona-free hamster oocytes in a dose-dependent manner. Finally, expression levels of ERp57 were associated with fertility; they were decreased dramatically in IVF patients with low fertilization rates compared with those with high rates or to fertile sperm donors. Taken together, these results show that ERp57 is a component of human sperm acrosome proteins, which play a critical role in gamete fusion. Furthermore, ERp57 could be a novel phenotype marker for male infertility and has the potential to be used to assess sperm selection for IVF.
Key words: ERp57/testes/capacitation/gamete fusion/spermatozoa
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Introduction |
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Approximately half of diagnosed infertility cases are caused by male-related factors (Shefi and Turek, 2006). This diagnosis is usually confirmed by assessing sperm characteristics including total count, motility, morphology and seminal parameters (e.g. pH and sperm antibodies). However these measures fail to explain all cases of male infertility and do not provide a precise prognosis for human fertility in vivo or in vitro (Wang et al., 1988). Furthermore, diagnosing defective sperm function is difficult because oocyte fertilization requires diverse biological properties, including acrosomal reactions, zona recognition, signal transduction and cell fusion, to remain intact for its success.
Fertilization depends on sperm–oocyte interactions, and much effort has been made to find sperm-associated molecules that mediate this process. To date, candidate molecules have been reported, such as Izumo (Inoue et al., 2005), calmegin (Ikawa et al., 1997), fertilinß (Cho et al., 1998), sp56 (Bookbinder et al., 1995), etc. Their roles in fertility have been suggested by results from knockout mouse models for these proteins; e.g. Izumo–/– mice were healthy but males were sterile. In the case of Izumo, more support for its role in fertilization is that it is present in human sperm and adding antibodies against human Izumo rendered sperm unable to fuse with zona-free hamster oocytes. Although many of these molecules appear to play a role in fertilization, it is evident that many more such proteins exist and that the mechanisms by which they are involved have yet to be elucidated.
To further explore the molecules involved in human spermatogenesis and sperm function, we constructed a human testis protein profile using the two-dimensional polyacrylamide gel electrophoresis (PAGE) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF) techniques and identified many such proteins (data not shown). Particular attention was paid to one protein, namely ERp57, because it is a multifunctional thiol-disulfide oxidoreductase that can efficiently catalyze disulfide reduction, disulfide isomerization and dithiol oxidation in substrate proteins (Frickel et al., 2004). Interestingly, ERp57 is located in the developing acrosome of spermatids during rat spermatogenesis and on the adult mouse sperm membrane (Ohtani et al., 1993; Ellerman et al., 2006). Furthermore, it has been shown to play a role in gamete fusion. These results indicate that ERp57 contributes importantly to the rodent fertility processes; however, at present, no information exists regarding whether ERp57 is crucial for the human sperm fertilization or regarding its clinical relevance in human spermatozoa.
In our testis protein profile, we identified for the first time in the human testis three forms of ERp57 protein with the same molecular weight but different isoelectric points (pIs = 5.3, 5.58, 5.72) and showed that ERp57 in human testis was phosphorylated through phosphorylation staining (data not shown). According to a bioinformation assay (http://www.phosphosite.org/), a cluster of phosphorylation sites were found, including serine phosphorylation (150th, 343th) and tyrosine phosphorylation (445th, 454th, 467th). Indeed, the phosphorylation site of ser150 has been proved in somatic cells as reported by Kita et al. (2006). On the basis of above information, we ascertained where ERp57 is located on human sperm, examined its modifications during sperm capacitation and explored its role during gamete fusion. Furthermore, the clinical relevance of ERp57 was determined by comparing its protein expression levels in the spermatozoa of fertile men and in infertile patients with high and low in vitro fertilization (IVF) fertility rates. We predict that human ERp57 could be a novel phenotype marker for male infertility and has the potential to be used to select sperm for IVF.
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Materials and Methods |
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Subjects and sample preparation
Approval for this study was granted by the ethics committee of Nanjing Medical University (China) prior to sample collection. All participants signed consent forms. Normal human adult testes specimens that were used for western blot analysis, and immunohistochemistry were obtained from the Body Donor Center (Nanjing Medical University, Nanjing, China). The donor died of accident. Semen samples from 18 male infertility patients undergoing IVF (Clinical Reproductive Center, Nanjing Medical University) and 6 sperm donors were analysed for ERp57 contents. The fertile donors (n =6) met the following criteria: had fathered at least one child and underwent routine semen analyses revealing that sperm was within the normal range according to WHO 1999 guidelines. The patients undergoing IVF were divided into two groups (n = 9 per group) according to their fertilization rate: high (fertilization rate > 60%) and low (fertilization rate < 60%). The fertilization rate in the high group was 95 ± 8.7 versus 20.4 ± 9.0 for the low group, a difference that was significant (P < 0.01). Patients in the high fertilization group had the following characteristics (all mean ± SD): sperm concentration (n x 106/ml): 82.2 ± 19.8; motility (%): 23.9 ± 8.2; viability (%): 67.2 ± 8.3; morphology (% abnormal): 25.9 ± 8.3. The low fertilization group had the following characteristics: sperm concentration (n x 106/ml): 86.1 ± 20.9; motility (%): 25.6 ± 5.8; viability (%): 68.3 ± 13.9; morphology (% abnormal): 28.4 ± 7.3. It is likely that females' symptoms would also have contributed to the fertilization rates in the patients; such factors included polycystic ovarian syndrome or low numbers of oocytes produced from superovulation. Therefore, the cases for which there were female reasons for infertility were excluded.
Semen samples were allowed to liquefy at 37°C for 30 min, and motile spermatozoa were collected from a discontinuous Percoll gradient, washed twice and resuspended in phosphate-buffered saline (PBS).
Protein extraction and western blot
Human testis and spermatozoa were washed with PBS and incubated in lysis buffer [7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2% (w/v) dithiothreitol (DTT)] containing a protease inhibitor cocktail (Pierce, USA) for 1 h at 4°C. Samples were then homogenized (Ultra Turrax, IKA, Germany) at 11 000 pm for 5 min on ice. After centrifugation at 10 000 g at 4°C for 30 min, the supernatant was collected and stored at –70°C until use.
The concentration of extracted protein was determined by Bradford microprotein assay using bovine serum albumin (BSA) as the standard protein. Protein samples were boiled in sodium dodecyl sulphate (SDS)–PAGE sample buffer [2% (v/v) mercaptoethaol, 2% (w/v) sucrose in 0.1875 M Tris, pH 6.8, with bromophenol blue] and resolved by SDS–PAGE on polyacrylamide gels followed by transfer onto nitrocellulose membrane under semi-dry conditions by means of Hoefer SemiPhor (Amersham Biosciences, Sweden). Membranes were blocked in 5% non-fat milk in Tris-buffered saline (TBS; pH 7.4) for 1 h before being incubated with rabbit ERp57 antibody (1:1000, Abcam, UK) diluted in blocking solution at 4°C overnight. Membranes were washed three times in TBS and then probed with peroxidase conjugated goat anti-rabbit immunoglobulin G (IgG) (1:1000; Beijing ZhongShan Biotechnology Co., China) for 1 h at 37°C. After washing, an enhanced chemoluminescence reaction kit (Amersham Biosciences, Sweden) was employed to detect the peroxidase activity and the image was captured by FluorChem 5500 (Alpha Innotech, USA).
ERp57 immunohistochemistry
Formalin-fixed human adult normal testis were embedded in paraffin, sectioned at 5 µm and mounted on silane-coated slides. Sections were de-waxed and re-hydrated through descending grades of alcohol to distilled water. Endogenous peroxidase was blocked by incubating the slides in 3% (v/v) hydrogen peroxidase in PBS, and then slides were subjected to microwave antigen retrieval in 0.02 M EDTA. Thereafter, they were washed in PBS and blocked with goat serum (Beijing ZhongShan Biotechnology Co.) for 2 h. They were subsequently incubated overnight at 4°C with rabbit anti-ERp57 at a 1:2000 dilution. After three washes in PBS, sections were incubated with goat anti-rabbit IgG conjugated to horse-radish peroxidase (Beijing ZhongShan Biotechnology Co.) for 1 h at room temperature. Immunoreactivity was revealed with di-aminobenzamidine. Sections were counterstained with hematoxylin and mounted onto coverslips. As a negative control, sections were incubated with normal rabbit serum (Santa Cruz, USA) in place of the primary antibody.
Immunofluorescence analysis
Spermatozoa samples from normal subjects were prepared as described above and air-dried onto poly-lysine-coated slides. Then samples were fixed with 4% paraformaldehyde in PBS for 30 min and permeabilized with 0.2% Triton X-100 in PBS for 20 min at 37°C. After three 5-min washes with PBS, slides were blocked in PBS containing goat serum (Beijing ZhongShan Biotechnology Co.) for 1 h and then incubated with a 1:50 dilution of ERp57 antibody overnight. After incubation with goat anti-rabbit immunoglobulin conjugated to FITC (Beijing ZhongShan Biotechnology Co.) at 1:1000 for 1 h, the slides were washed in PBS and coverslipped. They were viewed with a ZEISS fluorescent microscope, and images were captured with a digital camera. Negative controls were performed by replacing the primary antibody with normal rabbit serum (Santa Cruz, USA).
Dual-fluorescent stains for acrosome-reacted spermatozoa
The sperm prepared as described above were washed twice by centrifugation (10 min at 2000 rpm) in 8 ml volumes of Biggers–Whitten–Whittingham (BWW) medium (114 mM NaCl, 4.78 mM KCl, 1.71 mM CaCl2, 1.19 mM MgSO4, 1.19 mM KH2PO4, 21.58 mM sodium lactate, 5.56 mM glucose, 10 mM HEPES, 25.07 mM NaHCO3, 3 mg/ml BSA, pH 7.6). Sperm were capacitated at a concentration of 20 x 106 sperm/ml for 5 h in BWW medium. After inducing the acrosome reaction with calcium ionophore A23187
[GenBank]
(final concentration 10 µM; Sigma, USA), spermatozoa were washed with PBS, smeared on a glass microscope side and allowed to air-dry. Slides were then fixed with 4% paraformaldehyde in PBS for 30 min. The fixed sperm were placed in 30 µg/ml of fluorescein isothiocyanate (FITC)-labeled pisum sativum agglutinin (PSA, Sigma, USA) (Liu and Baker, 1988) for 1 h and then incubated with the ERp57 antibody overnight. After incubation with goat anti-rabbit immunoglobulin conjugated to TRITC (Beijing ZhongShan Biotechnology Co.) at 1:1000 for 1 h, the slides were washed in PBS and coverslipped.
Two-dimensional electrophoresis
Proteins from uncapacitated and capacitated human sperm of three fertile donors were prepared for two-dimensional electrophoresis. Isoelectric focussing (IEF) was performed using a commercially available, dedicated apparatus: IPGphor (GE Healthcare, Uppsala, Sweden). IPG strips, nonlinear pH 3–10, 24 cm long were used. Samples containing 80 µg protein were mixed with a re-hydration solution containing 8 M urea, 2% CHAPS, 20 mM DTT, 0.5% (v/v) IPG buffer (pH 3–10, NL) and 0.001% bromphenol blue. The linear ramping mode of the IEF voltage was applied according to the following paradigm: 30 V for 6 h, 60 V for 6 h, 500 V for 1 h, 1000 V for 1 h, followed by 8000 V to achieve 64 kVh at 20°C. Strips were then equilibrated at room temperature for 15 min in 8 ml equilibration solution [6 M urea, 50 mM Tris–HCl (pH 8.8), 30% (v/v) glycerol, 2% SDS, 1% (w/v) DTT] and incubated for another 15 min in an equilibration solution consisting of the same recipe with the exception that DTT was replaced with 2.5% (w/v) iodoacetamide. Second-dimension electrophoresis was performed on 12.5% SDS gels in Ettan DALTsix (Amersham Bioscience) with a constant power at 5 W per gel for the first 30 min followed by 12 W per gel until the bromphenol blue line reached the bottom of the gels. Gels were visualized by silver staining according to the published procedure except that glutardialdehyde was omitted in the sensitizing solution (Shevchenko et al., 1996).
Protein spot detection and statistical analysis
Two-dimensional electrophoresis of each sample was performed. Gels were scanned in Atrix scan 1010 plus (Microtek, Taiwan, China) and resulting images were analysed using the ImageMasterTM two-dimensional Platinum software (GE Healthcare, Uppsala, Sweden) for spot detection, quantification and comparative and statistical analyses. The amount of protein present in each spot was given as its volume, which was calculated as the volume above the spot border, situated at 75% of the spot intensity, as measured from the peak of the spot. To exclude variations due to protein loading and staining, we used relative volume (% volume), which normalized the spot volume as a percentage of the total volume of all the spots present in a gel; this normalization allowed protein expression differences to be evaluated between gels. The spot volumes from each experimental group were pooled respectively for the calculation of the mean ± SD, and an independent t-test was performed to determine the significant differences between two groups. P < 0.05 were considered statistically significant.
In-gel tryptic digestion and mass spectrometry
Protein spots were excised, dehydrated in acetonitrile and dried at room temperature. The proteins were reduced with 10 mM DTT and 25 mM NH4HCO3 at 56°C for 1 h and alkylated with 55 mM iodoacetamide and 25 mM NH4HCO3 in the dark at room temperature for 45 min in situ. Gel pieces were then thoroughly washed with 25 mM NH4HCO3, 50% acetonitrile and 100% acetonitrile in succession and were completely dried in a Speedvac. The dried gel pieces were reconstituted with 2–3 µl of trypsin solution (trypsin at a concentration of 10 ng/µl in 25 mM NH4HCO3), after incubation at 4°C for 30 min, excess liquid was discarded and gel plugs were incubated at 37°C for 12 h. Finally, TFA was added to a final concentration of 0.1% to stop the digestive reaction. MALDI-TOF analyses of trypsin digests were performed on a Bruker Biflex IV MALDI-TOF-MS (Bruker, Germany) equipped with an N2 laser (337 nm, 3-ns pulse length) in positive ion mode at an accelerating voltage of 19 kV. Peptide data were collected in the reflectron mode. Each spectrum was the accumulation of about 200 laser shots. External calibration for peptide analysis was performed using peptide calibration standards.
Assessment of acrosome reaction
Capacitated sperm were incubated for 30 min in BWW containing normal rabbit antibody (20 µg/ml) as a control or anti-ERp57 antibody (20 µg/ml). Calcium ionophore A23187
[GenBank]
was added simultaneously to induce the acrosome reaction. The percentage of acrosome reacted human sperm was evaluated by staining with 30 µg/ml of FITC–PSA. At least 200 sperm were counted under a fluorescence microscope with x400 magnification. The experiment was repeated three times.
Hamster oocyte penetration assay
Gamete incubations were performed in microdrops under paraffin oil at 37°C and 5% CO2. The sperm, prepared as described above, were washed twice by centrifugation (10 min at 2000 rpm) in 8 ml volumes of BWW medium. Different dilutions of ERp57 antibody (final concentration of 5, 10 and 20 µg/ml) and the control antibody (final concentration: 20 µg/ml) were added to the sperm suspension. Sperm were capacitated at a concentration of 20 x 106 sperm/ml for 5 h in BWW medium followed by 30 min incubation in calcium ionophore A23187
[GenBank]
(Sigma). Hamster oocytes were obtained from Golden Syrian hamsters and cumulus cells were removed by treating oocytes with 1 mg/ml hyaluronidase (Sigma). Zona pellucida were removed with 1 mg/ml pronase (Sigma) (Busso et al., 2005). Following sperm/antibody incubation, zona-free hamster oocytes were added directly to the sperm suspension, and the gametes were co-incubated for 3 h. Following gamete co-incubation, loosely bound sperm were removed from the oocytes by gentle pipetting. The oocytes were then treated with Hoechst for 10 min to stain the chromatin. To quantitate binding, oocytes were placed between a microscope slide and an elevated coverslip, and the number of sperm bound per oocyte was recorded. The number of sperm fused per oocyte was scored by counting the number of Hoechst-stainded sperm heads within each oocyte using fluorescent microscopy. The assay was repeated at least three times for each concentration of IgG used.
Statistical analysis
Data were expressed as means±SD. One way analysis of variance was used to analyse the data and significant results were examined with the least significant difference post hoc test.
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Localization of ERp57 in adult human testis and spermatozoa
The expression of ERp57 protein in human testis was determined via western blotting and immunohistochemistry. Immunoblots show that ERp57 was detected as a 57 kDa band that was highly and specially expressed in human testis and sperm (Fig. 1). Figure 2 shows intense ERp57 immunostaining in the human testes. Dense staining was observed in numerous cell types in various stages of spermatogenesis, from the primary spermatocyte to spermatozoa phases. Staining was also high in Leydig cells but relatively faint in Sertoli cells (Fig. 2).
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57 kDa in both HT and HS
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The expression of ERp57 protein in human spermatozoa was investigated in greater detail. Intense fluorescent staining was observed in the entire acrosome of human spermatozoa, including the anterior acrosomal region and the equatorial segment, and in the flagellum (Fig. 3a–d). Interestingly, ERp57 protein was expressed mostly on the equatorial segment and the flagellum of the acrosome-reacted spermatozoa, as shown through dual-fluorescent staining (Fig. 3e–g).
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Differential expression patterns of ERp57 before and after capacitation
We identified the phosphorylation style of ERp57 in human testis. Then we constructed proteomic expression maps of uncapacitated and capacitated human sperm from three fertile donors. Mass spectrum identification showed five spots as the ERp57 protein (Fig. 4a and b). All spots had the same molecular weight but differing pIs. Among the five spots, two showed significant changes in expression levels before and after capacitation. As shown in Fig. 4c and d, the expression level of Spot 1 decreased, whereas Spot 5 increased after capacitation. These results suggest that during human sperm capacitation, the protein undergoes post-translational modifications, perhaps phosphorylation.
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Effects of anti-ERp57 antibodies on human sperm acrosome reaction
Capacitated human sperm were incubated in BWW containing control antibody (20 µg/ml) and ERp57 antibody (20 µg/ml) and then assayed by FITC-PSA to determine the percentage of acrosome reacted sperm. In the A23187 [GenBank] -induced acrosome reaction, the percentage of acrosome reacted sperm was (50.83 ± 1.34)% in the medium containing ERp57 antibody, (50.70 ± 3.44)% in the medium containing control antibody and (50.77 ± 2.85)% in the medium containing only A23187. [GenBank] Thus, no inhibition was observed when ERp57 antibody was added to the medium, according to the statistical assay. The proportion of acrosome reacted sperm in the control was (4.93 ± 1.39)%, representing the degree of spontaneous acrosome reaction (Fig. 5).
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ERp57 antibody blocks human sperm from penetrating zona-free hamster oocytes
To assess the possible role of ERp57 in human gamete fusion during fertilization, we tested whether the ERp57 antibody could block human sperm from penetrating zona-free hamster oocytes. Sperm were incubated with ERp57 antibody or control antibody in BWW, and the number of oocytes with sperm penetration was calculated. Figure 6a shows sperm fused with the oocyte as visualized with the Hoechst stain for chromatin. The number of sperm penetrating zona-free hamster oocytes was 11.68 ± 0.94 with a control antibody, whereas at the concentration of 5 µg/ml, the ERp57 antibody reduced the number of penetrated sperm per oocyte to 5.74 ± 1.59 (P < 0.01 compared with controls) and at the increased concentration of 20 µg/ml, this number was reduced further to 1.23 ± 0.20 (P <0.01 compared with controls) (Fig. 6b).
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ERp57 expression levels differ in spermatozoa of IVF patients and fertile donors
To determine the clinical relevance of ERp57, its protein expression levels in the spermatozoa of fertile men and in infertile patients with high and low IVF fertility rates were compared. Western blotting was performed on a constant number of spermatozoa obtained from subjects in each group described above. Densitometric data were expressed as a ratio of ERp57 to tubulin. The fertile sperm donor group had ratios that ranged from 1 to 1.6. In the IVF patients, those with a high fertilization rate had ratios that ranged between 0.7 and 1.3, whereas those with a low fertilization rate had ratios between 0.1 and 0.9 (Fig. 7). Statistical analysis showed that ERp57 expression in sperm of IVF patients with a high fertilization rate did not differ from that in fertile sperm donors, but IVF patients with a low rate had significantly lower expression levels than did fertile sperm donors (P < 0.01) and IVF patients with a high fertilization rate (P < 0.01).
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Discussion |
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Recent work from our laboratory identified numerous proteins related to human spermatogenesis and sperm function via a human testis protein profile of our construct. The current study focussed on one of these newly recognized proteins, namely ERp57, in order to determine its function during the human fertilization process and its clinical relevance in spermatozoa.
The expression pattern of the ERp57 protein indicates that it may play a role in the human fertilization process. ERp57 was highly expressed in human testis, as also shown in our previous findings, and was located in cells from the primary spermatocyte to the spermatozoa phases of spermatogenesis. It was also prominent in Leydig cells but expressed at relatively modest levels in Sertoli cells. In addition to its expression in the testes, ERp57 was prevalent in human sperm, primarily in the acrosome and tail. Moreover, ERp57 was observed mostly in the equatorial segment and tail of acrosome reacted sperm. This is different from the position of ERp57 in mouse sperm (Ellerman et al., 2006). This difference may be due to the diverse species differences between human and mouse. This distribution of ERp57 suggests that it is important not only in spermatogenesis, but also in mature sperm function, especially during processes crucial for human fertilization, such as capacitation, the acrosome reaction and fusion with oocytes.
In addition to its localization, a possible function of ERp57 in fertilization is suggested by its patterns of post-translational processing. In the human testis protein profile, two sites in ERp57 that appear to undergo phosphorylation were previously identified. Moreover, bioinformatical analysis showed that ERp57 had several phosphorylation sites. During human sperm capacitation, we identified five spots as ERp57, all of which had identical molecular weights but different pIs. Two of the five spots (Spots 1 and 5) exhibited significant differences in expression levels between uncapacitated and capacitated sperm, and the other three spots remained unchanged. Changes in processing of ERp57 during capacitation suggest that it may undergo some kind of modification, perhaps phosphorylation. Protein phosphorylation is a necessary modification for capacitation that enables spermatozoa to fertilize oocytes (De et al., 1997; Visconti and Kopf, 1998). Other proteins, such as endoplasmin and heat shock protein 60, have been shown to be targets for phosphorylation on the surface of mouse spermatozoa after capacitation. Phosphorylating proteins may change their conformation, thus facilitating sperm-zona recognition (Asquith et al., 2005).
After capacitation, sperm should undergo the acrosome reaction. Through assessment of the acrosome reaction using FITC–PSA staining, ERp57 antibody was verified as not preventing A23187 [GenBank] -induced acrosome reaction. During the fertilization process, ERp57 may specifically act to mediate fusion between the sperm and oocyte. Blocking ERp57 using antibodies to the protein significantly inhibited the penetration of human sperm into oocytes, as assessed with the zona-free hamster oocyte assay. This assay is considered a measure of sperm–oocyte plasma membrane fusion and is proposed as a tool to identify male infertility (Yanagimachi et al., 1976). Other proteins, such as sp-10 (Hamatani et al., 2000), SOB2 (Lefevre et al., 1997), FLB1 (Boue et al., 1995) and FA-1 (Naz, 1987), that are recognized by monoclonal antibodies directed against human spermatozoa and have also been shown to be involved in oocyte penetration via the same assay. Here we demonstrate that ERp57, a sperm surface protein, is an important molecule involved in human gamete fusion.
The contribution of ERp57 to sperm function may be mediated via redox of thiol groups. ERp57 has been shown to efficiently catalyze disulfide reduction, disulfide isomerization and dithiol oxidation in substrate proteins in somatic cells and may also do so in sperm. Mammalian spermatozoa possess high concentrations of thiol groups, which participate in a number of physiological processes (Mercado et al., 1976). Thiol groups in sperm flagellum are involved in the maintenance of motility, and they play a role in increasing the stability of sperm heads and tails during maturation (Cornwall and Chang, 1990). Besides the above functions of thiol groups, redox of the fusion-related sperm thiol proteins may be related to sperm–oocyte fusion (Mammoto et al., 1997; Nivsarkar et al., 1998; De and Gagnon, 2003). Thus, ERp57 may influence fertilization through redox of sperm thiol-proteins.
The above findings suggest that ERp57 contributes to fusion between the sperm and oocyte during human fertilization; however, an important question arose as to whether it affected the ability of sperm to fertilize oocytes. This point is particularly important given the apparent rise in rates of infertility. Our results showed that fertile sperm donors and IVF patients with high rates of fertilization had similarly high levels of ERp57, whereas IVF patients with low fertility had markedly lower levels of the protein. Thus, levels of ERp57 were related to fertilization rate with low levels associated with low fertility and vice versa. This suggests that decreased levels of the protein may contribute causally to infertility. Many studies have attempted to discern the reasons for infertility and several assisted reproductive technologies (ATRs), such as IVF and intracytoplasmic sperm injection, have been applied to treat the condition (Diedrich et al., 1995). However, these technologies have a relatively low success rate resulting in 34.3% clinical pregnancies (Sullivan et al., 2006). Considering that this low success rate has major psychological and financial impacts, development of tests to predict the outcome of ATRs is a major interest. Assays with predictive value, including semen analysis, andrological assays, spermiogram value and functional assays, exist; however, they are cumbersome and have limited accuracy (Check et al., 2002). Recent attempts to develop predictive tests have focussed on identifying sperm-related proteins, such as P34H (Sullivan et al., 2006), HSPA2 (Ergur et al., 2002) and protamine (Aoki et al., 2006). Since ERp57 expression was decreased dramatically in the IVF patients with a low fertilization rate, its levels in sperm may predict its ability to fertilize oocytes and could be used to select sperm for IVF. This leads to the intriguing possibility that the levels of the protein may predict fertilization success.
In summary, ERp57 is located in the spermatogenic cell cytoplasm of the human testes as well as in the human sperm acrosome and tail. The protein is modified during sperm capacitation and may play an important role in gamete fusion. Although the molecular mechanisms of ERp57 in the male infertility need to be further studied, we predict that ERp57 could be a novel phenotype marker for male infertility and may have the potential to be used to assess sperm selection for IVF.
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The research was supported by grants from National Basic Research Program of China (2006CB504002 and 2006CB944002), National Natural Science Foundation of China (30630030) and Program of Changjiang Scholars and Innovative Research Team in University (IRT0631).
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Submitted on May 17, 2007; resubmitted on July 4, 2007; accepted on July 11, 2007.
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