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RESEARCH |
1 Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei 104, Taiwan2 Mackay Medicine, Nursing and Management College, Taipei 112, Taiwan3 Department of Medical Research, Mackay Memorial Hospital, Tamshui 251, Taipei County, Taiwan4 Department of Obstetrics and Gynecology, Taipei Medical University, Taipei 110, Taiwan5 Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
Correspondence should be addressed to S-H Li; Email: lsh{at}ms1.mmh.org.tw
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Abstract |
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Introduction |
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Imbalances between SPINK proteins and proteases may cause severe diseases, for example, mutations of human SPINK1 are thought to be associated with pancreatitis (Pfutzer et al. 2000, Kuwata et al. 2001), and Spink3 (a mouse orthologue gene) knockout mice have no pancreatic acinar cells owing to autophagic cell death and impaired regeneration (Ohmuraya et al. 2005).
The mouse SPINK3 protein, also called pancreatic secretory trypsin inhibitor or P12, is constitutively expressed in the pancreas (Mills et al. 1987); however, its expression is indeed prominent in sex accessory tissues of adult male mice, particularly in mouse seminal vesicles (Chen et al. 1998). It was demonstrated to bind to mouse sperm and shows an inhibitory effect against Ca2+ uptake by mouse sperm (Chen et al. 1998).
Seminal vesicle secretions (SVSs) constitute the major portion of seminal plasma. Removal of the seminal vesicle greatly reduces fertility (Pang et al. 1979, Peitz & Olds-Clarke 1986). Thus, the components of SVSs may affect sperm function. In fact, several proteins purified from murine SVSs have been demonstrated to affect sperm physiology (Chen et al. 1998, Huang et al. 1999, Luo et al. 2001, Li et al. 2005). However, other components of SVSs remain to be identified. Further investigation of the proteome may enhance our understanding of normal and abnormal aspects of male reproductive physiology. In addition to SPINK3, we found a SPINK-like (SPINKL) protein in the SVS. In this study, we report its purification and characterization and examine its protease inhibitory activities and effects on sperm in vitro.
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Results |
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8 kDa proteins (Fig. 1D, lane 7). The mass analyses revealed that at least six species with molecular masses of 9033.4–9852.7 kDa in the mixed peak a-to-c samples (Fig. 2A) manifested unusual mobility on reducing SDS-PAGE. Thus, peak a-to-c proteins apparently were glycoproteins with differential N-linked carbohydrate moieties.
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Automated Edman degradation of each peak of the a-to-c samples for 15 cycles gave reliable data, which were assembled into the N-terminal sequences. Two amino acids were detected in each cycle during the protein analysis of each of the peaks a–c. The actual yields of the two sequences in an individual cycle were such that the ratios of the major sequence to the minor ones were estimated to be 1.7–2.9:1. Assembly of the major and minor sequences produced peptide sequences of VNIFAGPENVIKEPX and AGPENVIKEPXXTMY respectively with the latter being the major one. The two N-terminal peptides were completely confirmed in the hypothetical protein consisting of 90 amino acid residues in all positions, except that X (asparagine or cysteine) was not identified in the protein sequence (Fig. 1F). This asparagine residue is the only potential site for N-linked carbohydrates in this protein. Post-translational cleavage at the peptide bond between Ser and Val or between Phe and Ala in the signal peptide of the hypothetical protein sequence gave rise to the peak a-to-c proteins. As a result, peaks a–c share two protein cores with different N-linked glycans. They were estimated to be 4–5 mg/ml in the seminal vesicle fluid. Thereafter, we combined them to produce antibodies. Among SVS protein components on the SDS-PAGE gel, the antibody against SPINKL immunoreacted only to a broad 12–15 kDa band corresponding to the antigen, thus indicating the high specificity of the antibody (Fig. 1E).
A BLASTP search of the non-redundant protein database using the hypothetical protein sequence as the query revealed that this protein is similar to a group of SPINK proteins, with the highest identity (64%) found to be with mouse SPINK11. Thus, we named it a serine protease inhibitor Kazal-type-like protein, abbreviated to SPINKL protein. A computer-annotated UniProtKB/TrEMBL entry (Q8CEK3) was previously assigned according to the translation of a coding sequence corresponding to Spinkl mRNA. We have submitted the MS and Edman data of SPINKL to SwissProt to update this entry. From the entry, we found that the SPINKL protein was encoded by the 9530002K18Rik gene and was mapped to chromosome 18 B3. Interestingly, Spink3 and Spink11 also have the same chromosome location, even though Spink11 is immediately adjacent to 9530002K18Rik.
Alignment of the SPINKL protein with several murine SPINK proteins retrieved from SwissProt revealed that the SPINKL protein nearly completely matched the putative Kazal signature except for an additional residue inserted between C2 and C3, indicating that the SPINKL protein is potentially a member of the SPINK protein family (Fig. 3).
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0.6 kb band corresponding to Spinkl mRNA was exclusively detected in seminal vesicles (Fig. 5A). Several characteristic forms of the SPINKL protein were detected by Western blotting (Fig. 5B). When an equal amount of total RNA from the homogenate of a non-reproductive organ was compared with that of the seminal vesicle, no Spinkl mRNA was found in the brain, heart, lungs, liver, spleen, kidneys, stomach, small intestines, muscle, skin, thymus, placenta, or bladder (data not shown).
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To evaluate the effect of testosterone on Spinkl gene expression in seminal vesicles, we compared the findings in castrated mice. The relative levels of Spinkl mRNA in seminal vesicles were significantly lower in castrated mice treated only with vehicle compared with castrated mice treated with testosterone (data not shown). The immunohistochemical results revealed that the SPINKL protein intensity in the mucosal epithelium of castrated mice treated with only corn oil (Fig. 5E) was obviously lower than that in normal adults or castrated mice treated with testosterone (Fig. 5D and F). Taken together, these results suggest that Spinkl gene expression in the seminal vesicles is modulated by androgen.
Enhancement of sperm motility by SPINKL in vitro
We used indirect fluorescence staining to assess the binding of SPINKL to epididymal sperm. Weak fluorescence was seen on the epididymal sperm after they had been consecutively treated with the SPINKL antibody and the fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG, indicating that a small amount of the SPINKL protein exists on the sperm surface (Fig. 6A and B). In contrast, when spermatozoa were preincubated with SPINKL proteins, the FITC fluorescence was prominent on the middle piece, relatively weak on the tail, and not found on the head (Fig. 6C and D). Thus, spermatozoa have a SPINKL-binding zone on their surface. Moreover, when the ejaculated sperm on the slides were immunostained with the antiserum pretreated with SPINKL protein, no fluorescence signal was detected (Fig. 6E and F). However, the SPINKL protein was immunodetected on the surface of the ejaculated sperm despite a high fluorescence background due to a large amount of SPINKL protein in semen that was difficult to remove completely during the sperm preparation (Fig. 6G and H).
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58%) after supplement of 3 mg/ml of BSA. However, SPINKL inhibited BSA-induced sperm capacitation with significant decreases after addition of 0.2 or 0.6 mg/ml SPINKL to the BSA-containing medium (Fig. 9A). Even an 82% decrease was found in the incubation with 1 mg/ml of SPINKL (not shown). These observations were correspondent to that inhibition of BSA-induced tyrosine phosphorylation by SPINKL (Fig. 8).
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We subsequently used in vitro fertilization (IVF) to test the effect of SPINKL on sperm fertility. The epididymal sperm had a lower 20% fertilization rate, which may result from spontaneous acrosome-reacted sperm, in the medium without any proteins. Addition of SPINKL to the medium did not seem to influence the sperm fertilization rate. As usual, BSA-capacitated epididymal spermatozoa largely fertilized the cumulus-intact eggs. By contrast, SPINKL significantly impaired the fertilization rate of BSA-capacitated epididymal spermatozoa with 36 and 52% decreases when treatment with 0.2 and 0.6 mg/ml SPINKL respectively (Fig. 10). Therefore, the SPINKL protein disturbed the IVF process between spermatozoa and oocytes.
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Discussion |
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The evidence that the SPINKL protein is a secreted member of the SPINK family includes its purification from SVSs and its expression in the lumen and apical region of the mucosal epithelium (Fig. 5). Edman sequencing indicated that the SPINKL protein has a 23- or 27-amino acid signal peptide (Fig. 1F). Removal of the hydrophobic leader sequence left a protein core consisting of 63 or 67 amino acid residues with a molecular mass of 7379 or 7853 kDa respectively. However, mass data revealed several SPINK products in the SVS with molecular masses of 9033–9852 kDa (Fig. 2A).
The results of PAS staining and N-glycosidase F treatment demonstrated that the SPINKL protein is a glycoprotein. The protein molecule contains only one potential N-glycosylation site (Fig. 1F). The results of Edman sequencing suggested that it is an N-glycosylated site. In contrast to these findings, SPINK3 is not a glycoprotein (our unpublished data).
There are several similarities between SPINKL and SPINK3, including the same gene locus in chromosome 18, gene regulation by testosterone, tissue-specific expression and localization in the mouse seminal vesicles, and conserved cysteine positions that are a feature of the SPINK protein family (Chen et al. 1998 and herein). Nonetheless, they differ in terms of glycosylation nature, protease inhibitory activities, and low sequence identities.
The growth and differentiation of seminal vesicles are closely associated with androgens (Chai 1956, Morley & Wright 1972). Many gene expressions in this tissue have been demonstrated to be regulated by androgen (Chen et al. 1987, 1998, Yu et al. 1993, Li et al. 2005, 2006). In this study, regulation of the Spinkl gene was also the case (Fig. 5). Androgen and its receptor form a complex that interacts with androgen response elements (AREs) in androgen-responsive genes and regulates their expression. We employed the MatInspector program (Cartharius et al. 2005) to predict the potential AREs in the promoter to a part of the intron 1 region of 9530002K18Rik corresponding to nucleotides 70 000 to 72 486 of GenBank accession no. AC124181. We found one conserved ARE in the promoter and another one in the intron 1 region. Thus, it is possible that androgen acts directly on Spinkl to regulate its gene expression.
The SPINKL protein can enhance sperm motility, especially the movement velocity and the LIN of cell tracks as they swim (Fig. 7). The molecular mechanism of how it enhances sperm motility deserves our further studies. It is noted that SPINKL protein inhibits BSA-induced in vitro capacitation and thus blocks sperm–oocyte interactions (Figs 8–10
). Therefore, SPINKL may inhibit premature sperm capacitation and serve as a decapacitation factor.
Several murine decapacitation factors have been identified, including a 40 kDa glycoprotein (Fraser 1998), a phosphatidylethanolamine-binding protein (Nixon et al. 2006), and two secreted seminal vesicle proteins, SVA (Huang et al. 2000) and SVS2 (Kawano & Yoshida 2007). The seminal vesicles secrete primarily basic proteins (Gerhardt et al. 1985). However, SPINKL and SVA are the acidic seminal vesicle-secreted proteins. Although they all inhibit BSA-stimulated sperm capacitation in vitro, their effects on sperm motility are very different. SPINKL enhances sperm motility, while SVA inhibits motility (Huang et al. 1999). The differences remain to be determined.
Although proteases have been implicated in the reproduction, only a few are associated with the process of capacitation (Zheng et al. 2001, Kawakami et al. 2004). As for serine protease inhibitors, there also seem few reports to point out the direct action on the process of sperm capacitation. A protease inhibitor of seminal vesicle origin was shown to block sperm's zona binding and acrosome reaction, thus has been suggested to be a decapacitation factor (Boettger-Tong et al. 1992). SPINKL may also be a decapacitation factor but with no protease inhibitor activity. It is of interest to note that SPINK3, another seminal vesicle protease inhibitor, possesses trypsin inhibitor activity but has no effect on capacitation (Lin et al. 2006). Thus, for a serine protease, the inhibitor activity and the role as a decapacitation factor may be not necessarily related.
Several signal transduction pathways have been demonstrated to be associated with sperm capacitation, including the extracellular signal-regulated kinase pathway (Luconi et al. 1998, de Lamirande & Gagnon 2002), the cyclic AMP/protein kinase A pathway (Visconti et al. 1995b, Breitbart 2003), and the reactive oxygen species affected pathway (de Lamirande & O'Flaherty 2008). We can speculate that SPINKL may affect the capacitation-related signal transduction machinery of sperm. To uncover its mechanism of action, we may start from searching the binding partners of SPINKL on the sperm plasma membrane by chemical cross-linking reagents or examining the activity of various kinases associated with capacitation. Whether the components of the pathways mentioned above are involved in the inhibition of capacitation by SPINKL remains to be investigated.
In conclusion, we have purified and identified a novel protein, SPINKL, which has the characteristic signature structure of the Kazal domain of SPINK, and we demonstrated its tissue-specific expression in mouse seminal vesicles. It is interesting to note that the SPINKL protein has no inhibitory activities against common serine proteases. The amount of the SPINKL protein in seminal vesicles is modulated by androgen and is correlated with the stage of animal maturation. SPINKL is a sperm-binding protein and is able to enhance sperm motility. Furthermore, it is able to inhibit BSA-induced sperm capacitation and impair sperm–oocyte interactions in vitro.
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Materials and Methods |
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Protein purification
Normal adult mice (10–12 weeks old) were killed by cervical dislocation. The seminal vesicles of 50 mice per separation were carefully dissected to free them from the adjacent coagulating glands, and the secretions were directly squeezed into 50 ml of ice-cold 10 mmol/l Tris–HCl in the presence of 1 mmol/l phenylmethyl sulfonyl fluoride at pH 8.0. After centrifugation at 10 000 g for 15 min, the supernatant was resolved by ion-exchange chromatography on a diethylaminoethyl (DEAE)-Sephacel (Amersham Pharmacia Biotech, Uppsala, Sweden) column (12x2.6 cm) into four fractions (Fig. 1A), as described previously (Li et al. 2005, 2006). Fraction IV collected from three separations was concentrated and subjected to a Sephadex G-75 (Sigma Chemical Co.) column (2.6x120 cm) pre-equilibrated with 50 mmol/l Tris–HCl and 150 mmol/l NaCl (pH 7.4). The column was eluted in the same buffer at a flow rate of 10 ml/h; fractions (2 ml) were collected, and the absorbance records are shown in Fig. 1B. Among them, peak 3 was further subjected to ion exchange HPLC on a Q HyperD 10 (Beckman, Fullerton, CA, USA) column (4.6 mmx10 cm). The column was eluted with a linear gradient of 0–15% (w/v) 1.0 mol/l NaCl in 25 mmol/l Tris–HCl (pH 8.2) at a flow rate of 1.0 ml/min for 40 min (Fig. 1C). SPINK3 was purified following a previously described method (Lai et al. 1991).
Mass spectrometry
The molecular mass was determined on an Ultraflex II MALDI-TOF/TOF mass spectrometer (Bruker Daltonik, Bremen, Germany). A sample was dissolved in 0.1% (v/v) trifluoroacetic acid (TFA) to a final concentration of 100 µmol/l. A sample of 1 µl was mixed with 1 µl matrix (50 mmol/l 2,5-dihydroxybenzoic acid in 50% (v/v) acetonitrile), then spotted onto a plate for mass analyses. For protein identification, protein bands on the SDS-PAGE gel were excised, washed in a solution containing acetonitrile and 100 mmol/l NH4HCO3 (1:1, v/v), and subjected to in-gel digestion with trypsin overnight at 37 °C. The tryptic peptides were then extracted with a solution of 50% (v/v) acetonitrile and 1% (v/v) TFA, lyophilized, resuspended in 0.1% (v/v) TFA, and analyzed by LC/MS/MS equipped with Agilent 1100 Series HPLC (Agilent Technologies, Palo Alto, CA, USA) and an LTQ FT hybrid mass spectrometer (Thermo Electron, San Jose, CA, USA). The MS/MS data were used for protein identification using the MASCOT search engine (http://www.matrixscience.com) based on the International Protein Index databases (http://www.ebi.ac.uk/IPI).
Protein analysis
Removal of the N-glycoconjugate from a glycoprotein was performed using N-glycosidase F (New England BioLabs, Beverly, MA, USA). PAS staining of the glycoproteins was performed using the Gelcode glycoprotein staining kit (Pierce, Rockford, IL, USA). The protein concentration was determined using a bicinchoninic acid protein assay (Smith et al. 1985). The amino acid sequence was determined using automated Edman degradation with a 492-protein sequencer and an online 140 C analyzer (Applied Biosystems, Foster City, CA, USA). A database search was performed using the Basic Local Alignment Search Tool (BLAST) algorithms against the non-redundant database up to October 2006 (http://www.ncbi.nlm.nih.gov/BLAST). Multiple protein sequence alignments were performed with ClustalW (http://www.ebi.ac.uk/clustalw). AREs in a promoter were analyzed by MatInspector (http://www.genomatix.de; Cartharius et al. 2005) against the matrix family library version 6.2 (October 2006).
Assay of inhibitory activity
The inhibitory activities of the SPINKL protein towards serine protease, trypsin, 
-chymotrypsin, subtilisin Carlsberg, and elastase (Sigma) were assayed by measuring the absorbance of p-nitroaniline produced by these proteases at 410 nm following previously described methods (Hergenhahn et al. 1987, Somprasong et al. 2006).
Antibody production and Western blotting
Antisera against SPINKL were produced in two New Zealand white rabbits. Preimmune serum was collected before the immunization. For the immunization, the purified SPINKL protein in normal saline (0.4 mg/ml) was emulsified with Freund's complete adjuvant (1:1, v/v). In total, 2 ml of the mixture was injected subcutaneously in multiple sites in one rabbit. Rabbits were boosted twice every 3 weeks with the mixture of the same amount of purified protein and Freund's incomplete adjuvant (1:1, v/v). Antiserum was collected in 10 days after the last injection.
One hundred micrograms of the purified SPINKL proteins were conjugated to AminoLink beads according to the manufacturer's instructions (Pierce). Antisera against SPINKL were adsorbed by the conjugated beads to remove the specific antibody to SPINKL. The treated antiserum was used as the control antiserum.
Proteins were resolved using SDS-PAGE on a 15% gel slab (8.2x7.3x0.075 cm) and were stained with Coomassie brilliant blue or transferred to a nitrocellulose membrane for immunostaining according to previously described methods (Towbin et al. 1979). Membranes were blocked with 5% (w/v) skim milk in PBS (blocking solution) for 2 h, and then incubated with anti-SPINKL antiserum (1:10 000) in blocking solution for 1 h at room temperature. After gentle agitation in four changes of PBS for 15 min each, they were immunoreacted with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Amersham Pharmacia Biotech) diluted to 1:10 000 in the blocking solution for 1 h. Immunoreactive bands were revealed using an enhanced ECL substrate according to the manufacturer's instructions (Pierce).
Immunohistochemical staining
Immunostaining was performed according to our previously described methods (Li et al. 2005, 2006). In brief, tissues slides were blocked with 10% (v/v) normal goat serum in PBS (blocking solution) for 1 h at room temperature and then incubated with anti-SPINKL antiserum diluted 1:1000 in the blocking solution at 4 °C for 16 h. After washing, the slides were treated with biotin-conjugated goat anti-rabbit IgG (1 µg/ml; Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) in the blocking solution for 1 h at room temperature. The slides were washed again and then incubated with HRP-conjugated streptavidin (1 µg/ml; Zymed Laboratories, South San Francisco, CA, USA) in the blocking solution for 40 min at room temperature. Protein signals were detected by 3-amino-9-ethylcarbazole staining (Zymed Laboratories). The slides were then counterstained with hematoxylin (Vector Laboratories, Burlingame, CA, USA) and photographed using an Olympus BX 40 microscope (Olympus, Tokyo, Japan) equipped with an Olympus DP-70 digital camera.
RNA isolation and northern blot analysis
Total RNA was extracted from tissue homogenates using an RNeasy Mini kit (Qiagen). PCR-amplified fragments of Spinkl cDNA (454 bp) or glyceraldehyde-3-phosphate dehydrogenase (Gapdh) cDNA (557 bp) were used as the templates to prepare the 32P-labeled cDNA probe using a random-priming kit (Promega). RNA samples (20 µg) were subjected to 1.0% (w/v) agarose–formaldehyde gel electrophoresis, blotted onto nylon membranes by capillary transfer and hybridized with a probe as previously described (Maniatis et al. 1989). In brief, blotted membranes were first hybridized with Spinkl probes overnight at 45 °C, and then the free probes were washed away. Messenger RNA signals on the membranes were examined by phosphoimaging. Subsequently, membranes were examined with the Gapdh probes in the same way after removal of the Spinkl probes. Thus, hybridization with the Spinkl and Gapdh probes was performed on the same membranes.
Immunostaining of sperm
Freshly prepared epididymal spermatozoa (106 cells/ml) were blocked in PBS containing 10% (v/v) normal goat serum (blocking solution) for 30 min at room temperature. The cells were further incubated with or without 0.5 µmol/l SPINKL for 1 h. At the end of the incubation, the cells were centrifuged and the cell pellets were washed with PBS to remove the unbound ligands. The cells were air-dried on a glass slide and washed twice with PBS. The slides were incubated with the anti-SPINKL antiserum (1:1000) in the blocking solution at 4 °C for 16 h. The slides were washed thrice with PBS to remove any excess antibodies before they were incubated with FITC-conjugated goat anti-rabbit IgG (Vector Laboratories) diluted to 1:3000 in blocking solution for 1 h. All slides were then washed with PBS, counterstained with 5 µg/ml Hoechst 33 258, and mounted in 100 µl of ProLong Gold antifade medium (Invitrogen Molecular Probes). Slides were examined by laser scanning confocal microscopy using a Leica TCS SP (Leica-Microsystems, Heidelberg, Germany) equipped with an argon/krypton ion laser and u.v. laser with the appropriate filter spectra adjusted to detect the FITC and Hoechst fluorescence.
The ejaculated sperm were collected from semen in the uterine cavity of three female mice with plugged vaginas. After extensive washing with PBS, the ejaculated sperm without incubation with the exogenous SPINKL protein were smeared on slides for immunolocalization of SPINKL as mentioned above.
Analysis of sperm motility
Sperm (106 cells/ml) from the cauda epididymis of 12- to 14-week-old males were capacitated in 150 µl of a modified simple-optimized medium (mKSOM; Summers et al. 2000), but with a modified 3 mg/ml of BSA under mineral oil at 37 °C in an atmosphere of 5% (v/v) CO2 in humidified air. To analyze the effect of SPINKL on sperm motility, the medium was supplemented with 0.2 mg/ml SPINKL or the BSA was replaced with 0.2 mg/ml SPINKL. Sperm motility was determined using a computer-assisted sperm assay with a sperm motility analyzer (IVOS version 10; Hamilton-Thorne Research, Beverly, MA, USA). A 10 µl sample was placed in a 10 µm deep Makler chamber at 37 °C for analyses at 30 min intervals up to 150 min. Motility parameters, including movement velocities of VCL, VAP, and VSL, the percentages of sperm motility (VAP >7 µm/s) and progressive motility (VAP >50 µm/s and STR >80%, where STR=VSL/VAP), and the LIN (LIN=VSL/VCL), which measures the departure of the cell track from a straight line, were measured. The analyzer settings were described previously (Li et al. 2005). For each assay, five to ten fields with a range of 350–400 sperm were analyzed.
Evaluation of sperm capacitation and acrosome reaction
Sperm capacitation in the molecular basis was examined by detecting the capacitation accompanied increase in the protein tyrosine phosphorylation of a subset of proteins of molecular weight 40 000–120 000 according to the previously described method (Visconti et al. 1995a). In brief, about 4x107 spermatozoa/ml were incubated in a modified Krebs–Ringer bicarbonate medium (Lee & Storey 1986) with or without BSA, as the positive or negative control respectively, or the medium supplemented with SPINKL, or BSA replaced with SPINKL at 37 °C in an atmosphere of 5% (v/v) CO2 in humidified air for 90 min. Then, the soluble fraction of sperm protein extracts was subjected to SDS-PAGE on an 8% gel slab. The proteins on the gel were electrotransferred onto a nitrocellulose paper. Western blot analyses were performed by using anti-phosphotyrosine antibody according to a previous method (Visconti et al. 1995a).
CTC fluorescence-staining method, as previously described (Ward & Storey 1984, Lee & Storey 1985), was conducted to morphologically assess sperm capacitation. In this experiment, we used the mKSOM medium instead of original modified Tyrode medium. Freshly prepared epididymal spermatozoa (106 cells/ml) were capacitated in 150 µl mKSOM medium with or without BSA (3 mg/ml), as the positive or negative control respectively at 37 °C in an atmosphere of 5% (v/v) CO2 in humidified air for 90 min. The medium was supplemented with SPINKL or the BSA was replaced with SPINKL to analyze the effects of SPINKL on sperm capacitation in vitro. The CTC staining of sperm was carried out following the original method and examined using a fluorescence microscope (BX 40, Olympus).
For analyzing the sperm acrosome reaction, the capacitated sperm described above was treated with 10 µmol/l A23187 [GenBank] in DMSO (0.2%) at 37 °C for 30 min. Sperm were smeared on the slide and fixed with methanol for 30 s. The sperm acrosomal status was assessed by staining with 5 µg/ml of Alexa Fluor 488-conjugated peanut agglutinin lectin (PNA; Invitrogen Molecular Probes) at dark for 10 min. The slides were immediately examined with a fluorescence microscope (BX 40, Olympus).
IVF
Epididymal sperm (2x105 cells/ml) in 150 µl mKSOM medium under mineral oil with or without BSA or/and SPINKL were capacitated for 90 min at 37 °C in an atmosphere of 5% (v/v) CO2 in humidified air. Oocyte–cumulus complexes collected by superovulation treatment (Gates & Bozarth 1978) were added to the same medium. After 6 h of insemination, the oocytes were washed with mKSOM medium, fixed on the slide with 4% (w/v) paraformaldehyde, and stained in 5 µg/ml Hoechst 33258 for 3 min. The slides were observed with a fluorescence microscope (BX 40, Olympus). Two pronuclei embryos were scored as fertilized.
Statistical analysis
Data are presented as the mean±S.D. Differences were analyzed by the Bonferroni post hoc test followed by one-way ANOVA using InStat software (GraphPad, San Diego, CA, USA). A P value of <0.05 was considered to be significant.
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Declaration of interest |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestFundingReferences |
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestFundingReferences |
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Footnotes |
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Received 14 August 2007
First decision 10 October 2007
Revised manuscript received 8 August 2008
Accepted 20 August 2008
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References |
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) against trypsin was found (A and B), in contrast with the positive control of SPINK3 (
), a known serine protease inhibitor towards trypsin (A). Data represent the mean of three repeated experiments and error bars represent the S.D.
), or the medium was supplemented with 0.2 mg/ml SPINKL (
