Hum. Reprod. Advance Access originally published online on March 8, 2007
Human Reproduction 2007 22(5):1420-1430; doi:10.1093/humrep/dem023
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Effect of fibronectin on proteasome activity, acrosome reaction, tyrosine phosphorylation and intracellular calcium concentrations of human sperm
Department of Biomedicine, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile
1 To whom correspondence should be addressed at: Department of Biomedicine, Faculty of Health Sciences, University of Antofagasta, PO Box 170, Antofagasta, Chile. E-mail: pmorales{at}uantof.cl
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Abstract |
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BACKGROUND: Previously we showed that the human sperm proteasome plays significant roles during mammalian fertilization. Here we studied the effect of fibronectin (Fn), an extracellular matrix protein present in the cumulus oophorus of the oocyte, on proteasome activity, acrosome reaction, intracellular calcium concentration ([Ca2+]i) and protein tyrosine phosphorylation of human sperm.
METHODS: Aliquots of motile sperm were incubated for 15 min (T0), 5 h (T5) and 18 h (T18), at 37°C, 5% CO2 and 95% air with Fn (0–100 µg/ml). The chymotrypsin- and trypsin-like activity of the proteasome was measured using the fluorogenic substrates, Suc-Leu-Leu-Val-Tyr-AMC and Boc-Gln-Ala-Arg-AMC, respectively. At T18, sperm aliquots were incubated for 15 min with Fn and/or progesterone in the presence or absence of epoxomicin (a proteasome inhibitor). The percentage of viable acrosome reacted sperm was evaluated using the Fluorescein isothiocyanate (FITC)-labeled Pisum sativum agglutinin. Tyrosine phosphorylation was evaluated by western blot and [Ca2+]i using fura 2.
RESULTS: Fn stimulated both enzymatic activities of the proteasome and the acrosome reaction of human sperm. Progesterone enhanced and epoxomicin drastically inhibited the effect of Fn. Fn treatment also increased the [Ca2+]i. Western blot analysis revealed that Fn increased tyrosine protein phosphorylation and that some proteasome subunits became tyrosine phosphorylated upon Fn treatment.
CONCLUSIONS: These results suggest that Fn activates the proteasome and induces the acrosome reaction in human sperm. This effect may involve binding with specific receptors (integrins) on the sperm surface and the activation of tyrosine kinases.
Key words: acrosome reaction/extracellular matrix/human sperm/proteasome/tyrosine phosphorylation
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Introduction |
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Mammalian fertilization is a specific cellular-recognition event that comprises sequential interactions between the fertilizing spermatozoon and the cumulus oophorus, zona pellucida and oocyte plasma membrane (Yanagimachi, 1994
). At the time of ovulation, mammalian oocytes are surrounded by the cumulus oophorus and the zona pellucida. The cumulus oophorus consists of a group of approximately 3000–5000 cells and an extracellular matrix secreted by the cells (Zhuo and Kimata, 2001; Tanghe et al., 2002). The extracellular matrix is composed of several types of molecules, the most important being hyaluronic acid, dermatan sulphate and adhesive glycoproteins such as laminin, fibronectin (Fn) and type IV collagen (Salustri et al., 1992; Camaioni et al., 1996; Einspanier et al., 1999).
In several reports, the expression of a great variety of proteins on the surface of mammalian sperm, including on that of humans, has been demonstrated (Talbot et al., 2003). The cellular adhesion molecules, also known as integrins, are one of those proteins. The integrins connect the external environment (extracellular matrix) with the inside of the cell (cytoskeleton). Its ligands are glycoproteins like Fn, type IV collagen, laminin, vimentin, tenascin among others. The expression of several integrin subunits in human sperm has been detected by immunofluorescence and western blot, and they exhibit different localization that depends upon sperm state (freshly ejaculated, capacitated, or acrosome reacted) (Fusi et al., 1996; Glander et al., 1998). Upon binding to its ligands, the integrins induce adhesion and/or clustering of the complexes in the membrane plane, ultimately leading to enhanced protein tyrosine phosphorylation (Kornberg et al., 1991). This enhanced tyrosine kinase activity triggers signal transduction cascades, mainly the MAPK pathway (Juliano, 2002).
Freshly ejaculated mammalian sperm do not have the ability to fertilize an oocyte (Yanagimachi, 1994). This capacity is acquired while they reside in the female genital tract, a phenomenon known as capacitation. Once capacitated, the sperm are able to bind to the zona pellucida and undergo the acrosome reaction on its surface. The acrosome reaction is necessary for fertilization success since it helps the fertilizing spermatozoon to reach the perivitelline space and to fuse with the oocyte plasma membrane (Yanagimachi, 1994). Both events, capacitation and acrosomal exocytosis, are regulated by intracellular signals and associated with protein phosphorylation in Tyr and Ser/Thr (Naz and Rajesh, 2004). Almost all known signalling systems that operate in somatic cells, with the exception of nuclear activity, have been found on mammalian sperm (Baldi et al., 2002; Breitbart, 2003). However, the function of many of these signalling pathways in sperm is still not clear. In spite of this, current evidence suggests that Tyr kinases, the MAPK/ERK pathway and activation of PKA and PKC may all be involved in the occurrence of the acrosome reaction (Leyton et al., 1992; Visconti et al., 1995; Breitbart and Naor, 1999; de Lamirande and Gagnon, 2002). For instance, it has been shown that inhibition of protein Tyr phosphorylation blocks the acrosome reaction (Leyton et al., 1992) and that PKA has an important function in the regulation of Tyr protein phosphorylation in the sperm (Breitbart and Naor, 1999; Baldi et al., 2002; Urner and Sakkas, 2003). Integrins have the capacity to activate all these signal transduction routes (Juliano, 2002).
Another protein, susceptible to be phosphorylated in the sperm, is proteasome. The proteasome is a complex multienzymatic threonine protease, tightly regulated, and is a highly substrate-specific housekeeping system, in charge of the removal of cytosolic and nuclear proteins, previously labeled by ubiquitin molecules. The 26S proteasome has a molecular mass of 
2000 kDa and is composed of a proteolytic core complex, termed the 20S proteasome (700 kDa), along with two polar 19S complexes that contain ATPase and non-ATPase regulators. The 19S particle is made of at least 17 proteasomal regulatory complex subunits, including ATP-dependent and ATP-independent ones, which are estimated to be 29–112 kDa in size. Some of the 19S subunits are thought to have polyubiquitin-chain binding and de-ubiquitinating capabilities. The 20S core is composed of 14 small subunits, 7 of 
-type, and 7 of 
-type, with molecular masses of 21–32 kDa. The -subunits possess regulatory functions and the -subunits catalytic functions (Coux et al., 1996; Ciechanover, 1998; Fenteany and Schreiber, 1998). Upon binding to 19S complex, the polyubiquitin chain is removed from substrate protein and recycled into reusable monoubiquitin molecules by de-ubiquitinating enzymes, the ubiquitin C-terminal hydrolases. The substrate is then unfolded and threaded through the hollow 20S proteasomal core, in which it is hydrolyzed into small peptides and released to be disassembled into single amino acids by cytosolic endopeptidases (Ciechanover, 1998; Fenteany and Schreiber, 1998). The 26S proteasome has been shown to be present in sperm of several species (Tipler et al., 1997; Pizarro et al., 2004), including humans (Tipler et al., 1997; Wojcik et al., 2000). It is known that an important mechanism in the functional regulation of the proteasome is the phosphorylation/de-phosphorylation of some of its subunits (Mason et al., 1998; Bose et al., 1999; Fernandez Murray et al., 2002).
Several reports have indicated that the sperm proteasome is involved in various steps of the fertilization process in marine invertebrates (Mykles, 1998). In mammals, however, there is limited information regarding the role of the sperm proteasome during fertilization (Morales et al., 2003).
We have hypothesized that Fn, an extracellular matrix protein present in the oocyte cumulus complex, upon binding to the integrins on the sperm surface, triggers intracellular signals in the spermatozoon. Among other events, these intracellular signals culminate in the phosphorylation of some proteasome subunits, in its activation, and in the later exocytosis of the acrosome, thus modulating the fertilizing capacity of the sperm. Therefore, the aim of this work was to evaluate the effect of Fn on proteasome activity, acrosome reaction, tyrosine phosphorylation and intracellular calcium concentration ([Ca2+]i) in human sperm.
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Materials and methods |
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Reagents
The following compounds were purchased from Sigma Chemical Co. (St. Louis, MO, USA): the trypsin inhibitor N-p-tosyl-L-lisine chloromethyl ketone hydrochloride (TLCK); the trypsin substrate Boc-Gln-Ala-Arg-AMC (BQAR-AMC); bovine serum albumin (BSA) (A7030); HEPES; dimethylsulfoxide (DMSO); Ponceau Red; progesterone; fura 2-AM; ethylenediaminetetraacetic acid (EDTA); EGTA; Digitonin; Hoechst 33258 (H258); Na3VO4; NaF; sodium desoxycholate; Igepal CA-630 (NP-40); phenylmethysulfonyl fluoride (PMSF); leupeptin; bestatin A; aprotinin; and monoclonal antibody anti
-actin (clone AC-15). Pisum sativum agglutinin (PSA)-FITC was purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). Fn was purchased from Gibco BRL, Life Technologies (Grand Island, NY, USA). The chymotrypsin substrate N-succinyl-Leu-Leu-Val-Tyr-7-AMC (SLLVY-AMC); the highly specific, cell permeable and irreversible proteasome inhibitor epoxomicin (Voorhees and Orlowski, 2006); the anti 4 proteasome antibody and the agarose-immobilized anti 4 proteasome subunit were obtained from Affinity Research Products (Biomol Research Laboratories, PA, USA). Monoclonal antibody against 5 integrin subunit and polyclonal anti human IgG were purchased from Chemicom (Temecula, CA, USA). Antibody to phosphotyrosine (clone 4G10) was obtained from Upstate Biotechnology (Lake Placid, NY, USA). Chemiluminiscence detection system was purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Immobilon P transfer membrane was obtained from Millipore Corporation (Bedford, MA, USA). DC-protein method was obtained from BioRad Laboratories Inc. (Hercules, CA, USA).
Deionized water used in these experiments was purified to >18 M
-cm with EASY-pure UV/UF ion-exchange system (Barnstead/Thermolyne, Dubuque, IA, USA). Stock solutions of substrates were prepared in DMSO. The final concentration of DMSO in the sperm suspensions was 0.1% (v/v). The concentration of Fn, progesterone, anti 5 integrin subunit antibody, and substrates used in the present study did not alter sperm motility in any way (scored according to World Health Organization guidelines 1999).
Sperm suspension preparation
Semen samples were obtained from normal donors after 2–3 days of sexual abstinence, with the approval of the Ethics Committee of the University of Antofagasta. All samples had normal semen parameters according to WHO guidelines (1999). The specimens were allowed to liquefy for 30–60 min at 37°C in a slide warmer. Motile spermatozoa were selected by centrifugation through a two-step (40/80%) percoll gradient as described previously (Morales et al., 1989). Briefly, aliquots of semen were layered over the upper step of the percoll gradient and centrifuged for 20 min at 300g. The pellet was diluted in 10 ml of modified Tyrode's medium consisting of 117.5 mM NaCl, 0.3 mM NaH2PO4, 8.6 mM KCl, 25 mM NaHCO3, 2.5 mM CaCl2, 0.5 mM MgCl2, 2 mM glucose, 0.25 mM Na-pyruvate, 19 mM Na-lactate, 70 µg/ml of both streptomycin and penicillin, phenol red, centrifuged again at 300g for 10 min and then resuspended in the appropriate medium. The sperm concentration was evaluated using a haemocytometer and adjusted as needed.
Preparation of sperm extracts
Highly motile sperm were incubated for 15 min (T0), 5 h (T5) and 18 h (T18) with different concentrations of Fn (0–100 µg/ml), at 37°C, 5% CO2 and 95% air. Other sperm aliquots were capacitated for 18 h and then incubated for 15 min with 50 µg/ml Fn or Fn solvent. Then, the sperm were washed twice in PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 4.3 mM Na2HPO4, pH 7.4), by centrifuging at 800g for 5 min. The resulting washed sperm pellet was resuspended in homogenization buffer (50 mM HEPES, 10% glycerol, pH 7.4) at a concentration of 25 x 106 sperm/ml (Morales et al., 1994). The sperm suspension was sonicated (Virsonic, Gardiner, NY) with six 60 W bursts for 20 s each, followed by centrifugation for 30 sec at 14 000g in a Beckman microfuge to remove nuclear and flagellar material. The supernatant was used as the enzyme stock preparation. All these procedures were performed at 4°C. The protein concentration in each sperm extract preparation, obtained using the DC-protein method, ranged between 0.5 and 1.5 mg/ml.
For western blot, at the end of incubation, cells were washed with PBS, resuspended at a concentration of 30 x 106 sperm/ml in 100 µl of radioimmunoprecipitation (RIPA) lysis buffer (containing 150 mM NaCl, 50 mM Tris, 1% Sodium dodecyl sulphate (SDS), 2 mM Na3VO4, 50 mM NaF, 2 mM EDTA, 1% sodium desoxycholate, 1% NP-40, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml bestatin A 10 µg/ml aprotinin and pH 7.4).
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Enzymatic activity |
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At all incubation times (T0, T5 and T18) the enzymatic activity of the sperm enzyme extracts was assayed using the fluorogenic substrates designed to evaluate the chymotrypsin-like and trypsin-like activity of the proteasome, SLLVY-AMC and BQAR-AMC, respectively. Fifty µl aliquots of enzyme extract were incubated in a final volume of 1 ml containing 50 mM HEPES, 10% glycerol, pH 7.4 and 10 µM substrate. The assay was run at 37°C and the fluorescence was monitored with excitation at 380 nm and emission at 460 nm in a Shimadzu 1501 (Kyoto, Japan) spectrofluorometer. Before adding the trypsin substrate, the sperm enzyme extract was incubated with 100 µM TLCK for 15 min to inhibit acrosin activity, as previously described (Morales et al., 2004
).
Acrosome reaction
To induce the acrosome reaction, motile sperm cells were obtained as described above, except that they were resuspended in modified Tyrode's medium, supplemented with 2.6% BSA. The sperm concentration was adjusted to 10 x 106 cells/ml and the cells incubated at 37°C, 5% CO2 and 95% air. After 18 h, the cells were incubated with 5 µM progesterone or different concentrations of Fn (0–100 µg/ml) for 15 min. Additional sperm aliquots were incubated with Fn in the presence of 5 µM progesterone or 10 µM epoxomicin for 15 min. At the end of this period, sperm viability and acrosomal status were evaluated using the supravital dye Hoechst 33258 (H258) and PSA-FITC, respectively, as described (Cross et al., 1986).
To test whether the Fn-induced acrosome reaction depends on the interaction with 51 integrin receptor, an immunoneutralization test was carried out with the antibody against the 5 subunit of the receptor. Sperm capacitated for 18 h were preincubated for 30 min at 37°C with the antibody (1 : 100 dilution) and then exposed to Fn (100 µg/ml) or progesterone (5 µM) for 30 min before determining acrosomal integrity, as described (Cross et al., 1986).
Measurement of [Ca2+]i concentration
Capacitated sperm suspensions were prepared for [Ca2+]i determination by loading with the acetoxy-methyl ether of fura 2 (3 µM) for 30 min at 37°C, 5% CO2 and 95% air. To remove the free fura 2, the cells were washed with Tyrode's medium supplemented with 2.6% BSA without phenol red and centrifuged twice at 300 g for 10 min. Then, the cells were resuspended in Tyrode's medium without phenol red at a final concentration of 7–8 x 106 cells/ml. Then, 1 ml sperm aliquots were used for spectrofluorometry resuspending directly into stirred fluorescence cuvettes. All these procedures were carried out in the dark to prevent sample photobleaching. Fluorescence caused by Ca2+ under various experimental conditions was monitored with a Shimazdu model 1501 spectrofluorometer at an excitation wavelength pair of 340/380 nm and emission wavelength of 510 nm. Spectrofluorometry was performed in a methylacrylate cuvette magnetically stirred and warmed to 37°C in a heated cuvette holder. After equilibration for 2 min, measurements of [Ca2+]i were started. At approximately 100 s after the beginning of each sample run, Fn (10–100 µg/ml) was added to the sperm suspension. In separate experiments, sperm were incubated with 10 µM epoxomicin or 2.5 mM EGTA (pH 8.0) before adding Fn. Sequential additions of 20 µM digitonin and 10 mM Tris-EGTA were made near the end of each experiment to facilitate determination of [Ca2+]i (Morales et al., 2000, 2002).
SDS-PAGE and western blotting
Non-denaturing polyacrylamide gel electrophoresis (PAGE) was performed with a 5% slab gel as described by Davis (1964), and proteins were stained with Coomassie brilliant blue. To carry out denaturing SDS-PAGE, sperm extracts were boiled for 5 min with sample buffer (500 µM Tris-HCl, 10% SDS, 30% glycerol, 0.5% -mercaptoethanol and 0.5% bromophenol blue, pH 6.8) and then immediately settled on ice. Samples (20 µg) were resolved in 12% SDS-PAGE (12% acrylamide/bisacrylamide for the resolving gel and 5% acrylamide/bisacrylamide for the stacking gel) in a Mini Protean Cell (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Gels were stained for proteins with Coomassie brilliant blue. After SDS-PAGE, gels were equilibrated in transfer buffer for 15 min and electrotransferred at 60 V for 150 min onto polyvinylidene difluoride (PVDF) membrane (Immobilon P) using a mini trans-blot cell (Bio-Rad Laboratories Inc, Hercules, CA, USA). Transfer was monitored by Ponceau red stain and then blocked with Tris-buffer saline (TBS)-Tween 20 (0.1%, v/v) with 3% (w/v) BSA and 3% non-fat dry milk for 90 min. Blots were probed with a mouse antibody against phosphotyrosine, clone 4G10 (1 : 1000). Then, blots were washed and incubated with an appropriated second biotinylated antibody. The reaction was enhanced with streptoavidin-peroxidase conjugates, and a chemiluminescence kit was used to detect the horseradish-peroxidase-labeled protein according to the manufacturer's instructions. Prestained protein standards with molecular mass range of approximately 149–14 kDa were used. The intensity of the autoradiographic bands was quantified by densitometry using the Scion Image software. Western blot analysis experiments were performed at least three times.
Immunoprecipitation procedure
For the immunoprecipitation procedure, the cell lysate (containing approximately 200 µg total protein), 5 µg antibody and 400 µl of RIPA buffer were added to a microcentrifuge tube. The reaction mixture was incubated on shaker overnight at 4°C. The immune complexes were obtained by centrifugation (15 000g, 30 s). The supernatants were discarded and the pellets were washed two times with RIPA buffer and once with PBS. The washed pellets were mixed with SDS-sample buffer (2X) and heated in a boiling water bath for 5 min and the supernatant was subjected to SDS-PAGE. The proteins on the gels were transferred to a PVDF membrane and then revealed using the clone 4G10 (1 : 1000) anti phosphotyrosine antibody, as described.
Stripping PVDF membranes
In order to confirm equal loading of protein, blots that had been probed for phosphotyrosine proteins were stripped and reprobed with an antibody against 4 proteasome subunit. For this procedure, approximately 30 ml of stripping buffer, consisting of 2% (w/v) SDS, 62.5 mM Tris, pH 6.7, 100 mM 2-mercaptoethanol, was added to the membrane for 1 h with constant shaking at 60°C. The membrane was then washed (3 x 10 min in TBS), blocked and probed with the primary antibody as described.
Statistics
Data were analyzed by the one-way analysis of variance and the Student-Newman-Keuls multiple comparison test for unequal replicates using an Instat program. A difference of P < 0.05 was considered significant. All data are presented as mean values ± SEM.
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Results |
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Fibronectin and proteasome activity
The results indicate that incubation with Fn stimulated both the chymotrypsin-like (Figure 1A) and trypsin-like (Figure 1B) activity of the sperm proteasome, in a dose dependent manner at all incubation times. For example, with respect to the control 100 µg/ml Fn increased the chymotrypsin-like activity of the proteasoma 1.5-fold at T0, 1.7-fold at T5 and 2-fold at T18. For the trypsin-like activity, the increase was 1.9-fold at T0, 1.7-fold at T5 and 2-fold at T18 (Figure 1B). In addition, neither the chymotrypsin-like nor the trypsin-like activity of the sperm proteasome increased over the capacitation period.
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A similar increase in the chymotrypsin-like (Figure 2A) and trypsin-like activity (Figure 2B) of the sperm proteasome was observed when sperm capacitated for 18 h were incubated for 15 min with different concentrations of Fn.
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Fibronectin and acrosome reaction
Sperm incubated with Fn also exhibited a dose dependent increase in the percentage of acrosome reactions (Figure 3). Thus, the percentage of acrosome reacted sperm rose from 13 ± 0.6% in the control to 35 ± 1.5% in the presence of 100 µg/ml Fn (P < 0.001). The Fn-induced increase in acrosome reactions was drastically inhibited to control levels in the presence of 10 µM epoxomicin (11 ± 1.8%). As a positive control, 5 µM progesterone increased the percentage of acrosome reacted sperm to 37 ± 1.2%. Sperm treatment with 10 µM epoxomicin also inhibited the progesterone-induced acrosome reaction (Figure 3). Previously, it was reported that proteasome inhibitors block the membrane fusion events of the human sperm acrosome reaction (Morales et al., 1994
).
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In addition, the ability of Fn to induce the human sperm acrosome reaction was further enhanced by the presence of progesterone. This was true for all concentrations of Fn tested (Figure 4). For example, while the percentage of acrosome reacted sperm was 24.5 ± 0.5% in the presence of 10 µg/ml Fn, it rose to 39 ± 3% with the simultaneous addition of progesterone (Table I) (P < 0.001). The percentage of acrosome reactions after sperm were treated with progesterone alone was 33 ± 1% (Table I and Figure 4).
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The addition of the antibody against the
5 subunit of the Fn receptor to the suspension of capacitated sperm prevented the stimulating effect of Fn (Figure 5). In contrast, the neutralization of the integrin was unable to affect the progesterone-induced acrosome reaction (Figure 5). Finally, the incubation of capacitated sperm with an anti human IgG antibody (control antibody) did not prevent the ability of Fn to induce the acrosome reaction (data not shown).
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Fibronectin and protein tyrosine phosphorylation
Western blot analysis revealed that incubation with Fn increased the level of protein tyrosine phosphorylation, both in freshly ejaculated (T0) and sperm incubated for 5 h (T5) and 18 h (T18) (Figure 6A and B). We noted a global increase in the tyrosine phosphorylation of proteins ranging in molecular mass between 12 and 150 kDa. In agreements with others, we detected phosphorylated bands at approximately 12, 30, 45, 85 and 95 a kDa (Leclerc et al., 1996
; Naz and Rajesh, 2004) (Figure 6, arrows). The level of tyrosine phosphorylation after 18 h capacitation was high in control sperm, therefore, it was difficult to demonstrate clearly further effects of Fn in whole cell protein extracts although the intensity of certain bands appeared to increase slightly (Figure 6B, bands at 30, 45 and 60 kDa). We did not detect the appearance of new proteins being phosphorylated by Fn treatment (Figure 6A and B).
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In the next series of experiments, sperm extracts were subjected to immunoprecipitation using a monoclonal antibody to 20S subunit
4 and the precipitated proteins were detected on a native, non-denaturing gel (Figure 7A) and in the presence of SDS and -mercaptoethanol (denaturating gel, Figure 7B). Under non-denaturating conditions, the precipitant revealed a single protein band (Figure 7A). When the precipitant was analyzed by SDS-PAGE, a characteristic ladder of at least 15 bands was observed in the molecular mass range of 12–150 kDa (Figure 7B). These results are in agreement with the presence of the 26S proteasome in human sperm (Tipler et al., 1997; Wojcik et al., 2000).
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To evaluate the influence of Fn upon the phosphorylation status of the human sperm proteasome, sperm extracts were subjected to immunoprecipitation using a monoclonal antibody to 20S subunit
4 and the precipitated proteins were tested on western blot with an anti-phosphotyrosine antibody. Sperm treated with Fn exhibited an increase in the content of tyrosine phosphorylated proteasome subunits (Figure 8). This was true whether the sperm were incubated for 18 h in the presence of Fn (Figure 8A) or if they were capacitated for 18 h and then treated with Fn for 15 min (Figure 8B). Such increase was not an artifact of unequal protein loading, as demonstrated by the 4 proteasome subunit control (Figure 8C).
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To discard non-specific immunoprecipitation, sperm extracts were immunoprecipitated as described, but the western blot was revealed with an anti
-actin antibody (Figure 8D) or with an anti 5 integrin subunit antibody (data not shown).
Fibronectin and [Ca2+]i
Regarding the effect on [Ca2+]i, the results show that Fn increased the free cytosolic calcium in the sperm cells. This effect depended upon the concentration of Fn used. Thus, different concentrations of Fn caused differences in the peak and plateau of the [Ca2+]i curve. The mazximum effect was reached with 100 µg/ml Fn and it was similar to that induced by 5 µM progesterone. The Fn-induced increase in [Ca2+]i was fast and transient and with 100 µg/ml this increase was from a basal value of 213 ± 12 nM to a peak value of 697 ± 14 nM (Figure 9). This represents a stimulation of 327 ± 8% in comparison to the basal calcium level. The peak value was reached about 30 s after Fn addition and slowly came back to a new basal value, which was slightly higher than before, about 150 s later (Figure 9).
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The effect of Fn upon [Ca2+]i on spermatozoa was inhibited by the prior addition of 2.5 mM EGTA or epoxomicin (Figure 10). The presence of 10 µM epoxomicin significantly reduced the plateau phase of the calcium curve (Figure 10). A similar result was found when the progesterone-induced increase in [Ca2+]i was studied (Morales et al., 2003
).
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Discussion |
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Previously, we demonstrated that the human sperm proteasome plays a significant role during several steps of the fertilization process (Morales et al., 1994
, 2003). In this work we have shown that Fn induces the human sperm acrosome reaction and that this effect depends upon proteasome activity. Epoxomicin, a potent and specific inhibitor of the proteasome (Voorhees and Orlowski, 2006), drastically inhibited the Fn-induced acrosome reaction. We also detected an increase in the enzymatic activity of the proteasome, measured as the ability to hydrolyze trypsin and chymotrypsin substrates, in the presence of Fn. This was true regardless of the capacitation status of the sperm.
What is the possible connection between a protein that is in the extracellular space with proteins that are located in the inside of the cell? Integrins are a family of cell surface glycoprotein receptors by which cells attach to other cells or extracellular matrices. Apart from functioning in adhesion, inside the cell integrins have a role in signal transduction. Thus, integrins can be viewed as two-way signalling molecules. An "inside-out" regulation that involves conformational changes in the external domain of the receptor by which the affinity or distribution of the integrin receptors is modulated by intracellular events, and an "outside-in" regulation that initiates after the binding of the ligand to the receptor and results in intracellular messengers formation (Miranti and Brugge, 2002). Both systems seem to be operative in mammalian sperm (Bronson and Fusi, 1996). In accord with this, we present evidence that sperm treatment with Fn increased the [Ca2+]i and the level of protein tyrosine phosphorylation. In addition, we found that the sperm proteasome was tyrosine phosphorylated upon Fn treatment. These events may be part of the signal transduction mechanism of Fn.
This is the first evidence that Fn, a protein of the extracellular matrix, is able to stimulate the acrosome reaction and increase the [Ca2+]i in human sperm. Previously, a couple of reports showed that laminin, another extracellular matrix protein, induced the acrosome reaction (Mattioli et al., 1998) and Ca2+ influx (Barboni et al., 2001) in boar sperm. In our study, that the effect of Fn was specifically mediated by interaction with its integrin receptor is supported by: (i) the expression of integrin subunits in human sperm, in particular the subunits 5 and 1, that corresponds to the Fn receptor has been demonstrated. These subunits are located mainly in the plasma membrane of the acrosomal region and in the equatorial segment (Fusi et al., 1996; Glander et al., 1998). In addition, by RT-PCR, it has been demonstrated that the expression of mRNA of the subunits 1, 3, 4 and 5 in human sperm (Rohwedder et al., 1996); and (ii) also consistent with the presence of this specific Fn binding integrin, are the results of the immunoneutralization test; the addition of the antibody against the 5 subunit of the Fn receptor to the suspension of capacitated sperm prevented the stimulating effect of Fn. In contrast, the neutralization of the 51 integrin was unable to affect the progesterone-induced acrosome reaction. The latter observation supports the notion that there must be some sperm whose membranes contain at least two different transduction machineries that are activated by specific agonists and lead to acrosome reaction, possibly by converging on the same downstream path within the sperm. This was further supported by the observation that progesterone enhanced the ability of Fn to induce the human sperm acrosome reaction. However, it seems that not all sperm express both receptors for progesterone and Fn on their membranes since the effect of treating the cells with both molecules was not additive. This notion needs further testing. Similar observations have been reported for the zona pellucida and progesterone-induced acrosome reaction (Baldi et al., 2002; Kirkman-Brown et al., 2002).
A number of studies on lymphocytes, granulocytes and osteoclasts have shown that ligand binding to integrin receptors in mammalian systems causes elevation of [Ca2+]i (Jaconi et al., 1991; Schwartz, 1993; Coppolino et al., 1997). In addition, Fn potentiates L-type Ca channels and L-type Ca current. The elevation of [Ca2+]i upon activation of integrins results from both inositol triphosphate-evoked Ca2+ release from sarcoplasmic/endoplasmic reticulum and extracellular Ca2+ influx through voltage-gated, L-type plasma membrane Ca2+ channels (Kwon et al., 2000). Our finding that Fn induces the acrosome reaction and Ca2+ influx, support the idea that in the sperm, 51 integrin has an outside-in signalling function. Besides, the existence of L-type calcium channels has already been reported in human sperm (Florman et al., 1992; Goodwin et al., 1997; Morales et al., 2000). In addition, we found that epoxomicin decreased the plateau phase of the Fn-induced calcium entry into the sperm cells. The initial transient peak was unaffected. These results suggest that proteasome activation may be upstream of the generation of the sustained phase of the [Ca2+]i increase. A similar finding was reported for the progesterone-induced increase in [Ca2+]i (Morales et al., 2003).
Current evidence suggests that multiple pathways are utilized by integrins to activate specific signalling proteins (reviewed in Miranti and Brugge, 2002). In many cell types, integrins activate the focal adhesion kinase (FAK) pathway (Giancotti and Erkki Ruoslahti, 1999). Upon activation, FAK autophosphorylates Tyr397, creating a binding site for the Src homology 2 (SH2) domain of Src (Schlaepfer et al., 1994). The Src kinase then phosphorylates a number of focal adhesion components. The major targets include paxillin and tensin (Richardson and Parsons, 1996). FAK also combines with, and may activate, phosphoinositide 3-OH kinase (PI 3-kinase) either directly or through the Src kinase (Chen et al., 1996). Finally, there is evidence that Src phosphorylates FAK at Tyr925, creating a binding site for the complex of the adapter Grb2 and Ras guanosine 5'-triphosphate exchange factor mSOS (Schlaepfer et al., 1994). Until now, however, there are no reports of the existence of FAK in mammalian sperm.
In addition to activating FAK, some 1 and v integrins, including the Fn receptor 51, may also directly activate the tyrosine kinase Fyn and, through it, the adapter protein Shc (Wary et al., 1998). In this pathway, the transmembrane protein caveolin-1 appears to function as an adapter, which couples the integrin subunit to Fyn. This function of caveolin-1 is consistent with its ability to bind cholesterol and glycosphingolipids and organize specialized plasma membrane "rafts" (Harder and Simons, 1997). Upon integrin binding to extracellular matrix proteins, Fyn becomes activated, and its SH3 domain interacts with a proline-rich site in Shc. Shc is then phosphorylated by Fyn at Tyr317 and combines with the Grb2-mSOS complex (Wary et al., 1998). In human sperm, several components of this pathway have been described, including the membrane lipid raft protein, caveolin-1 (Sousa et al., 2006); Fyn and c-Src tyrosine kinases (Kumar and Meizel, 2005); the SH2-containing Shc proteins (Morte et al., 1998; de Lamirande and Gagnon, 2002); PI 3-kinase (Fisher et al., 1998); the GRB2 adaptor protein (Morte et al. 1998; de Lamirande and Gagnon, 2002); and Rasp21, Raf and ERK1 and 2 (ERK1/2) (de Lamirande and Gagnon, 2002). Whether this is the pathway integrins use to activate the human sperm proteasome is under investigation.
Sperm capacitation and acrosome reaction are events regulated by intracellular signals, mainly induced by protein phosphorylation in Tyr and Ser/Thr residues (Visconti and Kopf, 1998; Urner and Sakkas, 2003). It has been demonstrated that protein phosphorylation in Tyr residues on the sperm head is necessary for the acrosome reaction (Naz and Rajesh, 2004). We observed that Fn induced an increase in the degree of protein phosphorylation in Tyr residues both in recently ejaculated and capacitated sperm. Moreover, some of the proteasome subunits exhibited an increase in tyrosine phosphorylation upon Fn stimulus. What is the molecular basis of the Fn-induced increase in proteasome activity? In this work, we have used small, synthetic peptides that do not need to be ubiquinated to be degraded by the proteasome. Therefore, the Fn-induced increase in substrate degradation is most probably due to differences in proteasome activity.
The possibility that we are suggesting is that the increase in phosphorylation of the proteasome is responsible for the increase in activity. Indeed, proteasome activity can be regulated by phosphorylation. For example, Yang et al. (1995) showed that phosphorylation events are necessary for the assembly of the 26S proteasome. Several subunits including MSS1, S4, S6 and S12 of the 19S regulatory complex have been shown to be phosphorylated (Mason et al., 1998). Recently, it was shown that assembly of the proteasome requires phosphorylation of Rpt6, an ATPase subunit (Satoh et al., 2001). Among the kinases, PKA greatly enhances the synaptic proteasome activity in both Aplysia and mouse (Upadhya et al., 2006) and we have evidence that the activity of the human sperm proteasome is regulated by PKA and tyrosine kinase (Morales et al., 2006). In addition, participation of the proteasome pathway has been reported in several aspects of integrin signal transductions. Thus, integrin activation upon interaction with their substrate activates ubiquitin ligase c-cbl (Levkowitz et al., 1999), promotes proteasome-mediated cleavage of erbB2 cytoplasmic domain, a member of the EGF receptor family (Shimizu et al., 2003), induced the proteasomal degradation of CDK inhibitors, p21Cip1 and p27Kip1 (Bao et al., 2002), and proteasome-dependent degradation of Raf-1 (Manenti et al., 2002) and of PDGF receptor (Baron and Schwartz, 2000). All these observations support the importance of the proteasome pathway in integrin signal transduction.
In most mammalian species, the cumulus oophorus still surrounds the oocyte when it arrives at the fertilization site in the ampulla of the oviduct (Hunter, 1988). The cumulus mass consists of a group of 3000–5000 cells and an extracellular matrix secreted by the cells (Zhuo and Kimata, 2001; Tanghe et al., 2002). The main component of the extracellular matrix is the glysosaminoglycan, hyaluronic acid, conjugated with several cell adhesion glycoproteins, including Fn and laminin (Salustri et al., 1992; Camaioni et al., 1996; Einspanier et al., 1999). Our observation that Fn, acting upon binding to its integrin receptor 51, can induce the acrosome reaction via calcium influx supports the hypothesis raised by Barboni and colleagues that the hyaluronic acid network of the expanded cumuli may be a preliminary sperm-oocyte recognition mechanism that precedes the one involving sperm-zona interaction (Barboni et al., 2001). Indeed, there are previous reports indicating that the cumulus contains and secretes other molecules that increase capacitation or the acrosome reaction in human sperm (e.g. progesterone, hyaluronic acid). (Suarez et al., 1986; Sabeur et al., 1998).
The sperm–cumulus interaction could serve at least two purposes. Cumulus-induced acrosome reactions might be used to eliminate supernumerary sperm that, after reacting with the cumulus, stick on it, as observed by Cummins and Yanagimachi (1986), and therefore cannot reach the oocyte. This may serve as a mechanism to prevent polyspermy. In contrast, only sperm still in the process of capacitation could cross this first vestment and interact with the zona. Upon reaching the zona pellucida, the sperm could activate the cascade of events leading to acrosomal exocytosis (Yanagimachi, 1994).
In conclusion, we present evidence that Fn, an extracellular matrix protein, present in the cumulus oophorus at the time of fertilization, induces the human sperm acrosome reaction by a mechanism that may depend upon proteasome phosphorylation and activation and calcium influx. This may represent a mechanism by which supernumerary sperm may be stimulated to undergo the acrosome reaction prematurely, before reaching the zona pellucida of the oocyte, thus decreasing the odds of polyspermy.
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This work was financed by FONDECYT 1040295 and DIRINV 1322-06 and 1316-06.
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