MOLECULAR REPRODUCTION AND DEVELOPMENT 69:87–93 (2004)

Proteasomal Activity in Mammalian SpermatozoaEDUARDO PIZARRO, CONSUELO PASTEN, MILENE KONG, AND PATRICIO MORALES*

Department of Biomedicine. Faculty of Health Sciences. University of Antofagasta, Antofagasta, Chile

ABSTRACT The proteasome is a multicataly-tic cellular complex, which possess three differentenzymatic activities, trypsin-like, chymotrypsin-like,and peptidylglutamyl peptidase. Its function is toremove abnormal or aged proteins. Recently, it hasbeen suggested the participation of the sperm protea-some during mammalian fertilization. In this study, wepresent evidence that indicates that sperm extractsfrom several mammalian species, including hamster,mice, rats, bovine, rabbits, and humans all possessproteasome activity. We characterized the threespecific activities of the proteasome using specific syn-thetic substrates and specific proteasome inhibitors.The results indicates that the highest specific activitydetected was in mouse sperm toward the trypsinsubstrates and it was 1,114% of the activity of humansperm toward the chymotrypsin substrate Suc-Leu-Leu-Val-Tyr-AMC (SLLVY-AMC, which was consideredas 100%). In all cases, the lowest activity was towardsubstrates for the peptidylglutamyl peptidase hydro-lyzing activity, and it was lowest for rabbit sperm(1.7% of the activity of human sperm toward thechymotrypsin substrate SLLVY-AMC). In addition,specific proteasome inhibitors were able to block allproteasome activities almost 100%, with the exceptionof clasto-Lactacystin b-lactone upon rat sperm. Allsperm extracts tested evidenced bands of about 29–32 kDa by Western blots using a monoclonal antibodyagainst proteasome subunits a1, 2, 3, 5, 6, and 7. Inconclusion, sperm from several mammals possessenzymatic activities that correspond to the protea-some. The proteasome from the different species holdsimilar but distinctive enzymatic characteristics. Mol.Reprod. Dev. 69: 87–93, 2004.� 2004 Wiley-Liss, Inc.

Key Words: mammalian sperm; proteasome; pro-teasome inhibitors; fertilization; proteases

INTRODUCTION

Proteases have been localized and characterized inspermatozoa of several species, including marine in-vertebrates and mammals. Several lines of evidencesuggest the involvement of these proteases duringfertilization (for reviews, see (Morales and Llanos,1995; Bedford, 1998; Mykles, 1998). Specifically, in seaurchin sperm proteases were implied in the egg jelly-induced acrosome reaction (Matsumura and Aketa,1989, 1991) and in the penetration through the egg

coats in ascidians (Hoshi et al., 1981; Sawada et al.,1982, 1983, 1984, 1986; Takizawa et al., 1993). Inmammals, for many years it was thought that thetrypsin-like protease acrosin was responsible for digest-ing the zona pellucida, enabling the sperm to penetratethrough it (McRorie and Williams, 1974). However,studies using acrosin-knockout mice revealed thatacrosinwas not essential for fertilization or spermpene-tration through the zona pellucida (Baba et al., 1994);rather, acrosin seems to be involved in acrosome dis-persal during the acrosome reaction (Yamagata et al.,1998). In humans, sperm proteases have been describedas having a role in the follicular fluid- and zonapellucida-induced acrosome reaction (Morales et al.,1994b).

Recently, it was shown that human andmouse spermpossess a multienzymatic protease called proteasome,which present a similar sedimentation coefficient to theproteasomes purified from other tissues, and possesscharacteristic trypsin-like, chymotrypsin-like an pepti-dylglutamyl peptide-hydrolyzing (PGPH) activities(Tipler et al., 1997; Wojcik et al., 2000; Morales et al.,2003).

The ubiquitin-proteasome pathway is responsible formost of the cell proteolysis. The proteasome degradesmostnuclearand cytosolic proteins, after theyhavebeencovalently labeled with ubiquitin molecules (Ciechan-over, 1998; Goldberg, 1995). This enzymatic complex iscomposed of a proteolytic body termed proteasome 20S(�700 kDa). When this catalytic subunit is associatedto regulatory proteins, ATPases, and activators, con-stitute the 26S proteasome (�2,500 kDa). The 26Sproteasome regulates the traffic of proteins that willbecomedegraded orprocessedand its activitydepends ofATP. Four stacked rings surrounding inner cavitiesform the 20S proteasome: two inner b-rings and twoouter a-rings. The a-subunits possesses regulatoryfunctions and the b-subunits catalytic functions (Couxet al., 1996). The b-subunits are specifically inhibited bylactacystin (Fenteany and Schreiber, 1998). From afunctional point of view, the access of the proteins to be

� 2004 WILEY-LISS, INC.

Grant sponsor: Fondecyt; Grant number: 1011051.

*Correspondence to: Patricio Morales, Unit of Reproductive Biology,Faculty of Health Sciences, University of Antofagasta, P.O. Box 170,Antofagasta, Chile. E-mail: pmorales@uantof.cl

Received 3 December 2003; Accepted 4 March 2004Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/mrd.20152

degraded to the catalytic chamber is restricted by theentry pore of �13 A. This means that before beingdegraded, a protein not only must be ubiquinated butalso denatured (Ciechanover, 1998). Free N-terminalthreonine (Thr) residues on three of the seven b-typesubunits act as nucleophiles and are essential inthe mechanism of catalysis (Seemuller et al., 1995;Fenteany and Schreiber, 1998).

In marine invertebrates, numerous reports indicatethat the sperm proteasome is involved in multiple stepsof the fertilization process, from acrosomal exocytosistriggered by the egg jelly to penetration of the vitelline-coat and fusionwith the egg plasmamembrane (Mykles,1998). Thus, in the solitary ascidian, Halocynthiaroretzi (order stolidobranch), the sperm proteasome isnecessary for sperm binding and penetration throughthe vitelline coat of the eggs (Hoshi et al., 1981; Sawadaet al., 1983, 2002a,b; Yokosawa et al., 1987). In thephlebobranch ascidian Ciona intestinalis, the spermproteasome is involved in the process of penetrationthrough the vitelline coat, probably functioning as alysine (Pinto et al., 1990; Sawada et al., 1998). In seaurchins (Strongylocentrotus intermedius), the spermproteasome is involved in Ca2þ channel activationleading to egg jelly-induced acrosomal exocytosis(Matsumura and Aketa, 1989).

In a previous report, we showed that human spermpossessed proteolytic activity that correspond to theproteasome and that this activity was involved inthe zona pellucida- and progesterone-induced acrosomereaction (Morales et al., 2003). However, the presence ofthis type of activity in spermatozoa of othermammalianspecies has not been clearly established. The presentstudy was designed to demonstrate the presence of pro-teasomal enzymatic activity in spermatozoa of severalmammalian species, including hamster, mice, rats,bovine, and rabbits.

MATERIALS AND METHODS

Chemicals

The following compounds were purchased fromSigma Chemical Co. (St. Louis, MO): the trypsininhibitorN-p-Tosyl-L-lysine chloromethyl ketonehydro-chloride (TLCK); the chymotrypsin inhibitorN-p-Tosyl-L-Phenylalanine-Chloromethyl Ketone (TPCK); thechymotrypsin substrates N-Succinyl-Ala-Ala-Pro-Phe-7-amido-4-methylcoumarin (SAAPF-AMC),N-Suc-Leu-Tyr-AMC (SLY-AMC), and N-Suc-Ala-Ala-Phe-AMC(SAAF-AMC); the trypsin substrate Boc-Gln-Ala-Arg-AMC (BQAR-AMC) and the elastase substrate N-Suc-Ala-Ala-Ala-AMC (SAAA-AMC); bovine serum albu-min (BSA, A7030); N-[2-Hydroxyethyl]piperazine-N0-[2-ethanesulfonic acid] (Hepes); trichloracetic acid;and dimethylsulfoxide (DMSO). The following com-poundswerepurchased fromAffinityResearchProducts(Exeter, UK): the proteasome inhibitors aclacinomycinA (aclarabucin), Carbobenzoxy-Leu-Leu-Leu-B(OH)2(MG262),Carbobenzoxy-Leu-Leu-Nva-H(MG115),Car-bobenzoxy-Leu-Leu-Leu-H (MG132), epoxomicin, Car-

bobenzoxy-Ile-Glu(OBut)-Ala-Leu-H (PSI), and clasto-Lactacystin b-lactone (clasto); the chymotrypsin sub-stratesN-Suc-Leu-Leu-Val-Tyr-7-AMC (SLLVY-AMC),Carbobenzoxy-Gly-Gly-Leu-AMC (ZGGL-AMC); thetrypsin substrate t-Butyloxycarbonyl-Leu-Arg-Arg-AMC (BLRR-AMC); and the anti-proteasome monoclo-nal antibody MCP231 that detects the proteasomesubunits a1, 2, 3, 5, 6, and 7. The following werepurchased from Peptides International (Louisville,KY): the thiol proteases (cysteinproteases) inhibitor[2S-3S]-trans-Epoxysuccinyl-L-leucylamido-3-methylbu-tane ethyl ester (E64d) and the PGPH substratesCarbobenzoxy-Leu-Leu-Glu-AMC (ZLLE-AMC) andCarbobenzoxy-Leu-Leu-Leu-AMC (ZLLL-AMC). Deio-nized water used in these experiments was purified to>18 MO-cm with an EASY-pure UV/UF ion-exchangesystem (Barnstead/Thermolyne, Dubuque, IA). Stocksolutions of substrates were prepared in DMSO. Thefinal concentration of DMSO in the sperm suspensionswas 0.1% (v/v).

Animals

Adult CF1 mice, golden hamsters, Sprague–Dawleyrats and white New Zealand rabbits bred on house wereused. The animals were maintained at 22–248C andwater and pelleted food were supplied ad libitum.

Sperm Source

Caudal epididymal sperm from golden hamster, rat,and mouse were obtained in the laboratory after theanimals were sacrificed in a CO2 gas chamber. Ejacu-lated sperm from rabbits were obtained using anartificial vagina (Fabro et al., 2002). Ejaculated andcryopreserved sperm from bull were obtained from AltaGenetics (Center of Artificial Insemination, Temuco,Chile). Human semen samples were obtained fromnormal donors after 2–3 days of sexual abstinence, withtheapproval of theBioethicsCommittee of theFaculty ofHealth Sciences of the University of Antofagasta. Allsamples had normal semen parameters according toWHO guidelines (WHO, 1999) and less than 1% of thespermatozoa had cytoplasmic droplets. The specimenswere allowed to liquefy for 30–60 min at 378C in a slidewarmer.

Sperm Suspension Preparation

Before obtaining the sperm extracts to be used as theenzyme stock preparation, the sperm were separatedfrom epidydimal fluid, seminal plasma, other cell typesand cellular debris by different sperm selection meth-ods. Human, bovine, rat, and rabbit sperm were centri-fuged through a two steps Percoll gradient, as describedpreviously (Suarez et al., 1986; Llanos et al., 1993;Morales et al., 1994a). Briefly, aliquots of spermsampleswere layered over the upper step of the Percoll gradientand centrifuged for 20 min at 300g. The pellet wasdiluted in 10ml of modified Tyrode’s medium consistingof 117.5 mM NaCl, 0.3 mM NaH2PO4, 8.6 mM KCl,25 mM NaHCO3, 2.5 mM CaCl2, 0.5 mM MgCl2,2mMglucose, 0.25mMNa pyruvate, 19mMNa lactate,

88 E. PIZARRO ET AL.

70 mg/ml of both streptomycin and penicillin, phenol redand 0.3% BSA, centrifuged again at 300g for 10min andthen resuspended in cold phosphate buffered saline(PBS). Mouse sperm were selected by centrifugationthrough a three steps Percoll gradient. The three stepsof the Percoll gradient were 4 ml of 90% in the lowerlayer, 3 ml of 67.5% in the middle layer, and 3 ml of45% in the upper layer. Sperm from six epidydimes,incubated for 60 min in 4 ml of T6 medium (99.4 mMNaCl, 0.36 mM NaHPO4� 12H2O, 1.42 mM KCl,25 mM NaHCO3, 0.78 mM CaCl2� 2H2O, 0.47 mMMgCl2�6H2O, 5.56mMglucose, 0.47mMNa pyruvate,24.9mMNa lactate, 50 mg/ml of streptomycin, 100UI/mlpenicillin, phenol red, and 20 mg/ml BSA, pH 7.4), werelayered over the Percoll gradient and then centrifugedfor 20 min at 500g. After selection from the Percollgradient, the resulting sperm pellet was washed twicewith PBS by centrifugation at 500g for 10 min. Epi-dydimal hamster spermwere suspended in PBS, passedthrough a glass beads column as described previously(Llanos et al., 1993), and washed twice with PBS bycentrifuging at 800g for 5 min.For Western blotting, washed sperm were concen-

trated by centrifugation at 5,000g for 1 min at roomtemperature. The sperm pellet was then suspended insample buffer (Laemmli, 1970) without mercaptoetha-nol and boiled for 5 min. After centrifugation at 5,000gfor 3 min, the supernatant was removed, 2-mercap-toethanol added to a final concentration of 5%, thesample boiled for 3 min, and then subjected to sodiumdodecyl sulphate–polyacrylamide gel electrophoresis(SDS–PAGE), as described below.

Preparation of Sperm Extracts

The resulting washed sperm pellet was resuspendedin homogenization buffer (50 mM Hepes and 10%glycerol, pH 7.4) at a concentration of 25�107 sperm/ml except in mice (5� 107 sperm/ml) (Morales et al.,1994a). The sperm suspension was then sonicated(Virsonic, Gardiner, NY) with seven 60 W bursts for20 s each, followed by centrifugation for 30 s at 14,000gin a Beckmanmicrofuge to remove nuclear and flagellarmaterial.The supernatantwasusedas theenzymestockpreparation. All these procedures were performed at48C. The protein concentration in each sperm extractpreparation, obtained using the Bradford method(Bradford, 1976), ranged between 0.5 and 1.5 mg/ml.

Enzyme Assay

Enzymatic activity was assayed using the fluorogenicsubstrates designed to evaluate the various specificactivities of the proteasome, such as chymotrypsin-like activity: ZGGL-AMC, SLY-AMC, SAAPF-AMC,SAAF-AMC, and SLLVY-AMC; trypsin-like activity:BLRR-AMC, BQAR-AMC; PGPH activity: ZLLE-AMC,ZLLL-AMC; elastase-like activity was also evaluatedusing the substrate SAAA-AMC. Aliquots of 100 ml ofenzyme extract were incubated for 15 min at 378C, 5%CO2 in a final volume of 2 ml containing 50 mM Hepes,10% glycerol, pH 7.4, and then 10 mM substrate was

added. Then, the assay was run at 378C and thefluorescence monitored with excitation at 380 nm andemission at 460 in a Shimadzu 1501 (Kyoto, Japan)spectrofluorometer. Before adding the trypsin sub-strates, the sperm enzyme extract was incubated with100 mM TLCK for 15 min to inhibit acrosin activity.

To test the effect of the inhibitors on the sperm extractproteolytic activity, 100 ml aliquots of each extract werepreincubated with the proteasome specific inhibitorsclasto-lactacystin (chymotrypsin and trypsin proteaso-mal activity inhibitor), epoxomicin, MG132, MG115,aclarubicin, and PSI (chymotrypsin proteasomal activ-ity inhibitor), MG262 (chymotrypsin and PGPH protea-somal activity inhibitor) or proteases inhibitors TPCK,(chymotrypsin serine proteases), TLCK (trypsin serineproteases), or E64d (cysteinproteases) for 15 min at378C. Then, the different substrates were added. Propercontrols were carried out with the inhibitor solvents.

SDS–PAGE and Immunoblotting

SDS–PAGE was performed using 12% gels accordingto an established method (Laemmli, 1970). Electro-phoretic transfer of proteins to a nitrocellulose mem-brane inall experimentswas carried out according to themethod of Towbin et al. (1979), at 80 V for 4 h at 48C.After transfer, the membrane was blocked (3% BSA inTris—buffered saline), washed three times, incubatedwith primary antibody, washed again three times, andincubated with secondary antibody conjugate. Horse-radish peroxidase detection was carried out usingstandard freshly made amplified Opti-4CN goat anti-mouse detection kit (BIO-RAD, Ph, PA) according to themanufacturer’s instructions.

RESULTS

The results show that sperm extracts of all speciestested possessed enzymatic activity toward all sub-strates used in this study, except rabbit sperm extractsfor substrates SLY-AMC and SAAF and hamster spermextracts for substrates SAAA-AMC and SLY-AMC. Inall cases, the three characteristic proteasome enzymaticactivities (trypsin-like, chymotrypsin-like, and PGPH)were present in all sperm extract studied (Table 1).In all species studied the highest specific activitydetected (nmol AMC hydrolyzed/mg protein/min) wastoward the trypsin substrates. It was greater towardthe substrate BQAR-AMC (19.6�6.8 human; 33.15�0.17 bovine; 7.54� 0.31 rat; 57.94� 3.5 mouse;44.23� 3.7 rabbit, and 8.02�0.23 hamster) than forthe substrate BLRR-AMC (10.2�4.2 human; 16.3�0.9 bovine; 4.57� 0.81 rat; 33.6� 1.2 mouse; 12.48�0.9rabbit, and 4.0�0.77 hamster). On the other hand,the chymotrypsin-like activity toward the substrateSLLVY-AMC, even though was lower than that ob-tained for trypsin substrates, was greater than for theother chymotrypsin substrates. However, for spermextracts of bovine, rat and rabbit the chymotrypsin-likeactivity was similar to the PGPH activity towardthe substrate ZLLE-AMC (Table 1). In the case ofPGPH activity, this was greater toward the substrate

MAMMALIAN SPERM PROTEASOMES 89

ZLLE-AMC than toward the substrate ZLLL-AMC,except for human sperm extracts where the specificenzymatic activity was identical for both substrates(Table 1). The highest enzymatic activity determined forall substrates studied was for mouse sperm extractsand the lowest was for hamster sperm extracts. Finally,with the exception of sperm extracts of mouse, hydro-lysis of the elastase substrate was always lower than allthe other substrates evaluated. A representative gra-phic of enzymatic activity toward the different sub-strates by human spermextracts is shown inFigure 1. Itis clear from Table 1 and Figure 1 that the trypsin-likespecific activity of the sperm extracts was significantlygreater than all other activities detected.

In addition, all the highly specific proteasome inhibi-tors (clasto, aclarabucin, MG262, MG115, MG132,epoxomicin, and PSI) inhibited the chymotrypsin-likeactivity of the sperm proteasome of all animal speciesstudied (Table 2). The lowest inhibition of proteasomalactivity was with clasto since the rest of inhibitor testedtotally inhibited this enzymatic activity. In addition, thechymotrypsin-like activity of the proteasome was onlypartially inhibited by the serine chymotrypsin inhibitorTPCK except for sperm extract of rat where inhibitionwas about 90% (2.2�0.5 nmol AMC hydrolyzed/mgprotein/min in absence of TPCK and 0.23�0.11 nmolAMC hydrolyzed/mg protein/min in the presence ofTPCK). Furthermore, the presence of E64d, a mem-brane-permeable inhibitor for cysteine proteases in-cluding cathepsins and calpains and of TLCK, a trypsinserine protease inhibitor, did not modify the chymo-trypsin-like activity of the different sperm extracts(Table 2). Finally, the specific proteasome inhibitorclasto also inhibited the hydrolysis of the trypsin sub-

strate BQAR-AMC by the sperm extracts (data notshown).

All these results strongly suggest that the proteolyticactivity of the different sperm extracts was due to theproteasome. In accord with this, the anti-proteasomemonoclonal antibody MCP231 detected 4–5 bands of�29 and 32 kDa in all species studied (insert Fig. 1).

DISCUSSION

In this study, we have demonstrated the presence ofenzymatic activity that correspond to the proteasome insperm of several mammalian species. Thus, we showedthat sperm from human, bovine, rat, mouse, hamster,and rabbit all possess proteasome activity, character-ized by the ability to hydrolyze specific substrates.Moreover, specific proteasome inhibitors dramaticallyinhibited the hydrolysis of chymotrypsin and trypsinsubstrates. On the contrary, the membrane-permeableinhibitor of cysteine proteasesE64ddid not significantlymodify the proteasome activity in any of the spermextracts tested. In addition, the trypsin-like proteaseinhibitor TLCK did not modify the activity of the dif-ferent sperm extracts tested upon hydrolysis of SLLVY-AMC and the chymotrypsin-like protease inhibitorTPCK inhibited the proteasome activity, although notas strongly as the more specific proteasome inhibitors.Wehavealsopresentedadditional evidence that confirmthe presence of proteasomal a-subunits in sperm ex-tracts of different species studied by means of Westernblots.

In eukaryotic cells, most proteins in the cytosol andnucleus are degraded via the ubiquitin-proteasomepathway. The proteasome catalyzes the selective degra-dation of short-lived regulatory proteins, such asmitotic

TABLE 1. Specific Enzymatic Activity of Sperm Extracts From Several Mammalian Species

Substrate Human Bovine Rat Mouse Rabbit Hamster

Boc-Gln-Ala-Arg-AMC(BQAR-AMC)

19.6� 6.8(377)

33.15� 0.17(638)

7.54� 0.31(145)

57.94� 3.5(1114)

44.23� 3.7(850)

8.02� 0.23(154)

Boc-Leu-Arg-Arg-AMC(BLRR-AMC)

10.2� 4.2(196)

16.30� 0.9(313)

4.57� 0.81(88)

33.60� 1.2(646)

12.48� 0.9(240)

4.0� 0.77(77)

Suc-Leu-Leu-Val-Tyr-AMC(SLLVY-AMC)

5.20� 1.10(100)

1.89� 0.21(36)

2.20� 0.49(42)

6.18� 0.90(119)

1.38� 0.35(27)

3.51� 0.35(68)

N-Suc-Ala-Ala-Phe-AMC(SAAF-AMC)

0.64� 0.20(12)

0.71� 0.05(14)

0.46� 0.23(9)

0.10� 0.08(29)

0.0� 0(0)

0.94� 0.28(18)

N-Suc-Ala-Ala-Pro-Phe-AMC(SAAPF-AMC)

0.41� 0.02(8)

0.15� 0.07(39)

0.03� 0.03(1)

0.69� 0.12(13)

1.08� 0.09(21)

0.02� 0.01(0.4)

Z-Gly-Gly-Leu-AMC(ZGGL-AMC)

0.20� 0.02(49)

0.30� 0.05(6)

0.04� 0.02(19)

0.55� 0.10(11)

0.26� 0.03(5)

0.03� 0.01(0.6)

N-Suc-Leu-Tyr-AMC(SLY-AMC)

0.07� 0.03(1)

0.12� 0.04(2)

0.06� 0.05(1)

0.49� 0.08(9)

0.0� 0(0)

0.0� 0(0)

Z-Leu-Leu-Glu-AMC(ZLLE-AMC)

0.90� 0.03(17)

1.77� 0.33(34)

1.93� 0.49(37)

3.25� 0.72(63)

1.82� 0.30(35)

1.54� 0.34(30)

Z-Leu-Leu-Leu-AMC(ZLLL-AMC)

0.90� 0.03(17)

0.86� 0.09(17)

0.31� 0.05(6)

1.62� 0.90(31)

0.09� 0.02(2)

0.29� 0.23(6)

N-Suc-Ala-Ala-Ala-AMC(SAAA-AMC)

0.03� 0.02(1)

0.03� 0.02(1)

0.04� 0.04(1)

0.63� 0.15(12)

0.11� 0.03(2)

0.0� 0(0)

The specific activity is expressed as nmol AMC hydrolyzed/mg protein/min. Results are the mean� sem of at least fiveexperiments conducted with different samples. In parenthesis is the specific activity expressed as a percentage of the activity ofhuman sperm toward the SLLVY-AMC substrate, which was considered as 100%.BQAR-AMCandBLRR-AMCare substrates for trypsin-like activity; SLLVY-AMC, SAAF-AMC, SAAPF-AMC, ZGGL-AMC, andSLY-AMC, are substrates for chymotrypsin-like activity; ZLLE-AMC and ZLLL-AMC are substrates for PGPH activity; SAAA-AMC is a substrate for elastase.

90 E. PIZARRO ET AL.

cyclins, and the rapid elimination of aged or mutat-ed proteins or proteins with abnormal conformation(Heinemeyer andWolf, 2000). In addition, proteins thatpossess regulatory functions such as NFkBI, the on-coprotein c-jun, the tumor suppressor protein p53, thea-subunit of the trimeric G protein, Mos kinases, etc.,are also degraded via proteasome (Palombella et al.,1994; Rock et al., 1994; Coux et al., 1996; Hershko,1996).The 26S proteasome is a 2,500 kDa molecule built

from approximate 31 different subunits and is the main

responsible for the degradation of multi-ubiquitinated,and some nonubiquitinated proteins. It contains abarrel-shaped proteolytic core complex, the 20S pro-teasome, consisting of 28 subunits in four stacked,heptameric rings with a C2 axis of symmetry (Vogeset al., 1999). The two outer rings comprise a-subunitsand the inner rings form the central cavity. They consistof b-subunits harboring the catalytically active b5 (X,LMP7), b1 (Y, LMP2), and b2 (Z, MECL–1) subunits,which belong to the family of N-terminal nucleophilehydrolases. They have functional threonine proteaseactive sites with chymotrypsin-like activity, PGPHactivity, and trypsin-like activity, respectively (Emmer-ich et al., 2000). When the proteasome 20S is capped atone or both ends by 19S regulatory complexes form the26S proteasome, recognize ubiquinated proteins and, inmost cases, cleave these protein substrates into smallpeptides varying between 3and 23amino acids in length(Kisselev et al., 1999).

The protease active sites face an inner cavity withinthe b-rings that can be accessed through a narrowchannel leading from the surface of the a-rings. Overall,the proteolytic body 20S can cleave peptide bonds afterany amino acid. However, each of the three active site-containing b-subunits preferentially cleaves after dif-ferent amino acids: b1 cleaves after acidic or smallhydrophobic amino acids, b2 cleaves after basic or smallhydrophobic amino acids, while b5 hydrolyzes thepeptide bond after hydrophobic residues whether bulkyor not (Dick et al., 1998). The rules that govern thecleavage rate of the same peptide bond can be signifi-cantly altered when put into the context of the primarystructure of the polypeptide. For instance, the specificitytoward a peptide bond between two amino acids can beaffected by the amino acids in flanking regions on eitherside, up to eight amino acids away. The location andidentity of each of these anchoring residues are differentfor different classes of peptide bonds (Holzhutter et al.,1999). Thus, in our study the specific enzymatic activity(nmol AMC release/mg protein/min) of tryptic activitywas greater for BQAR-AMC than for BLRR-AMCalthough in both cases the amino acid that is bound toAMC (P1) correspond to arginine (Arg), a basic aminoacid. Probably the amino acid in P2 position makes thedifference because in the substrate that had the highestspecific activity, it correspond to alanine (Ala), a small,hydrophobic amino acid that probably facilitates accessof the substrate to the proteolytic chamber. On the otherhand, proteolysis of the substrate BLRR-AMC may beimpairedby thepresence ofArg inP2, sinceArg is abasicand bulky amino acid.

Regarding the chymotrypsin-like activity of theproteasome, it was greater for substrates with theamino acid Tyr in the position previous to the AMC(P1), than for substrates with the other aromatic aminoacid, Phe, in this position although Phe is also smalland hydrophobic. Furthermore, it has been shown onnumerous occasions that SLLVY-AMC is a preferredsubstrate for the proteasomeand that it is hydrolyzed bythe sperm proteasome of marine invertebrates (Tipler

Fig. 1. Proteasome activity of human sperm extracts. Extracts offresh human sperm were prepared as described in ‘‘Materials andMethods.’’ Then, aliquots of these extracts were incubated withdifferent synthetic substrates and the enzyme activity measured in aspectrofluorometer. The results represent the mean�SEM of sixexperiments with different donors. The substrates used were: BQAR-AMC (–*–); BLRR-AMC (–*–); SLLVY-AMC (– –); ZLLE-AMC(–&–); ZLLL-AMC (–�–); SAAF-AMC (–&–); SAAPF-AMC (–~–);ZGGL-AMC (–~–); SLY-AMC (–}–); SAAA-AMC (–^–). In theinsert, the result of a typicalWestern blot is given. Sperm extracts frombovine (lane 1), rabbit (lane 2), hamster (lane 3), rat (lane 4), mouse(lane 5), and humans (lane 6) were probedwith amonoclonal antibodyagainst the proteasome a-subunits.

MAMMALIAN SPERM PROTEASOMES 91

et al., 1997; Mykles, 1998; Sawada et al., 1998, 2002b;Marino et al., 1999) and humans (Morales et al., 2003).In addition, the hydrophobicity of the amino acid in P2modified the chymotrypsin-like activity of the protea-some. This may explain that the higher enzymaticactivity was toward SLLVY-AMC. Decreasing enzy-matic activity was shown against SAAF-AMC and thenSAAPF, though both have Phe in P1 and the rest ofamino acids of the chain are small and hydrophobic.Probably the higher enzymatic activity toward SAAF-AMC in the majority of the cases occurred because thissubstratehaveone lessaminoacids in its chain; thismayallow better accessibility to the proteolytic chamber.However, this was not the case for mouse and rabbitsperm extracts, where SAAPF showed higher activitythan SAAF. The explanation of this is unknown to us.

Regarding the PGPH activity of the sperm extracts, itwas greater toward the ZLLE-AMC substrate than theZLLL-AMC substrate in all cases, except for humansperm extracts, where it was identical. Probably thiswas due to the fact that both peptidic substrates have anamino acids in P1 position with similar characteristicconditions to be degraded by the proteasome. In theZLLE-AMCsubstrate, it is glutamic acid (Glu), an acidicand small amino acid, and in the ZLLL-AMC substrate,it is leucine (Leu), a small and hidrophobic amino acid.However, the proteasome may prefer an acidic aminoacid to a hydrophobic amino acid in P1. Finally, thelowest activity of all the chymotrypsin substrates wastoward SLY-AMC probably because Tyr is not preferredto proteolysis by the proteasomewhen theP2 amino acidis the least hydrophobic of all amino acids mentioned.Similar results were reported previously for protea-somes of sea urchin (Matsumura and Aketa, 1991) andascidians (Saitoh et al., 1993; Sawada and Someno,1996; Sawada et al., 1998).

To our knowledge, this is the first study to show thatthere are proteasomal activities in sperm of differentmammalian species including human, bovine, rabbit,hamster, rat, and mouse. The proteasomes from thesedifferent species spermatozoa present similar but dis-tinctive characteristics, reflected in their specific enzy-

matic activity toward the various substrates evaluated.Recently, it was shown that there may be a pool ofproteasomes associated with the sperm plasma mem-brane surface, both in marine invertebrates and inmammals (Morales et al., 2004; Sawada et al., 2002a,b).In the solitary ascidianHalocynthia roretzi at least, thevitelline coat of the egg become multiubiquinated uponsperm–egg interaction; the sperm proteasome thendegrades this egg coat allowing fertilization (Sawadaet al., 2002a). Several experiments are under way in ourlaboratory to test the proteasome function in mamma-lian sperm cell.

REFERENCES

Baba T, Azuma S, Kashiwabara S, Toyoda Y. 1994. Sperm from micecarrying a targeted mutation of the acrosin gene can penetrate theoocyte zona pellucida and effect fertilization. J Biol Chem 269:31845–31849.

Bedford JM. 1998. Mammalian fertilization misread? Sperm penetra-tion of the eutherian zonapellucida is unlikely to be a lytic event. BiolReprod 59:1275–1287.

BradfordM. 1976.Rapidand sensitivemethods for thequantification ofmicrogram quantities of protein utilizing the principle of protein dyebinding. Anal Biochem 72:248–254.

Ciechanover A. 1998. The ubiquitin-proteasome pathway: On proteindeath and cell life. EMBO J 17:7151–7160.

Coux O, Tanaka K, Goldberg AL. 1996. Structure and function of the20S and 26S proteosomes. Ann Rev Biochem 65:801–847.

Dick TP, Nussbaum AK, Deeg M, Heinemeyer W, Groll M, Schirle M,Keilholz W, Stefanovic S, Wolf DH, Huber R, Rammenesee HG,Schild H. 1998. Contribution of proteasomal beta-subunits to thecleavage of peptide substrates analyzed with yeast mutants. J BiolChem 273:25637–25646.

Emmerich NPN, Nussbaum AK, Stevanovic S, Priemer M, Toes REM,Rammensee HG, Schild H. 2000. The human 26S and 20S protea-somes generate overlapping but different sets of peptide fragmentsfrom a model protein substrate. J Biol Chem 275:21140–21148.

FabroG,Rovasio RA,Civalero S, Frenkel AS, Caplan SR,EisenbachM,Giojalas LC. 2002. Chemotaxis of capacitated rabbit spermatozoa tofollicular fluid revealed by a novel directionality-based assay. BiolReprod 67:1565–1571.

FenteanyG,SchreiberSL. 1998.Lactacystin, proteasome function, andcell fate. J Biol Chem 273:8545–8548.

Goldberg AL. 1995. Functions of the proteasome: The lysis at the end ofthe tunnel. Science 268:522–523.

Heinemeyer W, Wolf DH. 2000. Active sites and assembly of the 20Sproteasome. In: Wolf DH, Hilt W, editors. Proteasomes: The world ofregulatoryproteolysis.Georgetown,TX:LandesBioscience.pp48–70.

TABLE 2. Effect of Several Inhibitors Upon the Chymotrypsin-Like Activity of the Proteasome FromSperm Extracts of Different Mammalian Species

Inhibitor Human Bovine Rat Mouse Rabbit Hamster

None 5.20� 1.10 1.89� 0.21 2.20� 0.49 6.18� 0.90 1.38� 0.35 3.51� 0.35E64d 4.8� 0.5 1.33� 0.12 1.56� 0.7 6.43� 1.2 1.33� 0.21 3.49� 0.54TLCK 4.7� 1.3 1.78� 0.3 1.89� 0.26 5.8� 0.4 1.29� 0.21 3.08� 0.22TPCK 3.94� 0.2 1.67� 0.19 0.23� 0.11 4.12� 0.9 0.92� 0.33 1.14� 0.18Clasto 0.12� 0.5 0.17� 0.12 1.52� 0.57 0.03� 0.01 0.155� 0.09 0.09� 0.02Aclarabucin 0.0 0.0 0.0 0.0 0.0 0.0MG 262 0.0 0.0 0.0 0 0.0 0.0MG 115 0.0 0.0 0.0 0 0.0 0.0MG 132 0.0 0.0 0.0 0 0.0 0.0Epoxomicin 0.0 0.0 0.0 0 0.0 0.0PSI 0.0 0.0 0.0 0 0.0 0.0

The specific activity is expressed as nmol AMChydrolyzed/mg protein/min. The chymotrypsin-like activity of the sperm extractswas tested using the Suc-Leu-Leu-Val-Tyr-AMC substrate. Results are the mean� sem of at least five experiments conductedwith different samples.

92 E. PIZARRO ET AL.

Hershko A. 1996. Mechanisms and regulation of ubiquitin-mediatedcyclin degradation. Adv Exp Med Biol 389:221–227.

Holzhutter HG, Froemmel C, Kloetzel PM. 1999. A theoreticalapproach towards the identification of cleavage-determining aminoacids motifs of the 20S proteasome. J Mol Biol 286:1251–1265.

Hoshi M, Numakunai T, Sawada H. 1981. Evidence for participationof sperm proteinases in fertilization of the solitary ascidian, Halo-cynthia roretzi: Effects of protease inhibitors. Dev Biol 86:117–121.

Kisselev AF, Akopian TN, Woo KM, Goldberg AL. 1999. The sizes ofpeptides generated from protein by mammalian 26 and 20Sproteasomes. J Biol Chem 274:3363–3371.

Laemmli UK. 1970. Cleavage of structural proteins during assembly ofbacteriophage T4. Nature 227:680–685.

Llanos MN, Morales P, Riffo MS. 1993. Studies of lysophospholipidsrelated to the hamster sperm acrosome reaction in vitro. J Exp Zool267:209–216.

Marino R, De Santis R, Giuliano P, Pinto MR. 1999. Follicle cellproteasome activity and acid extract from the egg vitelline coatprompt the onset of self-sterility in Ciona intestinalis oocytes. ProcNatl Acad Sci USA 96:9633–9636.

Matsumura K, Aketa K. 1989. Activation of Ca2þ channels during theacrosome reaction of sea urchin sperm is inhibited by inhibitors ofchymotrypsin-like proteases. Gamete Res 23:255–266.

Matsumura K, Aketa K. 1991. Proteasome (multicatalytic proteinase)of sea urchin sperm and its possible participation in the acrosomereaction. Mol Reprod Dev 29:189–199.

McRorie RA, Williams WL. 1974. Biochemistry of mammalianfertilization. Ann Rev Biochem 43:777–803.

Morales P, Llanos MN. 1995. Role of the acrosomal reaction in eggvestments and plasma membrane penetration. In: Fenichel P,Parinaud J, editors. Human sperm acrosome reaction. Collioure,France: Colloque INSERM/John Libbey Eurotext Ltd. pp 133–149.

Morales P, Socias T, Cortez J, Llanos MN. 1994a. Evidences for thepresence of a chymotrypsin-like activity in human spermatozoawith a role in the acrosome reaction. Mol Reprod Dev 38:222–230.

Morales P, Vigil P, Franken DR, Kaskar K, Coetzee K, Kruger TF.1994b. Sperm–oocyte interaction: Studies on the kinetics of zonapellucida binding and acrosome reaction of human spermatozoa.Andrologia 26:131–137.

Morales P, Kong M, Pizarro E, Pasten C. 2003. Participation of thesperm proteasome in human fertilization. Hum Reprod 18:1010–1017.

Morales P, Pizarro E, KongM, JaraM. 2004. Extracellular localizationof proteasomes in human sperm. Mol Reprod Dev (in press).

Mykles DL. 1998. Intracellular proteinases of invertebrates: Calcium-dependent and proteasome/ubiquitin-dependent systems. Int RevCytol 184:157–289.

Palombella VJ, Rando OJ, Goldberg AL, Maniatis T. 1994. Theubiquitin-proteasome pathway is required for processing the NF-kB1 precursor protein and the activation of NF-kB. Cell 78:773–785.

Pinto MR, Hoshi M, Marino R, Amoroso A, De Santis R. 1990.Chymotrypsin-like enzymes are involved in sperm penetrationthrough the vitelline coat of Ciona intestinalis egg. Mol Reprod Dev26:319–323.

Rock KL, GrammC, Rothstein L, Clark K, Stewin R, Dick L, Hwang D,Goldberg AL. 1994. Inhibitors of the proteasome block the degrada-tion of most cell proteins and the generation of peptides presented onMHC class I molecules. Cell 78:761–771.

Saitoh Y, Sawada H, Yokosawa H. 1993. High-molecular-weightprotease complexes (proteasomes) of sperm of the ascidian, Halo-

cynthia roretzi: Isolation, characterization, and physiological roles infertilization. Dev Biol 158:238–244.

Sawada H, Someno T. 1996. Substrate specificity of ascidian spermtrypsin-like proteases, spermosin and acrosin. Mol Reprod Dev45:240–243.

Sawada H, Yokosawa H, Hoshi M, Ishii S. 1982. Evidence for acrosin-like enzyme in sperm extract and its involvement in fertilization ofthe ascidian, Halocynthia roretzi. Gamete Res 5:291–301.

Sawada H, Yokosawa H, Hoshi M, Ishii S. 1983. Ascidian spermchymotrypsin-like enzyme; participation in fertilization. Experien-tia 39:377–378.

SawadaH, YokosawaH, Someno T, Saino T, Ishii S. 1984. Evidence forthe participation of two sperm proteases, spermosin and acrosin, infertilization of the ascidian,Halocynthia roretzi: Inhibitory effects ofleupeptin analogs on enzyme activities and fertilization. Dev Biol105:246–249.

Sawada H, Yokosawa H, Numakunai T, Ishii S. 1986. Timing ofaction of sperm proteases in ascidian fertilization. Experientia 42:74–75.

Sawada H, Pinto MR, De Santis R. 1998. Participation of spermproteasome in fertilization of the phlebobranch ascidian Cionaintestinalis. Mol Reprod Dev 50:493–498.

Sawada H, Sakai N, Abe Y, Tanaka E, Takahashi Y, Fujino J, KodamaE,TakizawaS,YokosawaH. 2002a.Extracellular ubiquitination andproteasome-mediated degradation of the ascidian sperm receptor.Proc Natl Acad Sci USA 99:1223–1228.

Sawada H, Takahashi Y, Fujino J, Flores SY, Yokosawa H. 2002b.Localization and roles in fertilization of sperm proteasomes in theascidian Halocynthia roretzi. Mol Reprod Dev 62:271–276.

Seemuller E, Lupas A, Stock D, Luwe J, Huber R, BaumeisterW. 1995.Proteosome from Thermoplasma acidophilun: A threonine protease.Science 268:579–581.

Suarez SS,Wolf DP,Meizel S. 1986. Induction of the acrosome reactionin human spermatozoa by a fraction of human follicular fluid.Gamete Res 14:107–121.

Takizawa S, Sawada H, Someno T, Saitoh Y, Yokosawa H, Hoshi M.1993. Effects of protease inhibitors on binding of sperm to thevitelline coat of ascidian eggs: Implications for participation of aproteasome (multicatalytic proteinase complex). J Exp Zool 267:86–91.

Tipler CP, Hutchon SP, Hendil K, Tanaka K, Fishel S,Mayer RJ. 1997.Purification and characterization of 26S proteasomes from humanand mouse spermatozoa. Mol Hum Reprod 3:1053–1060.

Towbin H, Staehelin T, Gordon J. 1979. Electrophoretic transfer ofproteins frompolyacrylamide gels to nitrocellulose sheets: Procedureand some applications. Proc Natl Acad Sci USA 76:4350–4354.

Voges D, Zwickl P, Baumeister W. 1999. The 26S Proteasome: Amolecular machine designed for controlled proteolysis. Annu RevBiochem 68:1015–1068.

WHO. 1999. Laboratory manual for the examination of human semenand sperm-cervical mucus interaction. Organization WH, editor:Cambridge University Press. 138p.

Wojcik C, Benchaib M, Lornage J, Czyba JC, Guerin JF. 2000.Proteasomes in human spermatozoa. Int J Androl 23:169–177.

Yamagata K, Murayama K, Okabe M, Toshimori K, Nakanishi T,Kashiwabara S, Baba T. 1998. Acrosin accelerates the dispersal ofsperm acrosomal proteins during acrosome reaction. J Biol Chem273:10470–10474.

Yokosawa H, Numakunai T, Murao S, Ishii S. 1987. Sperm chymo-trypsin-like enzymes of different inhibitor-susceptibility as lysin inascidians. Experientia 43:925–927.

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