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BOR - Papers in Press, published online ahead of print October 12, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.044644
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BIOLOGY OF REPRODUCTION 74, 275–287 (2006)
DOI: 10.1095/biolreprod.105.044644
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

The Identification of Mouse Sperm-Surface-Associated Proteins and Characterization of Their Ability to Act as Decapacitation Factors1

Brett Nixon 3, David A. MacIntyre 34 , Lisa A. Mitchell 3, Gerard M. Gibbs 5, Moira O'Bryan 5, and R. John Aitken 2 6

Reproductive Science Group,3 School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales 2308, Australia Mothers and Babies Research Centre,4 John Hunter Hospital, Newcastle, New South Wales 2310, Australia Monash Institute of Medical Research and ARC Centre of Excellence in Biotechnology and Development,5 Clayton, Victoria 3168, Australia ARC Centre of Excellence in Biotechnology and Development,6 School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales 2308, Australia

ABSTRACT

Mammalian spermatozoa must undergo capacitation before acquiring the ability to fertilize the oocyte. This process is believed to be initiated following the release of surface-associated decapacitation factors that are elaborated by both the epididymis and the male accessory organs. Herein, we report the identification of a number of proteins that are actively released from the surface of mouse spermatozoa during capacitation in vitro. As anticipated, the addition of these factors back to suspensions of mouse spermatozoa was shown to suppress several correlates of the capacitation process. Specifically, they induced a significant, dose-dependent inhibition of the ability of spermatozoa to undergo a progesterone-induced acrosome reaction and to bind to the zona pellucida in vitro. Inhibition of these functions was associated with the suppression of tyrosine phosphorylation in the sperm plasma membrane but had no effect on the phosphorylation of internal proteins in either the sperm head or tail. This inhibitory activity was attributed to a subset of the isolated proteins compromising at least four putative decapacitation factors. These proteins were identified via tandem-mass spectrometry amino acid sequence analysis as plasma membrane fatty acid binding protein, cysteine-rich secretory protein 1 (CRISP1), phosphatidylethanolamine binding protein 1 (PBP), and an unnamed protein product that we have termed decapacitation factor 10 (DF10). Of these proteins, PBP was identified as a primary candidate for a decapacitation factor.

epididymis, male reproductive tract, sperm capacitation

INTRODUCTION

Although spermatozoa attain morphological maturity within the testis, they must undergo two additional phases of functional maturation before gaining the competence to fertilize an egg. The first of these phases occurs as spermatozoa transit the epididymis, while the second is induced as these cells are exposed to poorly defined molecular stimuli during their ascent of the female reproductive tract. This final maturation step encompasses a series of elaborate cellular modifications that are collectively termed capacitation [1, 2].

In the 50 yr that have elapsed since capacitation was first described, a number of changes have been associated with this process, including extensive remodeling of the sperm plasma membrane and activation of an unusual cAMP-mediated tyrosine phosphorylation signal transduction cascade [3, 4]. There is now general agreement that capacitation is preceded by the loss or removal of surface-associated inhibitory factors that originate from the secretions of both the epididymis and accessory organs. The removal of such decapacitation factors from uncapacitated populations of spermatozoa has been shown to elicit a rapid increase in their fertilizing ability [5]. Furthermore, consistent with the notion that capacitation is a reversible process, the reintroduction of decapacitation factors to capacitated populations readily inhibits their fertilizing ability [6].

At present, the most well-characterized decapacitation factor is that identified by Fraser and colleagues and designated DF [5–7]. DF is a glycoprotein of 40 kDa that binds to a GPI-anchored membrane receptor located on the postacrosomal head region of uncapacitated caudal mouse spermatozoa [7]. In this position, DF is believed to positively regulate the activity of a plasma membrane Ca2+-ATPase. The dissociation of DF, in turn, reduces Ca2+-ATPase activity, resulting in a rise in intracellular Ca2+ levels and a concomitant stimulation of capacitation [8]. The inhibitory activity of a number of additional proteins, across a range of species, has also been documented [7, 9–20]. However, the identity of such proteins and the molecular mechanisms through which they act remains largely unresolved.

In light of these deficiencies in our knowledge, we have sought to characterize the complement of proteins that are passively released from the sperm surface during capacitation and identify those that possess inhibitory activity. These studies should help us produce an integrated model for sperm capacitation and shed light on the molecular mechanisms by which decapacitation factors prevent the premature onset of this process.

MATERIALS AND METHODS

Reagents

Unless otherwise stated, chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) and were of molecular-biology or research grade. Antiphosphotyrosine monoclonal antibody (clone 4G10), anti-phosphatidylethanolamine binding protein (PBP) polyclonal antibody (anti-RKIP), and anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP) conjugate were from Upstate Biotechnology (Lake Placid, NY). Biotin (Sulfo-NHS-LC-Biotin) was obtained from Pierce Chemical Co. (Rockford, IL). Streptavidin HRP conjugate was from Silenus (Melbourne, Australia).

Animals

All experimental procedures were carried out with the approval of the University of Newcastle's Animal Care and Ethics Committee (ACEC). Inbred Swiss mice were obtained from a breeding colony held at the institute's Central Animal House and maintained according to the recommendations prescribed by the ACEC. Mice were housed under a controlled-lighting regime (16L:8D) at 21–22°C and supplied with food and water ad libitum. Prior to dissection, animals were killed via CO2 inhalation.

Collection and Preparation of Spermatozoa

Immediately after adult male mice (>8 wk old) were killed, the epididymides were removed and carefully dissected free of fat and overlying connective tissue. The caudal region was isolated, blotted free of blood, and immersed under prewarmed water-saturated mineral oil. A small incision was made into the duct and sperm were teased out into a droplet of modified Biggers, Whitten, and Whittingham media (BWW; [21]) composed of 91.5 mM NaCl, 4.6 mM KCl, 1.7 mM CaCl20.2H2O, 1.2 mM KH2PO4, 1.2 mM MgSO40.7H2O, 25 mM NaHCO3, 5.6 mM D-glucose, 0.27 mM sodium pyruvate, 44 mM sodium lactate, 5 U/ml penicillin, 5 µg/ml streptomycin, 20 mM HEPES buffer, and 1 mg/ml polyvinyl alcohol (PVA), then allowed to disperse into the medium for 10 min. Where indicated, negative-control incubations were prepared using medium lacking NaHCO3, while positive-control incubations were conducted in media supplemented with 3 mM pentoxifylline (ptx) and 5 mM dibutyryl cyclic adenosine monophosphate (cAMP) and lacking CaCl2 but with additional NaCl incorporated to maintain an osmolarity of 300 mOsm/kg [22].

Following collection, sperm concentration was determined and the cells diluted as required. Sperm were then gently centrifuged (400 x g for 1 min) to separate epididymal plasma, resuspended in BWW, and assessed for cell vitality and motility. Samples in which either parameter was below 60% were discarded.

Unless otherwise stated, spermatozoa were capacitated by incubation for 90 min at 37°C under an atmosphere of 5% CO2:95% air. At regular intervals throughout the incubation, sperm suspensions were gently mixed to prevent settling of the cells and, at the end of the incubation, sperm vitality and motility were again assessed. Neither parameter was affected by any of the treatments reported in this study.

Biotinylation of Sperm-Surface Proteins

To confirm the surface orientation of the proteins isolated in this study, spermatozoa were vectorially labeled with sulfo-NHS-LC-biotin (Pierce), a membrane-impermeable derivative of biotin, in accordance with the manufacturer's instructions. Briefly, caudal spermatozoa were diluted to a concentration of 2 x 106 cells/ml and capacitated, as described above, in media supplemented with 0.5 mg/ml sulfo-NHS-LC-biotin. After 30 min, the biotinylation reaction was stopped by the addition of Tris (pH 7.4) to a final concentration of 1 mM and capacitation continued for a further 60 min. Following capacitation, spermatozoa were pelleted by centrifugation at 400 x g for 3 min and solubilized by incubation in 10 mM CHAPS ((3-[(3-Cholamidopropyl) dimethylammonio]-1propanesulfonate) for 1 h on ice. An aliquot of solubilized protein (2 µg) was resolved on a 10% SDS-PAGE gel and electrotransferred to a nitrocellulose membrane as described below. Affinity detection of biotinylated surface proteins was performed at room temperature using a streptavidin-HRP conjugated probe and an enhanced chemiluminescence (ECL) detection kit (Amersham).

The surface specificity and impact of the biotinylation reaction on capacitation was monitored by affinity labeling an aliquot of the sperm suspension with fluorescein isothiocynate (FITC)-conjugated streptavidin and by assessing the level of phosphotyrosine expression observed in treated cells, respectively, at various intervals throughout the incubation. Importantly, greater than 90% of biotinylated cells were characterized by a biotinylation labeling pattern consistent with that expected of intact cells and the kinetics and intensity of phosphotyrosine expression was indistinguishable from that seen in untreated samples (Nixon, unpublished observation).

Isolation of Released Sperm-Surface Proteins

Bulk isolation of proteins released from the sperm surface during capacitation was performed within a Transwell cell culture chamber system (Corning Costar Corporation, Cambridge, MA) consisting of an inner and outer well separated by a 0.4-µm polycarbonate membrane. The pore size of the membrane was selected to facilitate the passage of proteins released from the spermatozoa into the collection well while excluding whole cells. Following capacitation, the inner chamber, containing spermatozoa, was removed and the released proteins were recovered from the outer well. To remove excess salt and exclude molecular species with molecular weights below 10 kDa from the isolated proteins, they were transferred to dialysis membrane (molecular weight cut-off of 10 kDa) and dialyzed overnight at 4°C against three changes of H2O. The protein concentration was then estimated using a bicinchroninic acid (BCA) protein assay kit (Pierce) and the proteins aliquoted into appropriate volumes before being lyophilized. Proteins were then analyzed by either one (1D) and/or two-dimensional (2D) SDS-PAGE and used for the functional assays described below.

Analysis of Released Sperm-Surface Proteins

For the purpose of 1D SDS-PAGE, equivalent amounts of protein (2 µg) were denatured by boiling in SDS sample buffer [23] containing 2% ß-mercaptoethanol for 5 min and resolved on 10% SDS polyacrylamide gels according to the methods described by Laemmli [23]. For 2D SDS-PAGE, 10 µg of protein was precipitated with three volumes of ice-cold acetone at 4°C for 1 h, then resolubilized in 150 µl rehydration buffer (8 M Urea, 2% CHAPS, 10 mM DTT, 2% ampholytes [pH 3–10], 0.1% bromophenol blue) and used to rehydrate precast immobilized pH gradient acrylamide gels (IPG, 7 cm, pH 3–10; Amersham Biosciences, Buckinghamshire, U.K.). Isoelectric focusing was performed on a Multiphore II system (Amersham) using the following protocol: 200 V/30 min; 1500 V/2 h; 3000 V/13 h. At the completion of isoelectric focusing, IPG gels were placed in equilibration buffer (50 mM Tris, pH 6.8, 6 M urea, 30% glycerol, 2% SDS), containing 2% DTT for 10 min, then in equilibration buffer containing 2.5% iodoacetamide and 1% bromophenol blue for a further 10 min. Second-dimension protein separation was performed on large-format 10% SDS-PAGE gels.

Proteins resolved by either 1D or 2D SDS-PAGE were either electrotransferred to nitrocellulose membranes (Hybond Super-C; Amersham) under a constant current of 300 mA for 1 h [24] or silver stained [25]. Proteins of interest were excised from the gels, destained for 5 min with 0.3% potassium ferricyanide, and washed repeatedly with H2O. The gel slices were then dehydrated in actetonitrile before being rehydrated in a minimal volume of 20 mM ammonium bicarbonate containing 40 ng/µl trypsin for 16 h at 37°C. The resulting tryptic peptides were purified using a ZipTip and sequenced by electrosprayionization time of flight (ESI-TOF) tandem mass spectrometry (MS/MS) at the Australian Proteome Analysis Facility. The acquired peptide sequences were used to interrogate the NCBI/BLAST Swiss-Prot database using the FASTA program (http://fasta.bioch.virginia.edu) [26].

Fractionation of Isolation Sperm-Surface Proteins

Fractionation of isolated sperm proteins was performed by passing them over a Sephadex-G50 (Amersham) size exclusion column (inner diameter 15 mm, length 900 mm). Briefly, a concentrated 0.5-ml aliquot of the sample containing approximately 1 mg protein was applied to the column and eluted with 5 mM Tris (pH 7.4) maintained at a constant flow rate of 0.25 ml/min. The protein content of the eluant was monitored throughout the separation procedure by UV absorption. Fractions of 10 ml were collected, lyophilized, and analyzed by 1D SDS-PAGE electrophoresis before being used for functional assays.

Preparation of Recombinant PBP

Recombinant mouse PBP was expressed from the pOXP1 plasmid (derived from pET22b) in Escherichia coli BL21(DE3) and subsequently purified by IMAC affinity chromatography as described previously [27], except that washes and elutions were done in PBS with 2 mM ß-mercaptoethanol and 5 mM or 250 mM imidazol, respectively. Purified recombinant protein was buffer exchanged to PBS using Amicon Ultra 15 (Millipore) centrifugal filtration with a 5-kDa molecular-weight cut off. Recombinant PBP was at least 95% pure, as assessed by SDS-PAGE.

Inhibitory Activity of Isolated Sperm-Surface Proteins

The ability of the isolated sperm-surface proteins to inhibit sperm capacitation was assessed against a range of standard functional assays, including the ability of spermatozoa to tyrosine phosphorylate, bind to the zona pellucida, and undergo a progesterone-induced acrosome reaction. For the purpose of each of these studies, spermatozoa were capacitated in either BWW medium alone or in BWW medium supplemented with varying concentrations (100, 200, or 400 µg/ml) of the isolated proteins. The specificity of the inhibitory activity was confirmed by the inclusion of control samples treated with either 400 µg/ml BSA as a nonspecific protein or 400 µg/ml of the isolated proteins that were heat denatured by boiling at 100°C for 5 min.

In addition, the cholesterol content of each collection was accurately assessed using the Amplex Red Cholesterol Assay kit (Molecular Probes, Eugene, OR) in accordance with the manufacturer's instructions. Briefly, an aliquot of the dialyzed sample corresponding to the highest concentration of isolated proteins used for inhibitory studies (400 µg) was lyophilized, resuspended in 50 µl of reaction buffer, and mixed with a working solution composed of 300 µM Amplex Red reagent, 2 U/ml HRP, 2 U/ml cholesterol oxidase, and 2 U/ml cholesterol esterase in the wells of a 96-well microtiter plate. The plate was then incubated for 2 h at 37°C and the resulting fluorescence was measured using an Ultramark Microplate Imaging System (Biorad). The cholesterol content of each sample was estimated from a standard curve prepared using the supplied cholesterol reference standards. An equivalent amount of cholesterol sulfate was then added to a separate sperm sample (cholesterol control) to assess its inhibitory activity in the progesterone-induced acrosome reaction assay.

Detection of Protein Tyrosine Phosphorylation

Following capacitation of spermatozoa under the specified conditions, the cells were solubilized by boiling in extraction buffer (0.375 M Tris-HCl, pH 6.8, 2% SDS, 10% sucrose) supplemented with protease inhibitors and sodium orthovanadate (1 mM) for 5 min. Equivalent amounts of protein (2 µg) from the soluble fraction of the cell lysate were resolved by 1D SDS-PAGE and electro-transferred to nitrocellulose membranes as described above. Immunodetection of phosphotyrosine residues was performed at room temperature, as previously described [28], using an antiphosphotyrosine monoclonal antibody (clone 4G10; Upstate). Phosphorylated proteins were visualized using an ECL detection kit (Amersham) according to the manufacturer's instructions.

In addition to assessing the influence of the isolated proteins on global tyrosine phosphorylation, we also sought to examine the regulation of tyrosine phosphorylation within different sperm domains. For this purpose, sperm heads and flagella were separated essentially as described by Leclerc and Goupil [29]. Briefly, capacitated spermatozoa suspensions were placed on ice and sonicated three times for 30 sec, with a 30-sec rest interval between. Separation of sperm heads and flagella was confirmed by viewing the fragments under a microscope, and the sample was then layered onto a 75% Percoll cushion prepared in BWW. Following centrifugation (15 min, 700 x g), flagellar fragments were recovered at the surface of the Percoll layer, while the heads were isolated from the pellet. The supernatant was then centrifuged for 10 min (10 000 x g, 4°C) and the resulting supernatant was further centrifuged (1 h, 100000 x g, 4°C) to separate the membrane from the cytosolic fraction. An aliquot of each fraction (2 µl) was resolved by 1D SDS-PAGE and prepared for immunodetection of phosphotyrosine residues as indicated above. Importantly, to exclude protease and/or phosphatase action during this isolation procedure, all buffers were supplemented with protease inhibitors and sodium orthovanadate (1 mM).

Immunobead Detection of Surface Protein Tyrosine Phosphorylation

To correlate differences in membrane tyrosine phosporylation status with altered expression of phosphotyrosine residues on the outer leaflet of the sperm surface, an immunobead assay was performed essentially as described by Asquith et al. [22]. Briefly, protein G-coated magnetic beads (Dynabeads M-450; Dynal, Oslo, Norway) were washed 3 times in 0.1% BSA in PBS followed by conjugation with antiphosphotyrosine monoclonal antibody (clone 4G10) for 3 h at room temperature. Beads were then washed three times with BWW before incubation with the appropriate sperm treatments for 45 min at 37°C with continuous mixing. Wet mounts of the sperm/bead suspension were prepared and counterstained with propidium iodide for cell viability assessment. Coverslips were supported on pillars of paraffin wax and Vaseline [30], and the percentage of viable spermatozoa with bound antiphosphotyrosine coated beads was assessed by phase-contrast microscopy. Negative controls consisting of beads that were either uncoated or coated with an irrelevant antibody (anti-CD45; DB Transduction Laboratories, Lexington, KY) were included in each experiment. An additional treatment consisting of BWW supplemented with 400 µg BSA was also run in concert with each experiment to control for nonspecific masking of the antiphosphotyrosine binding sites.

Sperm-Zona Pellucida Binding Assay

Immature female mice (6 wk) were superovulated by an intraperitoneal (i.p.) injection of 10 IU of Folligon (eCG; Intervet, Sydney, Australia), followed by the i.p. administration of 10 IU of Chorulon (hCG; Intervet) 48 h later. Eggs were recovered from the oviducts of superovulated animals 15–20 h after Chorulon injection. Adherent cumulus cells were dispersed with 0.1% hyaluronidase solution and separated from oocytes by gentle pipetting. Oocytes were then washed through three changes of BWW and distributed among treatment groups. Aliquots containing 5 x 105 spermatozoa were diluted with the appropriate treatments and capacitated as above. Following capacitation, 10–20 oocytes were added to each treatment group and gametes were coincubated for 30 min at 37°C in an atmosphere 5% CO2:95% air. Using serial aspiration through a finely drawn pipette, unbound and loosely adhered spermatozoa were removed from oocytes. The oocytes were then fixed in 2% glutaraldehyde, mounted on slides, and the number of sperm bound to the zona pellucida counted using phase-contrast microscopy.

Progesterone-Induced Acrosome Reaction Assay

To examine the effect of the isolated sperm-surface proteins on the ability of sperm to undergo a progesterone-induced acrosome reaction, capacitated cells were incubated with 15 µM progesterone at 37°C for 15 min. Sperm suspensions were then washed, resuspended in hypoosmotic swelling (HOS) medium [31] and incubated for an additional 1 h. Following incubation, spermatozoa were washed, plated onto poly-L-lysine-coated slides, air-dried, and permeabilized by immersion in methanol for 10 min. The slides were then stained with fluorescein isothiocynate (FITC)-labeled Arachis hypogaea (1 µg/µl; Vector Laboratories Inc., CA) for 30 min at 4°C, mounted with 5 µl of antifade reagent (13% Mowiol4–88, 33% glycerol, 66 mM Tris [pH 8.5], 2.5% 1,4diazobcyclo-[2.2.2]octane), and the acrosomal status of viable cells was examined using a Zeiss Axioplan 2 fluorescence microscope (Zeiss, Jena, Germany).

Statistics

Experiments were replicated with material collected from at least three different animals, and the data are presented as mean ± SEM. Percentage data were subjected to arcsine transformation before performing an analysis of variance (ANOVA). The existence of statistically significant differences between group means was tested using the Fisher protected least significant difference (PLSD) test. Samples with a P-value <0.05 were considered statistically significant.

RESULTS

Characterization of Proteins Released from the Sperm-Surface During Capacitation

The collection of sperm decapacitation factors has traditionally relied on centrifugation of the cells following capacitation [6, 7]. However, the mechanical forces associated with centrifugation may promote the release of proteins from the sperm surface that would not be discharged under more physiological circumstances. To overcome the confounding effects of mechanical stress on the patterns of protein release, capacitation was conducted within a Transwell culture chamber. In Figure 1, we present the electrophoretic profile of proteins released from the surface of murine spermatozoa during capacitation under these conditions (Fig. 1, lane 1). For comparison, we also present the profile of proteins collected following centrifugation which, though similar, reveals distinct differences, particularly in higher molecular mass regions of the gels above 80 kDa (Fig. 1, lane 2). A total of 10 major proteins of approximate molecular weight 110, 96, 82, 70, 62, 55, 40, 32, 21, and 12 kDa were released from mouse spermatozoa during capacitation (Fig. 1, lane 1). The demonstration that each of these proteins was represented within the profile of biotinylated sperm-surface proteins (Fig. 1, lane 3) provides evidence that they were released from viable spermatozoa as opposed to damaged cells or those undergoing spontaneous acrosome reactions.


Figure 1
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  		FIG. 1. Electrophoretic profile of proteins isolated from mouse spermatozoa. Analytical 1D SDS-PAGE gels were used to resolve proteins released during capacitation of caudal spermatozoa incubated in Transwell culture chambers (lane 1) and those recovered following centrifugation of capacitated cauda epididymal sperm (lane 2). A Western blot of biotinylated sperm proteins is included to demonstrate the surface localization of each of the released proteins in intact cells (lane 3). Comparative gels of cauda epididymal fluid (lane 4) and the proteins released from the surface of caput epididymal cells are also presented for reference (lane 5). The 10 major proteins released from murine sperm during capacitation are indicated by an arrowhead and numbered for reference. This experiment was replicated five times and representative gels and blots are presented

With the exception of the two proteins exhibiting low molecular masses of 21 and 12 kDa, the remaining eight proteins resolved as a series of charge isoforms on analytical 2D SDS-PAGE gels (Fig. 2), a characteristic that is commonly associated with glycoproteins. A possible epididymal origin for these sperm-surface proteins was suggested by the similarity between the electrophoretic profile of caudal epididymal plasma (Fig. 1, lane 3) and the pattern of proteins shed from the surface of capacitating murine spermatozoa (Fig. 1, lane 1). The profile of proteins released from the surface of caput epididymal spermatozoa incubated under identical conditions appeared quite distinct from that observed with caudal cells (Fig. 1, lane 4), emphasizing the possible importance of the caudal epididymis as a source of putative decapacitation factors.


Figure 2
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  		FIG. 2. Two-dimensional SDS-PAGE profile of proteins isolated from the surface of capacitated mouse spermatozoa. A) Representative (n = 5) analytical 2D SDS-PAGE gel loaded with proteins that were actively released during capacitation and (B) reference gel indicating the position of the 10 major proteins

Inhibition of Sperm-Surface Protein Tyrosine Phosphorylation and Zona Binding

Recent studies from our laboratory and that of others [22, 32] have highlighted the importance of the tyrosine phosphorylation of multiple sperm proteins that accompanies capacitation, as a prerequisite for subsequent sperm-egg interaction. It was therefore hypothesized that the ability of decapacitation factors to suppress fertilization may be due, in part, to their ability to regulate components of the signal transduction cascade that drive tyrosine phosphorylation and hence capacitation. To explore this possibility, the putative decapacitation factors released from the surface of murine spermatozoa were examined for their ability to suppress sperm tyrosine phosphorylation and sperm-zona pellucida (ZP) binding. For this purpose, identical populations of spermatozoa were incubated in capacitating media supplemented with the pool of released proteins before assessing their protein tyrosine phosphorylation status and their ability to interact with the ZP in vitro.

In contrast with what we had anticipated, incubation of spermatozoa in capacitating media supplemented with putative decapacitation factors had no discernable effect on their ability to undergo tyrosine phosphorylation as assessed by Western blot analysis (Fig. 3A). Indeed, both the number of proteins tyrosine phosphorylated and the levels of phosphorylation achieved in all treatment groups were comparable with those observed in control samples incubated in capacitating media alone (Fig. 3A). The inability to suppress the overall levels of tyrosine phosphorylation was not restricted to sperm populations in which this event was stimulated by the inclusion of dbcAMP and ptx in the medium. In the absence of these pharmacologic agents, sperm tyrosine phosphorylation was still resistant to the addition of putative decapacitation factors (results not shown). However, despite the apparent inability of such factors to disrupt the tyrosine phosphorylation status of these cells, their presence had a significant (P < 0.001), dose-dependent inhibitory effect of their ability to bind to the ZP (Fig. 3B). At the highest concentration of isolated proteins (400 µg), the number of sperm bound to the ZP (13.3 ± 8.1) was reduced to approximately 20% of the controls (58.2 ± 14.1). In contrast, preincubation of spermatozoa in either a heat-denatured sample of the isolated proteins or in the presence of a nonspecific protein control (BSA) induced a relatively low level of suppression.


Figure 3
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  		FIG. 3. Proteins isolated from the surface of capacitated mouse spermatozoa inhibit sperm-ZP interaction but do not suppress tyrosine phosphorylation. Mouse cauda spermatozoa were incubated for 90 min in modified capacitation medium as described: BWW without NaHCO3 (negative control, lane 1), complete BWW (positive control supplemented with dbcAMP and ptx, lane 2), BWW containing varying concentrations of isolated proteins (lane 3 = 100 µg, lane 4 = 200 µg, lane 5 = 400 µg), BWW containing 400 µg of heat-denatured isolated proteins (lane 6), control incubation supplemented with BWW containing 400 µg of BSA (lane 7). Following treatment, the sperm sample was split and either (A) solubilized in extraction buffer and proteins resolved by 1D-SDS PAGE before being transferred to nitrocellulose membranes and probed with anti-phosphotyrosine or (B) incubated with oocytes for 30 min. Following incubation, the mean number of sperm bound to each zona was scored and expressed as a percentage of the control (complete BWW) for four replicates. Values represent means ± SEM. ** P < 0.001, * P < 0.05 compared with positive control. Arrowhead in A indicates the mouse sperm protein hexokinase, which is known to be constitutively phosphorylated in these cells and therefore served as an internal loading control in all experiments

To investigate the reason for the apparent dichotomy between sperm phosphorylation status and their zona binding ability, individual fractions consisting of sperm heads, flagella, and membranes were isolated (Fig. 4A) and the levels of tyrosine phosphorylation attained in each subcellular fraction was assessed. Commensurate with the results presented above, the tyrosine phosphorylation of proteins present within either sperm head or tail fractions proved insensitive to inhibition by the isolated proteins (Fig. 4B). In contrast, we did observe a dramatic reduction in the phosphorylation status of at least two high molecular-mass proteins (100 and 130 kDa) that were present with the sperm membrane fraction (Fig. 4B).


Figure 4
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  		FIG. 4. Proteins isolated from the surface of capacitated mouse spermatozoa inhibit the surface expression of phosphotyrosine residues. Mouse cauda spermatozoa were incubated for 90 min in modified capacitation medium as described: BWW without NaHCO3 (negative control, -ve), complete BWW (positive control, +ve), BWW containing 400 µg of isolated proteins (400 µg). Following treatment, the sperm sample was sonicated for a period of 15 sec, layered onto a 75% Percoll cushion, and centrifuged to facilitate the separation of sperm head, tail, and membrane fractions. The purity of the head and tail fractions was examined by phase microscopy (A, bar = 10 µm) and the proteins present in each were solubilized and resolved by 1D-SDS PAGE before being transferred to nitrocellulose and immunoblotted with anti-phosphotyrosine (B). The surface localization of phosphotyrosine residues was confirmed using an immunobead assay (C, D). Following incubation (as outlined above), spermatozoa were mixed with a suspension of magnetic beads conjugated to antiphosphotyrosine (C, bar = 10 µm). The number of sperm with beads attached in each of the respective treatments was scored (D). Each experiment was replicated three times. Values represent means ± SEM. ** P < 0.001, * P < 0.05 compared with positive control

The latter result was confirmed through the use of an immunobead assay that enabled us to localize phosphotyrosine residues on the surface of live spermatozoa. Consistent with previous reports [22], the use of this technique demonstrated that phosphotyrosine residues were expressed on the head, and no other region of approximately 10% of the viable, capacitated sperm population (Fig. 4C). Furthermore, preincubation of spermatozoa with the decapacitation factors released from the surface of murine spermatozoa significantly (P < 0.001) suppressed the percentage of labeled cells (Fig. 4D).

Inhibition of Progesterone-Induced Acrosome Reaction

Based on the premise that only capacitated spermatozoa are capable of undergoing an induced acrosome reaction [4], we next examined the ability of the decapacitation factor preparations to regulate acrosomal exocytosis. For the purpose of this study, the capacitation medium was again supplemented with a range of concentrations of the isolated proteins (100–400 µg). Following a capacitation period of 90 min, spermatozoa were successively incubated with progesterone and HOS media as described, to enable us to assess the number of acrosome-reacted cells among the viable population. The results of these experiments demonstrated that the isolated proteins significantly (P < 0.05) suppressed the ability of spermatozoa to respond to the progesterone challenge (Fig. 5B). At the highest concentrations of protein used in this study (400 µg), the number of acrosome-reacted cells was reduced to approximately 20% of that observed in control samples. Interestingly, the inhibitory effect of the isolated protein preparation was abolished by heat denaturation of the sample before incubation with spermatozoa or by washing the sperm population by gentle centrifugation following their initial exposure to the proteins. Furthermore, addition of either an equal concentration of a nonspecific protein (400 µg BSA; data not shown) or an equivalent amount of cholesterol to that present in the isolated protein sample (50 µM) failed to elicit a similar inhibitory effect.


Figure 5
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  		FIG. 5. Proteins isolated from the surface of capacitated mouse spermatozoa inhibit the progesterone-induced acrosome reaction. Mouse caudal spermatozoa were incubated for 90 min in modified capacitation medium as described: BWW without NaHCO3 (-ve control), complete BWW containing dbcAMP and ptx (+ve control), complete BWW containing varying concentrations of isolated proteins (100–400 µg), complete BWW containing 400 µg of heat-denatured isolated proteins (400 µg), 50 µg cholesterol. Additional incubations were conducted as above in the presence of ß-cyclodextrin. Following incubation, spermatozoa were challenged with progesterone (or the DMSO vehicle) and incubated in HOS media as described. The acrosomal status of viable cells was assessed using a FITC labeled Arachis hypogaea lectin. A) Corresponding phase contrast and fluorescence microscopy images displaying categories of acrosome reacted spermatozoa. AR, =Acrosome reacted; AI, acrosome intact. Bar = 10 µm. B) Percentage of acrosome-reacted viable spermatozoa. Values represent means ± SEM (n=3). ** P < 0.001, * P < 0.05 compared with positive control

To investigate the nature of this inhibition, spermatozoa were treated with 2-hydroxypropyl-ß-cyclodextrin (HßCD), a cholesterol-binding heptasaccharide that promotes the release of cholesterol from the sperm plasma membrane [33]. As anticipated, the level of spontaneous acrosome reaction was significantly elevated by this treatment (Fig. 5B). However, supplementation of the capacitating media with the isolated proteins again suppressed the level of both spontaneous and induced acrosome reactions in a dose-dependent manner.

Identification of the Protein(s) Responsible for Regulating Mouse Sperm Capacitation

Given the inhibitory activity of the isolated proteins, preliminary attempts were made to determine if this effect was attributable to the entire complement of recovered proteins or a subset of these molecules. For this purpose, the isolated proteins were fractionated over a Sephadex-G50 size exclusion column. Monitoring of the eluant using UV spectroscopy revealed four dominant protein peaks (Fig. 6A). As anticipated, the larger proteins (molecular weights 110, 96, 82, 70, 62, 55 kDa) were eluted first, within peaks one and two. The third peak was predominantly comprised of the protein of 40 kDa and the remaining peak contained four proteins of 40, 32, 21, and 12 kDa (Fig. 6B). Each of the fractionated protein samples was then examined individually for its ability to suppress a progesterone-induced acrosome reaction. As demonstrated in Figure 6C, the fractions containing the high molecular-weight proteins (>40 kDa, fractions 1 and 2) had no apparent inhibitory activity. In contrast, fractions 3 and 4 (containing proteins of 40, 32, 21, and 12 kDa) did compromise the ability of sperm to respond to the progesterone stimulus. Given that the inhibitory activity appeared to reside primarily within the lower molecular-weight fraction, we sought to identify each of these proteins by excision from a preparative 1D SDS-PAGE gel, trypsin digestion, and MS/MS sequence analysis of the resulting peptides.


Figure 6
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  		FIG. 6. The inhibitory activity of the isolated proteins resides in a subset of low molecular-weight proteins.A) Isolated sperm-surface proteins were fractionated as described and separate fractions were (B) analyzed by 1D SDS PAGE. C) Mouse cauda spermatozoa were then incubated for 90 min in modified capacitating media containing proteins (100 µg) from each of the four major fractions (1–4) and the percentage of viable acrosome-reacted cells was assessed. Values represent means ± SEM (n = 3). * P < 0.05 compared with positive control

Identification of Candidate Decapacitation Factors

A minimum of three peptides were successfully sequenced for each of the proteins present within the lower molecular-weight fraction. These peptide sequence data were used to perform similarity searches through the NCBI/BLAST Swiss-Prot database using the FASTA program (http://fasta.bioch.virginia.edu). In all cases, significant sequence homology was established with known proteins (that is, the alignment between the sequenced peptides and published sequence data displayed greater than 95% identity): 12 kDa, unnamed protein product (accession no. BAC34202) hereafter referred to as DF10; 21 kDa, phosphatidylethanolamine binding protein-1 (PBP, accession no. NP061346); 32 kDa, cysteine rich secretory protein 1 (CRISP1, accession no. NP033768); 40 kDa, plasma membrane fatty acid binding protein (also known as glutamate oxaloacetate transaminase 2, GOT2, accession no. NP034455). Although attempts were also made to sequence the larger molecular weight proteins, only one of these proteins was successfully identified: 55 kDa, sperm antigen-36 (SA36, accession no. AAF22645) (Fig. 7). Because all of these proteins apart from DF10 have been previously characterized in the literature, we examined this particular molecule in more detail.


Figure 7
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  		FIG. 7. Identification of inhibitory sperm-surface proteins. Sperm-surface proteins were isolated from capacitated cells and resolved on 1D SDS-PAGE gels as described. The major low molecular-weight proteins (i.e., those present within the fraction that possessed inhibitory activity) were sequenced by tandem mass spectrometry. This led to the successful identification of five proteins. Sequences of peptides that aligned with those of the known proteins are underlined

DF10

Based on the full-length published sequence for DF10, the protein has a predicted molecular mass and pI of 10.97 kDa and 4.3, respectively, closely approximating that seen in the present study (12 kDa and 4.4). As a preliminary step toward the characterization of DF10, its published sequence was used to further interrogate the NCBI/BLAST Swiss-Prot database for homologous proteins. This search revealed significant homology between DF10 and the carboxy terminus of the mouse Golgi phosphoprotein 4 (GOLPH4; accession no. NP780402) and human and rat Golgi-localized calcium-binding proteins (accession nos. XP227268 and NP055313, respectively) (Fig. 8). Sequence alignment [34] revealed 100% identity between DF10 and the corresponding region of GOLPH4 (A562 - M655) (Fig. 8). It would therefore appear that DF10 in fact represents an alternative, truncated product of the Golph4 gene.


Figure 8
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  		FIG. 8. Sequence similarity of DF10. As a preliminary step toward the characterization of DF10, its published sequence was used to interrogate the NCBI/BLAST Swiss-Prot database for homologous proteins. This search revealed significant homology between DF10 and the carboxy terminus of the mouse Golgi phosphoprotein 4 (Mouse GOLPH4) and human and rat Golgi-localized calcium-binding proteins (GLCBP)

Characterization of the Role of PBP in Mouse Sperm Capacitation

Although each of the four proteins identified within the lower molecular weight fraction potentially possess decapacitating activity, PBP was selected for further characterization on the basis of previously published functional data. To initiate these studies, we first sought to confirm the identity of this protein in the sample of decapacitation factors isolated from capacitating spermatozoa using Western blot analysis with a commercial anti-PBP polyclonal antibody (anti-RKIP). As anticipated, a protein of the appropriate molecular weight was detected in both the decapacitation protein sample and within caudal epididymal fluid (Fig. 9A). To examine the contribution of PBP to the inhibition of sperm capacitation, the cells were preincubated with recombinant PBP at a concentration of 100 µg/ml before assessing their ability to tyrosine phosphorylate, bind to the zona pellucida, and acrosome react. Consistent with the results outlined above, PBP failed to suppress the level of tyrosine phosphorylation in either the entire sperm extract or in isolated head and tail fractions (Fig. 9B). The protein did, however, significantly suppress the expression of phosphotyrosine residues within the sperm membrane fraction to a level comparable with that achieved using the entire complement of isolated proteins (Fig. 9B). This result was again confirmed using an immunobead assay, which demonstrated that PBP significantly (P < 0.05) suppressed the percentage of cells displaying surface phosphotyrosine residues (Fig. 9C). Recombinant PBP also significantly inhibited sperm-zona interaction (P < 0.01) (Fig. 9D) and the ability of sperm to acrosome react in response to progesterone challenge (P < 0.05) (Fig. 9E). Although the level of inhibition achieved in the latter experiment was more modest than that observed with the entire complement of isolated proteins, it was nevertheless significantly lower than that seen in untreated samples. As anticipated, the inhibitory activity of PBP in each of the functional assays was abolished by heat denaturation of the recombinant protein (data not shown).


Figure 9
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  		FIG. 9. Recombinant PBP inhibits sperm capacitation. A) Proteins isolated from capacitated mouse caudal sperm (DF) and those present in cauda epididymal fluid (CuF) were resolved by 1D SDS-PAGE and immunblotted with anti-PBP antibodies. B) Mouse caudal spermatozoa were incubated for 90 min in modified capacitating medium as described: BWW without NaHCO3 (negative control, -ve), complete BWW containing dbcAMP and ptx (positive control, +ve), complete BWW containing 400 µg of isolated proteins (DF), complete BWW containing 100 µg of recombinant PBP. Following treatment, the sperm sample was sonicated for a period of 15 sec, layered onto a 75% Percoll cushion, and centrifuged to facilitate the separation of sperm head, tail, and membrane fractions. The proteins present in each fraction were solubilized and resolved by 1D SDS-PAGE before being transferred to nitrocellulose and immunoblotted with antiphosphotyrosine. C) An immunobead assay was used to confirm the suppression of surface phosphotyrosine residues. Following incubation (as outlined above), spermatozoa were mixed with a suspension of magnetic beads conjugated to antiphosphotyrosine. The number of sperm with beads attached in each of the respective treatments was scored. Each experiment was replicated three times. D) Spermatozoa were capacitated as above before being incubated with oocytes for 30 min. Following incubation, the mean number of sperm bound to each zona was scored and expressed as a percentage of the control (complete BWW) for 3 replicates. ** P < 0.001, * P < 0.05 compared with positive control. E) To examine the effect of PBP on the ability of sperm to acrosome react, caudal spermatozoa were incubated for 90 min in the treatments outlined above. Following incubation, spermatozoa were challenged with progesterone (or the DMSO vehicle) and incubated in HOS media as described. The acrosomal status of viable cells (curled tails) was assessed using an FITC-labeled Arachis hypogaea lectin and the percentage of acrosome-reacted viable spermatozoa recorded. Values represent means ± SEM (n = 3). ** P < 0.001, * P < 0.05 compared with positive control

DISCUSSION

A characteristic feature of mammalian sperm capacitation is the extensive remodeling of the cell-surface architecture that accompanies this process. Such changes reflect the redistribution and loss of both protein and lipid components from within the different membrane domains. Although these changes occur commensurate with the acquisition of the ability to bind to the ZP and undergo an acrosome reaction, important questions remain as to how they are integrated with the maturation of signaling pathways that endow spermatozoa with functional competence. This situation stems not least from a paucity of data concerning the molecular identity of the factors that orchestrate capacitation. The induction of this process appears to involve a dynamic interaction between stimulatory factors released by the female reproductive tract and inhibitory, decapacitation factors originating in the male. Attempts to characterize the latter have yielded important data on the ability of cholesterol to suppress the capacitation of mammalian spermatozoa [35]; however, more complex decapacitation factors, such as the polypeptide identified by Fraser et al. [6], have not been resolved at the molecular level.

Against this background, we have sought to develop a novel system for detecting proteins that are released from the surface of mammalian spermatozoa during capacitation in vitro. Using this system, we have demonstrated that at least 10 major proteins appear to be shed from the surface of capacitating mouse spermatozoa. Preliminary analysis of these proteins indicates that they are predominantly glycoproteins, a subset of which appear to be elaborated by the epididymis and presumably coat the surface of spermatozoa during their passage through this organ. Although we present several lines of evidence that argue against the release of these proteins from damaged or canescent cells, we acknowledge that this remote possibility cannot be definitively excluded on the basis of our data and thus warrants further investigation. Nonetheless, the physiological relevance of their transitory interaction with the sperm surface is evidenced by the ability of the isolated proteins to suppress two of the primary biological endpoints of sperm capacitation, sperm-zona binding, and the ability to undergo acrosomal exocytosis. Both of these functional attributes were significantly inhibited by the addition of media conditioned with factors released from populations of capacitating spermatozoa. These findings are consistent with the notion that the male tract plays a key role in elaborating decapacitation factors that are capable of maintaining a population of viable, stabilized spermatozoa during their extended storage in the cauda epididymis [6, 17, 36–38]. In an evolutionary sense, the physiological significance of such factors presumably lies with their ability to synchronize the onset of sperm activation with the delivery of spermatozoa to the oocyte [39].

It was of interest, however, that, despite their ability to inhibit two important hallmarks of capacitation, such preparations of decapacitation factors had no inhibitory effect on the signal transduction cascades that stimulate protein tyrosine phosphorylation in either the sperm head or flagellum. Such findings support the notion that, although tyrosine phosphorylation represents a key activation step during sperm capacitation, it is not, in itself, sufficient for its completion. An important precedent for this conclusion is the finding that incubation of human spermatozoa in seminal plasma, which is known to contain decapacitation factors [18, 40–44], suppresses their ability to undergo an induced acrosome reaction, without compromising the time-dependent increase in tyrosine phosphorylation seen in these cells [45]. However, these findings are contrary to a recent report by Roberts et al. [46] in which it is suggested that the addition of CRISP, an epididymal secretory protein, to rat spermatozoa inhibits tyrosine phosphorylation in a dose-dependent manner. Although the reason for this discrepancy is not immediately apparent, it is possible that it may reflect fundamental species-specific differences in the signal transduction pathways that control sperm tyrosine phosphorylation events associated with sperm capacitation. Such a conclusion is consistent with a recent report that the redox control of tyrosine phosphorylation in murine spermatozoa is of limited significance compared with that of other species, including the rat [28]. Such findings suggest that the functional endpoints of capacitation can be arrived at via different molecular routes and emphasize the need for caution in making generalizations across species.

While decapacitation factors did not suppress the global increase in tyrosine phosphorylation associated with sperm capacitation, largely centered in intracellular protein kinase A anchoring proteins [47], they did induce a dramatic reduction in phosphotyrosine expression in the sperm plasma membrane. Importantly, the surface exposure of such phosphoproteins, which include members of the molecular chaperone family, has recently been implicated in the assembly of a functional receptor complex that ultimately endows capacitating spermatozoa with the ability to bind to the zona pellucida [22]. In this context, it has been shown that almost all spermatozoa bound to the zona pellucida demonstrate a distinct pattern of phosphotyrosine expression on the exterior surface of the sperm head, compared with fewer than 15% of the free-swimming population. It is therefore tempting to speculate that addition of inhibitory decapacitation factors somehow impedes the phosphorylation-dependent assembly of such a complex in the plasma membrane overlying the sperm head and, in this way, disrupts the ability of spermatozoa to both bind to the ZP and undergo acrosomal exocytosis.

One of the mechanisms by which the assembly of such functional complexes in the sperm plasma membrane might be impeded is through an increase in membrane stability. The role of membrane destabilization during capacitation and the subsequent acrosome reaction are well documented. Such changes are facilitated, in part, by cholesterol efflux that results from exposure of the capacitating spermatozoa to a negative external cholesterol gradient and lipid-binding serum proteins (high-density lipoproteins and albumin) within the uterine environment [35, 44]. By virtue of its hydrophobic nature and relative rigidity, cholesterol is able to intercalate between phospholipids and thereby restrict the overall fluidity of the lipid bilayer. Although the precise physiological function of cholesterol remains an enigma, it is hypothesized that the removal of the cholesterol barrier, and the commensurate reduction in the overall cholesterol:phospholipid ratio, promotes a reversible expansion of the lipid bilayer, forming unstable, transient gaps in either leaflet that are capable of reannealing or fusion with neighboring membrane [35, 44, 48]. The physiological importance of cholesterol depletion efflux is highlighted by the fact that the addition of cholesterol sinks (BSA or cyclodextrins) promotes sperm capacitation [33, 49, 50]. Furthermore, cholesterol itself is capable of acting as a decapacitation factor to prevent the onset of an induced acrosome reaction in several species [12, 42, 44, 48, 51, 52].

Although we acknowledge the potential inhibitory effect of cholesterol present within our isolated samples, a number of steps were taken to preclude this possibility. First, all isolated samples were subjected to dialysis to remove molecular species of less than 10 kDa before use. Second, we included controls to assess the inhibitory activity of the isolated samples following heat denaturation in each of our functional assays. Finally, we measured the concentration of both free cholesterol and cholesteryl esters within the isolated samples and incorporated an additional control in which an equivalent amount of cholesterol sulfate was added back to spermatozoa. The collective data from these studies argues against a major contribution of cholesterol in the response we have observed. This is further supported by the fact that inhibitory activity was retained following fractionation of the decapacitation protein preparations by size-exclusion chromatography. This latter experiment revealed that the inhibitory activity resided in a fraction consisting of four predominant proteins identified as plasma membrane fatty acid binding protein, CRISP, PBP, and an unnamed protein product, referred to as DF10.

Interestingly, at least two of these proteins, plasma membrane fatty acid-binding protein and PBP, possess attributes compatible with those expected of proteins involved in membrane stabilization [53–58]. Plasma-membrane fatty acid-binding protein is a 40-kDa member of the lipid-binding protein superfamily that has recently been identified by immunohistological, surface labeling, and immunoprecipitation techniques on the plasma membranes of hepatocytes, adipocytes, and other cells with high transmembrane fluxes of long-chain free fatty acids [59–63]. Interestingly, sequencing of the protein has demonstrated that it is identical to the glutamate oxaloacetate transaminase (GOT2, also known as mitochondrial aspartate aminotransferase), an enzyme found in the mitochondria of virtually all cells. While the mitochondrial form plays an important role in amino acid metabolism, the surface-localized protein has been implicated as both a lipid carrier and binding protein [64–66]. While, to our knowledge, this is the first report of the presence of such a protein on the surface of spermatozoa, a functionally related molecule, fatty acid binding protein 9 (FABP9, formerly known as PERF15), has been identified as a major protein constituent of the rat perforatorium, a region found between the inner acrosomal and the outer face of the nuclear envelope of the sperm head [67]. In this location, the protein has been suggested to interact with the membrane of the acrosome and play a major role in the structural arrangement and stability of the organelle. It is therefore considered feasible that plasma-membrane fatty acid-binding protein fulfills a similar function within the sperm plasma membrane.

By analogy, a similar mode of action could also be put forward to account for the potent inhibitory activity of PBP identified in the present study. Indeed, in addition to our study, previous research has also implicated PBP in the regulation of sperm membrane fluidity and the formation and maintenance of functional domains within the cell [56, 57, 68]. Collectively, this information suggests that the concerted action of lipid-binding proteins may play an important role in maintaining the structural integrity of the sperm plasma membrane. Furthermore, their subsequent release could provide the stimulus for the obligatory loss of cholesterol and decrease in membrane lipid order that accompanies capacitation [35, 48]. This conclusion is supported by the fact that the isolated proteins also effectively suppressed the action of the cholesterol acceptor ß-cyclodextrin, a molecule that has previously been shown to support capacitation. Furthermore, such a proposition mirrors that originally proposed for acrosomal stabilizing factor (ASF), a glycoprotein synthesized in the corpus epididymidis of the rabbit that displays reversible decapacitation activity [17, 36–38, 69–71]. However, the limited molecular characterization of ASF has failed to shed significant light onto the mechanisms by which it influences membrane integrity [38, 71].

It is also noteworthy that, in addition to its affinity for phosphatidylethanolamine, PBP is also characterized by the presence of a functional Raf kinase inhibitory protein (RKIP) domain. As such, the protein has been implicated as a suppressor of RAF1, a serine threonine kinase that represents an important component of the extracellular signal-regulated kinase (ERK) family of mitogen-activated protein (MAP) kinases [72, 73]. This inhibitory activity has been attributed to the protein's ability to sequester inactive RAF1, preventing its interaction with the MAP kinase, MEK [73]. Interestingly, a number of the components of the ERK signaling pathway, including the MAP kinases MAPK1 and MAPK3, have been identified within human and mouse spermatozoa and implicated in the tyrosine phosphorylation events associated with sperm capacitation [74, 75]. Moreover, these proteins also appear to display a restricted pattern of localization within the sperm head [75]. Coupled with our data, such findings raise the possibility that PBP may be responsible for regulating an ERK signaling cascade, and that its loss induces the phosphorylation of a subset of membrane proteins destined for expression on the outer leaflet of the sperm head plasma membrane. This hypothesis accommodates the fact that PBP shows preferential binding for phosphatidylethanolamine residues that reside in the inner leaflet of the sperm plasma membrane [76], and would thus be ideally positioned to block the RAF1/MEK interface [77]. However, an important caveat to this suggestion is that a definite association between the MAPK phosphorylating enzymes and their target proteins has yet to be established. Furthermore, it is not immediately apparent whether the exogenous addition of recombinant PBP could lead to the protein being reincorporated into the sperm plasma membrane to exert an inhibitory effect. An alternative possibility is that PBP is involved in the regulation of protease activity in the sperm plasma membrane and that the latter is, in turn, essential for capacitation to occur. Consistent with this hypothesis, PBP and related molecules are known to inhibit serine proteases [78]. Furthermore, proteases have been found to promote capacitation [79], while protease inhibitors suppress this process [80]. PBP may therefore represent a multifunctional molecule responsible for regulating different aspects of mammalian sperm capacitation.

ACKNOWLEDGMENTS

The authors wish to thank the University of Newcastle and the ARC Centre of Excellence in Biotechnology and Development for their support of this research. We also wish to thank Duangporn Jamsai for her assistance in the production of recombinant PBP.

FOOTNOTES

1 Supported by the Research Management Committee, University of Newcastle, Australia, and ARC Centre of Excellence in Biotechnology and Development and the National Health and Medical Research Council of Australia. Back

2 Correspondence: FAX: 61 2 4921 6923; John.Aitken{at}newcastle.edu.au Back

Received: 16 June 2005.

First decision: 19 July 2005.

Accepted: 6 October 2005.

REFERENCES

  1. Austin CR, Observations on the penetration of the sperm into the mammalian egg. Aust J Sci Res 1951 4:581-596
  2. Chang MC, Fertilising capacity of spermatozoa deposited in the fallopian tubes. Nature 1951 997-998
  3. 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 1995 121:1139-1150[Abstract]
  4. Yanagimachi R, Mammalian fertilisation. In: Knobil E, Neill JD, (eds.). The physiology of reproduction, 2nd ed. New York: Raven Press 1994 135-185
  5. Fraser LR, Mouse sperm capacitation in vitro involves loss of a surface-associated inhibitory component. J Reprod Fertil 1984 72:373-384[Abstract]
  6. Fraser LR, Harrison RA, Herod JE, Characterization of a decapacitation factor associated with epididymal mouse spermatozoa. J Reprod Fertil 1990 89:135-148[Abstract]
  7. Fraser LR, Interactions between a decapacitation factor and mouse spermatozoa appear to involve fucose residues and a GPI-anchored receptor. Mol Reprod Dev 1998 51:193-202[CrossRef][Medline]
  8. Adeoya-Osiguwa SA, Fraser LR, Evidence for Ca(2+)-dependent ATPase activity, stimulated by decapacitation factor and calmodulin, in mouse sperm. Mol Reprod Dev 1996 44:111-120[CrossRef][Medline]
  9. Martins SG, Miranda PV, Brandelli A, Acrosome reaction inhibitor released during in vitro sperm capacitation. Int J Androl 2003 26:296-304[CrossRef][Medline]
  10. Aonuma S, Okabe M, Suzuki K, Matsumoto K, Kawai Y, Studies on sperm capacitation. V. Characterization of decapacitation factor from guinea-pig spermatozoa. Chem Pharm Bull 1976 24:907-911[Medline]
  11. Abney TO, Williams WL, Inhibition of sperm capacitation by intrauterine deposition of seminal plasma decapacitation factor. Biol Reprod 1970 2:14-17[Medline]
  12. Begley AJ, Quinn P, Decapacitation factors in semen. Clin Reprod Fertil 1982 1:167-175[Medline]
  13. Bonilla E, Velasco R, Casas E, Ducolomb Y, Betancourt M, Inhibition of the pig sperm acrosome reaction by a decapacitation factor from pig seminal plasma. Med Sci Res 1996 24:75-77
  14. Dukelow WR, Chernoff HN, Williams WL, Properties of decapacitation factor and presence in various species. J Reprod Fertil 1967 14:393-399[Medline]
  15. Dukelow WR, Chernoff HN, Williams WL, Enzymatic characterization of decapacitation factor. Proc Soc Exp Biol Med 1966 121:396-398[Medline]
  16. Dukelow WR, Chernoff HN, Williams WL, Stability of spermatozoan decapacitation factor. Am J Physiol 1966 211:826-828[Free Full Text]
  17. Oliphant G, Reynolds AB, Thomas TS, Sperm surface components involved in the control of the acrosome reaction. Am J Anat 1985 174:269-283[CrossRef][Medline]
  18. Pinsker MC, Williams WL, Spermatozoan decapacitation factor (DF) in human seminal plasma. Proc Soc Exp Biol Med 1968 129:446-448[Medline]
  19. Pinsker MC, Robertson RT, Chemistry of spermatozoa decapacitation factor. J Reprod Fertil 1969 18:175[Medline]
  20. Reyes A, Oliphant G, Brackett BG, Partial purification and identification of a reversible decapacitation factor from rabbit seminal plasma. Fertil Steril 1975 26:148-157[Medline]
  21. Biggers JD, Whitten WK, Whittingham DG, The culture of mouse embryos in vitro. In: J.C. Daniels (ed.), Methods in Mammalian Embryology. San Francisco, CA: Freeman Press; 1971:86–116
  22. Asquith KL, Baleato RM, McLaughlin EA, Nixon B, Aitken RJ, Analysis of the mechanisms by which tyrosine phosphorylation regulates sperm-zona recognition in the mouse: a chaperone-mediated event?. J Cell Sci 2004 117:3645-3657[Abstract/Free Full Text]
  23. Laemmli UK, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970 227:680-685[CrossRef][Medline]
  24. Towbin H, Staehelin T, Gordon J, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979 76:4350-4354[Abstract/Free Full Text]
  25. Blum H, Biere H, Gross HJ, Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 1987 8:93-99[CrossRef]
  26. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ, Basic local alignment search tool. J Mol Biol 1990 215:403-410[CrossRef][Medline]
  27. Hickox DM, Gibbs G, Morrison JR, Sebire K, Edgar K, Keah HH, Alter K, Loveland KL, Hearn MT, de Kretser DM, O'Bryan MK, Identification of a novel testis-specific member of the phosphatidylethanolamine binding protein family, pebp-2. Biol Reprod 2002 67:917-927[Abstract/Free Full Text]
  28. Ecroyd H, Asquith KL, Jones RC, Aitken RJ, The development of signal transduction pathways during epididymal maturation is calcium dependent. Dev Biol 2004 268:53-63[CrossRef][Medline]
  29. Leclerc P, Goupil S, Regulation of the human sperm tyrosine kinase c-yes. Activation by cyclic adenosine 3',5'-monophosphate and inhibition by Ca(2+). Biol Reprod 2002 67:301-307[Abstract/Free Full Text]
  30. WHO. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction Cambridge University Press 1999
  31. Jeyendran RS, Van der Ven HH, Perez-Pelaez M, Crabo BG, Zaneveld LJ, Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil 1984 70:219-228[Abstract]
  32. Urner F, Sakkas D, Protein phosphorylation in mammalian spermatozoa. Reproduction 2003 125:17-26[Abstract]
  33. Choi YH, Toyoda Y, Cyclodextrin removes cholesterol from mouse sperm and induces capacitation in a protein-free medium. Biol Reprod 1998 59:1328-1333[Abstract/Free Full Text]
  34. Higgins D, Thompson J, Gibson T, Thompson JD, Higgins DG, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994 22:4673-4680[Abstract/Free Full Text]
  35. Cross NL, Role of cholesterol in sperm capacitation. Biol Reprod 1998 59:7-11[Free Full Text]
  36. Oliphant G, Brackett BG, Capacitation of mouse spermatozoa in media with elevated ionic strength and reversible decapacitation with epididymal extracts. Fertil Steril 1973 24:948-955[Medline]
  37. Reynolds AB, Thomas TS, Wilson WL, Oliphant G, Concentration of acrosome stabilizing factor (ASF) in rabbit epididymal fluid and species specificity of anti-ASF antibodies. Biol Reprod 1989 40:673-680[Abstract]
  38. Wilson WL, Oliphant G, Isolation and biochemical characterisation of the subunits of the rabbit sperm acrosome stabilising factor. Biol Reprod 1987 37:159-169[Abstract]
  39. Bedford JM, Enigmas of mammalian gamete form and function. Biol Rev Camb Philos Soc 2004 79:429-460[Medline]
  40. Mahadevan M, Trounson AO, Nayuda RV, Human seminal lectin. I. Demonstration and association with male infertility. Fertil Steril 1980 34:490-495[Medline]
  41. Lopes CH, Mazzini MN, Tortorella H, Konrath RA, Brandelli A, Isolation, partial characterization and biological activity of mannosyl glycopeptides from seminal plasma. Glycoconj J 1998 15:477-481[CrossRef][Medline]
  42. Davis BK, Inhibitory effect of synthetic phospholipid vesicles containing cholesterol on the fertilizing ability of rabbit spermatozoa. Proc Soc Exp Biol Med 1976 152:257-261[Abstract]
  43. Davis BK, Byrne R, Hungund B, Studies on the mechanism of capacitation. II. Evidence for lipid transfer between plasma membrane of rat sperm and serum albumin during capacitation in vitro. Biochim Biophys Acta 1979 558:257-266[Medline]
  44. Davis BK, Timing of fertilization in mammals: sperm cholesterol/phospholipid ratio as a determinant of the capacitation interval. Proc Natl Acad Sci U S A 1981 78:7560-7564[Abstract/Free Full Text]
  45. Emiliozzi C, Fenichel P, Protein tyrosine phosphorylation is associated with capacitation of human sperm in vitro but is not sufficient for its completion. Biol Reprod 1997 56:674-679[Abstract]
  46. Roberts KP, Wamstad JA, Ensrud KM, Hamilton DW, Inhibition of capacitation-associated tyrosine phosphorylation signaling in rat sperm by epididymal protein Crisp-1. Biol Reprod 2003 69:572-581[Abstract/Free Full Text]
  47. Moss SB, Gertoin GL, A-kinase anchor proteins in endocrine systems and reproduction. Trends Endocrinol Metab 2001 12:434-440[CrossRef][Medline]
  48. Cross NL, Decrease in order of human sperm lipids during capacitation. Biol Reprod 2003 69:529-534[Abstract/Free Full Text]
  49. Cross NL, Effect of methyl-beta-cyclodextrin on the acrosomal responsiveness of human sperm. Mol Reprod Dev 1999 53:92-98[CrossRef][Medline]
  50. Visconti PE, Galantino-Homer H, Ning X, Moore GD, Valenzuela JP, Jorgez CJ, Alvarez JG, Kopf GS, Cholesterol efflux-mediated signal transduction in mammalian sperm. beta-cyclodextrins initiate transmembrane signaling leading to an increase in protein tyrosine phosphorylation and capacitation. J Biol Chem 1999 274:3235-3242[Abstract/Free Full Text]
  51. Cross NL, Effect of cholesterol and other sterols on human sperm acrosomal responsiveness. Mol Reprod Dev 1996 45:212-217[CrossRef][Medline]
  52. Cross NL, Human seminal plasma prevents sperm from becoming acrosomally responsive to the agonist, progesterone: cholesterol is the major inhibitor. Biol Reprod 1996 54:138-145[Abstract]
  53. Araki Y, Vierula ME, Rankin TL, Tulsiani DR, Orgebin-Crist MC, Isolation and characterization of a 25-kilodalton protein from mouse testis: sequence homology with a phospholipid-binding protein. Biol Reprod 1992 47:832-843[Abstract]
  54. Campbell FM, Dutta-Roy AK, Plasma membrane fatty acid-binding protein (FABPpm) is exclusively located in the maternal facing membranes of the human placenta. FEBS Lett 1995 375:227-230[CrossRef][Medline]
  55. Dutta-Roy AK, Cellular uptake of long-chain fatty acids: role of membrane-associated fatty-acid-binding/transport proteins. Cell Mol Life Sci 2000 57:1360-1372[CrossRef][Medline]
  56. Frayne J, McMillen A, Love S, Hall L, Expression of phosphatidylethanolamine-binding protein in the male reproductive tract: immunolocalisation and expression in prepubertal and adult rat testes and epididymides. Mol Reprod Dev 1998 49:454-460[CrossRef][Medline]
  57. Seddiqi N, Segretain D, Bucquoy S, Pineau C, Jegou B, Jolles P, Schoentgen F, Characterization and localization of the rat, mouse and human testicular phosphatidylethanolamine binding protein. Experientia 1996 52:101-110[CrossRef][Medline]
  58. Schwieterman W, Sorrentino D, Potter BJ, Rand J, Kiang CL, Stump D, Berk PD, Uptake of oleate by isolated rat adipocytes is mediated by a 40-kDa plasma membrane fatty acid binding protein closely related to that in liver and gut. Proc Natl Acad Sci U S A 1988 85:359-363[Abstract/Free Full Text]
  59. Hertzel AV, Bernlohr DA, The mammalian fatty acid-binding protein multigene family: molecular and genetic insights into function. Trends Endocrinol Metab 2000 11:175-180[CrossRef][Medline]
  60. Cermelli S, Zerega B, Carlevaro M, Gentili C, Thorp B, Farquharson C, Cancedda R, Cancedda FD, Extracellular fatty acid binding protein (Ex-FABP) modulation by inflammatory agents: "physiological" acute phase response in endochondral bone formation. Eur J Cell Biol 2000 79:155-164[CrossRef][Medline]
  61. Campbell FM, Taffesse S, Gordon MJ, Dutta-Roy AK, Plasma membrane fatty-acid-binding protein in human placenta: identification and characterization. Biochem Biophys Res Comm 1995 209:1011-1017