TY - JOUR
AU - Nixon, Brett
AB - Abstract A unique characteristic of mammalian spermatozoa is that, upon ejaculation, they are unable to recognize and bind to an ovulated oocyte. These functional attributes are only realized following the cells' ascent of the female reproductive tract whereupon they undergo a myriad of biochemical and biophysical changes collectively referred to as ‘capacitation’. We have previously shown that this functional transformation is, in part, engineered by the modification of the sperm surface architecture leading to the assembly and/or presentation of multimeric sperm–oocyte receptor complexes. In this study, we have extended our findings through the characterization of one such complex containing arylsulfatase A (ARSA), sperm adhesion molecule 1 (SPAM1) and the molecular chaperone, heat shock 70kDa protein 2 (HSPA2). Through the application of flow cytometry we revealed that this complex undergoes a capacitation-associated translocation to facilitate the repositioning of ARSA to the apical region of the human sperm head, a location compatible with a role in the mediation of sperm–zona pellucida (ZP) interactions. Conversely, SPAM1 appears to reorient away from the sperm surface, possibly reflecting its primary role in cumulus matrix dispersal preceding sperm–ZP recognition. The dramatic relocation of the complex was completely abolished by incubation of capacitating spermatozoa in exogenous cholesterol or broad spectrum protein kinase A (PKA) and tyrosine kinase inhibitors suggesting that it may be driven by alterations in membrane fluidity characteristics and concurrently by the activation of a capacitation-associated signal transduction pathway. Collectively these data afford novel insights into the sub-cellular localization and potential functions of multimeric protein complexes in human spermatozoa. capacitation, HSPA2, molecular chaperone, spermatozoa, zona pellucida Introduction Human infertility has increased to the point where it is now estimated that ∼15% of all couples experience difficulty conceiving (Bhasin, 2007). This alarming rate of infertility is, in part, attributed to an aging population, and in particular to women who choose to delay childbearing until a later age. However, a significant proportion of infertility has been attributed to a male factor. Indeed, the World Health Organization (WHO) estimates that ∼50% of all infertility is due to abnormalities associated with the structure or function of the male gamete (McLachlan and de Kretser, 2001). Of these abnormalities, one of the greatest problems appears to lie in an inability of spermatozoa to recognize and bind to the zona pellucida (ZP), a protective glycoprotein layer that surrounds the oocyte (Liu and Baker, 2000). Nevertheless, despite the importance of sperm–ZP adhesion in the etiology of male infertility, the molecular basis of this process is not well understood. There is general agreement that the initial interaction between the two gametes is predicated on the spermatozoa having completed two phases of post-testicular maturation, the first of which occurs within the male epididymis (Cooper, 1986), and the second, termed capacitation, occurs in the female reproductive tract (Austin, 1951; Chang, 1951). While many aspects of these fundamental maturational events remain unknown, the balance of evidence indicates that they occur in the complete absence of de novo protein translation. Thus, the functional transformation of spermatozoa must be driven by remodeling of their lipid and protein architecture (Myles and Primakoff, 1984; Yanagimachi, 1994; Harrison et al., 1996; Cross, 2003; Harrison and Gadella, 2005; Gadella et al., 2008; Baker et al., 2012), as well as the post-translational modification of their intrinsic proteome (Visconti et al., 1995a, b; Leclerc et al., 1996, 1998; Lefievre et al., 2002; Mitchell et al., 2008; Baker et al., 2010). Despite this knowledge, the primary interactions between the spermatozoon and the ZP have, until recently, been portrayed as being reliant on a single, constitutively expressed receptor on the sperm surface. Indeed, a myriad of potential receptor molecules have been proposed (Reid et al., 2010); however, failure of the associated knockouts to display the anticipated infertility phenotype (Lu and Shur, 1997; Cho et al., 1998; Shamsadin et al., 1999; Nishimura et al., 2001; Ensslin and Shur, 2003) suggests that sperm–egg recognition may be too important to entrust to a single molecule. An alternative model is that sperm–ZP recognition is a highly redundant process that is mediated by a complex interplay between a variety of receptors and complementary ligands on the respective gametes (reviewed in ref. Nixon et al., 2005). On the basis of emerging evidence, this concept has been refined to propose that the entire supramolecular structure of the cell surface may play a key role, with capacitation resulting in zona-binding molecules aggregating within the anterior aspect of the sperm head (Nixon et al., 2009; Jones et al., 2010) and forming multimeric protein complexes within lipid rafts in order to facilitate adhesion to homologous zonae pellucidae (Asquith et al., 2004; Nixon et al., 2007, 2011; Gadella, 2008a). Recently, we have demonstrated the presence of a number of multimeric protein complexes on the surface of human spermatozoa, several of which contain proteins implicated in the cascade of events that precede fertilization, including the TCP-1 chaperonin complex and the 20S proteasome (Redgrove et al., 2011). The current study has sought to elucidate the structural and functional characteristics of an additional complex comprising a recognized zona-receptor molecule (arylsulfatase A, ARSA) (Tantibhedhyangkul et al., 2002; Carmona et al., 2002a), a hyaluronidase (sperm adhesion molecule 1, SPAM1) involved in dispersal of the cumulus mass (Lathrop et al., 1990; Kimura et al., 2009) and a molecular chaperone (heat shock 70 kDa protein 2, HSPA2) that has been implicated in orchestrating the surface expression of protein complexes during human sperm capacitation (Redgrove et al., 2012). These analyses have provided additional support for our model of human sperm–ZP recognition as a capacitation-mediated event involving the assembly and presentation of large multimeric ZP recognition complexes on the sperm surface. Materials and Methods Reagents Unless specified, chemical reagents were obtained from Sigma (St. Louis, MO) and were of research grade. The following primary antibodies were purchased to characterize proteins of interest: anti-ARSA (Sigma Cat # HPA005554), anti-SPAM1 (Abnova, Cat # H00006677-A01, Taipei City, Taiwan), rabbit polyclonal anti-HSPA2 (Sigma Cat # HPA000798), mouse polyclonal anti-HSPA2 (Sigma Cat # SAB1405970) and anti-CD59 (Serotec, Dusseldorf, Germany). Prior to use, the specificity of these antibodies was examined by immunoblotting against human sperm lysates. As anticipated each antibody detected a single predominant band of the appropriate molecular weight for the target proteins (Supplementary data, Fig. S1). FITC and tetramethylrhodamine isothiocyanate (TRITC) conjugated secondary antibodies were purchased from Sigma. Similarly, TRITC-conjugated Arachis hypogaea lectin (PNA) was also from Sigma. Nitrocellulose was from Amersham (Buckinghamshire, UK). Highly pure Coomassie Brilliant Blue G250 was from Serva (Heidelberg, Germany). Ethical approval The experiments described in this study were conducted with human semen samples obtained from a panel of healthy normozoospermic donors in accordance with the Institutes' Human Ethics Committee guidelines. These samples were collected via masturbation into sterile specimen containers after an abstinence period of 48 h and delivered to the laboratory within 1 h of ejaculation. Preparation of human spermatozoa Purification of human spermatozoa from these samples was achieved using a 44 and 88% discontinuous Percoll (GE Healthcare, Piscataway, NJ) gradient as described previously (Mitchell et al., 2007). Purified spermatozoa were recovered from the base of the 88% Percoll fraction and resuspended in Biggers, Whitten and Whittingham medium (Biggers et al., 1971), composed of 91.5 mM NaCl, 4.6 mM KCl, 1.7 mM CaCl2.2H2O, 1.2 mM KH2PO4, 1.2 mM MgSO4.7H2O, 25 mM NaHCO3, 5.6 mM d-glucose, 0.27 mM sodium pyruvate, 44 mM sodium lactate, 5 U/ml penicillin, 5 mg/ml streptomycin, 20 mM HEPES buffer and 1 mg/ml polyvinyl alcohol (PVA) (osmolarity of 300 mOsm/kg). The cells were then pelleted by centrifugation at 500g for a further 15 min and finally resuspended at a concentration of 10 × 106 cells/ml before being assessed for cell motility and vitality. The latter was examined by staining spermatozoa with SYTOX Green (50 nM for 15 min), a high-affinity, fluorescent nucleic acid stain that is impermeant to live cells and thus an indicator of cell viability. Samples in which either parameter was <85% were discarded. Preparation of human oocytes Human oocytes were obtained with informed consent from patients of the Hunter IVF Clinic. These oocytes were either immature or had failed to be fertilized following ICSI. Prior to use the oocytes were fixed in a high salt medium consisting of 1.5 M MgCl2, 0.1% dextran, 0.01 mM HEPES buffer and 0.1% PVA and stored at 4°C. Notwithstanding the degeneration of the oocyte, this form of storage has been demonstrated to retain the biological characteristics of the ZP (Yanagimachi et al., 1979; Nixon et al., 2011; Redgrove et al., 2011). Capacitation of human spermatozoa Following dilution in Biggers–Whitten–Whittingham (BWW), purified spermatozoa were incubated at 37°C under an atmosphere of 5% CO2:95% air. Non-capacitated cells were incubated in BWW prepared without NaHCO3 (BWW-HCO3−). Capacitated cells were prepared by incubation in BWW supplemented with 3 mM pentoxifylline and 5 mM dibutyryl cyclic adenosine monophosphate. Incubations were conducted for a period of 3 h with gentle mixing at regular intervals to prevent settling of the cells. At the end of the incubation an aliquot of each sperm suspension was removed and assessed for cell motility and vitality. Importantly, neither parameter was adversely affected by any of the treatments used in this study (i.e. remained >85%). The remainder of the sperm sample was prepared for the various treatments outlined below. The capacitation conditions used have been shown to induce optimal capacitation as defined by tyrosine phosphorylation, hyperactivation and, critically, ZP binding (Mitchell et al., 2007, 2008). These conditions also yield similar levels of capacitation to those obtained using a standard human IVF system (Nixon et al., 2011; Redgrove et al., 2011). Immunolocalization of target proteins on fixed human spermatozoa Populations of capacitated and non-capacitated were prepared as described above and then fixed in 4% paraformaldehyde solution for 10 min at room temperature. The cells were then washed three times with PBS supplemented with 0.05 M glycine, and resuspended in PBS. In order to examine the behavior of the proteins of interest following the induction of acrosomal exocytosis, capacitated spermatozoa were incubated in the calcium ionophore, A23187, at a final concentration of 2.5 μM for 30 min at 37°C. Following centrifugation, the cells were resuspended in 500 μl hypo-osmotic swelling media (25 mM sodium citrate, 75 mM fructose), and incubated for 1 h at 37°C. After resuspension in PBS, the cells were plated onto poly-l-lysine-coated glass slides and air dried. They were then permeabilized with ice-cold methanol for 10 min and blocked with 10% serum/3% BSA for 1 h. Slides were washed three times with PBS for 5 min and incubated in a 1:100 dilution of primary antibody at 4°C overnight. Slides were then subjected to 3 × 5 min washes with PBS and incubated in a 1:100 dilution of an appropriate FITC-conjugated secondary antibody (1:300 for anti-mouse FITC) for 1 h at 37°C. Slides were again washed and mounted in 10% mowiol 4-88 (Calbiochem) with 30% glycerol in 0.2 M Tris (pH 8.5) with 2.5% 1,4-diazobicyclo-(2.2.2)-octane. Cells were examined using either a Zeiss Axioplan 2 fluorescence microscope or a Zeiss LSM510 laser scanning confocal microscope equipped with argon and helium/neon lasers (Carl Zeiss, Thornwood, NY). Immunodetection of target proteins on live sperm using a fluorescence-activated cell sorter Capacitation-associated changes in sperm membrane fluidity were assessed by staining the cells with merocyanine 540, a fluorescent dye that preferentially intercalates with unsaturated lipids and cholesterol-free domains in loosely packed and disordered membranes. For this purpose, spermatozoa were mixed with merocyanine, diluted to a final concentration of 3 µM, incubated 15 min at 37°C in the dark and washed twice by centrifugation in BWW. In order to investigate the role of cell signaling in promoting the surface expression of target proteins, populations of capacitating spermatozoa were individually incubated with H89 (10 µM), staurosporine (10 μM), herbimycin A (10 µM) or genistein (100 µM). Spermatozoa were then counterstained for 15 min with 50 nM SYTOX Green. After washing, the cells were either viewed by fluorescence microscopy or analyzed on a fluorescence-activated cell sorting (FACS) Vantage flow cytometer (Becton Dickinson, San Jose, CA) with an FL4 530/30 nm band-press filter, allowing the collection of fluorescence data in the logarithmic mode and light-scatter data in the linear mode. Ten thousand cells were counted in each sample at a rate of 50–500 events per second. Data were analyzed using the Cell Quest package. A similar approach was also employed to study the surface expression of target sperm proteins during capacitation. Following purification, spermatozoa were diluted to 1 × 106 cells/ml and incubated with appropriate primary antibodies (diluted 1:100) for 30 min at 37°C. The cells were subsequently washed twice with BWW and incubated with FITC-conjugated secondary antibody (1:200) for a further 10 min. Following additional washes with BWW, the cells were incubated with propidium iodide (20 mg/ml) and analyzed by FACS as described above. Blue native polyacrylamide gel electrophoresis Following incubation under either capacitating or non-capacitating conditions, the suspensions of 1 × 106 sperm/ml were lightly pelleted (300 g for 5 min) and resuspended in native protein lysis buffer consisting of 1% n-dodecyl β-d-maltoside which was adjusted to a final concentration below that of the critical micellar concentration, 0.5% Coomassie Blue G250 and a cocktail of protease inhibitors (Roche, Mannheim, Germany). The samples were gently mixed and then incubated at 4°C on an orbital rotator for 30 min. Following incubation, the lysate was recovered by centrifugation at 14 000g for 20 min at 4°C and dialyzed against the Blue native cathode buffer (Invitrogen) to remove any excess salts and detergents. Following dialysis, the sample was placed in a clean 1.5 ml tube and glycerol was added to a final concentration of 5% (v/v). For the purpose of one-dimensional blue native page (1D BN-PAGE), native protein lysates were loaded onto pre-cast blue native polyacrylamide gels (NativePAGE Novex 4–16%, Bis-Tris; Invitrogen, Carlsbad, CA) and resolved using the NativePAGE cathode and anode buffer system (Invitrogen) (Redgrove et al., 2011). The BN-PAGE electrophoresis apparatus was placed at 4°C and the samples separated at 100 V until the Coomassie dye front reached the bottom of the loading wells. The voltage was then increased to 200 V and the separation continued until the Coomassie dye front reached the bottom of the gel. The gels were then removed from the electrophoresis apparatus and stained sequentially with Coomassie G250 and then with silver staining reagents (to detect less abundant proteins). Alternatively, the gels were prepared for either western blotting or two-dimensional BN-PAGE (2D BN-PAGE). 2D BN-PAGE was conducted in order to resolve native protein complexes into their individual components. For this purpose, individual lanes of the 1D BN-PAGE gel were excised and then pre-equilibrated in SDS-PAGE sample buffer (2% w/v SDS, 10% w/v sucrose in 0.1875 M Tris, pH 6.8) supplemented with 0.5% v/v dithiothreitol (DTT) and 4% v/v iodoacetamide for 10 min. The lanes were then placed on the top of a 10% SDS-PAGE gel prepared without stacking wells and sealed in position using 0.5% molten agarose. The gel was placed in a small-format electrophoresis chamber (Bio-Rad Laboratories, Hercules, CA), immersed in SDS-PAGE running buffer and electrophoresed at 100 V until the Coomassie dye front reached the bottom of the gel. Gels were then removed from their cassettes and either stained with Coomassie G-250 or prepared for western blotting. 2D SDS-PAGE Populations of non-capacitated and capacitated human spermatozoa were prepared as described above. Aliquots of 100 × 106 sperm/ml were then lysed in 400 μl of lysis buffer (4% CHAPS, 7 M urea, 2 M thiourea) for 1 h at 4°C with constant rotation. The samples were then centrifuged at 10 000g for 15 min and the supernatant transferred to a clean tube. The extracted protein was subsequently quantified using a 2-D Quant Kit (GE Healthcare, Castlehill, Australia) and aliquots of either 50 or 500 μg (for western blot analysis and mass spectrometry sequencing, respectively) were precipitated using methanol/chloroform as previously described (Baker et al., 2004). To each sample, 1.25 μl of Pharmalytes (Amersham, Buckinghamshire, UK), 1.5 μl Destreak (Amersham) and trace amounts of bromophenol blue were added and the samples were used to rehydrate immobilized pH gradient (IPG) gel strips (pH 3–10, 7 cm, non-linear IPG gels; GE Healthcare), overnight. Isoelectric focusing of the samples was achieved on an IPGphor (GE Healthcare) with the protocol of 300 V for 0.9 kV h (step and hold), a gradient up to 1000 V for 3.9 kV h, followed by an additional gradient to 8000 V for 13.5 kV h. The sample was then held at 8000 V for a total of 77.7 kV h. After focusing, each IPG strip was pre-equilibrated in SDS equilibration buffer (50 mM Tris, 6 M urea, 30% w/v glycerol, 2% SDS, trace bromophenol blue) supplemented with 0.5% w/v DTT and 4% w/v iodoacetamide for 10 min with constant shaking. The strips were then placed on the top of a 10% SDS-PAGE gel prepared without stacking wells and sealed in position using 0.5% molten agarose. The gel was placed in a small-format electrophoresis chamber (Bio-Rad), immersed in SDS-PAGE running buffer and electrophoresed at 100 V until the Coomassie dye front reached the bottom of the gel. Gels were then removed from their cassettes and either stained with Coomassie G-250 or prepared for western blotting. Individual spots identified via western blotting were excised from a nitrocellulose membrane, placed in clean individual LoBind Eppendorf tubes (Eppendorf, Hamburg, Germany) and stripped of antibody using 0.2 M NaOH for 10 min at room temperature. After washing thoroughly in distilled water, the membranes were resuspended in 10 µl trypsin (Promega, Annandale, NSW, Australia) made up to the volume with 25 mM ammonium bicarbonate and incubated overnight at 4°C. The supernatant was removed and placed in fresh LoBind tubes, then centrifuged at 10 000g 10 min. The supernatant was removed and placed in glass vials before being prepared for MS. LC-MS/MS analysis LC-MS/MS analysis was performed at the University of Newcastle's ABRF Mass Spectrometry Unit. Separation of tryptic peptide mixtures was achieved by nanoscale reversed phase high-pressure liquid chromatography (HPLC), in combination with on-line ESI-MS. The mass spectrometric analysis was performed on an LTQ XL-linear ion trap system (Thermo Scientific, West Palm Beach, FL). Prior to ion trap analysis, HPLC separation was performed using a nano-AQUITY system (Waters, Rydalmere, Australia), employing a linear gradient of 2–50% buffer B (100% ACN, 0.1% formic acid) over 60 min. The C18 column system consisted of a trapping column (5 µm bead, 180 µm inner diameter × 20 mm length) and a separation column (1.7 uM bead, 100 µm inner diameter × 100 mm length). For online coupling, a nano-ion spray source was used, equipped with an ESI needle (10 µm silica tip). The needle voltage was 2.0 kV in a positive ion mode. The scan cycle consisted of a survey scan (mass range: 400–2000 atomic mass units) followed by MS/MS of the four most intense signals in the spectrum with an exclusion list for ion signals set to 60 s after two MS/MS occurrences. For CID analysis, normalized collision energy was set to 35; activation Q set to 0.25 with an activation time set to 100 ms and isolation width of m/z 1. Analysis was performed on CID only. Bioinformatics Thermo RAW files were converted into MASCOT Generic Format using MASCOT Daemon 2.3.2 and imported into ProteinScape bioinformatics platform (Bruker Daltonics, Bremen, Germany) for database searching. Searches were performed against the SwissProt database (version 57.15) using in-house licensed MASCOT server (version 2.3.02, Matrix Science), with the species subset set to Homo sapiens and the number of allowed trypsin missed cleavages set to 2. Carbamidomethylation of cysteine was set as a fixed modification, whereas deamination of asparagine and glutamine, oxidation of methionine and phosphorylation of serine, threonine and tyrosine were set as variable modifications. The parent ion tolerance was set to 1.4 Da with a fragment ion set to 0.7 Da. Peptide thresholds were set requiring a false-positive rate at <0.05% and a MASCOT score at >40. Those spectra meeting these criteria were validated by manual inspection on a residue-by-residue basis to ensure accurate y- and b-ion detection with overlapping sequence coverage. Western blotting Proteins resolved by either 1D or 2D BN-PAGE were transferred onto nitrocellulose membranes using conventional western blotting techniques (Towbin et al., 1979). In order to detect proteins of interest, membranes were blocked with 3% w/v BSA in Tris-buffered saline (TBS; pH 7.4) supplemented with 0.1% polyoxyethylenesorbitan monolaurate (Tween-20). Membranes were rinsed in TBS and then probed with appropriate primary antibodies (diluted 1:1000 dilution in TBS supplemented with 1% BSA and 0.1% Tween-20) for 2 h at room temperature. Following incubation, membranes were washed three times in TBS containing 0.1% Tween-20 (TBST) for 10 min. Membranes were then probed for 1 h with an appropriate HRP-conjugated secondary antibody (diluted between 1:3000 and 1:5000 in TBST/1% BSA) at room temperature. Following a further three washes in TBST, cross-reactive proteins were visualized using an enhanced chemiluminescence kit (GE Healthcare) according to the manufacturer's instructions. Duolink proximity ligation assay Duolink in situ primary ligation assays (PLAs) were conducted in accordance with the manufacturers' instructions (OLINK Biosciences, Uppsala, Sweden). Briefly, human spermatozoa were purified and capacitated as previously described, after which they were fixed in 2% paraformaldehyde and aliquoted onto poly-l-lysine slides. These cells were then incubated in blocking solution (OLINK Biosciences) for 1 h at 37°C, before target proteins were sequentially labeled with a pair of appropriate primary antibodies (anti-HSPA2 and anti-pY; anti-pY and anti-tubulin) raised in different species (or anti-pY and anti-HSPA2 alone as negative controls) overnight at 4°C in a humidified chamber. After washing, appropriate secondary antibodies (anti-mouse for HSPA2 and tubulin and anti-rabbit for pY) conjugated to complementary synthetic oligonucleotides (PLA probes, OLINK Biosciences) were then applied to the samples for 1 h at 37°C. The samples were then sequentially hybridized (15 min), washed and enzymatically ligated (15 min). If the target proteins reside in close proximity, this reaction leads to the production of a signal that appears as a discrete fluorescent dot. These signals were visualized with an Axio Imager A1 fluorescence microscope (Carl Zeiss) and pictures taken using an Olympus DP70 microscope camera (Olympus America, Center Valley, PA). Sperm–ZP binding assays To examine the physiological importance of protein complexes in relation to sperm–ZP interaction, capacitated spermatozoa were prepared and incubated with the appropriate antibodies (diluted 1:100) for 30 min at 37°C. The spermatozoa were then co-incubated with zona-intact human oocytes for 30 min at 37°C as previously described. After stringent washing of the oocytes by repeated aspiration through a fine bore pipette, the number of spermatozoa remaining bound to each ZP was counted. In separate experiments, purified spermatozoa were capacitated in the presence of exogenous cholesterol sulfate (100 μM) or in the presence of either H89 (10 µM) or herbimycin A (10 µM). The spermatozoa were then co-incubated with human oocytes for 30 min at 37°C and the number of spermatozoa bound to each oocyte was counted as described above. In these experiments, the sperm concentration was adjusted such that we observed a maximum of 1–2 sperm binding per ZP in the non-capacitated negative control samples, and between 30 and 35 spermatozoa binding in the capacitated-positive control samples. Co-immunoprecipitation An aliquot of 60 µl (per treatment) magnetic Protein G-coated Dynabeads (Dynal Biotech ASA, Oslo, Norway) was washed three times in washing and binding buffer [5 mM Tris–HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl]. This was followed by conjugation with 4 µg anti-ARSA antibody or 2 μg of anti-HSPA2 antibody at 4°C overnight with constant mixing. A control sample of beads was left non-conjugated and was incubated with washing and binding buffer only. Following conjugation, the beads were washed twice in washing and binding buffer. Capacitated spermatozoa were then lysed in IP lysis buffer [0.1% (v/v) Triton X-100, 300 mM NaCl, 20 mM Tris, pH 7.4 supplemented with protease inhibitor cocktail and a 1:100 dilution of HALT complete phosphatase inhibitor cocktail (Pierce)]. Approximately 100 µg of soluble lysate was added to the pre-adsorbed beads and left to incubate at 4°C overnight with constant mixing. Following incubation, the beads were washed twice in washing and binding buffer and resuspended in SDS sample buffer. The suspension was then boiled for 5 min, the beads removed and the precipitated proteins resolved on 10% polyacrylamide gels before being electro-transferred onto nitrocellulose membranes and immunoblotted as described previously. Control incubations were included where beads were incubated with sperm lysate in the absence of antibody, and also antibody-conjugated beads were incubated in the absence of cell lysate. These controls were processed as described above. Statistical analysis All experiments were replicated at least three times with independent samples and data are expressed as mean values ± SEM, the standard errors being calculated from the variance between samples. Statistical significance was determined using an analysis of variance (ANOVA). Differences were considered significant for P < 0.05. Results HSPA2 forms a stable complex with ARSA and SPAM1 in human spermatozoa In order to investigate the stable association of the HSPA2/ARSA/SPAM1 complex previously identified in human spermatozoa (Redgrove et al., 2012), a reciprocal co-immunoprecipitation strategy was adopted whereby sperm lysates were precipitated with either anti-ARSA (Fig. 1A–D) or anti-HSPA2 antibodies (Fig. 1E–H), and the eluates subsequently examined for the presence of each member of the complex. As illustrated in Fig. 1A and E, this technique effectively isolated the target proteins of ARSA and HSPA2, respectively. Furthermore, upon stripping and re-probing of these blots, predominant bands of the appropriate molecular weight were detected for SPAM1 (Fig. 1B) and HSPA2 (Fig. 1C) in the ARSA immunoprecipitation. Similarly, SPAM1 (Fig. 1F) and ARSA (Fig. 1G) were also detected in the HSPA2 immunoprecipitation. The specificity of both immunoprecipitations was confirmed through the incorporation of antibody and bead-only controls in addition to reprobing of the blots with secondary antibodies alone (Fig. 1D and 1H), none of which revealed the presence of the proteins of interest. Figure 1 View largeDownload slide Examination of HSPA2/ARSA/SPAM1 interaction in human spermatozoa. Lysates of capacitated human spermatozoa were incubated with protein G Dynabeads conjugated with either anti-ARSA antibodies (A–D) or anti-HSPA2 antibodies (E–H). The beads were washed, then bound proteins eluted and resolved on SDS-PAGE gels before being transferred to nitrocellulose membranes. Membranes were probed with (A) anti-ARSA antibodies to confirm the efficacy of immunoprecipitation before being stripped and reprobed with (B) SPAM1, (C) HSPA2 antibodies or (D) secondary alone. Alternatively, the HSPA2 immunoprecipitated proteins were probed with (E) anti-HSPA2 antibodies to confirm the efficacy of immunoprecipitation before being stripped and reprobed with (F) anti-SPAM1, (G) anti-ARSA or (H) secondary antibodies alone. Negative controls included an antibody only control (Ab only) in which antibody-conjugated beads were incubated in the absence of cell lysate and a bead only control (bead only) in which non-conjugated beads were incubated with sperm lysate. A whole sperm lysate was included to confirm the identity of the co-precipitated proteins as was the material recovered after washing the beads to confirm the specificity of the elution. The experiment was replicated three times using pooled semen samples and representative blots are depicted. Figure 1 View largeDownload slide Examination of HSPA2/ARSA/SPAM1 interaction in human spermatozoa. Lysates of capacitated human spermatozoa were incubated with protein G Dynabeads conjugated with either anti-ARSA antibodies (A–D) or anti-HSPA2 antibodies (E–H). The beads were washed, then bound proteins eluted and resolved on SDS-PAGE gels before being transferred to nitrocellulose membranes. Membranes were probed with (A) anti-ARSA antibodies to confirm the efficacy of immunoprecipitation before being stripped and reprobed with (B) SPAM1, (C) HSPA2 antibodies or (D) secondary alone. Alternatively, the HSPA2 immunoprecipitated proteins were probed with (E) anti-HSPA2 antibodies to confirm the efficacy of immunoprecipitation before being stripped and reprobed with (F) anti-SPAM1, (G) anti-ARSA or (H) secondary antibodies alone. Negative controls included an antibody only control (Ab only) in which antibody-conjugated beads were incubated in the absence of cell lysate and a bead only control (bead only) in which non-conjugated beads were incubated with sperm lysate. A whole sperm lysate was included to confirm the identity of the co-precipitated proteins as was the material recovered after washing the beads to confirm the specificity of the elution. The experiment was replicated three times using pooled semen samples and representative blots are depicted. The interaction between HSPA2/ARSA/SPAM1 was also investigated in populations of capacitated and non-capacitated spermatozoa using a dual-labeling strategy. As shown in Fig. 2, this approach revealed strong co-localization of HSPA2, ARSA and SPAM1 irrespective of the capacitation status of spermatozoa. Notwithstanding the differences in the labeling pattern observed with each antibody, overlapping fluorescence was primarily restricted to the peri-acrosomal region of the sperm head. Figure 2 View largeDownload slide Co-localization of HSPA2, SPAM1 and ARSA in human spermatozoa. Populations of non-capacitated and capacitated human spermatozoa were fixed in paraformaldehyde and allowed to settle onto poly-l-lysine slides. These samples were labeled with different combinations of anti-HSPA2, anti-SPAM1 or anti-ARSA antibodies followed by either appropriate TRITC- or FITC-conjugated secondary antibodies. The slides were then viewed using confocal microscopy. The merged images clearly show that the co-localization of the three proteins is primarily restricted to the peri-acrosomal region of the sperm head. Scale bar = 10 µM. These experiments were replicated three times using independent samples. Figure 2 View largeDownload slide Co-localization of HSPA2, SPAM1 and ARSA in human spermatozoa. Populations of non-capacitated and capacitated human spermatozoa were fixed in paraformaldehyde and allowed to settle onto poly-l-lysine slides. These samples were labeled with different combinations of anti-HSPA2, anti-SPAM1 or anti-ARSA antibodies followed by either appropriate TRITC- or FITC-conjugated secondary antibodies. The slides were then viewed using confocal microscopy. The merged images clearly show that the co-localization of the three proteins is primarily restricted to the peri-acrosomal region of the sperm head. Scale bar = 10 µM. These experiments were replicated three times using independent samples. The HSPA2/ARSA/SPAM1 complex participates in human sperm–oocyte interactions The functional significance of the HSPA2/ARSA/SPAM1 complex in relation to fertilization was explored by incubating capacitating populations of human spermatozoa with antibodies directed against each of the target proteins. These spermatozoa were then examined for their ability to adhere to the ZP of homologous oocytes. As anticipated on the basis of our previous work (Redgrove et al., 2012), antibodies against HSPA2 failed to abrogate binding of capacitated cells to the ZP. This result was confirmed with two different polyclonal antibodies raised in either the mouse or rabbit, potentially indicating an indirect role for this protein in the binding event (Fig. 3). In contrast, antibodies directed against either SPAM1 or ARSA were able to significantly suppress sperm–ZP adhesion (Fig. 3) without compromising either sperm viability or motility (data not shown). In this context, anti-SPAM1 and anti-ARSA antibodies reduced sperm–ZP binding to ∼40 and 15% of that observed in the control populations (capacitated spermatozoa), respectively. The specificity of this inhibition was demonstrated by the fact that antibodies against CD59, an alternative sperm surface protein that does not participate in sperm–ZP adhesion (Fenichel et al., 1994), failed to elicit any reduction in sperm binding. Figure 3 View largeDownload slide Investigation of the role of the members of the HSPA2/ARSA/SPAM1 complex in sperm–ZP binding. Capacitated human spermatozoa were incubated with anti-HSPA2 (mouse and rabbit polyclonal antibodies), anti-ARSA or anti-SPAM1 antibodies before being washed and introduced into a droplet containing 8–10 salt-stored oocytes. A negative control in which sperm were incubated with antibodies against a protein that does not participate in ZP interaction (CD59) was included. After stringent washing, the number of sperm bound to zonae in each treatment group was recorded: non-capacitated = 0.9 ± 0.3; capacitated = 30.5 ± 4.2; CD59 = 35.4 ± 5.7; mouse-HSPA2 = 34.5 ± 3.6; rabbit-HSPA2 = 30.3 ± 4.5; SPAM1 = 12 ± 3.6; ARSA = 3.9 ± 1.5. These data are expressed as a percentage of the positive control (capacitated spermatozoa). This experiment was replicated three times and the graphical data are presented as the mean ± SEM. *P < 0.05, **P < 0.001 compared with the capacitated spermatozoa control. Figure 3 View largeDownload slide Investigation of the role of the members of the HSPA2/ARSA/SPAM1 complex in sperm–ZP binding. Capacitated human spermatozoa were incubated with anti-HSPA2 (mouse and rabbit polyclonal antibodies), anti-ARSA or anti-SPAM1 antibodies before being washed and introduced into a droplet containing 8–10 salt-stored oocytes. A negative control in which sperm were incubated with antibodies against a protein that does not participate in ZP interaction (CD59) was included. After stringent washing, the number of sperm bound to zonae in each treatment group was recorded: non-capacitated = 0.9 ± 0.3; capacitated = 30.5 ± 4.2; CD59 = 35.4 ± 5.7; mouse-HSPA2 = 34.5 ± 3.6; rabbit-HSPA2 = 30.3 ± 4.5; SPAM1 = 12 ± 3.6; ARSA = 3.9 ± 1.5. These data are expressed as a percentage of the positive control (capacitated spermatozoa). This experiment was replicated three times and the graphical data are presented as the mean ± SEM. *P < 0.05, **P < 0.001 compared with the capacitated spermatozoa control. Components of the HSPA2/ARSA/SPAM1 complex are differentially localized on the surface of capacitating human spermatozoa Having confirmed the tight association of the HSPA2/ARSA/SPAM1 complex and its functional significance in relation to sperm–oocyte interactions, we next employed a flow cytometry assay to investigate the orientation of its components within the membrane of live spermatozoa and whether this is influenced by the capacitation process. In contrast to HSPA2, which has previously been shown to occupy an intracellular domain, we were able to demonstrate that both SPAM1 and ARSA are expressed on the surface of the live human spermatozoa (Fig. 4A). Interestingly however, we noted a reciprocal change in the expression of these proteins following the induction of capacitation (Fig. 4A). Thus, we observed a highly significant (P < 0.001) time-dependent increase in the number of sperm-expressing ARSA on their surface, from <5% in the non-capacitated population to ∼80% in cells incubated under capacitating conditions for 3 h (Fig. 4A). Following an opposite trend, SPAM1 was detected on the surface of >80% of non-capacitated spermatozoa but reduced such that it was detected in <40% of these cells following 3 h of capacitation (Fig. 4A). An initial change in the localization of both proteins was detected as early as 30 min after the induction of capacitation; however, the majority of this change did not occur until at least 60 min after this process was initiated. These results extend an earlier single time-point study in demonstrating that the reorientation of ARSA and SPAM1 is progressive and time-dependent. Although we considered the possibility that these altered expression profiles could be influenced by the loss or degradation of either protein, this did not appear to the case. Indeed, permeabilization of the cells prior to flow cytometry analysis demonstrated the presence of both ARSA and SPAM1 in virtually all spermatozoa irrespective of their capacitation status (Fig. 4B and C). Figure 4 View largeDownload slide Examination of the surface expression of SPAM1 and ARSA in human spermatozoa. Human spermatozoa were incubated in capacitating media and aliquots were sampled and assessed at 30, 60 and 180 min. The presence of SPAM1 and ARSA on the surface of live spermatozoa was assessed using anti-SPAM1 and anti-ARSA primary antibodies, followed by an FITC-conjugated secondary antibody and propidium iodide (PI) as a counterstain to assess cell viability; positive control incubations were labeled with anti-CD59. (A) Graphical representation of percentage of viable cells expressing the target proteins on their surface as detected using flow cytometry. (B and C) To ensure the change in surface expression did not reflect the loss or gain of either protein, flow cytometry was also used to assess the percentage of non-capacitated or capacitated spermatozoa labeled for either (B) ARSA or (C) SPAM1 before (non-permeabilized) and after permeabilization (permeabilized). All experiments were replicated three times with a minimum of 10 000 cells scored for each experiment. *P < 0.05, **P < 0.001 compared with non-capacitated spermatozoa. Figure 4 View largeDownload slide Examination of the surface expression of SPAM1 and ARSA in human spermatozoa. Human spermatozoa were incubated in capacitating media and aliquots were sampled and assessed at 30, 60 and 180 min. The presence of SPAM1 and ARSA on the surface of live spermatozoa was assessed using anti-SPAM1 and anti-ARSA primary antibodies, followed by an FITC-conjugated secondary antibody and propidium iodide (PI) as a counterstain to assess cell viability; positive control incubations were labeled with anti-CD59. (A) Graphical representation of percentage of viable cells expressing the target proteins on their surface as detected using flow cytometry. (B and C) To ensure the change in surface expression did not reflect the loss or gain of either protein, flow cytometry was also used to assess the percentage of non-capacitated or capacitated spermatozoa labeled for either (B) ARSA or (C) SPAM1 before (non-permeabilized) and after permeabilization (permeabilized). All experiments were replicated three times with a minimum of 10 000 cells scored for each experiment. *P < 0.05, **P < 0.001 compared with non-capacitated spermatozoa. Investigation of the molecular mechanisms that underpin the capacitation-associated reorientation of the HSPA2/ARSA/SPAM1 complex Among the most dramatic changes incurred in the sperm plasma membrane during capacitation is a regulated efflux of cholesterol to various acceptor molecules (Osheroff et al., 1999). The profound increase in the fluidity characteristics of the membrane that results from cholesterol efflux could conceivably promote the reorientation of the HSPA2/ARSA/SPAM1 complex. Thus, we examined whether we could suppress the movement of ARSA and SPAM1 through the inclusion of exogenous cholesterol sulfate in the capacitation media to prevent cholesterol being lost from the membrane environment. As a prelude to these studies we first demonstrated that this treatment significantly reduced the incorporation of merocyanine 540, a marker of membrane fluidity (Fig. 5A). Using these conditions we were also able to demonstrate that cholesterol effectively blocked the capacitation-associated movement of SPAM1 and ARSA within the sperm membrane (Fig. 5B). Indeed, in the presence of exogenous cholesterol the surface expression of both ARSA and SPAM1 remained essentially the same as that observed in non-capacitated cells (Fig. 5B). Figure 5 View largeDownload slide Effect of membrane fluidity on SPAM1 and ARSA surface expression. (A) Human spermatozoa were incubated in either capacitation media alone, or co-incubated with 100 μM cholesterol sulfate (ChS) for 3 h. The increase in membrane fluidity brought about by cholesterol loss was assessed using flow cytometry to monitor the incorporation of merocyanine 540. (B) The influence of exogenous cholesterol on the surface expression of ARSA and SPAM1 was assessed by incubating spermatozoa under capacitating conditions for 3 h in the presence of 100 μM ChS. Following the treatment, the cells were labeled with anti-SPAM1 or anti-ARSA antibodies, counterstained with propidium iodide and the percentage of live, positively labeled cells recorded. Positive control incubations were labeled with anti-CD59. All experiments were replicated three times with a minimum of 10 000 viable cells scored for each experiment. *P < 0.05, **P < 0.001. (C) The ability of exogenous cholesterol to abrogate additional hallmarks of the capacitation process was assessed by examining the levels of phosphotyrosine achieved in the presence of ChS. For this purpose, human spermatozoa were incubated in either capacitation media alone, or in the presence of 100 μM ChS for 3 h. Cell lysates were then prepared for immunoblotting with anti-phosphotyrosine antibodies (anti-pY). To confirm equal protein loading, immunoblots were stripped before being re-probed with anti-α-tubulin. Figure 5 View largeDownload slide Effect of membrane fluidity on SPAM1 and ARSA surface expression. (A) Human spermatozoa were incubated in either capacitation media alone, or co-incubated with 100 μM cholesterol sulfate (ChS) for 3 h. The increase in membrane fluidity brought about by cholesterol loss was assessed using flow cytometry to monitor the incorporation of merocyanine 540. (B) The influence of exogenous cholesterol on the surface expression of ARSA and SPAM1 was assessed by incubating spermatozoa under capacitating conditions for 3 h in the presence of 100 μM ChS. Following the treatment, the cells were labeled with anti-SPAM1 or anti-ARSA antibodies, counterstained with propidium iodide and the percentage of live, positively labeled cells recorded. Positive control incubations were labeled with anti-CD59. All experiments were replicated three times with a minimum of 10 000 viable cells scored for each experiment. *P < 0.05, **P < 0.001. (C) The ability of exogenous cholesterol to abrogate additional hallmarks of the capacitation process was assessed by examining the levels of phosphotyrosine achieved in the presence of ChS. For this purpose, human spermatozoa were incubated in either capacitation media alone, or in the presence of 100 μM ChS for 3 h. Cell lysates were then prepared for immunoblotting with anti-phosphotyrosine antibodies (anti-pY). To confirm equal protein loading, immunoblots were stripped before being re-probed with anti-α-tubulin. On the basis of these data we infer that cholesterol efflux and the associated increase in membrane fluidity are important requirements for the reorientation of the HSPA2/SPAM1/ARSA complex. However, given the dichotomy in timing between the rapid increase in membrane fluidity (<15 min) (Fig. 5A) and the reorientation of the complex, which does not occur at appreciable levels until between 1 and 3 h of capacitation (Fig. 4A), we anticipate that additional mechanisms must contribute to this phenomenon. Consistent with this notion, we were able to demonstrate that the addition of exogenous cholesterol not only led to a suppression of membrane fluidity but also inhibited tyrosine phosphorylation (Fig. 5C), a hallmark of the capacitation process. On the basis of these data, we therefore examined the degree to which canonical capacitation-associated signal transduction pathways (Fig. 6A) influenced this movement. For this purpose we assessed the surface expression of SPAM1 and ARSA following the inhibition of protein kinase A (PKA) with either broad spectrum (staurosporine) or specific (H89) pharmacological reagents, both of which were demonstrated to decrease tyrosine phosphorylation levels during capacitation (Supplementary data, Fig. S2). As shown in Fig. 6B, incubation of capacitating populations of sperm in either inhibitor significantly reduced the repositioning of SPAM1 and ARSA within the membrane. We were also able to elicit a similar response in the presence of broad spectrum inhibitors (herbimycin A and genistein) of phosphotyrosine kinases (PTKs) that act downstream of PKA. Indeed, as shown in Fig. 6C, pre-incubation of capacitating populations of sperm in either inhibitor effectively eliminated the re-orientation of SPAM1 and ARSA. Figure 6 View largeDownload slide Investigation of the role of capacitation-associated cell signaling in promoting the surface expression of SPAM1 and ARSA. (A) Primary canonical signaling pathway underpinning human sperm capacitation. (B) Human spermatozoa were incubated either in capacitation media alone, or co-incubated with 10 μM H89 or 10 μM staurosporine or (C) 10 μM herbimycin A or 100 μM genistein for 180 min. The presence of SPAM1 and ARSA on the surface of live spermatozoa was assessed by flow cytometry using anti-SPAM1 and anti-ARSA primary antibodies, followed by an FITC-conjugated secondary antibody and counterstaining with propidium iodide. Positive control incubations were labeled with anti-CD59. All experiments were replicated three times with a minimum of 10 000 viable cells scored for each experiment. *P < 0.05, **P < 0.001 compared with non-capacitated control. Figure 6 View largeDownload slide Investigation of the role of capacitation-associated cell signaling in promoting the surface expression of SPAM1 and ARSA. (A) Primary canonical signaling pathway underpinning human sperm capacitation. (B) Human spermatozoa were incubated either in capacitation media alone, or co-incubated with 10 μM H89 or 10 μM staurosporine or (C) 10 μM herbimycin A or 100 μM genistein for 180 min. The presence of SPAM1 and ARSA on the surface of live spermatozoa was assessed by flow cytometry using anti-SPAM1 and anti-ARSA primary antibodies, followed by an FITC-conjugated secondary antibody and counterstaining with propidium iodide. Positive control incubations were labeled with anti-CD59. All experiments were replicated three times with a minimum of 10 000 viable cells scored for each experiment. *P < 0.05, **P < 0.001 compared with non-capacitated control. To explore the physiological significance of these treatments, populations of spermatozoa capacitated in the presence of either cholesterol sulfate, H89 or herbimycin A were assessed for their ability to bind homologous zonae pellucidae. Despite a modest reduction in zona adhesion in cholesterol sulfate (P = 0.05) and herbimycin A (P = 0.06) treated spermatozoa, only those cells incubated in the presence of H89 displayed a significant loss of zona-binding ability (P < 0.05) (Fig. 7A). At the concentrations used in this study, none of the treatments had a negative impact on sperm motility or viability (data not shown). Similarly, as demonstrated by BN-PAGE, these treatments did not affect the tight association of the different members of the HSPA2/ARSA/SPAM1 complex (Fig. 7B and C), which was detected at the same molecular weight (∼200 kDa) as that previously reported by Redgrove et al. (2012). Figure 7 View largeDownload slide Investigation of the effect of suppression of capacitation-associated signaling on sperm–ZP binding and formation of the ARSA/SPAM1/HSPA2 complex. (A) Populations of human spermatozoa were capacitated in the presence of cholesterol sulfate (ChS, 100 µM), 10 μM herbimycin A or 10 μM H89 before being washed and introduced into a droplet containing 8–10 salt-stored oocytes. A vehicle control in which capacitated spermatozoa were incubated with DMSO was also included. After stringent washing, the number of sperm bound to zonae in each treatment group was recorded: non-capacitated = 1.2 ± 0.8; capacitated + DMSO = 32.4 ± 3.8; capacitated + ChS = 19.2 ± 1.5; capacitated + herbimycin A = 14.7 ± 2.4; capacitated + H89 = 12.3 ± 1.2. These data are expressed as a percentage of the capacitated + DMSO-treated spermatozoa. This experiment was replicated three times and the graphical data are presented as the mean ± SEM. *P < 0.05, **P < 0.001 compared with DMSO control. (B and C) Human spermatozoa were incubated either in capacitation media alone, or co-incubated with either 10 μM H89 or 10 μM herbimycin A. The cells were then solubilized in blue native lysis buffer and the extracted proteins were resolved on BN-PAGE gels. The gels were then (B) stained with Coomassie blue 250 or (C) prepared for immunoblotting with anti-HSPA2 antibodies. The arrowhead denotes the complex of ∼200 kDa containing HSPA2, ARSA and SPAM1 (Redgrove et al., 2012). Figure 7 View largeDownload slide Investigation of the effect of suppression of capacitation-associated signaling on sperm–ZP binding and formation of the ARSA/SPAM1/HSPA2 complex. (A) Populations of human spermatozoa were capacitated in the presence of cholesterol sulfate (ChS, 100 µM), 10 μM herbimycin A or 10 μM H89 before being washed and introduced into a droplet containing 8–10 salt-stored oocytes. A vehicle control in which capacitated spermatozoa were incubated with DMSO was also included. After stringent washing, the number of sperm bound to zonae in each treatment group was recorded: non-capacitated = 1.2 ± 0.8; capacitated + DMSO = 32.4 ± 3.8; capacitated + ChS = 19.2 ± 1.5; capacitated + herbimycin A = 14.7 ± 2.4; capacitated + H89 = 12.3 ± 1.2. These data are expressed as a percentage of the capacitated + DMSO-treated spermatozoa. This experiment was replicated three times and the graphical data are presented as the mean ± SEM. *P < 0.05, **P < 0.001 compared with DMSO control. (B and C) Human spermatozoa were incubated either in capacitation media alone, or co-incubated with either 10 μM H89 or 10 μM herbimycin A. The cells were then solubilized in blue native lysis buffer and the extracted proteins were resolved on BN-PAGE gels. The gels were then (B) stained with Coomassie blue 250 or (C) prepared for immunoblotting with anti-HSPA2 antibodies. The arrowhead denotes the complex of ∼200 kDa containing HSPA2, ARSA and SPAM1 (Redgrove et al., 2012). Tyrosine phosphorylation of the molecular chaperone, HSPA2 On the basis of our collective results we infer that the reorientation of the HSPA2/ARSA/SPAM1 complex may, in part, rely on the chaperoning activity of HSPA2. Furthermore, it is apparent that this activity is modulated by the cell capacitation status, raising the possibility that HSPA2 is a target for capacitation-associated tyrosine phosphorylation. An in silico analysis of HSPA2 revealed two consensus tyrosine phosphorylation motifs residing between residues 520–528 and 542–550. We therefore implemented a number of strategies to investigate the phosphorylation status of HSPA2 in human spermatozoa. Initial studies employed the use of a proximity ligation assay to determine whether HSPA2 and phosphorylated tyrosine residues co-localize in populations of non-capacitated or capacitated spermatozoa. The fluorescent signal produced as a consequence of this interaction appeared as a number of discrete spots that were present within the peri-acrosomal and post-acrosomal regions of the sperm head (Fig. 8A). This pattern resembles that previously described for HSPA2, ARSA and SPAM1 (Redgrove et al., 2012) and was detected in ∼70% of capacitated spermatozoa (Fig. 8B). In contrast, this fluorescent labeling was detected in <5% of the non-capacitated sperm population (Fig. 8B). The specificity of this labeling pattern was confirmed through the use of anti-pY and an antibody against α-tubulin. Similarly, negative controls in which sperm were labeled in the absence of either anti-HSPA2 and/or anti-pY also failed to produce any fluorescent signals in the sperm head (Fig. 8A and B). Figure 8 View largeDownload slide Tyrosine phosphorylation of HSPA2 during human spermatozoa capacitation. (A) Populations of non-capacitated (Non-Cap) and capacitated (Cap) human spermatozoa were fixed in paraformaldehyde and allowed to settle onto poly-l-lysine coated slides. These samples were then blocked in Duolink blocking solution, followed by incubation with primary antibodies (anti-HSPA2 and anti-pY; pY and tubulin; HSPA2 only; pY only) and oligonucleotide-conjugated secondary antibodies (PLA probes). The PLA probes were then ligated and the signal amplified according to the manufacturer's instructions before being viewed using fluorescence microscopy. (B) The number of cells expressing the PLA signal (red fluorescent dots) in the sperm head was recorded for each treatment. This experiment was repeated in triplicate with different donors and representative images are shown. *P < 0.05 compared with non-capacitated control. (C) Lysates of non-capacitated and capacitated human spermatozoa were focused on an IPG strip before being resolved on SDS-PAGE gels. These gels were transferred to nitrocellulose membranes and probed with either anti-HSPA2 or anti-pY antibodies. The red box indicates the region of the gel depicted in the lower panels and the red circle and arrowhead denote HSPA2 isoforms positively identified by LC-MS/MS. Figure 8 View largeDownload slide Tyrosine phosphorylation of HSPA2 during human spermatozoa capacitation. (A) Populations of non-capacitated (Non-Cap) and capacitated (Cap) human spermatozoa were fixed in paraformaldehyde and allowed to settle onto poly-l-lysine coated slides. These samples were then blocked in Duolink blocking solution, followed by incubation with primary antibodies (anti-HSPA2 and anti-pY; pY and tubulin; HSPA2 only; pY only) and oligonucleotide-conjugated secondary antibodies (PLA probes). The PLA probes were then ligated and the signal amplified according to the manufacturer's instructions before being viewed using fluorescence microscopy. (B) The number of cells expressing the PLA signal (red fluorescent dots) in the sperm head was recorded for each treatment. This experiment was repeated in triplicate with different donors and representative images are shown. *P < 0.05 compared with non-capacitated control. (C) Lysates of non-capacitated and capacitated human spermatozoa were focused on an IPG strip before being resolved on SDS-PAGE gels. These gels were transferred to nitrocellulose membranes and probed with either anti-HSPA2 or anti-pY antibodies. The red box indicates the region of the gel depicted in the lower panels and the red circle and arrowhead denote HSPA2 isoforms positively identified by LC-MS/MS. The inference that HSPA2 is tyrosine phosphorylated during capacitation was also supported by analysis of sperm lysates using 2D SDS-PAGE and immunoblotting (Fig. 8C). This approach revealed that the anticipated increase in protein tyrosine phosphorylation after capacitation was associated with proteins of the appropriate molecular weight and isoelectric point for HSPA2 (∼72 kDa, pI 5.6, respectively, Govin et al., 2006). Furthermore, HSPA2 antibodies labeled a number of additional isoforms in lysates prepared from capacitated spermatozoa compared with those of non-capacitated cells (Fig. 8C). Importantly, sequence analysis of one of the isoforms uniquely detected within the capacitated samples confirmed its identity as HSPA2 (Table I). Table I MS/MS identification of HSPA2 in protein spot excised from 2D SDS-PAGE gel. Protein (symbol)  UniProt accession number  Molecular weight (kDa)  Number of peptides matched  Peptide sequences  Mascot scores for individual peptides  Overall mascot score  Heat shock 70 kDa protein 2 (HSPA2)  P54652  70  8  K.NQVAMNPTNTIFDAK.R (O)  93.2  1205.1  R.QATKDAGTITGLNVLR.I  61.0  K.DAGTITGLNVLR.I  82.5  K.GQIQEIVLVGGSTR.I  99.0  K.GQIQEIVLVGGSTR.I (D)  59.9  K.FDLTGIPPAPR.G  72.5  K.ITITNDKGR.L (D)  51.0  R.LSKDDIDR.M  59.4  Protein (symbol)  UniProt accession number  Molecular weight (kDa)  Number of peptides matched  Peptide sequences  Mascot scores for individual peptides  Overall mascot score  Heat shock 70 kDa protein 2 (HSPA2)  P54652  70  8  K.NQVAMNPTNTIFDAK.R (O)  93.2  1205.1  R.QATKDAGTITGLNVLR.I  61.0  K.DAGTITGLNVLR.I  82.5  K.GQIQEIVLVGGSTR.I  99.0  K.GQIQEIVLVGGSTR.I (D)  59.9  K.FDLTGIPPAPR.G  72.5  K.ITITNDKGR.L (D)  51.0  R.LSKDDIDR.M  59.4  View Large Discussion It has long been known that mammalian spermatozoa must undergo capacitation within the female reproductive tract in order to acquire functional competence and express their ability to interact with the oocyte (Austin, 1951; Chang, 1951). However, many of the intricacies that underpin this process of capacitation, and the circumstances that allow for this fundamental recognition event to occur, remain largely unknown. One emerging concept, originally proposed by Asquith et al. (2004), suggests that sperm–egg recognition is a highly redundant process mediated by a suite of putative ZP receptors that are brought to the cell surface during capacitation under the influence of molecular chaperones (Asquith et al., 2004; Nixon et al., 2007). Recent refinement of this model has led to the proposal that chaperones mediate the insertion and/or stabilize the interaction of ZP recognition molecules with detergent-resistant membrane domains (lipid rafts). Such microdomains are of interest as they undergo a polarized migration during capacitation and may therefore provide a mechanism for the presentation of receptors on the anterior aspect of the sperm head where ZP contact will be initiated (Jones et al., 2010). These concepts have largely been developed on the basis of studies conducted in the mouse but are also supported by recent work in the human (Redgrove et al., 2011). Herein we have characterized the spatial and temporal behavior of one of the first chaperone assemblages to have been identified in human spermatozoa and linked to the fertility of these cells (Redgrove et al., 2012). This multimeric complex harbors two potential ZP-binding proteins, ARSA and SPAM1, in addition to a molecular chaperone HSPA2, that has also been indirectly implicated in ZP adhesion. In somatic cells, ARSA acts as a lysosomal enzyme that has the ability to desulfate various arylsulfates including the natural substrates of sulfogalactosylglycerolipid and sulfogalactosylcerebroside, in addition to a variety of small artificial substrates such as P-nitrocatechol sulfate and P-nitrophenylsulfate (Mehl and Jatzkewitz, 1968; Rahi and Srivastava, 1983). In addition to well-documented roles in the regulation of myelin sheath deposition in the central nervous system (Eckhardt, 2008), ARSA has also been identified within the acrosome of sperm from a variety of species including boar (Dudkiewicz, 1984; Tanphaichitr et al., 1998), mouse (Tanphaichitr et al., 1990; White et al., 2000; Tantibhedhyangkul et al., 2002; Weerachatyanukul et al., 2003) and human (Rattanachaiyanont et al., 2001; Tantibhedhyangkul et al., 2002). Among its proposed roles in sperm, ARSA has been implicated in the dispersion of the cumulus matrix of cumulus–oocyte complexes (Wu et al., 2007), as well as in the mediation of ZP adhesion (Rattanachaiyanont et al., 2001; Carmona et al., 2002b). Interestingly, these dual roles have also been ascribed to the other predominant component of the sperm complex characterized in this study, SPAM1 (previously known as PH-20), and again these roles appear to extend across a large number of species (Primakoff et al., 1985; Cowan et al., 1986; Phelps and Myles, 1987; Gmachl et al., 1993; Cherr et al., 1996; Hunnicutt et al., 1996; Sabeur et al., 1997; Baba et al., 2002). However, despite these overlapping functions the cooperative action of these proteins has not been studied nor are they well characterized in human spermatozoa. Herein we demonstrate the ability of SPAM1 and ARSA to form a stable complex that is dynamically reoriented during the capacitation of human spermatozoa. Consistent with proposed roles in cumulus dispersion and sperm–ZP interaction (Sabeur et al., 1997; Rattanachaiyanont et al., 2001; Tantibhedhyangkul et al., 2002), the proteins were co-localized within the peri-acrosomal region of the sperm head. Interestingly, the regulated alteration in the surface expression of both of these proteins does not occur until the latter stages of capacitation. Indeed, the results clearly demonstrate that the observed shift is not complete until after at least 60 min of capacitation (Fig. 4A). Importantly, neither of these proteins was lost from the plasma membrane of spermatozoa during capacitation (Fig. 4B and C), indicating that the change in the surface expression observed was indeed indicative of a reorientation or reorganization event. It is therefore tempting to speculate that the complex under study is initially orientated to facilitate SPAM1 exposure and promote the dispersal of the cumulus cells. However, after the cells have breached the cumulus barrier and become fully capacitated, the complex is re-orientated leading to exposure of ARSA in preparation for ZP adhesion. This model is in keeping with previous demonstrations of the capacitation-dependent appearance of ARSA on the sperm surface (Tantibhedhyangkul et al., 2002) and with compelling clinical data that has identified a positive correlation between the competence to bind the cumulus mass and the ZP (Huszar et al., 2007). Indeed, the binding of human spermatozoa to hyaluronic acid polymers is currently used as a diagnostic test in selecting high-quality spermatozoa for assisted conception therapy (Huszar et al., 2006, 2007). The present results offer a rational explanation as to why these interactions may be functionally linked. This is corroborated by the results of our sperm–ZP binding assays, which demonstrated that ARSA is probably the more dominant binding partner for the ZP (Fig. 3). Previous work on sperm ARSA has led to the proposal that it initiates sperm–ZP engagement by virtue of its ability to act as a receptor for sulfated sugar ligands that decorate the ZP glycoproteins (Tanphaichitr et al., 1993; White et al., 2000; Weerachatyanukul et al., 2001; Tantibhedhyangkul et al., 2002; Carmona et al., 2002a). This notion is supported by the demonstration that ARSA has the ability to bind to polysulfated glycans (Huang et al., 1984; O'Rand et al., 1988; Jones and Williams, 1990; Jones et al., 1996) and that the components of the ZP are highly sulfated in nature (Prasad et al., 2000). Furthermore, it has been shown that a range of synthetic sulfated substrates are capable of competitively inhibiting the fertilization of hamster oocytes in vitro at the level of sperm–zona binding (Ahuja and Gilburt, 1985). Electrostatic analyses of the structural characteristics of ARSA have revealed the presence of an additional domain adjacent to the active site that could account for the ability of the enzyme to bind to the ZP when the active site being concurrently occupied with alternate substrates (Schenk et al., 2009). Furthermore, recent studies have indicated that mutation of the active site does not inhibit the ability of ARSA to bind to mouse ZP2 and ZP3 (Xu et al., 2012). The more modest reduction in sperm–ZP binding observed after incubation of human spermatozoa with anti-SPAM1 antibodies (Fig. 3) suggests that this protein may fulfill an alternative function either upstream or downstream of ZP adhesion. This interpretation is consistent with the fact that SPAM1 is not essential for fertilization (Kang et al., 2010) but has been proposed to participate in the dispersal of the cumulus mass surrounding the ovulated oocyte. This is thought to be achieved via catalytic degradation and dispersal of the hyaluronan (or hyaluronic acid) matrix that the cumulus cells are embedded in Cherr et al. (2001), thus allowing sperm penetration through to the ZP. If this model is accurate, then the ability of the ARSA/SPAM1 complex to perform its apparently distinct roles relies heavily on the dynamic re-organization of the plasma membrane that occurs as spermatozoa undergo capacitation (Huszar et al., 1997; Cross, 2004; Gadella et al., 2008; Gadella, 2008b). One of the fundamental requirements for this remodeling is the cholesterol efflux that the plasma membrane experiences in response to HCO3− stimulus (Harrison et al., 1996; de Vries et al., 2003). The rapid loss of the stabilizing influence of cholesterol results in a higher state of membrane fluidity, and has been shown to drive the aggregation and presentation of key ZP-binding molecules to the apical surface of the sperm cell (Cross, 1998, 2003, 2004; Visconti et al., 1999; Travis and Kopf, 2002; Shadan et al., 2004; Gadella et al., 2008; Jones et al., 2010). In support of this model, our results indicate that cholesterol efflux is a prerequisite for the re-orientation of the HSPA2/ARSA/SPAM1 complex (Fig. 5B). However, while cholesterol efflux clearly plays an important role in promoting the reorientation of the complex, it is considered unlikely to be the only cellular mechanism underpinning the event. As stated above, the loss of cholesterol from the membrane occurs almost immediately upon addition of the sperm to media containing bicarbonate, which agrees with previous reports that an influx of bicarbonate and subsequent disordering of the membrane are early events in capacitation (Harrison and Miller, 2000). However, the reorientation of ARSA and SPAM1 does not appear to be complete until the later stages of capacitation. This timing coincides with the completion of the canonical capacitation-associated signaling events, thus raising the possibility that reorientation of the complex is intimately tied to the activation of PKA (Visconti et al., 1995a, b). Our results demonstrate that the inhibition of PKA through the use of either broad spectrum (staurosporine) or specific (H89) inhibitors prevents the movement of SPAM1 and ARSA within the membrane, as well as impeding tyrosine phosphorylation. However, since the activation of PKA is also an early event in the capacitation process (Harrison and Miller 2000; Lefievre et al., 2002; Visconti et al., 2002; Harrison, 2004) it is considered more likely that the reorientation phenomenon relies on the key downstream targets that become tyrosine phosphorylated during the later stages of the capacitation cascade. Indeed, bicarbonate-induced activation of PKA has been shown to peak in human spermatozoa ∼30 min after the initiation of fetal cord serum ultrafiltrate-induced capacitation (Lefievre et al., 2002), whereas increased tyrosine phosphorylation is not observed until after 1–2 h of capacitation (de Lamirande et al., 1997). This lag period has been attributed to the requisite ‘cross-talk’ of the two signaling pathways, with PKA first increasing the serine/threonine phosphorylation of a multitude of proteins, including promiscuous PTKs such as SRC (Baker et al., 2006), before these targets are able to effect tyrosine phosphorylation (de Lamirande et al., 1997). Importantly, our results clearly show that the inhibition of PTK through the use of individual broad-spectrum inhibitors, herbimycin A and genistein, abrogates the ability of SPAM1 and ARSA to reorient within the plasma membrane. It is therefore possible that tyrosine phosphorylation of the component(s) of the complex may be critical in driving the change in its surface orientation. Based on the accumulated evidence from previous studies of mouse (Asquith et al., 2004; Dun et al., 2011) and human spermatozoa (Redgrove et al., 2011) the most compelling candidate for directing the reorientation of the HSPA2/ARSA/SPAM1 complex is the resident molecular chaperone, HSPA2. Although HSPA2 has not previously been identified as a target for phosphorylation, it adds to a growing list of chaperones that appear to be phosphorylated during sperm maturation (Ecroyd et al., 2003; Asquith et al., 2004). Such modifications have been shown to regulate the activity and oligomerization of small chaperone proteins such as the HSPB members. Larger chaperones, such as HSPA and HSPC, are thought to be regulated in a similar manner, although this often occurs in conjunction with the presence of a co-chaperone (Panaretou et al., 2002; Vos et al., 2008). In addition to our preliminary evidence that HSPA2 is a target for capacitation-associated tyrosine phosphorylation, it has also been causally linked to male fertility. Indeed, the relative levels of HSPA2 expression in human spermatozoa has been used as a highly accurate diagnostic marker of male infertility (Huszar et al., 2006). Specifically, reduced HSPA2 has been positively correlated with defects in ZP adhesion (Huszar et al., 1994, 2007). Given that HSPA2 is present in a number of large molecular mass complexes in human spermatozoa (Redgrove et al., 2012) it remains to be established if this defect is solely attributed to the complex we have characterized in this study. In conclusion, this study has established an intriguing link between capacitation-associated membrane remodelling and the acquisition of ZP-binding competence. Further characterization of the receptors described in this study and those in the additional HSPA2 complexes, together with the analyses of the molecular mechanisms regulating their surface expression, are critical questions that constitute the focus of our ongoing research program. Supplementary data Supplementary data are available athttp://molehr.oxfordjournals.org/. Authors' roles K.A.R. was responsible for the acquisition, analysis and interpretation of the data, in addition to manuscript and figure drafting. A.L.A. was involved in data acquisition and analysis. E.A.M., M.K.O., R.J.A. and B.N. were involved in the study design, the analysis of data and manuscript drafting. Funding This work was supported by grants from the National Health and Medical Research Council of Australia (NHMRC Project Grants 401267, 56923, 1046346) and Hunter Medical Research Institute (HMRI 08-15). Conflict of interest None declared. Acknowledgements The authors are extremely grateful for the assistance of the staff and donors of the Hunter IVF clinic for the supply of oocytes to facilitate this study, and to the University of Newcastle's Analytical and Biomolecular Research Facility (ABRF) for protein identification via mass spectrometry. References Ahuja KK,  Gilbert DJ.  Involvement of sperm sulphatases in early sperm-zona interactions in the hamster,  J Cell Sci ,  1985, vol.  78 (pg.  247- 261) Google Scholar PubMed  Asquith KL,  Baleato RM,  McLaughlin EA,  Nixon B,  Aitken RJ.  Tyrosine phosphorylation activates surface chaperones facilitating sperm–zona recognition,  J Cell Sci ,  2004, vol.  117 (pg.  3645- 3657) Google Scholar CrossRef Search ADS PubMed  Austin CR.  Observations on the penetration of the sperm in the mammalian egg,  Aust J Sci Res ,  1951, vol.  4 (pg.  581- 596) Baba D,  Kashiwabara S,  Honda A,  Yamagata K,  Wu Q,  Ikawa M,  Okabe M,  Baba T.  Mouse sperm lacking cell surface hyaluronidase PH-20 can pass through the layer of cumulus cells and fertilize the egg,  J Biol Chem ,  2002, vol.  277 (pg.  30310- 30314) Google Scholar CrossRef Search ADS PubMed  Baker MA,  Lane DJ,  Ly JD,  De Pinto V,  Lawen A.  VDAC1 is a transplasma membrane NADH-ferricyanide reductase,  J Biol Chem ,  2004, vol.  279 (pg.  4811- 4819) Google Scholar CrossRef Search ADS PubMed  Baker MA,  Hetherington L,  Aitken RJ.  Identification of SRC as a key PKA-stimulated tyrosine kinase involved in the capacitation-associated hyperactivation of murine spermatozoa,  J Cell Sci ,  2006, vol.  119 (pg.  3182- 3192) Google Scholar CrossRef Search ADS PubMed  Baker MA,  Reeves G,  Hetherington L,  Aitken RJ.  Analysis of proteomic changes associated with sperm capacitation through the combined use of IPG-strip pre-fractionation followed by RP chromatography LC-MS/MS analysis,  Proteomics ,  2010, vol.  10 (pg.  482- 495) Google Scholar CrossRef Search ADS PubMed  Baker MA,  Nixon B,  Naumovski N,  Aitken RJ.  Proteomic insights into the maturation and capacitation of mammalian spermatozoa,  Syst Biol Reprod Med ,  2012, vol.  58 (pg.  211- 217) Google Scholar CrossRef Search ADS PubMed  Bhasin S.  Approach to the infertile man,  J Clin Endocrinol Metab ,  2007, vol.  92 (pg.  1995- 2004) Google Scholar CrossRef Search ADS PubMed  Biggers JD,  Whitten WK,  Whittingham DG.  Daniel JCJ.  The culture of mouse embryos in vitro,  Methods in Mammalian Embryology ,  1971 San Francisco, CA Freeman Press(pg.  86- 116) Carmona E,  Weerachatyanukul W,  Soboloff T,  Fluharty AL,  White D,  Promdee L,  Ekker M,  Berger T,  Buhr M,  Tanphaichitr N.  Arylsulfatase a is present on the pig sperm surface and is involved in sperm–zona pellucida binding,  Dev Biol ,  2002a, vol.  247 (pg.  182- 196) Google Scholar CrossRef Search ADS   Carmona E,  Weerachatyanukul W,  Xu H,  Fluharty A,  Anupriwan A,  Shoushtarian A,  Chakrabandhu K,  Tanphaichitr N.  Binding of arylsulfatase A to mouse sperm inhibits gamete interaction and induces the acrosome reaction,  Biol Reprod ,  2002b, vol.  66 (pg.  1820- 1827) Google Scholar CrossRef Search ADS   Chang MC.  Fertilizing capacity of spermatozoa deposited into the fallopian tubes,  Nature ,  1951, vol.  168 (pg.  697- 698) Google Scholar CrossRef Search ADS PubMed  Cherr GN,  Meyers SA,  Yudin AI,  VandeVoort CA,  Myles DG,  Primakoff P,  Overstreet JW.  The PH-20 protein in cynomolgus macaque spermatozoa: identification of two different forms exhibiting hyaluronidase activity,  Dev Bio ,  1996, vol.  175 (pg.  142- 153) Google Scholar CrossRef Search ADS   Cherr GN,  Yudin AI,  Overstreet JW.  The dual functions of GPI-anchored PH-20: hyaluronidase and intracellular signaling,  Matrix Biol ,  2001, vol.  20 (pg.  515- 525) Google Scholar CrossRef Search ADS PubMed  Cho C,  Bunch DO,  Faure JE,  Goulding EH,  Eddy EM,  Primakoff P,  Myles DG.  Fertilization defects in sperm from mice lacking fertilin beta,  Science ,  1998, vol.  281 (pg.  1857- 1859) Google Scholar CrossRef Search ADS PubMed  Cooper TG. ,  The Epididymis, Sperm Maturation and Fertilisation ,  1986 1st edn Berlin, Germany Springer-Verlag Cowan AE,  Primakoff P,  Myles DG.  Sperm exocytosis increases the amount of PH-20 antigen on the surface of guinea pig sperm,  J Cell Biol ,  1986, vol.  103 (pg.  1289- 1297) Google Scholar CrossRef Search ADS PubMed  Cross NL.  Role of cholesterol in sperm capacitation,  Biol Reprod ,  1998, vol.  59 (pg.  7- 11) Google Scholar CrossRef Search ADS PubMed  Cross NL.  Decrease in order of human sperm lipids during capacitation,  Biol Reprod ,  2003, vol.  69 (pg.  529- 534) Google Scholar CrossRef Search ADS PubMed  Cross NL.  Reorganization of lipid rafts during capacitation of human sperm,  Biol Reprod ,  2004, vol.  71 (pg.  1367- 1373) Google Scholar CrossRef Search ADS PubMed  de Lamirande E,  Leclerc P,  Gagnon C.  Capacitation as a regulatory event that primes spermatozoa for the acrosome reaction and fertilization,  Mol Hum Reprod ,  1997, vol.  3 (pg.  175- 194) Google Scholar CrossRef Search ADS PubMed  de Vries KJ,  Wiedmer T,  Sims PJ,  Gadella BM.  Caspase-independent exposure of aminophospholipids and tyrosine phosphorylation in bicarbonate responsive human sperm cells,  Biol Reprod ,  2003, vol.  68 (pg.  2122- 2134) Google Scholar CrossRef Search ADS PubMed  Dudkiewicz AB.  Purification of boar acrosomal arylsulfatase A and possible role in the penetration of cumulus cells,  Biol Reprod ,  1984, vol.  30 (pg.  1005- 1014) Google Scholar CrossRef Search ADS PubMed  Dun MD,  Smith ND,  Baker MA,  Lin M,  Aitken RJ,  Nixon B.  The chaperonin containing TCP1 complex (CCT/TRiC) is involved in mediating sperm–oocyte interaction,  J Biol Chem ,  2011, vol.  286 (pg.  36875- 36887) Google Scholar CrossRef Search ADS PubMed  Eckhardt M.  The role and metabolism of sulfatide in the nervous system,  Mol Neurobiol ,  2008, vol.  37 (pg.  93- 103) Google Scholar CrossRef Search ADS PubMed  Ecroyd H,  Jones RC,  Aitken RJ.  Tyrosine phosphorylation of HSP-90 during mammalian sperm capacitation,  Biol Reprod ,  2003, vol.  69 (pg.  1801- 1807) Google Scholar CrossRef Search ADS PubMed  Ensslin MA,  Shur BD.  Identification of mouse sperm SED1, a bimotif EGF repeat and discoidin-domain protein involved in sperm–egg binding,  Cell ,  2003, vol.  114 (pg.  405- 417) Google Scholar CrossRef Search ADS PubMed  Fenichel P,  Cervoni F,  Hofmann P,  Deckert M,  Emiliozzi C,  Hsi BL,  Rossi B.  Expression of the complement regulatory protein CD59 on human spermatozoa: characterization and role in gametic interaction,  Mol Reprod Dev ,  1994, vol.  38 (pg.  338- 346) Google Scholar CrossRef Search ADS PubMed  Gadella BM.  The assembly of a zona pellucida binding protein complex in sperm,  Reprod Dom Anim Zuch ,  2008a, vol.  43  Suppl. 5(pg.  12- 19) Google Scholar CrossRef Search ADS   Gadella BM.  Sperm membrane physiology and relevance for fertilization,  Anim Reprod Sci ,  2008b, vol.  107 (pg.  229- 236) Google Scholar CrossRef Search ADS   Gadella BM,  Tsai PS,  Boerke A,  Brewis IA.  Sperm head membrane reorganisation during capacitation,  Int J Dev Biol ,  2008, vol.  52 (pg.  473- 480) Google Scholar CrossRef Search ADS PubMed  Gmachl M,  Sagan S,  Ketter S,  Kreil G.  The human sperm protein PH-20 has hyaluronidase activity,  FEBS Lett ,  1993, vol.  336 (pg.  545- 548) Google Scholar CrossRef Search ADS PubMed  Govin J,  Caron C,  Escoffier E,  Ferro M,  Kuhn L,  Rousseaux S,  Eddy EM,  Garin J,  Khochbin S.  Post-meiotic shifts in HSPA2/HSP70.2 chaperone activity during mouse spermatogenesis,  J Biol Chem ,  2006, vol.  281 (pg.  37888- 37892) Google Scholar CrossRef Search ADS PubMed  Harrison RA.  Rapid PKA-catalysed phosphorylation of boar sperm proteins induced by the capacitating agent bicarbonate,  Mol Reprod Dev ,  2004, vol.  67 (pg.  337- 352) Google Scholar CrossRef Search ADS PubMed  Harrison RA,  Gadella BM.  Bicarbonate-induced membrane processing in sperm capacitation,  Theriogenology ,  2005, vol.  63 (pg.  342- 351) Google Scholar CrossRef Search ADS PubMed  Harrison RA,  Miller NG.  cAMP-dependent protein kinase control of plasma membrane lipid architecture in boar sperm,  Mol Reprod Dev ,  2000, vol.  55 (pg.  220- 228) Google Scholar CrossRef Search ADS PubMed  Harrison RA,  Ashworth PJ,  Miller NG.  Bicarbonate/CO2, an effector of capacitation, induces a rapid and reversible change in the lipid architecture of boar sperm plasma membranes,  Mol Reprod Dev ,  1996, vol.  45 (pg.  378- 391) Google Scholar CrossRef Search ADS PubMed  Huang TT,  Kosower NS,  Yanagimachi R.  Localization of thiol and disulfide groups in guinea pig spermatozoa during maturation and capacitation using bimane fluorescent labels,  Biol Reprod ,  1984, vol.  31 (pg.  797- 809) Google Scholar CrossRef Search ADS PubMed  Hunnicutt GR,  Primakoff P,  Myles DG.  Sperm surface protein PH-20 is bifunctional: one activity is a hyaluronidase and a second, distinct activity is required in secondary sperm–zona binding,  Biol Reprod ,  1996, vol.  55 (pg.  80- 86) Google Scholar CrossRef Search ADS PubMed  Huszar G,  Vigue L,  Oehninger S.  Creatine kinase immunocytochemistry of human sperm–hemizona complexes: selective binding of sperm with mature creatine kinase-staining pattern,  Fert Steril ,  1994, vol.  61 (pg.  136- 142) Google Scholar CrossRef Search ADS   Huszar G,  Sbracia M,  Vigue L,  Miller DJ,  Shur BD.  Sperm plasma membrane remodeling during spermiogenetic maturation in men: relationship among plasma membrane beta 1,4-galactosyltransferase, cytoplasmic creatine phosphokinase, and creatine phosphokinase isoform ratios,  Biol Reprod ,  1997, vol.  56 (pg.  1020- 1024) Google Scholar CrossRef Search ADS PubMed  Huszar G,  Ozkavukcu S,  Jakab A,  Celik-Ozenci C,  Sati GL,  Cayli S.  Hyaluronic acid binding ability of human sperm reflects cellular maturity and fertilizing potential: selection of sperm for intracytoplasmic sperm injection,  Curr Opin Obstet Gynecol ,  2006, vol.  18 (pg.  260- 267) Google Scholar CrossRef Search ADS PubMed  Huszar G,  Jakab A,  Sakkas D,  Ozenci CC,  Cayli S,  Delpiano E,  Ozkavukcu S.  Fertility testing and ICSI sperm selection by hyaluronic acid binding: clinical and genetic aspects,  Reprod Biomed Online ,  2007, vol.  14 (pg.  650- 663) Google Scholar CrossRef Search ADS PubMed  Jones R,  Williams RM.  Identification of zona- and fucoidan-binding proteins in guinea-pig spermatozoa and mechanism of recognition,  Development ,  1990, vol.  109 (pg.  41- 50) Google Scholar PubMed  Jones R,  Parry R,  Lo Leggio L,  Nickel P.  Inhibition of sperm–zona binding by suramin, a potential ‘lead’ compound for design of new anti-fertility agents,  Mol Hum Reprod ,  1996, vol.  2 (pg.  597- 605) Google Scholar CrossRef Search ADS PubMed  Jones R,  Howes E,  Dunne PD,  James P,  Bruckbauer A,  Klenerman D.  Tracking diffusion of GM1 gangliosides and zona pellucida binding molecules in sperm plasma membranes following cholesterol efflux,  Dev Biol ,  2010, vol.  339 (pg.  398- 406) Google Scholar CrossRef Search ADS PubMed  Kang W,  Zhou C,  Koga Y,  Baba T.  Hyaluronan-degrading activity of mouse sperm hyaluronidase is not required for fertilization?,  J Reprod Dev ,  2010, vol.  56 (pg.  140- 144) Google Scholar CrossRef Search ADS PubMed  Kimura M,  Kim E,  Kang W,  Yamashita M,  Saigo M,  Yamazaki T,  Nakanishi T,  Kashiwabara S,  Baba T.  Functional roles of mouse sperm hyaluronidases, HYAL5 and SPAM1, in fertilization,  Biol Reprod ,  2009, vol.  81 (pg.  939- 947) Google Scholar CrossRef Search ADS PubMed  Lathrop WF,  Carmichael EP,  Myles DG,  Primakoff P.  cDNA cloning reveals the molecular structure of a sperm surface protein, PH-20, involved in sperm–egg adhesion and the wide distribution of its gene among mammals,  J Cell Biol ,  1990, vol.  111 (pg.  2939- 2949) Google Scholar CrossRef Search ADS PubMed  Leclerc P,  de Lamirande E,  Gagnon C.  Cyclic adenosine 3′,5′ monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility,  Biol Reprod ,  1996, vol.  55 (pg.  684- 692) Google Scholar CrossRef Search ADS PubMed  Leclerc P,  de Lamirande E,  Gagnon C.  Interaction between Ca2+, cyclic 3′,5′ adenosine monophosphate, the superoxide anion, and tyrosine phosphorylation pathways in the regulation of human sperm capacitation,  J Androl ,  1998, vol.  19 (pg.  434- 443) Google Scholar PubMed  Lefievre L,  Jha KN,  de Lamirande E,  Visconti PE,  Gagnon C.  Activation of protein kinase A during human sperm capacitation and acrosome reaction,  J Androl ,  2002, vol.  23 (pg.  709- 716) Google Scholar PubMed  Liu DY,  Baker HW.  Defective sperm–zona pellucida interaction: a major cause of failure of fertilization in clinical in-vitro fertilization,  Hum Reprod ,  2000, vol.  15 (pg.  702- 708) Google Scholar CrossRef Search ADS PubMed  Lu Q,  Shur BD.  Sperm from beta 1,4-galactosyltransferase-null mice are refractory to ZP3-induced acrosome reactions and penetrate the zona pellucida poorly,  Development ,  1997, vol.  124 (pg.  4121- 4131) Google Scholar PubMed  McLachlan RI,  de Kretser DM.  Male infertility: the case for continued research,  Med J Aust ,  2001, vol.  174 (pg.  116- 117) Google Scholar PubMed  Mehl E,  Jatzkewitz H.  Cerebroside 3-sulfate as a physiological substrate of arylsulfatase A,  Biochim Biophys Acta ,  1968, vol.  151 (pg.  619- 627) Google Scholar CrossRef Search ADS PubMed  Mitchell LA,  Nixon B,  Aitken RJ.  Analysis of chaperone proteins associated with human spermatozoa during capacitation,  Mol Human Reprod ,  2007, vol.  13 (pg.  605- 613) Google Scholar CrossRef Search ADS   Mitchell LA,  Nixon B,  Baker MA,  Aitken RJ.  Investigation of the role of SRC in capacitation-associated tyrosine phosphorylation of human spermatozoa,  Mol Hum Reprod ,  2008, vol.  14 (pg.  235- 243) Google Scholar CrossRef Search ADS PubMed  Myles DG,  Primakoff P.  Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization,  J Cell Biol ,  1984, vol.  99 (pg.  1634- 1641) Google Scholar CrossRef Search ADS PubMed  Nishimura H,  Cho C,  Branciforte DR,  Myles DG,  Primakoff P.  Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin beta,  Dev Biol ,  2001, vol.  233 (pg.  204- 213) Google Scholar CrossRef Search ADS PubMed  Nixon B,  Aitken RJ,  McLaughlin EA.  New insights into the molecular mechanisms of sperm–egg interaction,  Cell Mol Life Sci ,  2007, vol.  64 (pg.  1805- 1823) Google Scholar CrossRef Search ADS PubMed  Nixon B,  Bielanowicz A,  McLaughlin EA,  Tanphaichitr N,  Ensslin MA,  Aitken RJ.  Composition and significance of detergent resistant membranes in mouse spermatozoa,  J Cell Physiol ,  2009, vol.  218 (pg.  122- 134) Google Scholar CrossRef Search ADS PubMed  Nixon B,  Mitchell LA,  Anderson AL,  McLaughlin EA,  O'Bryan MK,  Aitken RJ.  Proteomic and functional analysis of human sperm detergent resistant membranes,  J Cell Physiol ,  2011, vol.  226 (pg.  2651- 2665) Google Scholar CrossRef Search ADS PubMed  Nixon B,  Paul JW,  Spiller CM,  Attwell-Heap AG,  Ashman LK,  Aitken RJ.  Evidence for the involvement of PECAM-1 in a receptor mediated signal-transduction pathway regulating capacitation-associated tyrosine phosphorylation in human spermatozoa,  J Cell Sci ,  2005, vol.  118 (pg.  4865- 4877) Google Scholar CrossRef Search ADS PubMed  O'Rand MG,  Widgren EE,  Fisher SJ.  Characterization of the rabbit sperm membrane autoantigen, RSA, as a lectin-like zona binding protein,  Dev Biol ,  1988, vol.  129 (pg.  231- 240) Google Scholar CrossRef Search ADS PubMed  Osheroff JE,  Visconti PE,  Valenzuela JP,  Travis AJ,  Alvarez J,  Kopf GS.  Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinase A-mediated up-regulation of protein tyrosine phosphorylation,  Mol Hum Reprod ,  1999, vol.  5 (pg.  1017- 1026) Google Scholar CrossRef Search ADS PubMed  Panaretou B,  Siligardi G,  Meyer P,  Maloney A,  Sullivan JK,  Singh S,  Millson SH,  Clarke PA,  Naaby-Hansen S,  Stein R, et al.  Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1,  Mol Cell ,  2002, vol.  10 (pg.  1307- 1318) Google Scholar CrossRef Search ADS PubMed  Phelps BM,  Myles DG.  The guinea pig sperm plasma membrane protein, PH-20, reaches the surface via two transport pathways and becomes localized to a domain after an initial uniform distribution,  Dev Biol ,  1987, vol.  123 (pg.  63- 72) Google Scholar CrossRef Search ADS PubMed  Prasad SV,  Skinner SM,  Carino C,  Wang N,  Cartwright J,  Dunbar BS.  Structure and function of the proteins of the mammalian Zona pellucida,  Cells Tissues Organs ,  2000, vol.  166 (pg.  148- 164) Google Scholar CrossRef Search ADS PubMed  Primakoff P,  Hyatt H,  Myles DG.  A role for the migrating sperm surface antigen PH-20 in guinea pig sperm binding to the egg zona pellucida,  J Cell Biol ,  1985, vol.  101 (pg.  2239- 2244) Google Scholar CrossRef Search ADS PubMed  Rahi H,  Srivastava PN.  Isolation and characterization of the pig endometrial arylsulphatase A,  Biochem J ,  1983, vol.  211 (pg.  649- 659) Google Scholar CrossRef Search ADS PubMed  Rattanachaiyanont M,  Weerachatyanukul W,  Leveille MC,  Taylor T,  D'Amours D,  Rivers D,  Leader A,  Tanphaichitr N.  Anti-SLIP1-reactive proteins exist on human spermatozoa and are involved in zona pellucida binding,  Mol Hum Reprod ,  2001, vol.  7 (pg.  633- 640) Google Scholar CrossRef Search ADS PubMed  Redgrove KA,  Anderson AL,  Dun MD,  McLaughlin EA,  O'Bryan MK,  Aitken RJ,  Nixon B.  Involvement of multimeric protein complexes in mediating the capacitation-dependent binding of human spermatozoa to homologous zonae pellucidae,  Dev Biol ,  2011, vol.  356 (pg.  460- 474) Google Scholar CrossRef Search ADS PubMed  Redgrove KA,  Nixon B,  Baker MA,  Hetherington L,  Baker G,  De-Yi L,  Aitken RJ.  The molecular chaperone HSPA2 plays a key role in regulating the expression of sperm surface receptors that mediate sperm–egg recognition,  PLoS One ,  2012, vol.  7 pg.  e50851  Google Scholar CrossRef Search ADS PubMed  Reid AT,  Redgrove K,  Aitken RJ,  Nixon B.  Cellular mechanisms regulating sperm–zona pellucida interaction,  Asian J Androl ,  2010, vol.  13 (pg.  88- 96) Google Scholar CrossRef Search ADS PubMed  Sabeur K,  Cherr GN,  Yudin AI,  Primakoff P,  Li MW,  Overstreet JW.  The PH-20 protein in human spermatozoa,  J Androl ,  1997, vol.  18 (pg.  151- 158) Google Scholar PubMed  Schenk M,  Koppisetty CA,  Santos DC,  Carmona E,  Bhatia S,  Nyholm PG,  Tanphaichitr N.  Interaction of arylsulfatase-A (ASA) with its natural sulfoglycolipid substrates: a computational and site-directed mutagenesis study,  Glycoconj J ,  2009, vol.  26 (pg.  1029- 1045) Google Scholar CrossRef Search ADS PubMed  Shadan S,  James PS,  Howes EA,  Jones R.  Cholesterol efflux alters lipid raft stability and distribution during capacitation of boar spermatozoa,  Biol Reprod ,  2004, vol.  71 (pg.  253- 265) Google Scholar CrossRef Search ADS PubMed  Shamsadin R,  Adham IM,  Nayernia K,  Heinlein UA,  Oberwinkler H,  Engel W.  Male mice deficient for germ-cell cyritestin are infertile,  Biol Reprod ,  1999, vol.  61 (pg.  1445- 1451) Google Scholar CrossRef Search ADS PubMed  Tanphaichitr N,  Smith J,  Kates M.  Levels of sulfogalactosylglycerolipid in capacitated motile and immotile mouse spermatozoa,  Biochem Cell Biol ,  1990, vol.  68 (pg.  528- 535) Google Scholar CrossRef Search ADS PubMed  Tanphaichitr N,  Smith J,  Mongkolsirikieart S,  Gradil C,  Lingwood CA.  Role of a gamete-specific sulfoglycolipid immobilizing protein on mouse sperm–egg binding,  Dev Biol ,  1993, vol.  156 (pg.  164- 175) Google Scholar CrossRef Search ADS PubMed  Tanphaichitr N,  Moase C,  Taylor T,  Surewicz K,  Hansen C,  Namking M,  Berube B,  Kamolvarin N,  Lingwood CA,  Sullivan R, et al.  Isolation of antiSLIP1-reactive boar sperm P68/62 and its binding to mammalian zona pellucida,  Mol Reprod Dev ,  1998, vol.  49 (pg.  203- 216) Google Scholar CrossRef Search ADS PubMed  Tantibhedhyangkul J,  Weerachatyanukul W,  Carmona E,  Xu H,  Anupriwan A,  Michaud D,  Tanphaichitr N.  Role of sperm surface arylsulfatase A in mouse sperm–zona pellucida binding,  Biol Reprod ,  2002, vol.  67 (pg.  212- 219) Google Scholar CrossRef Search ADS PubMed  Towbin H,  Staehelin T,  Gordon J.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications,  Proc Natl Acad Sci USA ,  1979, vol.  76 (pg.  4350- 4354) Google Scholar CrossRef Search ADS PubMed  Travis AJ,  Kopf GS.  The role of cholesterol efflux in regulating the fertilization potential of mammalian spermatozoa,  J Clin Invest ,  2002, vol.  110 (pg.  731- 736) Google Scholar CrossRef Search ADS PubMed  Visconti PE,  Bailey JL,  Moore GD,  Pan D,  Olds-Clarke P,  Kopf GS.  Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation,  Development ,  1995a, vol.  121 (pg.  1129- 1137) Visconti PE,  Moore GD,  Bailey JL,  Leclerc P,  Connors SA,  Pan D,  Olds-Clarke P,  Kopf GS.  Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway,  Development ,  1995b, vol.  121 (pg.  1139- 1150) Visconti PE,  Ning X,  Fornes MW,  Alvarez JG,  Stein P,  Connors SA,  Kopf GS.  Cholesterol efflux-mediated signal transduction in mammalian sperm: cholesterol release signals an increase in protein tyrosine phosphorylation during mouse sperm capacitation,  Dev Biol ,  1999, vol.  214 (pg.  429- 443) Google Scholar CrossRef Search ADS PubMed  Visconti PE,  Westbrook VA,  Chertihin O,  Demarco I,  Sleight S,  Diekman AB.  Novel signaling pathways involved in sperm acquisition of fertilizing capacity,  J Reprod Immunol ,  2002, vol.  53 (pg.  133- 150) Google Scholar CrossRef Search ADS PubMed  Vos MJ,  Hageman J,  Carra S,  Kampinga HH.  Structural and functional diversities between members of the human HSPB, HSPH, HSPA, and DNAJ chaperone families,  Biochemistry ,  2008, vol.  47 (pg.  7001- 7011) Google Scholar CrossRef Search ADS PubMed  Weerachatyanukul W,  Rattanachaiyanont M,  Carmona E,  Furimsky A,  Mai A,  Shoushtarian A,  Sirichotiyakul S,  Ballakier H,  Leader A,  Tanphaichitr N.  Sulfogalactosylglycerolipid is involved in human gamete interaction,  Mol Reprod Dev ,  2001, vol.  60 (pg.  569- 578) Google Scholar CrossRef Search ADS PubMed  Weerachatyanukul W,  Xu H,  Anupriwan A,  Carmona E,  Wade M,  Hermo L,  da Silva SM,  Rippstein P,  Sobhon P,  Sretarugsa P, et al.  Acquisition of arylsulfatase A onto the mouse sperm surface during epididymal transit,  Biol Reprod ,  2003, vol.  69 (pg.  1183- 1192) Google Scholar CrossRef Search ADS PubMed  White D,  Weerachatyanukul W,  Gadella B,  Kamolvarin N,  Attar M,  Tanphaichitr N.  Role of sperm sulfogalactosylglycerolipid in mouse sperm–zona pellucida binding,  Biol Reprod ,  2000, vol.  63 (pg.  147- 155) Google Scholar CrossRef Search ADS PubMed  Wu A,  Anupriwan A,  Iamsaard S,  Chakrabandhu K,  Santos DC,  Rupar T,  Tsang BK,  Carmona E,  Tanphaichitr N.  Sperm surface arylsulfatase A can disperse the cumulus matrix of cumulus oocyte complexes,  J Cell Physiol ,  2007, vol.  213 (pg.  201- 211) Google Scholar CrossRef Search ADS PubMed  Xu H,  Liu F,  Srakaew N,  Koppisetty C,  Nyholm PG,  Carmona E,  Tanphaichitr N.  Sperm arylsulfatase A binds to mZP2 and mZP3 glycoproteins in a nonenzymatic manner,  Reproduction ,  2012, vol.  144 (pg.  209- 219) Google Scholar CrossRef Search ADS PubMed  Yanagimachi R.  Fertility of mammalian spermatozoa: its development and relativity,  Zygote ,  1994, vol.  2 (pg.  371- 372) Google Scholar CrossRef Search ADS PubMed  Yanagimachi R,  Lopata A,  Odom CB,  Bronson RA,  Mahi CA,  Nicolson GL.  Retention of biologic characteristics of zona pellucida in highly concentrated salt solution: the use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa,  Fert Steril ,  1979, vol.  31 (pg.  562- 574) Google Scholar CrossRef Search ADS   © The Author 2012. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com
TI - Investigation of the mechanisms by which the molecular chaperone HSPA2 regulates the expression of sperm surface receptors involved in human sperm–oocyte recognition
JF - Molecular Human Reproduction
DO - 10.1093/molehr/gas064
DA - 2012-12-17
UR - https://www.deepdyve.com/lp/oxford-university-press/investigation-of-the-mechanisms-by-which-the-molecular-chaperone-hspa2-1tcNSE8hSt
SP - 120
EP - 135
VL - 19
IS - 3
DP - DeepDyve
ER -