BIOLOGY OF REPRODUCTION 80, 1026–1035 (2009)Published online before print 14 January 2009.DOI 10.1095/biolreprod.108.073924

Participation of the Human Sperm Proteasome in the Capacitation Processand Its Regulation by Protein Kinase A and Tyrosine Kinase1

Milene Kong, Emilce S. Diaz, and Patricio Morales2

Unit of Reproductive Biology, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile

ABSTRACT

The proteasome is a multicatalytic cellular complex present inhuman sperm that plays a significant role during several steps ofmammalian fertilization. Here, we present evidence that theproteasome is involved in human sperm capacitation. Aliquots ofhighly motile sperm were incubated with proteasome inhibitorsMG132 or epoxomicin. The percentage of capacitated sperm,the chymotrypsin-like activity of the proteasome, cAMP content,and the pattern of protein phosphorylation were assayed byusing the chlortetracycline hydrochloride assay, a fluorogenicsubstrate, the cAMP enzyme immunoassay kit, and Western blotanalysis, respectively. Our results indicate that treatment ofsperm with proteasome inhibitors blocks the capacitationprocess, does not alter cAMP concentration, and changes thepattern of protein phosphorylation. To elucidate how protea-some activity is regulated during capacitation, sperm wereincubated with: 1) tyrosine kinase (TK) inhibitors (genistein orherbimycin A); 2) protein kinase (PK) A inhibitors or activators(H89 and Rp-cAMPS, and 8-Br-cAMP, respectively); or 3) PKCinhibitors (tamoxifen or staurosporin) at different capacitationtimes. The chymotrypsin-like activity and degree of phosphor-ylation of the proteasome were then assayed. The resultsindicate that sperm treatment with TK and PKA inhibitorssignificantly decreases the chymotrypsin-like activity of theproteasome during capacitation. Immunoprecipitation andWestern blot results suggest that the proteasome is phosphor-ylated during capacitation in a TK- and PKA-dependent pathway.In conclusion, we suggest that the sperm proteasome partici-pates in the capacitation process, and that its activity ismodulated by PKs.

capacitation, fertilization, gamete biology, kinases, proteasome,protein kinases, protein phosphorylation, sperm capacitation

INTRODUCTION

Sperm capacitation involves a series of complex biochem-ical and physiological events that occur while the cells aretraveling through the female genital tract [1]. Some of thechanges that take place during this process include an increasein sperm plasma membrane fluidity due to cholesterol loss [2],an increase in ionic influx by membrane hyperpolarization [3],changes in cAMP concentration [4], and an increase in thedegree of protein phosphorylation [5–9]. These changes,among others, enable the sperm to change its movement

pattern to ‘‘hyperactivated,’’ and to be able to undergo theacrosome reaction. These events are crucial for the fertilizingspermatozoon to penetrate the zona pellucida and fuse with theoocyte plasma membrane [1].

Although the intracellular signaling pathways that lead toincreased protein phosphorylation during sperm capacitationare not completely clear, different protein kinases (PKs) havebeen described in mammalian sperm, including tyrosinekinases (TK) [10], PKA (PRKA) [11], PKC (PRKC) [10,12], and glycogen synthase kinase (GSK) 3b [13]. Theseenzymes regulate the phosphorylation of several proteins attyrosine and serine/threonine residues, thus regulating spermfunction [8, 9, 13, 14]. In particular, it has been described that,during human sperm capacitation, there is an increase in thedegree of tyrosine phosphorylation in proteins of 80 and 105kDa (see [15] and references therein). The major proteinsphosphorylated at tyrosine residues correspond to fibroussheath proteins [16]. On the other hand, little is known aboutthe phosphorylation of proteins at serine and threonine residuesin mammalian sperm.

One protein complex susceptible to modification byregulated phosphorylation is the proteasome. The proteasomeis a multienzymatic complex involved in nonlysosomalproteolysis of cytosolic and nuclear proteins that are abnormal,aged, or short lived [17] after they have been covalently labeledwith ubiquitin molecules [18]. The proteolytic core of theproteasome is called the 20S proteasome, and is approximately600 kDa. When this complex is associated with ATPaseregulator and activator proteins, it constitutes the 26Sproteasome of approximately 2000 kDa. The 20S core particleis made up of two copies each of seven different a and sevendifferent b subunits arranged into four stacked rings. The twoouter a rings are catalytically inactive, whereas three of theseven inner b subunits are catalytically active [19] with trypsin-like, chymotrypsin-like, and peptidylglutamyl peptide hydro-lyzing (PGPH) activities [20, 21]. The 20S proteasome canfully degrade unfolded proteins in the absence of ATP andubiquitin [22]; however, the 26S proteasome degrades proteinsin an ATP-dependent manner, and, in most cases, requires thepresence of a ubiquitin chain on the substrate protein [23].

Although little is known about proteasome regulation,numerous studies carried out in different cell types indicatethat several subunits of the proteasome are phosphorylated atserine and tyrosine residues [24–26]. It is not clear, however, ifdifferential phosphorylation of its subunits modulates protea-some activity, thereby regulating protein degradation. Somestudies have shown that this phosphorylation is essential for theassembly of the regulator complex, 19S, with the 20S complex[27, 28]. In addition, there are reports indicating thatphosphorylation of many proteins is a signal that triggersdegradation by the proteasome pathway (see review in [18]). Incontrast, there are a few proteins (e.g., MOS, Fos and Jun) inwhich phosphorylation inhibits proteolysis, implying thatnonphosphorylated forms are metabolically labile in the cell[29–31].

1Supported by the Fondo Nacional de Desarrollo Cientıfico yTecnologico de Chile ([FONDECYT] 1080028 and 11070051), and bythe University of Antofagasta (PEI 1322-06).2Correspondence: FAX: 55 5655 637 802; e-mail: pmorales@uantof.cl

Received: 2 October 2008.First decision: 23 November 2008.Accepted: 30 December 2008.� 2009 by the Society for the Study of Reproduction, Inc.eISSN: 1259-7268 http://www.biolreprod.orgISSN: 0006-3363

1026

The existence and function of the proteasome have beenwell characterized in spermatozoa of several species of marineinvertebrates. In these species, the proteasome is tightlyinvolved in multiple steps of fertilization, from the acrosomereaction induced by the egg jelly, to penetration of the vitelinecoat, and fusion with the plasma membrane of the femalegamete [32, 33]. Recently, studies by Wojcik et al. [34] andPizarro et al. [20] demonstrated the presence of the proteasomein mammalian sperm, including human sperm, and showed thatthis multienzymatic complex takes part in many events ofmammalian fertilization. The sperm proteasome plays an activerole during the zona pellucida- and progesterone-inducedacrosome reaction and the calcium influx that precedes it [35].It is still unknown, however, whether the sperm proteasome isinvolved during mammalian capacitation, including thechanges in the protein phosphorylation pattern that takes placeduring this process. In addition, there are no studies related tothe regulation of the sperm proteasome by PKs in spermatozoa.

The aim of the present work was to characterize the role ofthe proteasome during human sperm capacitation. In addition,we studied the relationship between proteasome activity andprotein phosphorylation during this process, and whether thechymotrypsin-like activity of the sperm proteasome isregulated by PKs.

MATERIALS AND METHODS

Reagents and Antibodies

The following reagents were purchased from Sigma Chemical Co. (St.Louis, MO): progesterone; Na-Tosyl-Lys-chloromethylketone�HCl (TLCK);bovine serum albumin (BSA; A7030); Hoechst 33258; 1,4-diazabicy-clo[2.2.2.]octane (DABCO); Hepes; dimethylsulfoxide (DMSO); [Z]-1-[p-dimethylaminoethoxyphenyl]-1–2-diphenyl-1-butene (tamoxifen); 7-chlorote-tracycline hydrochloride (CTC); and 40,5,7-trihydroxyisoflavone (genistein).The following compounds were purchased from Affinity Research Products(Biomol Research Laboratories, Plymouth Meeting, PA): N-acetyl-N-methyl-L-isoleucyl-L-isoleucyl-N-[(1S)-3-methyl-1-[[(2R)-2-methyloxiranyl]carbo-nyl]butyl]-L-threoninamide (epoxomicin); Z-Leu-Leu-Leu-CHO); Suc-LLVY-AMC (N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (MG132); N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide�2HCl (H89);Rp-adenosine-30,50-cyclic monophosphorothioate (Rp-cAMPS); staurosporine(St); herbimycin A; and 8-bromoadenosine-3 0,5 0-cyclic monophosphate,sodium salt (8-Br-cAMP). The chemiluminescence detection and cAMP DirectBiotrak enzyme immunoassay (EIA) system kit no. RPN225 was purchasedfrom Amersham Pharmacia Biotech (Piscataway, NJ). Immobilon P transfermembrane was obtained from Millipore Corporation (Bedford, MA). The RCDC Protein Assay was obtained from BioRad Laboratories, Inc. (Hercules,CA). Pisum sativum agglutinin (PSA)-FITC was purchased from VectorLaboratories, Inc. (Burlingame, CA).

The following antibodies were used: clone 4G10 anti-phosphotyrosineantibody (Upstate Biotechnology, Lake Placid, NY); mouse anti-phospho-threonine (42H4) no. 9386 (Cell Signaling, Danvers, MA); poly-Z-PS1 rabbitanti-phosphoserine (Zymed Laboratories Inc.); goat anti-rabbit biotinylatedantibody, goat anti-mouse biotinylated antibody, and goat anti-mousebiotinylated immunoglobulin M antibody (Chemicon International, Temecula,CA). The anti-a-4 proteasome antibody and the agarose-immobilized anti-a-4proteasome subunit were obtained from Affinity Research Products (BiomolResearch Laboratories).

The deionized water used in these experiments was purified to .18 MX-cmwith an EASY-pure UV/UF ion exchange system (Barnstead/Thermolyne,Dubuque, IA). Vectashield was purchased from Vector laboratories.

Semen Samples

Semen samples were obtained with the approval of the Ethics Committee ofthe University of Antofagasta after 2–3 days of sexual abstinence. All donorssigned a form consenting to the use of their sperm cells for research purposes.All samples used were normal according to World Health Organization criteria[36]. Motile sperm were separated using a double Percoll gradient, as describedpreviously [35]. Briefly, aliquots of semen were layered over the upper step ofthe Percoll gradient and centrifuged at 300 3 g for 20 min. The pellet was thenresuspended in 10 ml of modified Tyrode medium consisting of 117.5 mM

NaCl, 0.3 mM NaH2PO

4, 8.6 mM KCl, 25 mM NaHCO

3, 2.5 mM CaCl

2, 0.5

mM MgCl2, 2 mM glucose, 0.25 mM sodium pyruvate, 19 mM sodium lactate,

70 lg/ml penicillin and streptomycin, phenol red, and 0.3 % BSA, andcentrifuged again at 300 3 g for 10 min. Finally, the sperm pellet wasresuspended in the same medium supplemented with 2.6% BSA. The spermconcentration was adjusted to 10 3 106 cells/ml, and the suspension wasincubated at 378C, with 5% CO

2in air for 0.5 (T0), 5 (T5), or 18 (T18) h.

Evaluation of Sperm Capacitation

To elucidate the role of the sperm proteasome during capacitation, weevaluated this process directly by using the CTC fluorescence assay, andindirectly by testing the ability of the sperm to undergo the acrosome reactionwhen stimulated with progesterone. It is well known that only capacitatedsperm will undergo the acrosome reaction when challenged with aphysiological stimulus [1].

CTC Assay

The capacitation state of the spermatozoa was assessed using the CTCfluorescence assay method, as described previously [37, 38]. Briefly, CTCsolution was prepared on the day of use and contained 750 lM CTC in a bufferof 130 mM NaCl, 5 mM cysteine, and 20 mM Tris-HCl, pH adjusted to 7.8.This solution was kept wrapped in foil at 48C until just before use. A 5-llaliquot of a sperm suspension treated with 10 lg/ml Hoechst was placed on aslide at 378C; 5 ll of CTC stock solution was then rapidly added, followedwithin 30 sec by 0.5 ll of 2% glutaraldehyde in 1 M Tris buffer (pH 7.8). Onedrop of DABCO mounting medium was carefully mixed in to retard fading ofthe fluorescence, and a cover slip was placed on top. Cells were assessed fortheir living/dead state using ultraviolet light, as described previously [39]. Ineach sample, 200 live cells were assessed for CTC staining patterns, and, in allcases, the proportion of dead cells was very low. There were three mainpatterns of CTC fluorescence that could be identified: F, with uniformfluorescence over the entire head, characteristic of uncapacitated, acrosome-intact cells; B, with a fluorescence-free band in the postacrosomal region,characteristic of capacitated, acrosome-intact cells; and AR, with dull or absentfluorescence over the sperm head, characteristic of capacitated, acrosome-reacted cells [37, 38]. At all three stages, bright fluorescence on the midpiececould be seen.

Progesterone-Induced Acrosome Reactions

Aliquots of highly motile spermatozoa were incubated for 18 h with 10 lMMG132 or its solvent (0.1% DMSO). At the end of the incubation period, thesperm were washed and resuspended twice in the same medium and thenincubated for another 15 min with 7 lM progesterone or its solvent (0.1%DMSO). The acrosomal state and sperm viability were evaluated using FITC-labeled PSA and the Hoechst 33258 dye, respectively, as described previously[39]. In this assay, the inhibitor was present only during capacitation; during theacrosome reaction stimulus, the sperm were incubated in inhibitor-free media.

Measurement of Total cAMP

Sperm aliquots (10 3 106/ml) were incubated for 18 h in the presence orabsence of 10 lM epoxomicin at 378C, 5% CO

2. At the end of the incubation

period, total cAMP production was quantified using the cAMP Direct BiotrakEIA system kit according to the manufacturer’s instructions. Results areexpressed as fmol/10 3 106 sperm/ml.

Preparation of Sperm Extracts

To measure the enzymatic activity of the proteasome, different spermaliquots were washed in PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH

2PO

4,

4.3 mM Na2PO

4, pH 7.4). The pellet was then resuspended in homogenization

buffer (50 mM Hepes, 10% glycerol, 200 lM TLCK, pH 7.4) at a concentrationof 200 3 106 cells/ml, and the sperm suspension was sonicated (Virsonic,Gardiner, NY) with seven 60-W bursts of 30 sec each, and centrifuged for 30sec at 5000 3 g in a Beckman microfuge to remove nuclear and flagellarmaterial [40]. The supernatant was used as the enzyme stock preparation. All ofthese procedures were performed at 48C.

For Western blot analysis and immunoprecipitation, protein extracts wereprepared in radioimmunoprecipitation (RIPA) lysis buffer (150 mM NaCl, 50mM Tris, 1% SDS, 2 mM Na

3VO

4, 50 mM NaF, 2 mM EDTA, 1% sodium

desoxycholate, 1% NP-40, 1 mM PMSF, 10 lg/ml leupeptin, 10 lg/mlaprotinin, 10 lg/ml bestatin A, pH 7.4). All of these procedures were performedat 48C. According to previous work from our laboratory, none of the protease

SPERM PROTEASOME, CAPACITATION, AND PROTEIN PHOSPHORYLATION 1027

inhibitors used, including TLCK, has a significant effect on the chymotrypsin-like activity of the sperm proteasome [20, 40]. The protein concentration ineach sperm extract preparation was measured using the RC DC protein assay.

Enzymatic Activity

To determine the enzymatic activity of the sperm proteasome, motile spermwere incubated with 10 lM MG132 or its solvent. In others experiments, thesperm were incubated with 10 lM herbimycin A, 50 lM genistein, 10 lM H89,50 lM tamoxifen, 1 nM St, 200 lM Rp-cAMPS or 500 lM 8-Br-cAMP for 30min (T0), 5 h (T5) or 18 h (T18) at 378C, 5% CO

2. Chymotrypsin-like activity

was assayed using the fluorogenic substrate Suc-LLVY-AMC [35]. Briefly, 50-ll aliquots were incubated in a final volume of 995 ll homogenization bufferfor 15 min at 378C, 5% CO

2, before adding the 10-lM substrate. The assay was

run at 378C, and the fluorescence was monitored with excitation at 380 nm andemission at 460 in a Shimadzu 1501 spectrofluorometer (Kyoto, Japan), asdescribed previously [35].

SDS-PAGE and Immunoblotting

Aliquots of highly motile sperm, selected by Percoll gradient, were dividedinto four groups: 1) noncapacitated (T0); 2) incubated for 5 h at 378C, 5% CO

2

(T5); 3) incubated for 18 h under the same conditions (T18); and 4) incubatedwith 10 lM epoxomicin during capacitation (T18 þ E). Controls aliquots weretreated with the inhibitor solvent.

To carry out denaturing SDS-PAGE, sperm extracts were boiled for 5 minwith sample buffer (500 lM Tris-HCl, 10% SDS, 30% glycerol, 0.5% b-mercaptoethanol, and 0.5% bromophenol blue, pH 6.8) and then immediatelysettled on ice. Samples (20 lg) were resolved on 12% SDS-PAGE (12%acrylamide/bisacrylamide for the resolving gel and 5% acrylamide/bisacryla-mide for the stacking gel) in a Mini Protean Cell (Bio-Rad Laboratories, Inc.,Hercules, CA) [41].

Electrophoretic transfer of proteins to polyvinylidene fluoride (PVDF)membranes in all experiments was carried out according to the method ofTowbin et al. [42], at 150 V for 1 h at 48C. Transfer was monitored by Ponceaured stain. The membrane was then blocked (5% BSA in Tris-buffered saline[TBS]; 3% milk and 5% BSA in TBS; or 3% milk in PBS), washed six times,and incubated with primary antibody at 48C overnight. Blots were washed sixtimes and incubated with the appropriate biotinylated secondary antibody for 1h at room temperature. The membrane was again washed as described above,and then the phosphorylated proteins were detected using an enhancedchemiluminescence kit (Amersham Corp., Sydney, Australia), according to themanufacturer’s instructions. Prestained protein standards were used with amolecular mass range of approximately 149–14 kDa. Finally, the imageanalysis system, Scion Image 4.0.2 (Center for Information Technology, NIH,Bethesda, MD), was used to quantify the changes in intensity of various bands[43].

Immunoprecipitation

Sperm at T0, T18, and at T18 with 10 lM H89 or 50 lM genistein wereimmunoprecipitated with an anti-proteasome subunit a-4 antibody immobilizedwith agarose at 7 ll/400 lg of protein, as described previously [43]. Thereaction mixture was incubated on an orbital shaker overnight at 48C. Theimmune complex was obtained by centrifugation (15 000 3 g, 30 sec). Thesupernatants were discarded, and the pellets were washed twice with RIPAbuffer and once with TBS. The washed pellets were mixed with SDS samplebuffer (23) and heated in a boiling water bath for 5 min, and the supernatantwas subjected to SDS-PAGE. The proteins on the gels were transferred to aPVDF membrane and detected as described above.

Stripping PVDF

To confirm equal protein loading, blots were stripped and reprobed with anantibody against the a-4 proteasome subunit. For this procedure, approximately20 ml of stripping buffer (2% [w/v] SDS, 62.5 mM Tris, pH 6.7, and 100 mM2-mercaptoethanol) was added to the membrane for 30 min with constantshaking at 608C. The membrane was then washed, blocked, and probed withthe primary antibody, as previously described [43].

Statistical Analysis

Data were analyzed using one-way analysis of variance and the t-test forunequal replicate using the Instat Program (GraphPad, La Jolla, CA). Adifference with P , 0.05 was considered significant.

RESULTS

Proteasome and Sperm Capacitation

To elucidate the role of the sperm proteasome duringcapacitation, we evaluated this process using the CTC assay(Fig. 1). There was a significant increase in the percent ofcapacitated sperm at T18, and a significant reduction whencapacitation took place in the presence of epoxomicin orMG132, potent and specific inhibitors of proteasomal activity[44] (Fig. 1). These results agree with those obtained when thecapacitation state of the spermatozoa was evaluated by theirability to undergo the progesterone-induced acrosome reaction(Fig. 2). Sperm were incubated for 18 h with MG132, areversible proteasome inhibitor, or its solvent. Aliquots werethen washed and resuspended twice in fresh medium to remove

FIG. 1. Effect of the proteasome inhibitors epoxomicin (E) and MG132on human sperm capacitation. Sperm were incubated for 18 h with 0.1%DMSO (T18), 10 lM epoxomicin (T18 þ E) or 10 lM MG132 (T18 þMG132). Additional sperm aliquots were obtained immediately afterPercoll selection (Control). The capacitation state of the sperm was thenevaluated by means of the chlortetracycline fluorescence assay. Resultsare the mean 6 SEM of five experiments. *P , 0.01 in comparison to theT18 group.

FIG. 2. Effect of the proteasome inhibitor MG132 on human spermcapacitation. Sperm were incubated for 18 h with 10 lM MG132,washed, resuspended in fresh medium and treated with 7 lMprogesterone for 15 min (MG132w þ P). Control sperm were incubatedwith 0.1% DMSO for 18 h (Control) or with 0.1% DMSO for 18 h,washed, resuspended in fresh medium, and treated with 7 lMprogesterone for 15 min (P). Results are the mean 6 SEM of sixexperiments. *P , 0.01 in comparison to the progesterone-treated group.

1028 KONG ET AL.

the inhibitor before adding 7 lM progesterone. The resultsdemonstrate that, when the proteasome was inhibited duringcapacitation, the sperm were not able to undergo theprogesterone-induced acrosome reaction (Fig. 2).

In parallel experiments, we measured the enzymatic activityof the sperm proteasome during treatment with both inhibitors(Fig. 3). The chymotrypsin-like activity decreased to less than5% of the control when the sperm were incubated in thepresence of either inhibitor; however, in agreement with thereversible nature of MG132, the enzymatic activity of theproteasome was not different from the control when MG132was removed shortly before the assay (Fig. 3, MG132w). Thisexperiment indicates that both compounds are potent andspecific inhibitors of the sperm proteasome, that spermwashing was successful in removing the proteasome inhibitorMG132, and that the chymotrypsin-like activity of spermatozoais entirely due to the proteasome. Since similar results withregard to capacitation and proteasome activity were obtainedwith MG132 and epoxomicin, only epoxomicin was used in thesubsequent experiments.

To examine whether the proteasome is involved in thechanges in protein phosphorylation that take place duringcapacitation, cells were incubated for 18 h in the presence of 10lM epoxomicin, and sperm extracts were evaluated todetermine protein phosphorylation (Fig. 4). Western blotanalysis revealed that T0 sperm exhibited several proteinbands phosphorylated at serine residues, with molecular massesof approximately 134, 120, 107, 99, 42, and 27 kDa. Theproteins phosphorylated at serine residues increased duringcapacitation at both T5 and T18, especially in the highmolecular mass range. However, when sperm were treated withepoxomicin during capacitation (T18 þ E), they did not exhibitthis increase to the degree of protein phosphorylation at serineresidues (Fig. 4A).

With regard to the pattern of protein phosphorylation atthreonine residues, densitometric analysis revealed a gradualincrease in the degree of phosphorylation during capacitationof bands in the range of 124–23 kDa. When the sperm werecapacitated in the presence of epoxomicin, however, the degreeof protein phosphorylation at threonine residues increased,especially in bands of 124 and 115 kDa (Fig. 4B).

When the phosphorylation pattern at tyrosine residues wasanalyzed during capacitation, a clear increase in phosphoryla-tion was seen for bands in the range of 115–27 kDa. Thisphosphorylation pattern was not affected by capacitating thesperm in the presence of the proteasome inhibitor (Fig. 4C).This result was not surprising, given that sperm proteasomesare concentrated in the neck region near the centrioles [34].Some proteasome labeling has also been detected at theperiphery of the head, acrosomal, and postacrosomal regions,and in the connecting piece [45], but there are no proteasomeslocated in the flagellum.

These results strongly suggest that the sperm proteasome isinvolved in the capacitation process. The proteasome maymodulate protein phosphorylation at serine and threonineresidues, directly or indirectly.

On the other hand, we wanted to establish whether thiscatalytic complex is involved in the cAMP production that hasbeen reported to take place during capacitation. To this end, wemeasured the total cAMP concentration with the Direct BiotrakEIA system kit. The results demonstrate that the concentrationof cAMP rose during the 18 h of incubation (48 6 3.5 fmol/103 106 sperm/ml at T0 to 138 6 8 fmol/10 3 106 sperm/ml atT18). This increase in cAMP production during spermcapacitation, however, did not change when the sperm were

treated with epoxomicin (120 6 8 fmol/10 3 106 sperm/ml atT18 þ epoxomicin).

PKs and the Sperm Proteasome

Next, we evaluated the role of PKs during capacitation (Fig.5) and their involvement in regulating the enzymatic activity ofthe proteasome (Fig. 6). As reported by others [46], PKA,PKC, and TK inhibitors prevented human sperm capacitation(Fig. 5). In addition, the results indicate that incubation with 10lM H89, 10 lM herbimycin A, and 50 lM genistein inhibitedthe chymotrypsin-like activity of the sperm proteasome at allcapacitation times (P , 0.05) (Fig. 6, A and B). PKCinhibitors, such as tamoxifen or St, did not affect spermproteasomal activity (Fig. 6C). The importance of PKA in themodulation of proteasomal activity was corroborated by the useof Rp-cAMPS (a PKA inhibitor) and 8-Br-cAMP (a PKAactivator). When the sperm were incubated for 18 h with Rp-cAMPS, the chymotrypsin-like activity of the sperm protea-some was significantly decreased in comparison with thecontrol (Fig. 6A). In contrast, when the sperm were incubatedfor 18 h with 8-Br-cAMP, the chymotrypsin-like activity of thesperm proteasome was significantly increased in comparison tothe control (Fig. 6A).

Finally, the next experiments were designed to determinewhether the chymotrypsin-like activity of the sperm protea-some is modulated by the degree of phosphorylation of itssubunits during capacitation (Fig. 7). T0 or T18 sperm aliquotswere incubated in the presence or absence of H89 orherbimycin A. The chymotrypsin-like activity of the protea-some was then evaluated (Fig. 7, upper panel), and the degreeof phosphorylation of the proteasome subunits or its associat-ing proteins was probed with different antibodies followingimmunoprecipitation (Fig. 7, lower panel). The results revealthat the chymotrypsin-like activity of the proteasome doubledduring capacitation, and that this increase was prevented byH89 and herbimycin A (Fig. 7, upper panel). With regard to thephosphorylation pattern, bands of 67 and 24 kDa werephosphorylated at serine residues (Fig. 7A), bands of 94 and77 kDa were slightly phosphorylated at threonine residues, and

FIG. 3. Effect of the proteasome inhibitor MG132 and epoxomicin onthe chymotrypsin-like activity of the sperm proteasome. Sperm wereincubated for 18 h with 0.1% DMSO (Control) or with 10 lM MG132(MG132), washed, and resuspended in fresh medium (MG132w) or with10 lM epoxomicin. At the end of the incubation period, thechymotrypsin-like activity of the proteasome was measured in spermextracts using the fluorogenic substrate Suc-LLVY-AMC. Results are themean 6 SEM of six experiments. *P , 0.01 in comparison to othergroups.

SPERM PROTEASOME, CAPACITATION, AND PROTEIN PHOSPHORYLATION 1029

bands of 101 and 43 kDa were faintly phosphorylated attyrosine residues in T0 sperm (Fig. 7, B and C). With regard toserine, the degree of phosphorylation increased slightly during18 h of incubation. On the other hand, phosphorylation atthreonine and tyrosine residues exhibited great increases duringcapacitation. In all three cases, an increase in proteasomesubunit phosphorylation during capacitation did not take placewhen the sperm were treated with H89 or herbimycin A (Fig.7).

DISCUSSION

In this work, we present evidence strongly suggesting thatthe proteasome plays a significant role during human spermcapacitation. The capacitation state was evaluated directly withthe CTC assay, and indirectly by assessing the ability of thesperm to undergo the acrosome reaction when stimulated withprogesterone. The CTC assay indicated that about 40% of thesperm were capacitated after 18 h of incubation. Similar resultshave been reported by others [37, 38]. The percentage of

capacitated sperm only increased to about 13%, however, whenthe cells were incubated in the presence of the proteasomeinhibitors, MG132 or epoxomicin. In addition, the progester-one-induced acrosome reaction assay confirmed similarinhibition of capacitation by MG132. When the potent,reversible, and cell-permeable proteasome inhibitor, MG132[44], was present during capacitation and induction of theacrosome reaction, the sperm did not undergo exocytosis of theacrosome. More importantly, after the sperm were washed freeof MG132, resuspended in fresh medium, and treated withprogesterone, they were still unable to undergo the acrosomereaction. The chymotrypsin-like activity of the sperm protea-some was significantly reduced by the presence of bothinhibitors. MG132 is a peptide aldehyde that inhibits ubiquitin-mediated proteolysis by binding to and inactivating the 20Sand 26S proteasome [44], and its activity is reversible [47].Epoxomicin is a cell-permeable, natural product that potently,selectively, and irreversibly inhibits proteasome activity.Epoxomicin modifies four catalytic subunits of the 20Sproteasome, primarily resulting in the inhibition of the

FIG. 4. Effect of the proteasome inhibitorepoxomicin (E) on the phosphoproteincontent of sperm extracts. Proteins wereextracted from noncapacitated sperm (T0),sperm capacitated for 5 h (T5), and spermcapacitated for 18 h in the presence (T18 þE) or absence of epoxomicin (T18). Thephosphoserine (A), phosphothreonine (B),and phosphotyrosine (C) contents were thenanalyzed by immunoblotting with the anti-bodies described in the Materials andMethods. The numbers on the left-hand sideof the immunoblots indicate the molecularmass (kDa) of the major phosphoprotein-containing bands. On the right-hand side,the densitometric analysis of the bands isshown (mean 6 SEM of three experiments).*P , 0.01 in comparison to the T18 group.A representative gel of three is shown.

1030 KONG ET AL.

chymotrypsin-like activity [48]. Thus, the results of these twoassays confirmed the involvement of the proteasome duringhuman sperm capacitation.

In the next series of experiments, we confirmed thehypothesis that the sperm proteasome is involved in theprotein phosphorylation that takes place during human spermcapacitation. In fact, treatment with epoxomicin duringcapacitation modified the pattern of protein phosphorylationat both threonine and serine residues. Moreover, there seemedto be a bidirectional relationship between the proteasome andPKA, since inhibition of PKA caused a decrease in proteasomeactivity, as well as a decrease in the degree of phosphorylationof proteasome subunits (or its associating proteins).

In this work, we did not find evidence that the spermproteasome is involved in protein tyrosine phosphorylation. Ithas previously been shown that the major phosphotyrosine-containing proteins that become phosphorylated during capac-itation are located in the fibrous sheath of the sperm flagellum[7, 49]. The proteasome is not located in the flagellum;therefore, it was not surprising that we did not find arelationship between this protease and the major proteins thatbecome phosphorylated at tyrosine residues during capacita-tion.

Because of methodological problems, phosphorylation ofserine and threonine is difficult to detect. Only one articledealing with activation of the serine/threonine phosphorylationcascade during capacitation has been published [50]. In thepresent work, several proteins showed increased phosphoryla-tion at serine and threonine residues during capacitation. Someof these proteins coincided with those described by Naz [50]—specifically those of 27, 42, 99, and 107–120 kDa for serine,and those of 23, 36, 57, 93, and 100–124 kDa for threonine.

The proteasome is a multicatalytic complex present inmammalian sperm of several species, including humans [20,21, 34, 35, 51]. Previous reports have established that theproteasome actively participates in calcium influx and theacrosome reaction induced by both the zona pellucida andprogesterone [35, 52]. To the best of our knowledge, however,this is the first report to show that the proteasome has a roleduring human sperm capacitation.

Sperm capacitation is a process that is correlated withmodifications at the molecular and immunological levels,which prepare the sperm for physiological changes, such as‘‘hyperactivation’’ and the acrosome reaction [1]. Some of thechanges described as taking place during human spermcapacitation include an increase in membrane fluidity,cholesterol efflux [2], ion fluxes resulting in membranehyperpolarization [3], and an increase in the degree of proteinphosphorylation at tyrosine and serine/threonine residues,

FIG. 6. Effect of PK inhibitors on the chymotrypsin-like activity of thehuman sperm proteasome during capacitation. Sperm aliquots wereincubated with PKA inhibitors or an activator (A), TK inhibitors (B), andPKC inhibitors (C) for 30 min (T0), 5 h (T5), or 18 h (T18). Control spermaliquots were incubated with the appropriate solvents. The chymotrypsin-like activity of the sperm proteasome was then measured using thefluorogenic substrate Suc-LLVY-AMC. Results are the mean 6 SEM of sixexperiments. *P , 0.01 in comparison to the control group at each time.

FIG. 5. The effect of PK inhibitors on human sperm capacitationmeasured using the CTC assay. Sperm aliquots were incubated with 10 lMH89 (black bars), 10 lM herbimycin A (horizontally hatched bars), or 50lM tamoxifen (dotted bars) for 30 min (T0), 5 h (T5), or 18 h (T18). Controlsperm aliquots were incubated with the appropriate solvents (white bars).The capacitation state of the sperm was then evaluated by means of thechlortetracycline fluorescence assay. Results are the mean 6 SEM of fiveexperiments. *P , 0.01 in comparison to the control group.

SPERM PROTEASOME, CAPACITATION, AND PROTEIN PHOSPHORYLATION 1031

which appears to be crucial for the sperm to fertilize an oocyte[8, 9, 14]. A complex balance between kinase and phosphataseactivities regulates sperm protein phosphorylation. DifferentPKs have been described in mammalian sperm, including TK[10], PKA (PRKA) [11], PKC (PRKC) [10, 12], glycogensynthase kinase 3b (GSK3) [13], casein kinase II (CK2,CSNK2) [53], extracellular signal-regulated kinase (MAPK)[54], and Ca2þ/calmodulin-dependent protein kinase (CAMkinase, CAMK) [55]. In addition, serine/threonine-specificprotein phosphatase (PP) activity has been identified inspermatozoa from different species, and is involved in theregulation of sperm motility [56]. The Ca2þ/calmodulin-dependent phosphatase calcineurin (PP2B, PPP3C) has beendetected in dog sperm [57], among others.

The intracellular signaling pathways that lead to an increasein the degree of sperm protein phosphorylation duringcapacitation are not fully understood. Here, we presentevidence that the proteasome may be modulating thisphosphorylation. We have shown a decrease in phosphoryla-tion at serine residues in proteins between 134 and 99 kDawhen the sperm are treated with epoxomicin during capacita-

tion. This suggests that the proteasome may be involved in thedegradation of a serine phosphatase during capacitation. On theother hand, proteins containing phosphothreonine residuesincreased in bands in the range of 124–115 kDa and 73–29 kDawhen the sperm were treated with epoxomicin duringcapacitation. This suggests that the proteasome may beinvolved in the degradation of threonine kinases duringcapacitation. Alternatively, the proteasome may be involvedin the degradation of several proteins that are phosphorylated atthreonine residues as a trigger for proteolysis.

There is information in the literature regarding themodulation of kinase and phosphatase proteins by theproteasome [58–60]. In salmon sperm, the proteasomes arelocated along the flagella, where they may be involved incAMP-dependent phosphorylation of the axonemal protein of22 kDa, dynein light chain [61, 62]. In Aplysia neurons,regulatory subunits of PKA have been shown to be a substratefor degradation via the ubiquitin-proteasome system [63]. Thesame condition was described for the G protein-coupledreceptor kinase 2 (GRK2, ADRBK1), which is degraded byactivation of b2-adrenergic receptors in human embryonickidney-293 cells [64]. Other authors have demonstrated thatERK1/2 (mitogen-activated PK [MAPK] 3/1) activation canenhance MAPK phosphatase 1 (MKP-1, DUSP1) [65] andMKP-3 [66] degradation by the proteasome. On the other hand,several studies have shown that protein phosphorylation atserine residues is a signal that triggers proteolysis by theproteasome [66–68]. In 1999, Yano et al. [69] reported a newfunction for the purified 20S proteasome obtained from ahuman lymphoblastoma cell line. They demonstrated that theproteasome exhibits intrinsic nucleoside diphosphate kinase-like activity. Whether this finding is related to a diminution inthe degree of phosphorylation at serine residues observed insperm treated with epoxomicin in our study is currentlyunknown.

Now that we know that the sperm proteasome has a role indegrading or modulating necessary proteins so that thecapacitation process can take place, it is important toinvestigate the mechanisms of activation or maintenance ofits enzymatic activity during this stage. Our results show thatthe chymotrypsin-like activity of the proteasome increasedduring capacitation, and that, when the sperm were incubatedwith PKA and TK inhibitors, this enzymatic activity decreased.Nevertheless, the enzymatic activity was not modified whenthe sperm were treated with PKC inhibitors. This observation isin agreement with the work of Marambaud et al. [70], whichsuggested a role for PKA during proteasome phosphorylationin HK 293 cells, and showed that incubation of the cells withforskolin increased proteasome activity. More recently, it wasreported that the chymotrypsin-like and trypsin-like activitiesof the proteasome are directly stimulated by PKA phosphor-ylation at serine 120 of the ATPase subunit of the AAA type(Rpt6) in normal rat kidney cells [71]. On the other hand, arecent report did not find an increase in the PGPH activity ofthe human sperm proteasome during capacitation [72]. We alsofailed to find an increase in the sperm proteasome PGPHactivity during capacitation (data not shown).

Numerous studies have shown that proteasome subunits arephosphorylated in cells of several species. For example, the a-3, a-5, and a-6 subunits of Candida albicans, the a-3 subunitof yeast [24], and the C9 subunit in mammalian cells [73] areall phosphorylated. The work of Satoh et al. [28] has indicatedthat the phosphorylation of subunits in the 19S complex (p45)might be important for 26S proteasome stability. Likewise,Bose et al. [27] showed that the phosphorylation of the C8 a-7subunit in the 20S proteasome affected the assembly of the 19S

FIG. 7. The effect of H89 (H89) and herbimycin A (HA) on thechymotrypsin-like activity (upper panel) and on serine, threonine, andtyrosine phosphorylation (lower panels) of the human sperm proteasomeduring capacitation. Sperm aliquots were incubated in culture medium for30 min (T0) or 18 h (T18) in the presence of 10 lM H89 (black bar), 10 lMherbimycin A (striped bar) or 0.1% DMSO (control, white bars). Thechymotrypsin-like activity of the sperm proteasome was then measuredusing the fluorogenic substrate, Suc-LLVY-AMC. *P , 0.01 in comparisonto the control group. In the lower panels, extracts of noncapacitated sperm(T0), sperm capacitated for 18 h (T18), and sperm capacitated for 18 h inthe presence of 10 lM H89 (H89) or 10 lM herbimycin A (HA) wereimmunoprecipitated with an anti-proteasome antibody and detected withan anti-phosphoserine antibody (A), an anti-phosphothreonine antibody(B), or an anti-phosphotyrosine antibody (C). The membrane was thenstripped and reprobed by using an anti-a4 proteasome antibody. TheWestern blot is representative of two independent experiments. Thenumbers on the left-hand side of the immunoblots indicate the molecularmass (kDa) of the major phosphoprotein-containing bands.

1032 KONG ET AL.

complex to the 20S proteasome, via changes in the charge ofthe C-terminal or local conformational changes.

In the present report, we present evidence, for the first time,that the human sperm proteasome (or the proteasome-associating proteins) becomes phosphorylated at serine,threonine, and tyrosine residues during capacitation, and thatthis process is modulated by PKA and TK. We also recentlyreported that, during the fibronectin-induced acrosome reac-tion, the human sperm proteasome becomes phosphorylated attyrosine residues [43]. In addition, it was recently shown thatthe proteasome a subunit type 6b undergoes tyrosinephosphorylation during capacitation in the mouse [74].

There are some reports related to the effect of TK onphosphorylation at serine/threonine residues. On the otherhand, regulation of protein tyrosine phosphorylation by PKA iswell known in spermatozoa. In fact, the increase in tyrosinephosphorylation of proteins of 105 and 80 kDa during humansperm capacitation is associated with the activity of cAMP/PKA [49, 75–78], and the phosphorylation of PKA substrates(Arg-X-X-Ser/Thr) is blocked by TK inhibitors, but not byPKC inhibitors [79]. In knockout mice that lack C2a, thetesticle-specific catalytic subunit of PKA, sperm cannot moveactively, and cannot undergo the normal increase in tyrosinephosphorylation observed in wild-type sperm [80]. In addition,mouse spermatozoa that lack soluble adenylate cyclase do notexhibit changes in the pattern of protein tyrosine phosphory-lation during capacitation [81, 82]. With regard to the influenceof TK upon serine/threonine phosphorylation, it was reportedin cardiac myocytes that treatment with endothelin-1 increasedphosphorylation of PKCd (PRKCD) at tyrosine residues; thisincrease was correlated with an increase in diacylglycerol-independent activity of the enzyme [83]. There is also evidencethat adenylate cyclase can be activated by TK [84], thusincreasing the generation of cAMP and activating PKA. TK-dependent activation of adenylate cyclase, independent of Gprotein activation, has been reported in human mononuclearleukocytes and vascular smooth muscle cells, and appears toinvolve raf kinase-mediated serine phosphorylation [85].Whether the soluble adenylate cyclase, characterized as thepredominant form in mammalian sperm [86], is sensitive toactivation by TK is not known. Finally, it has been reportedthat PP1C is phosphorylated in vitro by the protein TK, pp60[87], which decreases phosphatase activity by 50%, whilePP2A is subject to tyrosine phosphorylation at Tyr307 [88].Tyrosine phosphorylation potently inhibits PP2A activity [88].

Finally, we observed that, while the phosphorylation levelof the proteasome increased during capacitation, the chymo-trypsin-like activity increased concomitantly between non-capacitated and capacitated sperm. When proteasomephosphorylation was inhibited by PKA and TK inhibitors, itsactivity decreased, and capacitation did not take place. We stilldo not know the exact relationship between phosphorylation ofproteasome subunits and its enzymatic activity. Further studiesare needed to understand which subunits become phosphory-lated, and how this affects enzymatic activity.

In summary, we report here that the proteasome plays a roleduring human sperm capacitation, and that PKs are involved inthe regulation of its activity.

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