evidences for the presence of chymotrypsin-like activity in human spermatozoa with a role in the...

MOLECULAR REPRODUCTION AND DEVELOPMENT 38222230 (1994)

Evidences for the Presence of Chymotrypsin-Like Activity in Human Spermatozoa With a Role in the Acrosome Reaction PATRICIO MORALES,l TERESA SOCIAS,' JACQUELINE CORTEZ,' AND MIGUEL N. LLANOS2 'Unit of Reproduction and Development, Faculty of Biological Sciences, P. Catholic University of Chile, and 'Unit of Reproductive Biology, INTA, University of Chile, Santiago, Chile

ABSTRACT The effect of chymotrypsin inhibitors and substrates on the human sperm acrosome reaction stimulated by the human zonae pellucidae or follicular fluid were evaluated. Motile spermatozoa, selected by a Percoll gradient, were incubated at 1 x lo7 cells/ml, 37"C, and 5% C02. After 4.5 hr, the chyrnotrypsin inhibitor TPCK (N-Tosyl-L-Phenylalanine-Chloromethyl Ketone) or the substrate ATEE (N-Acetyl-L-Tyrosine Ethyl Ester) were added for 30 min. Then, four oocytes were added and the percentage of acrosome-reacted spermatozoa on the zona was determined. TPCK and ATEE inhibited the zona pellucida-induced acrosome reaction. The chymotrypsin inhibitors TPCK and chymostatin and the chymotrypsin substrates ATEE, BTEE (N-Benzoyl-L-Tyrosine Ethyl Ester), Succinyl-Ala-Ala-Phe-7-Amido-4-Methyl-Cournarin (Suc-Ala- Ala-Phe-AMC), and SuccinyCLeu-Leu-Val-Tyr-7-Amido-4- Methyl-Coumarin (Suc-Leu-Leu-Val-Tyr-AMC) inhibited the human follicular fluid-induced acrosome reaction. Sperm extracts exhibited hydrolytic activity toward Suc-Ala-Ala- Phe-AMC and Suc-Leu-Leu-Val-Tyr-AMC. This enzyme activity was abolished by TPCK and chymostatin, was independent of Ca2+, and was not modified by 1 , l O phenanthroline. In addition, the activity was present in the supernatant after the acrosome reaction was induced with calcium ionophore and in epididymal spermatozoa recovered from the cauda region. Electron microscopic observations indicated that the inhibitors prevented the membrane events of the acrosome reaction. These data suggest an association between human spermatozoa and chymotrypsin-like activity with a possible role in the acrosome reaction. 0 1994 Wiley-Liss, Inc.

Key Words: Chymotrypsin-like, Sperm proteases, Fer- tilization, Zona pellucida, Epididymal sperm

sperm AR by inhibiting the membrane events of this process. Other investigators have suggested, however, that a trypsin-like activity is involved in the dispersal of the acrosomal matrix (Shams-Borhan and Harrison, 1981; Perreault et al., 1982). Recently, it was shown that trypsin inhibitors can block the human sperm AR (De Jonge et al., 1989; Pillai and Meizel, 1991), includ- ing the reaction that takes place on the surface of the human zona pellucida (hZP) (Llanos et al., 1993). Sev- eral other protease activities have been reported to be present in the human spermatozoa. Indeed, a metalloendoprotease and a calpain-like activity have been implied in the fusion between human spermatozoa and zona-free hamster oocytes (Moretti-Rojas et al., 1991; Diaz-PCrez and Meizel, 1992).

Recent work with marine invertebrates has suggested a role for a sperm chymotrypsin-like activity during fertilization (Yokosawa et al., 1987; Pinto et al., 1990). A chymotrypsin-like protease was involved in the activation of calcium channels during the sea urchin sperm AR. Inhibitors and substrates of the enzyme were able to block both calcium influx and the AR (Matsumura and Aketa, 1989).

To date, however, there are no reports suggesting an association between a chymotrypsin-like activity and mammalian fertilization. In this study we present evidence that chymotrypsin inhibitors and substrates can block the human sperm AR induced by hZP or human follicular fluid (hFF). In addition, we found that extracts of human spermatozoa contain a hydrolytic activity toward specific chymotrypsin substrates. Our results are the first to suggest a role for a chymotrypsin- like activity in the molecular mechanism of the human sperm AR.

MATERIALS AND METHODS Chemicals

The following reagents were purchased from Sigma Chemical Co. (St. Louis, MO): chymotrypsin inhibitors

INTRODUCTION The acrosome reaction (AR) is a n exocytotic event

that involves fusion and fenestration of the outer acrosomal membrane with the overlying plasma membrane (Yanagimachi, 1988). The role of hydrolytic enzymes, particularly proteases, in this process has

been described for spermatozoa Of different spe- ties (Zaneveld et ale, 1991). Dravland et al. (1984) showed that trypsin inhibitors blocked the hamster

Received November 8, 1993; accepted January 5,1994. Address reprint requests to Patricio Morales, Unit of Reproduction and Development, Faculty of Biological Sciences, P. Catholic Univer- sity of Chile, P.0. Box 114-D, Santiago, Chile.

0 1994 WILEY-LISS, INC.

CHYMOTRY PSIN-LIKE ACTIVITY AND THE ACROSOME REACTION 223

TPCK (N-Tosyl-L-Phenylalanine-Chloromethyl Ke- tone) and chymostatin; metalloendoprotease inhibitor 1 , l O phenanthroline; aminopeptidase inhibitor bestatin; trypsin inhibitor benzamidine; chymotrypsin substrates ATEE (N-Acetyl-L-Tyrosine Ethyl Ester), BTEE (N-Benzoyl-L-Tyrosine Ethyl Ester), Succinyl- Ala-Ala-Phe-7-Amino-4-Methyl-Coumarin (Suc-Ala- Ala-Phe-AMC), and Suc-Leu-Leu-Val-Tyr-AMC; the product of the enzyme cleavage of ATEE, N-Acetyl-L- Tyr; EGTA ([Ethylenebis [oxyethylenenitrilo]] tet- raacetic acid); FITC (fluorescein isothi0cyanate)-conju- gated goat antirabbit IgG; Hoechst 33258; BSA (bovine serum albumin, A7030); Hepes, (4-[2-Hydroxyethyll-l- piperazineethanesulfonic acid); and DABCO (1 ,4-diaz- abicyclo 12.2.2.1 octane). PSA (Pisum sativum aggluti- nin)-FITC was purchased from Vector Laboratories, (Burlingame, CAI. The calcium ionophore A23187 was purchased from Calbiochem Biochemicals (La Jolla, CAI, and the calcium standards were from Merck (Darm- stadt, Germany).

Sperm Preparation Normal semen samples, obtained for diagnostic pur-

poses at the Center for the Study of Reproductive Biol- ogy (CEBRE) of the P. Catholic University of Chile, were used. All samples had normal semen parameters, according to World Health Organization (WHO) guidelines, and <1% of the spermatozoa had cytoplasmic droplets. The specimens were allowed to liquefy for 30-60 min at room temperature. Motile spermatozoa were selected by centrifugation through a two-step Percoll gradient as described previously (Suarez et al., 1986, Llanos et al., 1993). The pellet was diluted in modified Tyrode’s medium consisting of 117.5 mM NaC1,0.3 mM NaH,PO,, 8.6 mM KCl, 25 mM NaHCO,, 2.5 mM CaCl,, 0.5 mM MgCl,, 2 mM Glucose, 0.25 mM Na Pyruvate, 19 mM Na Lactate, and 70 Fg/ml of both streptomycin and penicillin, supplemented with 2.6% BSA. One ml sperm aliquots, a t 1 x 107cells/ml, were incubated in 1.5 ml Micro Test tubes (Brand, Germany) for various time periods at 37°C in 5% CO,, 95% air.

Stock solutions of TPCK, chymostatin, BTEE, Suc- Ala-Ala-Phe-AMC, and Suc-Leu-Leu-Val-Tyr-AMC were prepared in dimethyl sulfoxide. Stock solutions of ATEE, N-Acetyl-L-Tyr, and 1,lO phenanthroline were dissolved in ethanol. Bestatine was dissolved in distilled water. The final concentration of dimethyl sulfoxide and ethanol in the sperm suspensions was 0.1% (v/v). A23187 (10 mM) was dissolved in dimethyl sulfoxide and stored a -20°C until use.

Collection of Human Oocytes Human oocytes were dissected from ovarian tissue

obtained from cadavers and stored at -80°C as previously described (Cross et al., 1988). After thawing, the oocytes were freed of remaining cumulus cells by pass- ing them through a narrow bore pipette. As a result of freezing and thawing, these oocytes were not viable.

Collection of Human Follicular Fluid The fluids used in this study were collected from pre-

ovulatory follicles after gonadotrophin stimulation and were kept frozen in aliquots a t -20°C until use. The hFF used here was selected based on its effectiveness in inducing the AR in previous experiments (Morales et al., 1992).

Effect of Chymotrypsin Inhibitors and Substrates on the AR

To evaluate the effect of chymotrypsin inhibitors and substrates on the acrosome reaction, hZP or hFF were used to induce it (see below). The concentrations of inhibitors and substrates used in the present study were the highest possible that did not inhibit sperm motility (scored according to WHO guidelines).

Zona Pellucida-Induced AR After the spermatozoa were incubated for 4.5 hr,

some aliquots were treated for 30 min with TPCK or ATEE. Control aliquots were incubated with dimethyl sulfoxide and ethanol, respectively. Four human oocytes were then added to each aliquot and incubation continued another 30 min. The oocytes with adhered spermatozoa were fixed in ethanol and the acrosomal status of the bound spermatozoa was determined as described below. In some experiments, sperm suspensions treated with TPCK and ATEE were washed and resuspended in fresh media before adding the zonae.

Follicular Fluid-Induced AR After the spermatozoa were incubated for 20 hr, some

aliquots were treated for 30 min with TPCK, ATEE, BTEE, Suc-Ala-Ala-Phe-AMC, or Suc-Leu-Leu-Val- Tyr-AMC. Other sperm aliquots were incubated with chymostatin for 1 hr. Sperm suspensions incubated with TPCK were always washed and resuspended in inhibitor-free media before additional treatment. Con- trol sperm suspensions were incubated with dimethyl sulfoxide or ethanol. Additional controls included treatment of the sperm cells with N-Acetyl-L-Tyr as described above. The various sperm aliquots were then treated with 20% hFF (v/v) for 15 min to induce the AR. The AR was detected as described below.

Ionophore-Induced AR To induce the AR with the calcium ionophore

A23187, cells were capacitated as described above, except that the sperm concentration was 2 x 107/ml. After 20 hr, the sperm suspensions were diluted with Ty- rode’s medium without BSA, resulting in a medium containing 6.5 mg/ml BSA and 0.5 x lo7 celldml. These sperm suspensions were incubated with TPCK, ATEE, or BTEE as described above. The suspensions were then incubated with 10 pM calcium ionophore A23187 or 20% hFF (v/v) for an additional 15 min. Proper controls

224 P. MORALES ET AL.

included the incubation of the inhibitor-treated spermatozoa with the solvent for A23187. At the end of this period, the percentage of acrosome reacted cells was determined.

Detection of the AR The AR of spermatozoa in suspension was detected

using the PSA lectin. The AR of spermatozoa bound to the hZP was detected using an antisperm antiserum, obtained as described previously (Cross et al., 1986). Both probes are directed mainly against intraacroso- ma1 contents and provide equivalent assessments of acrosomal status (Cross et al., 1986; Morales et al., 1989).

Electron Microscopy In other experiments, spermatozoa treated with

ATEE (0.25 mM) or with TPCK (80 pM) and then treated with hFF were processed for transmission electron microscopy. Control spermatozoa were incubated with dimethyl sulfoxide or hFF (Llanos et al., 1993). A total of 100 spermatozoa were scored in each group.

Preparation of Sperm Extracts Semen samples from several men, obtained as de-

scribed above, were allowed to liquefy for 30-60 min and then were pooled. Sperm were separated from seminal plasma, other cell types and cellular debris by centrifugation through a column of Percoll as described above, except that the Percoll was prepared in 50 mM Hepes, 191 mM NaC1, pH 7.4 (Gottlieb and Meizel, 1987). The resulting sperm pellet was washed by centrifugation a t 500 x g for 10 min and then resuspended in homogenization buffer (1 mM benzamidine, 50 mM Hepes, 10% glycerol, pH 7.4) at a concentration of 25 x lo7 sperm/ml. The sperm suspension was then soni- cated (Virsonic, Gardiner, NY) with three 20-Watt bursts for 10 sec each, followed by centrifugation for 30 sec a t 14,OOOxg in a Beckman microfuge to remove nuclear and flagellar material. Other extracts were obtained in the presence of 1 mM 1,lO phenanthroline and 1 mM EGTA. The supernatant was used as the enzyme stock preparation. All these procedures were performed a t 4°C. The protein concentration in each sperm extract preparation, obtained using the Bradford (1976) method, ranged between 0.3 and 0.8 mg/ml.

Spermatozoa were also obtained from the cauda region of epididymis of recently deceased men. Once obtained, the testes were deposited in a sterile recipient over a filter paper wetted with Tyrode’s medium at 37°C to avoid the dehydration and damage of the organ. The testes were then cooled slowly during transport to the laboratory by placing the sterile recipient on ice. The spermatozoa were then aspirated from the cauda epididymis, passed through a Percoll gradient, and son- icated as described above. The time between donor dead and preparation of the sperm extract was <6 hr.

Another source of sperm enzyme activity consisted of the supernatant of spermatozoa induced to undergo the

AR. Sperm incubated for 4 h r were treated with calcium ionophore as described above. The supernatant was then collected, resuspended in homogenization buffer containing 1 , l O phenanthroline and EGTA, son- icated as above, and used as an enzyme stock solution. Control spermatozoa were incubated with dimethyl sulfoxide (0.1%).

Chymotrypsin-like activity was assayed using the fluorogenic substrates Suc-Ala-Ala-Phe-AMC and Suc- Leu-Leu-Val-Tyr-AMC. Because a crude extract was used as an enzyme preparation, benzamidine and bestatin were added to the assay medium. Aliquots of 100 pl of enzyme extract were incubated in a final volume of 2.5 ml containing 10 mM CaCl,, 50 mM Hepes, pH 7.4, 10 pg/ml bestatin, 1 mM benzamidine, and 50 pM substrate. The assay was run a t 25°C and the fluores- cence was monitored with excitation at 380 nm and emission at 460 in a Hitachi-Merck F-1050 spectrofluo- rometer.

To test the effect of chymotrypsin inhibitors on the sperm chymotrypsin-like activity, 100 p1 aliquots of the extract were preincubated with TPCK or chymostatin for 15 min at 4°C. Proper controls were carried out with the inhibitor solvents. In additional studies we tested the effect of various additions on chymotrypsin-like activity; 1 , l O phenanthroline (1 mM) was dissolved in ethanol, EGTA (1 mM), CaC1, (10 mM), and NaCl (26 mM) were dissolved in distilled water. Controls con- tained the same solvents.

Calcium levels in sperm extracts and in the enzyme assay buffer were determined using atomic absorption spectrophotometry. Samples were atomized with a mix- ture of aceti1ene:air and measured at 423 nm in a Per- kin-Elmer model 403.

Statistics All percentages were subjected to arc-sine transfor-

mation before analysis. The Bartlett’s test for homoge- neity, 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 spermatozoa in the control and treated groups. Differences were considered significant at the 0.05 level of confi- dence.

RESULTS Effect of Chymotrypsin Inhibitors and

Substrates on the AR A series of experiments were designed to investigate

whether the AR could be inhibited by chymotrypsin inhibitors and/or substrates. First, we studied the effect of TPCK and ATEE on the AR induced by the hZP. After 30 min of gamete co-incubation, the percentage of acrosome reactions among the zona bound spermatozoa was 27.4 * 2.9% in the control group (mean * sem). Pretreatment of the spermatozoa with TPCK significantly reduced the percentage of reacted spermatozoa on the zona (Table 1). The inhibitory effect of TPCK on the zona-induced AR depended upon the dose of inhibi-


TABLE 1. Effect of TPCK and ATEE on Number and Percentage of Acrosome-Reacted Sperm on the

Surface of the Human Zona Pellucidat

Percentage of acrosome

Pretreatment reactions

None 21.4 * 2.9 TPCK (25 pM) 12.2 * 3.0* TPCK (50 p.M) 9.4 * 2.4* TPCK (80 pM) 6.8 2 0.6* TPCK (80 pM) 6.3 * 1.2*

and washa ATEE (1 mM) 2.1 * 1.0* ATEE (1 mM)

and washa 22.7 * 2.8

Percentage of acrosome reaction

inhibition

Number of sperm

boundhona

0 51 5 7 65 * 6 74 f 5 72 * 3

92 * 3 3 r 0.2

70 * 9 94 -t 8 59 2 10 57 * 9 54 * 8

62 * 9 58 * 7

?Results represent the mean * sem of five experiments with different males. In each experiment, four zonae pellucidae were used. asperm were treated with TPCK or ATEE for 30 min. Then, they were washed and resuspended in fresh medium before addition of the zonae-pellucidae. *Significantly lower than control value (P < 0.001).

tor used. The maximum effect was observed at 80 pM TPCK. Higher concentrations of the inhibitor affected sperm motility. The inhibitory effect of TPCK re- mained after the spermatozoa were washed and resuspended in fresh medium (Table 1). The effect of TPCK was specific on the spermatozoa, since incubation of the zonae with the inhibitor did not block the occurrence of the acrosome reaction (data not shown). Pretreatment of the spermatozoa with ATEE also decreased the percentage of acrosome reacted spermatozoa on the zona surface (Table 1). However, when the ATEE-treated spermatozoa were washed and resuspended in substrate-free medium, the inhibition was reversed. This suggested that ATEE treatment did not affect the sperm cells in a nonspecific way (Table 1).

The lower percentage of acrosome reacted spermatozoa observed on the zona surface after TPCK or ATEE treatment could be explained, a t least in part, by a preferential detachment of the reacted spermatozoa from the zona. This was not the case, however, since the total number of spermatozoa bound to the zona pellucida was not affected by the presence of the inhibitor or substrate (Table 1).

Due to scarcity of human zona pellucida material, hFF was used to induce the AR in the second series of experiments. After treatment of the spermatozoa with hFF, the percentage of acrosome reactions increased from a baseline of 7 * 1.4% (control) to 28.4 * 2.3% (P < 0.001, Fig. 1). Spermatozoa treated with ATEE, BTEE, Suc-Ala-Ala-Phe-AMC, Suc-Leu-Leu-Val-Tyr- AMC exhibited a significant inhibition of the hFF-induced AR (range of inhibition from 242 2% to 97 2 2%, P < 0.001, Fig. 1A). Treatment of the spermatozoa with TPCK or chymostatin before the addition of hFF also caused a significant inhibition of the AR (range of inhibition from 24 k 3, to 97 * 2%, P < 0.001, Fig. 1B).

In another study, acrosomal loss in spermatozoa treated with hFF before incubation with ATEE or TPCK was also counted by transmission electron microscopy (Fig. 2). The AR results (0.25 mM ATEE + hFF = 14%; 80 pM TPCK + hFF = 17%, hFF alone = 35%; control = 10%) were similar to those seen in the immunofluorescence studies. There were significantly fewer acrosome reactions in the presence of the inhibitors, with most spermatozoa having intact acrosomal and plasma membranes (Fig. 2). We did not detect intermediate stages of the AR in the ATEE- or TPCK- inhibited spermatozoa.

To test whether the inhibitory effect of ATEE was due to a competitive inhibition of the chymotrypsin- like activity or to an effect of the cleavage products of this peptide, we tested the effect of N-Acetyl-L-Tyr and ethanol upon the ability of hFF to induce the AR. One mM N-Acetyl-L-Tyr did not have any inhibitory effect on the AR (Table 2). Ethanol (1%) was also without effect on the hFF-induced AR (Table 2) .

The AR induced by the calcium ionophore A23187 was not blocked by TPCK, ATEE, or BTEE (Table 3). Under these conditions, there was no detrimental effect on sperm motility.

Demonstration of Chymotrypsin-like Activity in Spermatozoa

The chymotrypsin-like activity in sperm extracts was assessed using the specific fluorogenic substrates, Suc- Leu-Leu-Val-Tyr-AMC and Suc-Ala-Ala-Phe-AMC. Sperm extracts were able to hydrolyze both substrates (Fig. 3). The specific activity toward Suc-Leu-Leu-Val- Tyr-AMC was 3.1 nmol AMC hydrolyzedlmg protein/ min (Fig. 3A), and the specific activity toward Suc-Ala- Ala-Phe-AMC was 0.18 nmol AMC hydrolyzed/mg proteidmin (Fig. 3B). The rate of hydrolysis was linear for up to -15 min. Then, the activity of the extracts started to decrease. In addition, the sperm enzyme activity was markedly reduced after storage of the extracts for 2 hr at -4°C. This loss of activity was prevented by the addition of 1 mM EGTA plus 1 mM 1,lO phenanthroline to the sperm extracts (data not shown). There was a decrease of 40 ? 2% in the rate of substrate hydrolysis when the enzyme assays were done in Ca2+- free buffer (Fig. 3), whether or not 1 mM EGTA was added (data not shown). The total calcium concentration in the Ca2+-free enzyme assay was 13.4 pM, as detected by atomic absorption spectrophotometry. In the presence of EGTA, the maximum concentration of free Ca2+ was calculated to be 1.85 x M; 1,lO phenanthroline did not change the rate of hydrolysis of the substrates in any condition (data not shown). In addition, the results obtained in Ca2+-free media were not due to a change in the ionic strength of the assay buffer, since the addition of NaCl(26 mM) to the assay buffer did not modify the results (data not shown).

The supernatant collected after the spermatozoa were treated with calcium ionophore had chymotrypsin-like activity (2.1 nmol AMC hydrolyzed/mg protein/


A B

# O , 0 10 100 1000

1 , $? O J t . 0 10 100 1000

SUBSTRATE CONCENTRATION (pM) INHIBITOR CONCENTRATION (pM)

Fig. 1. Effect of different concentrations of chymotrypsin substrates and inhibitors on the human follicular fluid (hFF)-induced AR. A. Sperm were treated with different concentrations of Suc-Leu-Leu-Val- Tyr-AMC (o), Suc-Ala-Ala-Phe-AMC (O), ATEE (a), or BTEE (e), and then, hFF was added. B. Sperm were treated with different concentra-

tions of TPCK (A) or chymostatin ( A ) before adding hFF. The percentage of acrosome reacted sperm was assessed after hFF addition as described in Materials and Methods. Results are the mean 2 sem of eight experiments. The baseline percentage of AR was 7 t 1.4%.

Fig. 2. Morphology of capacitated human spermatozoa treated with human fopllicular fluid prior incubation with (A) 0.25 mM ATEE or (B) 80 pM TPCK. Note the intact sperm head plasma membrane and the outer acrosomal membrane. There were significantly fewer acrosome reacted spermatozoa in the presence of ATEE or TPCK. IAM, inner acrosomal membrane; OAM, outer acrosomal membrane; PM, sperm head flasma membrane. (~25,000).

min). The supernatant in the control group did not have detectable chymotrypsin-like activity. The percentage of acrosome reacted spermatozoa was 26% and 4% in the ionophore and control groups, respectively.

When the sperm extracts were incubated with 80 pM TPCK or 1 mM chymostatin, the enzyme activity to-

ward Suc-Leu-Leu-Val-Tyr-AMC was significantly reduced (P < 0.01, Fig. 4). In the presence of Ca2+, the percentage of inhibition was 58 -t- 2% for TPCK and 98 2 0.1% for chymostatin (Fig. 4A). When the assay was done in Ca2+-free buffer supplemented with EGTA and 1,lO phenanthroline the inhibition was 59 5 1% for

CHYMOTRYPSIN-LIKE ACTIVITY AND THE ACROSOME REACTION 227

TABLE 2. Effect of N-Acetyl-L-Tyr and Ethanol on the Human Follicular Fluid-induced Acrosome Reaction?

Percentage of Pretreatmenta Treatment acrosome reactions

N-Acetyl-L-Tyr (1 mM) Medium 8.1 t 2.8 N-Acetyl-L-Tyr (1 mM) hFF 26.8 * 2.9* 1% Ethanol Medium 7.4 t 1.9 1% Ethanol hFF 25.5 2 2.6*

tResults represent the mean * sem of four experiments with different males. asperm were treated with either 1 mM N-Acetyl-L-Tyr or 1% ethanol for 30 min. Then, 20% human follicular fluid (hFF) or medium was added for 15 min. *Significantly higher than the control (P < 0.001).

TABLE 3. Effect of TPCK, ATEE, and BTEE on Calcium IonoPhore-Induced Acrosome Reaction*

Percentage of acrosome reactions Pretreatmenta hFF" A23187

None 24.2 % 2.6 35.9 * 4.9 TPCK (80uM) 8.3 t 2.1 40.3 2 3.8 ATEE (1 mM) 9.2 t 1.7 41.1 * 6.5 BTEE (0.5 mM) 11.7 * 1.8 36.3 t 2.8

*Results represent the mean 2 sem of seven experiments with different males. The percentage of background acrosome reactions was 7 t 2. "Sperm were treated with TPCK, ATEE, or BTEE for 30 min. Then. 20% human follicular fluid (hFF) or calcium ionoDhore A23187 (10 pM) was added for 15 min.

TPCK and 97 5 1% for chymostatin (Fig. 4B). A similar percentage of inhibition was obtained when the substrate Suc-Ala-Ala-Phe-AMC was used (data not shown).

Sperm recovered from the cauda portion of the epididymis also were able to hydrolyze the synthetic chymotrypsin substrate Suc-Leu-Leu-Val-Tyr-AMC. The specific activity of epididymal spermatozoa toward this substrate was 1.53 nmol AMC hydrolyzedmg proteintmin.

DISCUSSION In this study we have shown that chymotrypsin in-

hibitors and substrates can prevent the human sperm AR. This was the case whether hZP or hFF were used to induce the AR. In addition, sperm extracts and head material released after the acrosome reaction had oc- curred exhibited hydrolytic activity toward synthetic chymotrypsin substrates. These results demonstrate for the first time the association of a chymotrypsin-like activity with the head of human spermatozoa and suggest that this activity may participate in the AR.

The electron microscopy studies suggest that the membrane events of the AR were inhibited in the presence of chymotrypsin inhibitors and substrates, as it has been previously shown for trypsin-like inhibitors (Pillai and Meizel, 1991; Llanos et al., 1993). We did not find intermediate stages of the AR in the spermatozoa treated with ATEE or TPCK, i.e., most spermatozoa had intact sperm head plasma and outer acrosomal membranes. It is still unclear whether the chymotryp-

sin-like activity is involved in the dispersal of the acrosomal matrix, as previously suggested for the trypsin-like activity (Shams-Borhan and Harrison, 1981; Perreault et al., 1982).

In this study, treatment of the spermatozoa with TPCK, BTEE, or ATEE inhibited the AR induced by the hZP or hFF but not the AR induced by A23187. Similar findings were reported for trypsin inhibitors on the progesterone-induced AR (Pillai and Meizel, 1991). All these observations suggest that trypsin-like and chymotrypsin-like activities may be involved in the early events of the human sperm AR, perhaps before stimulation of calcium entry into the cells. It is not yet known whether chymotrypsin inhibitors or substrates can block the calcium influx required for the human sperm AR, as was reported for trypsin inhibitors (Pillai and Meizel, 1991).

We have also found that extracts of human spermatozoa can hydrolyze specific chymotrypsin substrates. This activity was assessed using the sensitive fluorogenic substrates Suc-Ala-Ala-Phe-AMC and Suc-Leu- Leu-Val-Tyr-AMC. Both substrates are hydrolyzed by chymotrypsin-like enzymes from invertebrate spermatozoa (Sawada et al., 1983; Pinto et al., 1990; Mat- sumura and Aketa, 1991). The observations that con- tribute to support that this activity corresponds to a chymotrypsin-like enzyme(s) are: 1) the activity was totally inhibited by chymostatin and partially inhibited by TPCK. Both inhibitors are chymotrypsin inhibitors (Umezawa, 1982; Bond and Butler, 1987). 2) Although chymostatin and TPCK can also inhibit cysteine proteases (Whitaker and Perez-Villasenor, 1968; Chase and Shaw, 1970; Bond and Butler, 19871, the activity was present without Ca2+ added and not modified by the further addition of EGTA. Under these conditions, possible substrate hydrolysis due to a calpain-like activity was precluded since calpains are Ca2+-dependent cysteine proteases (Bond and Butler, 1987). Moverover, only calpain 11, a high (0.5-1 mM)- calcium concentration requiring enzyme, has been detected in bovine spermatozoa (Schollmeyer, 1986). In our enzyme assay in the presence of EGTA, calcium was in the picomolar range. At this level of calcium the calpains, included calpain I, are not active. 3) The activity was retained in the presence of 1,lO phenanthroline, a powerful metalloendoprotease inhibitor (Umezawa, 1982; Bond and Butler, 1987; Gottlieb and Meizel, 1987). 4) Although AMC could be released as a result of successive digestion by an endopeptidase and an aminopeptidase (Pinto et al., 19901, bestatin in the assay medium prevented hydrolysis of the substrates by aminopeptidases. An aminopeptidase-like activity in mammalian spermatozoa was described by Meizel and Cotham (1972). This activity, however, was not inhibited by TPCK. All these observations support the idea that this activity must be ascribed to a chymotrypsin-like enzyme. To our knowledge, this is the first study to show an association between human spermatozoa and a chymotrypsin-like activity with a functional role in the AR. Stambaugh and Buckley (1970) reported


A

3 6 9 12

TIME (rnin)

B 2.01

3 6 9 12

TIME (rnin)

Fig. 3. Hydrolysis of the chymotrypsin substrates Suc-Leu-Leu-Val-Tyr-AMC (A) and Suc-Ala-Ala- Phe-AMC (B) by sperm extracts. The extracts were incubated with calcium (0, .) or without calcium plus EGTA (0, 0) for the times indicated. The amount of free AMC released was spectrofluorometrically assayed. Results are the mean k sem of three experiments.

3 6 9 12 TIME (rnin)

3 6 9 12 TIME (rnin)

Fig. 4. Effect of chymotrypsin inhibitors on the hydrolysis of the substrate Suc-Leu-Leu-Val-Tyr-AMC by sperm extracts. The assays were carried out with calcium (A) or without calcium plus EGTA and 1 , l O phenanthroline (B). The circles (0 , 0) represent hydrolysis in control conditions, the triangles (A, A ) represent hydrolysis in the presence of 80 p M TPCK, and the squares (m, n) represent hydrolysis in the presence of 1 mM chymostatin.

the presence of a chymotrypsin-like activity in mammalian spermatozoa. However, the method used to pre- pare their sperm extract did not permit them to con- clude clearly whether the activity was associated with the spermatozoa, the seminal plasma, or other cell types. Our preliminary results indicate that human spermatozoa recovered from the cauda epididymis also exhibited chymotrypsin-like activity. This is in clear discrepancy with the work of Stambaugh and Buckley (1970) and suggests that the activity is not derived from the seminal plasma.

In this study, the substrates were used as competitive inhibitors of the putative, endogenous, chymotrypsin- like activity. The results indicate that ATEE did not inhibit the AR by causing nonspecific irreversible cell damage since washing the spermatozoa reversed the inhibition. In contrast, the inhibitory effect of TPCK was irreversible. The difference in reversibility of these reagents is related to their type of inhibitory action

upon chymotrypsin (Schoellmann and Shaw, 1963). It could be argued that the inhibition of the AR by the substrates was due to an action of the products of the enzyme cleavage. The present results show that at least the products of ATEE cleavage, N-Acetyl-L-Tyr and ethanol did not have any inhibitory effect upon the AR.

Evidence for the involvement of a chymotrypsin-like activity in the AR has been reported for invertebrate spermatozoa. In sea urchin, chymotrypsin inhibitors and substrates block the AR induced by the egg jelly but not the AR induced by calcium ionophore (Mat- sumura and Aketa, 1989). In Pleurogona and Entero- gona ascidians, it has been reported that fertilization is inhibited by treating the spermatozoa with chymotrypsin inhibitors and substrates (Yokosawa et al., 1987; Matsumura and Aketa, 1989; Pinto et al., 1990). The mechanism by which this inhibition was accomplished is not fully understood. It was suggested that the chymotrypsin-like activity may be involved in spermato-


zoa binding andlor penetration through the vitelline coat.

It is not known how this chymotrypsin-like activity may be acting during the human sperm AR. The results with calcium ionophore A23187 and the observation that the chymotrypsin-like activity is Ca2+-independent and still present after the additions of benzamidine, 1 , l O phenanthroline, and bestatin suggest that the activation of this enzyme does not require proteolytic cleavage. In addition, the activity of this enzyme could be prior to and necessary to activate a trypsin- like enzyme, which in turn would participate in calcium influx required for the AR (Pillai and Meizel, 1991). Regulation of this chymotrypsin-like activity re- mains to be elucidated.

This chymotrypsin-like activity may be part of a large multicatalytic complex known as proteasome (Ar- rigo et al., 1988). Proteasomes are ubiquitously distrib- uted in a variety of eukaryote cells, ranging from human to yeast (Tanaka et al., 1988). All proteasomes have common properties such as latent forms and different peptidase activities associated to at least three independent active sites within a single complex (Wilks and Orlowski, 1983; Rivett, 1989). One of the active sites always possesses chymotrypsin-like activity (Riv- ett, 1989). This type of enzyme complex was recently found to be involved in the sea urchin sperm AR (Mat- sumura and Aketa, 1991). The proteasome may be ex- actly what a spermatozoon requires to efficiently carry out some key, irreversible steps of fertilization, such as the AR and zona pellucida penetration (Yanagimachi, 1988). Undoubtedly, this is an attractive hypothesis that, together with our results, opens new areas of research related to mammalian sperm biochemistry and physiology.

ACKNOWLEDGMENTS This research was financed by grants RF GAPS 9110

and FONDECYT 688/93. J.C. was a fellow from the Rockefeller Foundation. We thank Dr. Stanley Meizel for helpful and encouraging advice, and Dr. Horacio Croxatto for his comments on the manuscript. The skillful technical assistance of Ms. Ana Maria Salgado is greatly appreciated.

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