Participation of the sperm proteasome in human fertilization

Patricio Morales1, Milene Kong, Eduardo Pizarro and Consuelo Pasten

Department of Biomedicine, Faculty of Health Sciences, University of Antofagasta, Avda, Angamos 601, P.O. Box 170, Antofagasta, Chile

1 To whom correspondence should be addressed. e-mail: pmorales{at}uantof.cl


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Fertilization in mammals comprises the sequential interactions of the sperm with the cumulus oophorus, zona pellucida, and oocyte plasma membrane. Here we investigate proteasome activity in human sperm and its possible involvement during the fertilization process. METHODS: Proteasome activity was measured in intact sperm and in sperm extracts using the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC, in the presence or absence of the specific proteasome inhibitor, clasto-lactacystin {beta}-lactone. The participation of the proteasome was evaluated during (i) sperm–zona binding using the hemizona assay; (ii) zona pellucida-induced acrosome reactions with a pulse and chase design; (iii) progesterone-induced acrosome reactions incubating overnight capacitated sperm with progesterone; and (iv) progesterone-induced Ca2+ influx using fura-2AM. RESULTS: Intact sperm and sperm extracts possessed proteasome activity, which was susceptible to inhibition by clasto-lactacystin {beta}-lactone. Sperm–zona binding was not inhibited by clasto-lactacystin {beta}-lactone. However, both zona pellucida- and progesterone-induced acrosome reactions were inhibited by clasto-lactacystin {beta}-lactone. The proteasome inhibitor also blocked the sustained phase of the Ca2+ influx provoked by progesterone but not the peak. CONCLUSION: The human sperm proteasome is involved in the exocytosis of the acrosome, perhaps in events upstream of the plateau phase of the Ca2+ influx.

Key words: calcium influx/human zona pellucida/proteases/proteasome/sperm–zona binding


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fertilization is a highly specific cellular-recognition event, that comprises the sequential interactions between the sperm and cumulus oophorus, zona pellucida and oocyte plasma membrane (Yanagimachi, 1994Go). The participation of acrosomal proteases in these processes has been described in several species, including marine invertebrates and mammals (for reviews, see Morales and Llanos, 1995Go; Bedford, 1998Go; Mykles, 1998Go). In mammals, numerous reports indicate that one or several acrosomal proteases may have a role during fertilization. In particular, for many years it was thought that the trypsin-like protease acrosin was responsible for digesting the zona pellucida, enabling the sperm to penetrate through this thick egg-coat (McRorie and Williams, 1974Go). However, recent studies using acrosin-knockout mice revealed that acrosin was not essential for fertilization or sperm penetration through the zona pellucida (Baba et al., 1994Go); rather, acrosin seems to be involved in acrosome dispersal during the acrosome reaction (Yamagata et al., 1998Go).

Recently, it was shown that human and mouse sperm possess a multi-enzymatic protease complex or proteasome, which presents a similar sedimentation coefficient to the proteasomes purified from other tissues, and possess trypsin-like, chymotrypsin-like and peptidylglutamyl peptide-hydrolyzing activities (Tipler et al., 1997Go; Wojcik et al., 2000Go). The ubiquitin-proteasome pathway is responsible for most of the cell proteolysis. The proteasome degrades most nuclear and cytosolic proteins, after they have been co-valently labelled to ubiquitin molecules (Goldberg, 1995Go; Ciechanover, 1998Go). This enzymatic complex is composed of a proteolytic core complex termed the 20S proteasome (~700 kDa). When the 20S proteasome is associated with the PA700 proteasome activator (19S cap), composed of several ATPases and regulatory proteins, it constitutes the 26S proteasome (~2000 kDa). The 26S proteasome regulates the traffic of proteins that will become degraded or processed and its activity depends on ATP. Four stacked rings surrounding inner cavities form the 20S proteasome—two inner {beta}-rings and two outer {alpha}-rings. The {alpha}-subunits possess regulatory functions and the {beta}-subunits catalytic functions (Coux et al., 1996Go). The {beta}-subunits are specifically inhibited by lactacystin (Fenteany and Schreiber, 1998Go). From a functional point of view, the access of the proteins to be degraded to the catalytic chamber is restricted by the entry pore of ~13 A°. This means that before being degraded, a protein must not only be ubiquinated but also denatured (Ciechanover, 1998Go). Free N-terminal threonine residues on three of the seven {beta}-type subunits act as nucleophiles and are essential in the mechanism of catalysis (Seemuller et al., 1995Go; Fenteany and Schreiber, 1998Go).

There are numerous reports indicating that in marine invertebrates, the sperm proteasome is involved in multiple steps of the fertilization process, from acrosomal exocytosis triggered by the egg jelly to penetration of the vitelline coat and fusion with the egg plasma membrane (Mykles, 1998Go). Thus, in the solitary ascidian, Halocynthia roretzi (order stolidobranch), the sperm proteasome is necessary for sperm binding and penetration through the vitelline coat of the eggs (Hoshi et al., 1981Go; Sawada et al., 1983Go; Yokosawa et al., 1987Go; Sawada et al., 2002bGo). In the phlebobranch ascidian Ciona intestinalis, the sperm proteasome is involved in the process of penetration through the vitelline coat, probably functioning as a lysin (Sawada et al., 1998Go), in addition to a lower molecular mass chymotrypsin-like protease (Pinto et al., 1990Go; Marino et al., 1992Go). In sea urchins (Strongylocentrotus intermedius), the sperm proteasome is involved in Ca2+ channel activation leading to egg jelly-induced acrosomal exocytosis (Matsumura and Aketa, 1989Go). To date, however, the physiological role of the sperm proteasome in mammals remains unknown.

In this work we investigate the presence of proteolytic activity in human sperm that may correspond to the proteasome. Additionally, we studied whether the proteasome is involved in sperm binding to the human zona pellucida (hZP) and in the hZP- and progesterone-induced acrosome reactions. The involvement of Ca2+ influx in the progesterone-induced acrosome reaction (AR) is also discussed.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
The following reagents were purchased from Sigma Chemical Co. (St Louis, MO, USA): progesterone, fura 2-AM, the chymotrypsin inhibitor N-p-Tosyl-L-phenylalanine-chloromethyl ketone (TPCK); the chymotrypsin substrate N-Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC); the cysteine proteases inhibitor [2S-3S]-trans-Epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester (E64d); Hoechst 33258; bovine serum albumin, (BSA) A7030; Hepes; ionomycin and 1,4-Diazabicyclo[2.2.2.] octane (DABCO). Pisum sativum agglutinin (PSA)-FITC was purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). Pure nitrocellulose transfer and immobilization membrane was from Schleicher and Schuell (Keene, NH, USA). The proteasome inhibitor clasto-lactacystin (clasto-lactacystin {beta}-lactone) and the anti-proteasome antibodies MCP20, MCP34, MCP168, MCP205, and MCP231 were from Affinity Research Products (Exeter, UK). MCP20 detects the {alpha}6 subunit (HC2), MCP34 detects the {alpha}4 subunit (XAPC7), MCP168 detects the subunit {beta}2 (Z), MCP205 detects the subunit {beta}7 (HN3), and MCP231 detects the proteasome subunits {alpha}1, 2, 3, 5, 6 and 7. Deionized water used in these experiments was purified to >=18 M{Omega}-cm with an EASY-pure UV/UF ion-exchange system (Barnstead/Thermolyne, Dubuque, IA, USA). Stock solutions of TPCK, Suc-LLVY-AMC and ionomycin were prepared in dimethyl sulphoxide (DMSO). Stock solution of E64d was dissolved in methanol:water (1:1). The final concentration of DMSO and methanol in the sperm suspensions was 0.1% (v/v).

Human zonae pellucidae
Human oocytes were dissected from ovarian tissue and stored at –80°C as previously described (Morales et al., 1989Go, 1994b). Briefly, ovarian tissue was placed on ice and dissected immediately following the protocol of Overstreet et al. (1980Go). Zona-intact, immature oocytes were denuded of granulosa cells, placed in capillary tubes containing 2 mol/l DMSO in phosphate buffered saline (PBS), and stored at –80°C. After thawing, the oocytes were freed of remaining cumulus cells by passing through a narrow bore pipette. As a result of freezing and thawing these oocytes were never viable. The oocytes thus obtained were used for sperm–zona binding and for sperm–acrosome reaction (see below).

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 World Health Organization guidelines (World Health Organization, 1999Go) and <1% of the sperm had cytoplasmic droplets. The specimens were allowed to liquefy for 30–60 min at 37°C in a slide warmer. Motile sperm were selected by centrifugation through a two-step Percoll gradient (40/80%) as described previously (Suarez et al., 1986Go). Briefly, aliquots of semen were layered over the upper step of the Percoll gradient and centrifuged for 20 min at 300 g. The pellet was diluted in 10 ml of modified Tyrodes medium consisting of 117.5 mmol/l NaCl, 0.3 mmol/l NaH2PO4, 8.6 mmol/l KCl, 25 mmol/l NaHCO3, 2.5 mmol/l CaCl2, 0.5 mmol/l MgCl2, 2 mmol/l glucose, 0.25 mmol/l Na pyruvate, 19 mmol/l Na lactate, 70 µg/ml of both streptomycin and penicillin, phenol red and 0.3% BSA, centrifuged again at 300 g for 10 min and then resuspended in the same medium but with 2.6% BSA. The sperm concentration was adjusted to 20x106 cells/ml and the cells incubated at 37°C, 5% CO2 for different times, to promote capacitation (Aitken et al., 1996Go). The sperm were then evaluated for their ability to bind to the hZP, to undergo the AR, and to increase the intracellular Ca2+ concentration ([Ca2+]i) (see below).

For Western blotting, sperm incubated for 4.5 h were concentrated by centrifugation at 5000 g for 1 min at room temperature and then washed twice in 1 ml PBS at room temperature. The sperm pellet was then suspended in sample buffer (Laemmli, 1970Go) without mercaptoethanol and boiled for 5 min. After centrifugation at 5000 g for 3 min, the supernatant was removed, 2-mercaptoethanol added to a final concentration of 5%, the sample boiled for 3 min, and then subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), as described below.

Preparation of sperm extracts
Semen samples were obtained as described above and allowed to liquefy for 30–60 min. The sperm were then separated from seminal plasma, other cell types and cellular debris by centrifugation through a column of Percoll (40/80%) as described above except that the Percoll was prepared in 50 mmol/l Hepes, 191 mmol/l NaCl, pH 7.4 (Morales et al., 1994aGo). The resulting sperm pellet was washed two times by centrifugation at 300 g for 10 min and then resuspended in homogenization buffer (50 mmol/l Hepes, 10% glycerol, pH 7.4) at a concentration of 25x107 sperm/ml. The sperm suspension was then sonicated (Virsonic, Gardiner, NY, USA) with six 60 watt bursts for 20 s each, followed by centrifugation for 30 s at 5000 g 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 Bradford method (Bradford, 1976Go), ranged between 0.3–0.8 mg/ml.

The chymotrypsin-like activity of the proteasome was assayed using the fluorogenic substrate Suc-LLVY-AMC. Aliquots of 100 µl of enzyme extract were incubated in a final volume of 2 ml containing 10 mmol/l CaCl2, 50 mmol/l Hepes, pH 7.4, and 10 µmol/l 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.

To test the effect of the protease/proteasome inhibitors on the sperm proteolytic activity, 100 µl aliquots of the extract were preincubated with TPCK, clasto-lactacystin, or E64d for 15 min at 37°C before adding the substrate. Proper controls were carried out with the inhibitor solvents. In some experiments, free-swimming sperm were incubated with the inhibitors prior to preparing the extract.

Sperm-hZP binding assay
The sperm capacity to bind to the hZP was evaluated using the hemizona assay (Burkman, 1988Go; Franken et al., 1989Go). Briefly, 99 µl droplets of sperm, suspended in modified Tyrode’s medium containing 2.6% BSA, were treated by adding 1 µl of test or control solutions, under oil in a plastic petri-dish. Then, one hemizonae was added to the control sperm droplet and the matching hemizona was added to the test sperm droplet. Control and test sperm droplets containing hemizonae were incubated for 10 min at 37°C, 5% CO2. After incubation, each hemizona was removed and gently washed with a wide bore pipette. The tightly bound sperm on the outer surface of each hemizona were counted under a phase-contrast microscope. These procedures have been described in detail elsewhere (Morales et al., 1989, 1994b, 1999Go).

Effect of the proteasome inhibitor clasto-lactacystin on the AR
To evaluate the effect of clasto-lactacystin on the sperm acrosome reaction, hZP, progesterone or the Ca2+ ionophore ionomycin were used as the stimulus.

hZP-induced acrosome reaction
A pulse and chase protocol was used (Cross et al., 1988Go). Sperm, at a concentration of 50x106 cells/ml, were treated for 15 min with 10 µmol/l of clasto-lactacystin or DMSO (control). Then, oocytes were added to the sperm suspensions and incubated for 1 min (pulse). The oocytes with bound sperm were then removed and transferred to a sperm-free droplet. Some oocytes were immediately processed to determine the acrosomal status of the bound sperm; the rest of the oocytes were incubated for 30 min (chase), at 37°C, 5% CO2 in air. After the chase, the oocytes were removed and processed to determine the acrosomal status of the bound sperm.

Progesterone and ionophore-induced AR
To induce the AR with progesterone or ionomycin, cells were capacitated as described above, except that the sperm concentration was 10x106/ml. After 20 h, the sperm suspensions were treated for 15 min with 10 µmol/l clasto-lactacystin or DMSO (control). The suspensions were then incubated with 10 µmol/l ionomycin or 0.69 µmol/l progesterone for an additional 15 min. Proper controls included the incubation of the inhibitor treated sperm with the solvent for ionomycin. At the end of this period the percentage of acrosome reacted cells was determined.

Detection of the AR
The acrosomal status of zona-bound sperm and sperm in suspension was determined as previously described (Cross et al., 1986Go; Liu and Baker, 1996Go). Briefly, zona-bound sperm were removed by vigorous aspiration in and out of a narrow bore pipette, slightly smaller than the oocyte, and deposited into a 5 µl drop of PBS (0.2% BSA) in a 0.1% poly-L-lysine-treated glass slide. The sperm in suspension were treated with Hoechst 33258 (1 µg/ml) for 5 min and then deposited into a glass slide. The slides were then left to air dry and fixed in 95% ethanol for 30 min. Finally, the sperm were treated with FITC-PSA (100 µg/ml) for 10 min, rinsed in PBS, mounted with DABCO, and the sperm evaluated with fluorescence microscopy.

Measurement of [Ca2+]i
Capacitated sperm suspensions were prepared for [Ca2+]i determination by loading with the acetoxy-methyl (AM) ester of fura 2 (3 µmol/l final extracellular concentration) for 30 min at 37°C and 5% CO2, as described before (Garcia and Meizel, 1999Go; Morales et al., 2000Go). After the free fura 2 was removed, sperm aliquots were used for spectrofluorometry resuspending directly into stirred fluorescence cuvettes. Fluorescence caused by [Ca2+]i under various experimental conditions was monitored using a Shimadzu 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 100 s after the beginning of each sample run, progesterone (0.69 µmol/l) was added to the sperm suspension. To test whether the proteasome was involved in the sperm response to progesterone, sperm aliquots were incubated with 10 µmol/l clasto-lactacystin for 15 min at 37°C, 5% CO2, before the addition of 0.69 µmol/l progesterone. Sequential additions of 20 µmol/l digitonin and 10 mmol/l Tris-EGTA were made near the end of each experiment to facilitate determination of [Ca2+]i, as previously described (Garcia and Meizel, 1999Go; Morales et al., 2000Go), using a Kd of 285 nmol/l for fura 2 at 37°C (Garcia and Meizel, 1999Go).

SDS-PAGE and immunoblotting
SDS-PAGE was performed using 12% gels according to an established method (Laemmli, 1970Go). Electrophoretic transfer of proteins to a nitrocellulose membrane in all experiments was carried out according to the method of Towbin et al. (1979), at 80 v for 4 h at 4°C. After transfer, the membrane was blocked (3% BSA in Tris-buffered saline), washed three times, incubated with primary antibody, washed again three times, and incubated with secondary antibody conjugate. Horseradish peroxidase detection was carried out using standard freshly made amplified Opti-4CN goat anti-mouse detection kit according to the manufacturer’s instructions.

Statistics
All percentages were subjected to arc-sine transformation before analysis. Bartlett’s test for homogeneity, followed by the F-test and then the paired t-test and/or Dunnett’s multiple comparison tests were used to compare the percentage of acrosome reacted sperm in the control and treated groups. Differences were considered significant at the 0.05 level of confidence.


    Results
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 Materials and methods
 Results
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 References
 
Human sperm extracts possessed chymotrypsin-like activity, detected using the chymotrypsin substrate Suc-LLVY-AMC (Figure 1A). The specific activity of the sperm extracts toward this substrate was 1.85 ± 0.5 nmol AMC hydrolyzed/mg protein/min. This protease activity was only partially inhibited by the chymotrypsin inhibitor TPCK. Thus, the specific activity in the presence of 80 and 160 µmol/l TPCK was 1.34 ± 0.2 and 0.89 ± 0.2 nmol AMC hydrolyzed/mg protein/min respectively. On the other hand, this protease activity was drastically inhibited by the presence of the specific proteasome inhibitor, clasto-lactacystin. Thus, the specific enzyme activity in the presence of 1 and 10 µmol/l clasto-lactacystin was 0.31 ± 0.01 and 0.12 ± 0.09 nmol AMC hydrolyzed/mg protein/min respectively. Sperm extracts treated with 50 µmol/l and 100 µmol/l clasto-lactacystin were totally devoid of chymotrypsin-like activity. In addition, the presence of E64d, a membrane-permeable inhibitor for cysteine proteases including cathepsins and calpains, did not modify the chymotrypsin-like activity of the sperm extracts (Figure 1A). Treatment of intact, free-swimming sperm with clasto-lactacystin also inhibited the chymotrypsin-like activity of the proteasome (2.84 ± 0.9 versus 0.31 ± 0.2 nmol AMC hydrolyzed/mg protein/min, control and clasto-lactacystin treated sperm respectively), confirming that this inhibitor is cell permeable (Fenteany and Schreiber, 1998Go). These results strongly suggest that this proteolytic activity was due to the proteasome. In accord with this, the anti-proteasome monoclonal antibody MCP231 detected two bands of ~29 and 32 kDa (Figure 1B, lane 1). The monoclonal antibodies MCP20, MCP34, MCP205 and MCP168 detected single bands of the expected molecular size (Figure 1B, lanes 2–4).



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Figure 1. Presence of proteasomes in human sperm. (A) Chymotrypsin-like activity of human sperm extracts incubated with the proteasome inhibitor clasto-lactacystin {beta}-lactone (1 µmol/l open triangles and 10 µmol/l, dark triangles respectively), with the chymotrypsin inhibitor TPCK (80 µmol/l, dark diamonds and 160 µmol/l, open diamonds respectively), with the calpain inhibitor E64d (10 µmol/l, open circles), or with solvent (dark circles). Then, the synthetic chymotrypsin substrate, Suc-Leu-Leu-Val-Tyr-AMC (10 µmol/l) was added. Results are the mean ± SEM of six experiments conducted with five different semen samples. (B) Western blotting of lysates obtained from purified sperm using anti-proteasome monoclonal antibodies. The positions of molecular size markers are indicated on the left.

 
The incubation of human sperm in capacitating conditions for 4.5 and 20 h did not substantially change the chymotrypsin-like activity of the proteasome, in comparison with non-capacitated sperm samples (Figure 2). Thus, there were no significant differences in the specific enzymatic activity of the sperm extracts during the incubation period (Figure 2). Clasto-lactacystin also inhibited the chymotrypsin-like activity of the proteasome of sperm capacitated for 4.5 and 20 h (Figure 2).



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Figure 2. Proteasome activity of extracts of capacitated human sperm. Extracts of human sperm capacitated for different times were incubated with the synthetic chymotrypsin substrate, Suc-Leu-Leu-Val-Tyr-AMC (10 µmol/l), in the presence (dark symbols) or absence of the proteasome inhibitor clasto-lactacystin {beta}-lactone (open symbols). The circles represent sperm capacitated for 4.5 h; the squares represent non-capacitated sperm; and the triangles represent sperm capacitated for 20 h. Results are the mean ± SEM of six experiments conducted with five different semen samples.

 
Similarly, there were no significant differences in the number of zona-bound sperm (Figure 3) or in the percentage of motile sperm (data not shown) in the presence of increasing concentrations of clasto-lactacystin. This suggests that the proteasome was not involved in sperm binding to the hZP or in sperm motility. However, when we evaluated the hZP-induced AR, striking results were found (Figure 4). In the control group the percentage of acrosome reacted sperm among those bound to the hZP rose from 8.8 ± 1.7% after the pulse to 23.2 ± 2.4% after the chase (P < 0.001). However, in the clasto-lactacystin-treated group, the percentage of acrosome reacted sperm among those bound to the hZP was 9.1 ± 1.4% after the pulse and only 12.5 ± 2.1% after the chase (NS) (Figure 4).



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Figure 3. Number of sperm bound to the human zona pellucida in the presence of various concentrations of the proteasome inhibitor clasto-lactacystin {beta}-lactone. The results are expressed as a percentage (mean ± SEM) of the control hemizonae and they represent the average ± SEM of six experiments conducted with five different semen samples.

 


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Figure 4. Zona-pellucida-induced sperm acrosomal exocytosis in the presence (open circles) and absence (dark circles) of the proteasome inhibitor clasto-lactacystin {beta}-lactone. Sperm and oocytes were incubated for 1 min (pulse). The oocytes were then withdrawn from the sperm suspension and some of them were immediately processed to determine the acrosomal status of the bound sperm. The other oocytes were transferred to a sperm-free drop and incubated for 30 min (chase). After that time, the acrosomal status of the bound sperm was determined. Results represent the mean ± SEM of five experiments conducted with five different semen samples; in each experiment 3–4 oocytes were used.

 
We also evaluated the progesterone-induced AR (Figure 5). Treatment with 0.69 µmol/l progesterone stimulated the sperm to undergo the AR from a basal level of 12 ± 3% to 42 ± 5% (P < 0.001). When the sperm were treated with 10 µmol/l clasto-lactacystin, however, they were unable to undergo the AR in response to the progesterone stimulus (Figure 5). On the other hand, treatment with clasto-lactacystin did not inhibit the response of the sperm to the calcium ionophore ionomycin (50.3 ± 55% ionomycin versus 48 ± 5% clasto-lactacystin plus ionomycin, NS) (Figure 5).



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Figure 5. Progesterone-induced sperm acrosomal exocytosis in the presence or absence of the proteasome inhibitor clasto-lactacystin {beta}-lactone. Sperm aliquots were treated with progesterone alone (P, 0.69 µmol/l), with clasto-lactacystin-{beta}-lactone (10 µmol/l) and then with progesterone (Clasto + P), with ionomycin alone (10 µmol/l), or with clasto-lactacystin-{beta}-lactone and then with ionomycin (Clasto + ionomycin) to stimulate acrosomal exocytosis. Results are the mean ± SEM of six experiments conducted with five different semen samples.

 
Finally, we assessed whether the progesterone-induced Ca2+ influx was modified by treatment with clasto-lactacystin. Addition of progesterone to the sperm suspensions elicited a rapid response, associated with a peak phase and a plateau phase (Figure 6). While the peak phase was not modified by sperm treatment with clasto-lactacystin, the plateau phase was significantly reduced. This was also evident when the area under the Ca2+ curve was evaluated (Figure 6, insert. P < 0.01).



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Figure 6. Progesterone-induced increase in [Ca2+]i in the presence or absence of clasto-lactacystin {beta}-lactone, measured using fura 2 AM. The dark circles represent the control sperm suspension and the open circles the sperm suspension treated with clasto-lactacystin {beta}-lactone (10 µmol/l). Progesterone (P, 0.69 µmol/l) was added at 100 s. A representative experiment from five is shown. In the insert, the area under the curve (mm2) for the progesterone-induced increase in [Ca2+]i for control and clasto-lactacystin sperm suspensions is shown.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this report we have shown that the highly specific proteasome inhibitor, clasto-lactacystin {beta}-lactone, can prevent the human sperm AR. This was the case whether hZP or progesterone was used to stimulate acrosomal sperm exocytosis. We also present evidence that the sperm proteasome was involved in the progesterone-induced Ca2+ influx, specifically diminishing the plateau phase. In addition, sperm extracts exhibited hydrolytic activity toward a synthetic chymotrypsin substrate and this activity was specifically inhibited by clasto-lactacystin. Thus, these results strongly suggest the participation of the human sperm proteasome in the exocytosis of the acrosome.

In the present study, treatment of the sperm with clasto-lactacystin inhibited the AR induced by hZP or progesterone but not the AR induced by the Ca2+ ionophore ionomycin. The latter result is in agreement with that shown by Wojcik et al. (2000Go). Also in agreement with the results of Wojcik and colleagues (Wojcik et al., 2000Go), we found that clasto-lactacystin did not affect sperm motility or capacity to bind to hZP. In addition to their observations, we found that clasto-lactacystin interfered with the progesterone-induced Ca2+ entry into the sperm cells. However, only the plateau phase of the calcium entry was decreased by clasto-lactacystin treatment. The initial transient peak was unaffected. Thus, these results suggest that the activation of the proteasome is upstream of the generation of the sustained phase of the [Ca2+]i increase. All these observations indicate that the sperm proteasome is involved in human fertilization, specifically in the AR induced by the hZP. Previously, it was reported that trypsin and chymotrypsin inhibitors blocked the zona pellucida- and progesterone-induced AR (Pillai and Meizel, 1991Go; Llanos et al., 1993Go; Morales et al., 1994aGo). Although it was not clear at that time whether the inhibitors blocked the proteasome or some other sperm protease, the suggestion was raised that this proteolytic activity could be part of a larger multi-catalytic complex, the proteasome (Morales et al., 1994aGo).

We have also found that human sperm extracts hydrolyzed the specific proteasome substrate, Suc-LLVY-AMC. It has been shown on numerous occasions that this is a preferred substrate for the proteasome and it is hydrolyzed by the sperm proteasome of marine invertebrates (Tipler et al., 1997Go; Mykles, 1998Go; Sawada et al., 1998Go; Marino et al., 1999Go; Sawada et al., 2002bGo). In support of the view that this activity corresponded to the proteasome was the observation that it was inhibited by the highly specific proteasome inhibitor, clasto-lactacystin (Fenteany and Schreiber, 1998Go). Clasto-lactacystin {beta}-lactone is a potent, highly specific and cell-permeable inhibitor of the proteasome (Fenteany and Schreiber, 1998Go). The natural product lactacystin exerts its cellular effects through a mechanism involving acylation and inhibition of the proteasome, a cytosolic proteinase complex that is an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In vitro, lactacystin does not react with the proteasome; rather it undergoes a spontaneous conversion (lactonization) to the active proteasome inhibitor, clasto-lactacystin {beta}-lactone (Fenteany et al., 1995Go; Fenteany and Schreiber, 1998Go). Clasto-lactacystin {beta}-lactone inhibits, in an irreversible manner, the trypsin- and chymotrypsin-like activities and in a reversible manner the peptidylglutamyl peptide-hydrolyzing activity of the proteasome. Additionally, participation of sperm cysteine proteases, including cathepsins and calpains, in the enzymatic degradation of Suc-LLVY-AMC was excluded because the inhibitor E64d did not affect proteolysis of the substrate. Recently, it has been suggested that clasto-lactacystin may also inhibit cathepsin A-like proteases (Ostrowska et al., 2000Go). However, we have found no report indicating the presence of cathepsin A in mammalian sperm (Luedtke et al., 2000Go). In addition, cathepsin A is a serine carboxypeptidase, characterized by having carboxypeptidase activity toward peptides and proteins at acidic pH, and esterase activity toward amino acid esters at neutral pH. Thus, our assay conditions, carried out at pH 7.4, should have prevented the activity of a possible sperm cathepsin A toward the substrate Suc-LLVT-AMC (Matsuzaki et al., 1998Go).

To our knowledge, this is the first study to show that the proteasome in human sperm has a functional role in the AR. However, it is still not know how the proteasome may be acting during the human sperm AR. The results with ionomycin and the observation that the plateau phase of the progesterone-induced Ca2+ influx was inhibited by clasto-lactacystin, suggest that the proteasome may be acting upstream to the plateau phase of the Ca2+ influx required for the AR. A similar finding was reported by Bonaccorsi et al. (1995Go). They observed that tyrosine kinase inhibitors blocked the plateau phase of the progesterone-mediated increase in [Ca2+]i, and suggested that the activation of tyrosine kinase by progesterone is upstream of the generation of the sustained phase of the increase in [Ca2+]i (Bonaccorsi et al., 1995Go). Therefore, our results could be interpreted as follows. The proteasome inhibitor blocked a proteasome-mediated step necessary for the hZP- and progesterone-initiated AR. On the other hand, ionomycin stimulated the AR by permitting the necessary increase in sperm [Ca2+]i even in the presence of clasto-lactacystin. This suggests that the ionophore bypassed steps required for the sustained plateau phase of the Ca2+ wave are dependent directly or indirectly on the sperm proteasome activity. The inhibitor may therefore be blocking the hZP- and progesterone-initiated AR by preventing the proteasome-mediated plateau phase of the increase in [Ca2+]i. In sea urchin sperm, proteasome inhibitors blocked the Ca2+ influx that was prior to and required for the egg jelly-induced AR (Matsumura and Aketa, 1991Go).

Evidence for the involvement of the sperm proteasome in the AR has been reported in several marine invertebrates. In the stolidobranch ascidian H. roretzy, the sperm proteasome is necessary for sperm binding and digestion/penetration of the vitelline coat of the egg (Saitoh et al., 1993Go; Sawada et al., 1996Go). Proteasome inhibitors block the binding of the sperm to the egg and, consequently the vitelline coat digestion and fertilization (Saitoh et al., 1993Go; Sawada et al., 1996Go). The proteasome may be secreted from the sperm when it becomes activated (equivalent to the AR of mammals) (Saitoh et al., 1993Go), although recent evidence suggests that the proteasome may be located on the cell surface of the head region of H. roretzi sperm (Sawada et al., 2002bGo). Moreover, in H. roretzi sperm two chymotrypsin-like activities were detected. One of them is related to the proteasome 20S and the other one to a protease complex of higher molecular weight (930 kDa), the 26S-like proteasome. In sperm of the phlebobranch ascidian, Ciona intestinalis, there is a 20S proteasome. It has been suggested that its function is at the level of binding (Pinto et al., 1990Go; Marino et al., 1992Go) and penetration (Sawada et al., 1998Go) through the vitelline coat of the egg. As pointed out previously, in sea urchin sperm proteasome inhibitors block the egg jelly-induced AR. Moreover, these inhibitors block the Ca2+ influx that is prior to and required for the AR induced by the egg jelly but not the Ca2+ influx provoked by a Ca2+ ionophore (Matsumura and Aketa, 1989Go; Matsumura and Aketa, 1991Go). With this evidence, the authors proposed that the proteasome may participate in activating Ca2+ channels and thus allowing Ca2+ influx (Matsumura and Aketa, 1991Go; Inaba et al., 1992Go).

Recently, it was reported that in H. roretzi the vitelline coat is multi-ubiquitinated and then degraded by the sperm ubiquitin-proteasome system during fertilization. The sperm ubiquitin-proteasome system would be released upon sperm activation (Sawada et al., 2002aGo). Thus, in these animals the proteasome seems to play an extracellular role. This finding contrasts remarkably with the generally accepted concept that the ubiquitin-proteasome system functions intracellularly in protein breakdown in various biological events.

We now have evidence that the proteasome is present in human and mouse sperm (Tipler et al., 1997Go; Wojcik et al., 2000Go). According to Tipler et al. (1997Go) human sperm express specific isoforms of proteasome subunits. The location of the proteasome in mature human sperm was restricted to the neck region and the acrosomal and postacrosomal regions; the proteasome was not found in the flagellum (Wojcik et al., 2000Go; Bialy et al., 2001Go). This is in agreement with our results indicating that the proteasome inhibitor clasto-lactacystin did not affect sperm motility. One interesting finding of the work of Tipler and colleagues was that although in mature sperm ubiquitin-protein conjugates and the ubiquitin-conjugating enzymes (E2) are almost absent, the proteasome activity is high in relation to elongated spermatids, where it reached a peak (Tipler et al., 1997Go). Thus, they raised the possibility that the proteasome in mature human sperm may have a role other than the degradation of ubiquitin-protein conjugates. Thus, whether mammalian sperm proteasomes have similar biological roles to the proteasomes from marine invertebrate sperm is still under investigation.


    Acknowledgement
 
This work was financed by Fondecyt 1011051.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on July 30, 2002; accepted on November 19, 2002.