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Gamete Biology; |
Department of Anatomy4 and Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp),5 Faculty of Science, Mahidol University, Bangkok 10400, Thailand
Chronic Disease,6 Ottawa Health Research Institute, Ottawa, Ontario K1Y 4E9, Canada
Departments of Biochemistry/Microbiology/Immunology,7 Medicine,8 and Obstetrics and Gynecology,9 University of Ottawa, Ottawa, Ontario K1H 8M5, Canada
ABSTRACT
Trypsin-like enzymes in egg water (EW), a natural acrosome reaction (AR) inducer, are known for their importance in shrimp AR. In this report, we describe a unique phenomenon of the AR of black tiger shrimp (Penaeus monodon) sperm. It was completed within 45–60 sec and comprised only the acrosomal exocytosis and depolymerization of the sperm head anterior spike. We used peptidyl fluorogenic substrates to show the presence of trypsin-like enzymes in P. monodon EW and sperm, but minimal activities of chymotrypsin-like enzymes. In sperm, these trypsin-like enzymes existed both on the sperm surface and in the acrosome. The acrosomal enzyme was revealed as a 45-kDa band by fluorogenic substrate in-gel zymography. Although EW possessed high trypsin-like enzyme activities, they were not essential for the AR induction; EW pretreated with an irreversible trypsin inhibitor, or heat-inactivated EW (HI-EW), to abolish the trypsin-like activities could still induce the AR. The HI-EW-induced AR was inhibited by the presence of a membrane impermeant soybean trypsin inhibitor (SBTI) in the sperm suspension, indicating the significance of sperm-borne trypsin-like enzymes (on the surface and/or in the acrosome) in this AR process. However, pretreatment of sperm with SBTI followed by its removal from the suspension still allowed the AR to occur within 5 min of sperm exposure to HI-EW. Since trypsin-like activity of the SBTI-pretreated sperm surface at 5 min after SBTI removal was at the minimal level, our results suggest the importance of the acrosomal trypsin-like enzyme in the AR process.
acrosome reaction, egg water, fertilization, gamete biology, P. monodon, sperm, trypsin
In penaeoid shrimp, spermatophores have to be inserted into the female seminal receptacle (called thelycum) before the release of sperm together with eggs during spawning [1]. It has been shown previously that sperm gain fertilizing ability during their storage in the thelycum [2, 3]. Unlike sperm of other marine invertebrates, penaeoid sperm are nonflagellated and immotile [4, 5]. The success of fertilization in these shrimp thus relies on two key steps: contact of the sperm anterior spike with the egg outer vestment and the subsequent acrosome reaction (AR). The AR process involves a triggered fusion of the sperm plasma membrane with the outer acrosomal membrane, leading to the release of the acrosomal vesicles and soluble content. Thereafter, the AR spermatozoon interacts, fuses, and incorporates into the egg proper. In Sicyonia ingentis, the AR has been shown to be a biphasic process. During the first phase, the anterior spike of the sperm becomes depolymerized upon contacting the vitelline envelope (VE), leading to an acrosomal exocytosis. The second phase involves the formation of the acrosomal filament, which binds to the egg plasma membrane, followed by gamete plasma membrane fusion [5–7]. These two distinct events are temporally separated, and can be induced by two different inducers [7]. While the event in the first AR phase is common in all penaeoid sperm, formation of the acrosomal filament during the second AR phase cannot be detected in some penaeoid shrimp, including Penaeus aztecus, Penaeus setiferus, and Penaeus stylirostris [8].
In Penaeus monodon, detailed information of the AR has not yet been described. Our preliminary work indicates that the AR in P. monodon differs drastically from that of S. ingentis in both timing and morphological alterations [9]. The AR occurring as part of the fertilization process of P. monodon takes only a few minutes compared with 
45–60 min in S. ingentis [10]. This rapid process makes it difficult to detect the various steps of morphological changes during the AR in this species. Overall structural alterations during the AR in P. monodon, as detected at the light microscopic level, include depolymerization of the anterior spike, in conjunction with the swelling and rupturing of the acrosomal vesicle. However, the formation of the acrosomal filament during the AR is not notable in P. monodon sperm. Therefore, spike depolymerization and rupture of the acrosomal vesicle are used to mark completion of AR in this species.
Egg water (EW), a flocculent material in seawater that is generated during egg spawning, has been commonly used for in vitro induction of sperm AR in several aquatic species, including sea urchins [11, 12], sturgeons [13], and shrimp [10]. EW of penaeoid shrimp is made up of components released from the female during spawning, including the outermost layer of the eggs, VE, cortical rods (CRs), which are the egg jelly components embedded in the egg surface crypts, and some thelycal (T) substances. In S. ingentis, the trypsin-like proteolytic activity in EW is important for acrosomal filament formation during the second AR phase [7, 14]. The given explanation is that trypsin may hydrolyze enzymes or channel proteins in the inner acrosomal membrane, thus activating filament formation [14]. Whether or not these EW enzymes are involved in any steps of the AR induction in P. monodon remains to be addressed. In this study, we determined whether EW trypsin-like enzymes were involved in any steps of the AR induction in P. monodon. Alternatively, EW may be involved in the initial part of AR induction, simply through its binding to the sperm surface. Furthermore, since sperm of many invertebrate species have been known to contain trypsin-like enzymes [14–16], we investigated their existence and possible involvement in P. monodon sperm AR induction.
Collection of EW was carried out at the Bangkok Aquaculture Farm Company, Nakhon Sri Thammarat Province, Thailand. Female shrimp possessing mature ovaries (stage IV) were individually placed in a 500-L plastic tank. In order to exclude sperm factors in the EW, only shrimp that had not been inseminated were used in this study. The shrimp were held firmly over a 500-ml container and allowed to spawn their eggs into filtered seawater. After swirling gently to settle the eggs to the bottom, seawater without spawned eggs was collected and designated as EW. This EW was centrifuged (10 000 x g, 15 min, 4°C) to remove particulates, and kept at –80°C until use. Proteins in the EW were quantified with a Bradford's reagent (Sigma, St. Louis, MO) [17].
To collect T sperm , inseminated females were anesthetized under ice, and the thelyca located at the fifth pair of walking legs were carefully removed and placed in calcium-free artificial seawater (CFASW; 423 mM NaCl, 9 mM KCl, 23 mM MgCl2, 9.3 mM MgSO4, 2.1 mM NaHCO3, pH 7.8). Subsequently, they were teased with dissecting forceps to release sperm masses and fluid. The T-sperm suspension was filtered through a 212-µm metal sieve (Endecotts, London, UK) to remove aggregates, washed (500 x g, 5 min), and resuspended in CFASW at the final concentration of 1 x 107 sperm/ml before use. T fluid (TF) was also collected from the female shrimp, which had not been inseminated, following the dissecting method described above. This fluid was centrifuged (12 000 x g, 10 min, 4°C) to remove any small particulates, assayed for the protein concentration as mentioned above and kept frozen at –80°C until use.
CRs were isolated from mature ovaries according to the method described by Lynn and Clark [18]. Briefly, pieces of ovaries were homogenized in an isolation medium (IM; 500 mM NaCl, 9 mM CaCl2, 14 mM KCl, 15 mM MgCl2, and 10 mM Tris-HCl, pH 7.6) containing 30% sucrose. The homogenate was centrifuged (1000 x g, 5 min, 4°C). The pellet containing CRs was resuspended in IM, and the suspension was overlaid onto 60% sucrose in IM and centrifuged (8000 x g, 60 min, 4°C). The white pellet containing mainly CRs was washed four times with IM. As in physiological conditions, isolated CRs were left overnight (4°C) in artificial seawater (ASW; same compositions as CFASW, but with 9.3 mM CaCl2, pH 7.8) to obtain soluble materials. Solubilization of CRs was also facilitated by brief sonications (100 W, 15 sec, 4°C, three times) before the overnight incubation. Protein concentration of the solubilized CRs was measured as described above.
Induction of the Sperm AR with EW
Isolated T-sperm in CFASW were pelleted and resuspended in ASW. The sperm were then treated with various concentrations of EW (1–64 µg/ml) for 5 min or with 16 µg/ml EW for various time periods (0–600 sec) at room temperature to induce the AR. Alternatively, EW treated with amidino-PMSF (APMSF; a trypsin inhibitor) (APMSF-EW) or heat-inactivated EW (HI-EW) (see below) was used in place of native EW in the AR induction. Treated sperm were fixed with 4% paraformaldehyde in ASW and the percentages of unreacted (with an intact long anterior spike) and reacted (without the anterior spike) sperm were scored under a phase contrast microscope. The percentage of spontaneous AR was assessed from sperm treated with ASW. Approximately 200 sperm were counted for each data point. Each experiment was repeated at least three times with different sperm samples.
Electron Microscopy of Acrosome Intact and Reacted Sperm
Sperm were fixed with 2% glutaraldehyde and 4% paraformaledehyde in ASW (pH 7.8, 2 h, 4°C). They were then postfixed in 1% OsO4 in ASW, dehydrated in increasing percentages of cold ethanol, and finally embedded in Spurr's resin (EMS, Fort Washington, PA). Thin sections (70 nm) were cut and mounted on formvar-coated copper grids and counterstained with lead citrate and uranyl acetate before being viewed under an FEI Tecni-20 transmission electron microscope (FEI-USA, Hillboro, OR) at 80 kV.
Binding of Fluorescently Labeled EW to the Sperm Surface
EW was conjugated to an Alexa-488 fluorescent reactive dye (Molecular Probes, Eugene, OR) according to the manufacturer's procedures. Since EW initiated AR very rapidly (see Fig. 1C), we used aldehyde-fixed AI sperm for the EW-sperm binding experiments. T-sperm collected in CFASW were fixed with 4% paraformaldehyde in ASW (1 h, room temperature). Fixed sperm were washed twice with ASW and blocked with 4% BSA containing 0.1 M glycine in ASW (30 min, room temperature). The sperm were incubated (15 min, room temperature) with 16 µg/ml fluorescently labeled EW, washed twice (500 x g, 10 min) to eliminate unbound EW, plated on a slide, and topped with a coverslip before being observed under a Nikon Eclipse epifluorescent microscope equipped with a Nikon DXM 1200 CCD camera (Nikon Corp., Kanagawa, Japan). T-sperm were also incubated with Alexa-488 EW in the presence of a 50-fold unlabeled EW.
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Effects of Protease Inhibitors on the EW-Induced AR
To test whether AR induction was dependent on protease activities, various concentrations of protease inhibitors (all from Sigma) were preadded to EW prior to coincubation with T-sperm resuspended in ASW. These inhibitors included the following: 1) serine protease inhibitors, PMSF (0.1–1 mM), APMSF (0.2–1 mM), soybean trypsin inhibitor (SBTI; 0.1–0.4 mM); 2) cysteine protease inhibitor, E-64 (1–100 µM); 3) aspartic protease inhibitor, pepstatin A (1–10 µM); and 4) metalloprotease inhibitor, EGTA (0.25–1 mM). The percentage of the AR response was analyzed according to the aforementioned protocol.
To determine whether EW-derived proteases, especially trypsin-like ones, were mediators of AR, HI-EW or APMSF-EW was used in place of native EW to treat sperm. HI-EW was prepared by heating EW to 100°C for 2 h, followed by freezing at –20°C overnight. The activity of this HI-EW was examined with a trypsin-specific fluorogenic substrate (as described below) in parallel with native EW. The remaining activity of HI-EW was compared with that of native EW. APMSF-EW was prepared by preincubating EW with 0.5 mM APMSF at 4°C for 2 h. The mixture was washed and concentrated (10 000 x g, 15–30 min, 4°C) three times with ASW through a Microcon YM-10 centrifugal device (Millipore, Bedford, MA) to remove unbound APMSF, which came down in the flow-through fraction. Native EW that washed and concentrated in a similar fashion as APMSF-EW served as an experimental sham control. The remaining activity of APMSF-EW was then determined and compared with that of the EW sham control. HI-EW, APMSF-EW, and sham-EW were used to treat the sperm in place of native EW and the number of AR sperm was scored as described above.
In an alternative experiment, sperm-borne trypsin-like enzymes of acrosome-intact (AI) sperm were inhibited with 0.4 mM membrane impermeant SBTI (15 min, room temperature). The excess amount of the inhibitor was washed from sperm by centrifugation (500 x g, 5 min, twice). Aliquots of this sperm suspension were then induced to undergo AR with 16 µg/ml HI-EW (5 min, room temperature), and assayed for the AR responses following the protocol described above. Another aliquot of sperm was used for trypsin-like enzyme assays at various time points after the removal of SBTI.
Serine protease assay was performed according to the previously described method [19] with some modifications. Fluorogenic-4-methylcoumarin-7-amide (MCA) substrates were used in these assays. These included a final concentration of 20 µM of Boc-Gln-Ala-Arg-MCA (Boc = t-butyloxycarbonyl; Peptide Institute, Louisville, KY) for trypsin-like enzymes, and Suc-Ala-Ala-Pro-Phe-MCA (Suc = succinyl; Bachem, Torrance, CA) for chymotrypsin-like enzymes. All assays were performed at room temperature in a final volume of 100 µl in a 96-well, flat-bottom black plate (Corning Inc., Corning, NY). The samples included EW-related samples (native EW, HI-EW, and APMSF-EW) and T-sperm-related samples (untreated live sperm and SBTI-treated live sperm, AR sperm, and acrosomal vesicles and content). The reaction mixture was comprised of 10 µl of each sample, 10 µl of 0.2 mM substrate solution in 10% dimethyl sulfoxide, and 80 µl of buffer A (10 mM CaCl2, 0.001% Triton X-100 in 50 mM Tris-HCl, pH 7.5) for trypsin-like enzyme assay, or buffer B (10 mM CaCl2 in 50 mM Tris-HCl, pH 8.0) for chymotrypsin-like enzyme assay. Fluorescent 7-amino-4-methylcoumarin (AMC), the released product from substrate hydrolysis, was monitored spectrofluorometrically at various time points with a Spectra Max Gemini XS (Molecular Dynamics, Sunnydale, CA), with excitation and emission wavelengths of 360 and 470 nm, respectively. The measured raw fluorescence units were converted to the amount of free AMC released by using an AMC standard curve. One unit of the enzyme activity was defined as a picomole of AMC released per hour at 25°C from Boc-Gln-Ala-Arg-MCA for trypsin-like activity or from Suc-Ala-Ala-Pro-Phe-MCA for chymotrypsin-like activity. Specific activity of the enzyme was defined as unit activity per microgram protein.
Characterization of a Trypsin-Like Acrosomal Enzyme by Fluorogenic Substrate in-Gel Zymography
The released acrosomal vesicles and content of P. monodon sperm were collected as a supernatant of T-sperm that were induced to undergo the AR by HI-EW, as described above. This AR supernatant collected from 5 x 106 sperm possessed 1500 U of trypsin like activity, when Boc-Gln-Ala-Arg-MCA was used as the substrate (see the enzyme assay method above). The supernatant of AI sperm (sham-treated with ASW) was also collected from the same number of sperm and used as a negative control; it contained only 100 U of trypsin-like activity. The zymography method described by Yasothornsrikul and Hook [20] was used to characterize the trypsin-like enzyme in the supernatant of AR sperm. An SDS-10% polyacrylamide gel was copolymerized with 200 µM Boc-Gln-Ala-Arg-MCA in 0.375 Tris-HCl, pH 8.8, and 0.4% SDS. The separation gel was allowed to polymerize in the dark at room temperature. When polymerization of the separation gel was complete, the stacking gel, consisting of 4% polyacrylamide, 0.330 mM Tris-HCl, pH 6.8, and 0.1% SDS, was cast and polymerized. Subsequently, 10 µl of the supernatant from AR or AI sperm, or purified trypsin (0.5 ng of 8000 U; Roche Applied Science, Indianapolis, IN) was mixed with 10 µl of 2x Laemmli sample loading buffer [21] without heating, and then loaded into the gel well. Electrophoresis was performed in the dark at 4°C at a constant current of 10 mA until completed. The gel was then washed in 2.5% Triton X-100 solution to remove SDS for 15 min at room temperature, followed by washing seven times in cold distilled water (5 min for each wash). The gel was subsequently incubated in trypsin zymography developing buffer (200 mM NaCl, pH 7.5, 20 mM Hepes, 10 mM CaCl2 and 0.005% Triton X-100) at 37°C for 30 min. Fluorescent bands indicating trypsin activity were immediately observed and recorded with an Ultraviolet Transilluminator (Alpha Innotech Inc, San Leandro, CA).
The morphology of the AI and AR P. monodon sperm are shown in Figure 1A. Transmission electron microscopy revealed the presence of electron-dense materials in the anterior spike, and moderately electron-dense materials in the acrosome and subacrosomal region of the AI sperm (Fig. 1A, a). Within 5 min of EW addition, the anterior spike completely disappeared along with the rupture of the acrosome (Fig. 1A, b). The subacrosomal region of the AR sperm became somewhat larger and more electron dense compared with that of the AI sperm (Fig. 1A, b versus Fig. 1A, a). There was no evidence for the acrosomal filament formation (as seen in S. ingentis) [22], even at the longer incubation time period (>10 min) (data not shown). At the light microscopic level, the disappearance of the anterior spike and the presence of dark subacrosomal materials in the AR sperm were evident. These features were thus used to differentiate between AI and reacted sperm in all experiments described below.
The ability of a natural inducer, EW, to induce the AR in P. monodon sperm was concentration dependent (Fig. 1B). Within 5 min of sperm treatment with 1 and 4 µg/ml EW, the AR levels were 54.9 ± 15.7% and 76.1 ± 11.0%, respectively. At 16 µg/ml of EW, the maximal AR response was reached (90.1 ± 9.9%). This EW concentration was therefore selected for all subsequent experiments. When ASW was used in place of EW, 20% of sperm underwent spontaneous AR.
Time-dependent AR induction of P. monodon sperm by EW is demonstrated in Figure 1C. Within 15 sec of the EW treatment, 71.9 ± 0.9% of sperm underwent AR, whereas only 25.0 ± 2.0% of sperm treated with ASW became AR. The percentage of AR sperm increased to the maximal value of 89.3 ± 4.9% within 30 sec of EW incubation time, and remained constant afterwards. In contrast, the spontaneous AR rate of sperm incubated with ASW remained at 25%–30% within 5 min of the ASW incubation. We therefore chose the 5-min incubation period for subsequent experiments.
Trypsin-Like Proteases Mediated AR Induction in Shrimp Sperm
We investigated the possible involvement of proteases in sperm AR induction. Results shown in Figure 2 indicate that inhibitors of trypsin (PMSF, APMSF, and SBTI) inhibited EW-induced AR in a dose-dependent manner. Notably, SBTI at 0.4 mM decreased EW-induced AR to only 12% of the control values. PMSF and APMSF at 1 mM also exerted marked inhibition (75%–85%) of the EW-induced AR. Inclusion of EGTA, a specific Ca2+ chelator and metalloprotease inhibitor, also showed a dose-dependent inhibitory effect, and, at 1.0 mM EGTA, the inhibition of the EW-induced AR was 80%. E-64 and pepstatin A, inhibitors of cysteine and aspartic proteases, respectively, exerted no or very low inhibitory effects on sperm AR induction at all concentrations tested.
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Existence of Trypsin-Like Enzymes in EW
Among serine proteases, trypsin and chymotrypsin have been shown to be significant for sperm AR induction [15, 23]. We thus investigated the existence and physiological functions of these two proteases in EW. Results in Table 1 show that EW demonstrated trypsin-like activity. It selectively hydrolyzed the trypsin-specific substrate (Boc-Gln-Ala-Arg-MCA), with a specific activity of 30.7 U/µg protein. In contrast, EW possessed minimal chymotrypsin-like activity when testing with suc-Ala-Ala-Pro-Phe-MCA as the substrate (i.e., 0.2 U/µg protein).
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Further attempts were made to determine the source of trypsin-like activity in the two major components of EW, CRs, and TF. Comparatively, high trypsin-like activity (171.0 U/µg protein) was detected in TF, whereas minimal trypsin-like activity (2.4 U/µg protein) was present in isolated CRs (Table 1). These results suggest that the enzymatic activity detected in EW was mainly derived from TF.
Trypsin-Like Enzymes in EW Were Not Involved in the AR Induction
Previous evidence has indicated that EW trypsin-like enzymes mediate the AR induction in S. ingentis [7]. This prompted us to determine whether this mechanism also held true in P. monodon sperm. Activity of trypsin-like enzymes in EW was abolished by either HI-EW or APMSF-EW. The remaining trypsin-like activities of HI-EW and APMSF-EW were 1.5 and 1.9 U/µg protein, respectively, compared with that of native EW (Table 1). Surprisingly, treatment of sperm with HI-EW or APMSF-EW produced AR responses of 84% and 82%, respectively, a result that was similar to that observed in sperm treated with native EW (90%) (Fig. 3). The percentage of spontaneous AR in these experiments was 16%. This result indicated that trypsin-like activity involved in AR induction was not derived from EW.
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Binding of EW to the Sperm Surface
Binding of the EW to the sperm surface may initiate sperm signaling events leading to the AR. Our results showed that fluorescently labeled EW bound to the entire sperm surface as well as the anterior spike (Fig. 4, a and b) in the overall sperm population. A slight difference in fluorescence intensity was noted among individual sperm. A background level of fluorescent staining was observed when an excess amount of unlabeled EW was included in the sperm-EW coincubates to compete with fluorescently labeled EW in sperm binding (Fig. 4, c and d); this indicated specific interaction of EW to the sperm surface.
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Presence of Sperm Trypsin-Like Enzymes and Their Putative Roles in AR Induction
Inhibitor studies indicated the significance of trypsin-like enzymes in sperm AR induction (Fig. 2). However, since APMSF-EW and HI-EW could still induce the AR, the trypsin-like enzymes participating in the AR induction would have belonged to sperm, and they could be localized, either on the sperm surface or in the acrosome. Results show that live AI sperm did possess trypsin-like activity (77.1 ± 11.4 U/106 sperm), which would reflect the presence of trypsin-like enzymes on the AI sperm surface. The supernatant of AI sperm contained trypsin-like activity of only 11.8 ± 9.9 U/106 sperm, and this may reflect a low degree of trypsin-like enzyme release from sperm. When these live AI sperm were treated with a membrane-impermeable SBTI, the trypsin-like activity measured at 30 sec after SBTI removal was 2.2 ± 0.6 U/106 (Table 2), indicating the marked inhibition of SBTI on sperm surface trypsin. At 5 and 10 min after the removal of the excess amount of SBTI from sperm, the sperm surface trypsin-like activity was still relatively low (i.e., 6.8 ± 3.4 and 12.9 ± 6.0 U/106 sperm, respectively [<16% of the activity of untreated control sperm]). However, this enzyme activity was restored to 46.4 ± 13.8 and 71.7 ± 8.5 U/106 sperm 30 and 60 min post-SBTI removal, respectively (Table 2). AR sperm induced by HI-EW also possessed trypsin-like enzyme activity, but to a lesser extent than AI sperm (53.2 ± 8.5 U/106 sperm). However, the supernatant of these HI-EW-treated sperm (containing acrosomal vesicles and soluble content) showed a significant level of trypsin-like activity (84.0 ± 27.9 U/106 sperm).
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To determine which trypsin-like enzymes (i.e., those at the sperm surface versus those in the acrosome) were significant for AR induction, the sperm surface enzymes were inhibited by pretreating AI live sperm with 0.4 mM of cell-impermeant SBTI. These SBTI-treated sperm still contained low surface trypsin activity within 5–10 min after SBTI removal by centrifugation (Table 2). Significantly, the SBTI-pretreated sperm were still responsive to HI-EW following 5-min exposure to this inducer; 88.3% of these sperm underwent AR (Fig. 5, fourth bar), a rate that was comparable to that observed with untreated sperm incubated with either HI-EW or native EW (Fig. 5, second bar; Fig. 3, first bar). However, when SBTI was present throughout the HI-EW-sperm coincubation, the AR rate was only 12%, similar to the spontaneous AR rate (Fig. 5, first and third bars). All of these results suggest that the sperm surface trypsin-like enzymes may not be essential for the AR induction. Instead, the trypsin-like enzyme(s) in the acrosome would play a more pertinent role in inducing AR in P. monodon sperm.
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To further characterize the trypsin-like enzyme(s) in the acrosome, the supernatant of the HI-EW-treated sperm, containing the acrosomal vesicles and soluble content, was subjected to fluorogenic substrate in-gel zymography. Figure 6 indicates that this supernatant generated a major fluorescent band (indicating the presence of a trypsin-like activity) of a 45-kDa molecular mass. As expected, purified trypsin generated a major fluorescent band at 21 kDa and a minor fluorescent band at 69 kDa (possibly due to the polymerization of the enzyme). In contrast, the supernatant of the AI sperm treated with ASW showed no fluorescent bands in the zymogram (Fig. 6).
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We report here that the AR induction in P. monodon sperm involved EW binding and enzymatic action of sperm trypsin-like enzymes. In spite of an extremely short period of AR in this species, two morphological changes—spike depolymerization and the rupture of the acrosome (Fig. 1A)—were recognized and, therefore, used as criteria for determining a complete AR in P. monodon. Serine proteases, particularly trypsin- and chymotrypsin-like enzymes, have been shown to play significant roles in sperm AR induction [15, 23]. In this study, we also demonstrated the physiological relevance of trypsin-like proteases in this process in P. monodon sperm (Fig. 2). This was deduced from our results showing that the EW-induced AR was inhibited by trypsin inhibitors (APMSF, PMSF, and SBTI). We used fluorogenic substrates specific for trypsin and chymotrypsin to show that P. monodon sperm and their acrosomal vesicles and content contained substantial levels of trypsin-like activity, but minimal amounts of chymotrypsin-like activity. The results of inhibitor studies suggest that metalloproteases (an inhibitor of which is EGTA) may also be important for sperm AR induction. However, this inhibition can also be due to the effect of EGTA on chelating extracellular calcium, the role of which during the AR process has been extensively documented [22, 24].
The significance of trypsin-like proteases in AR induction has long been reported in many invertebrates, including sea urchins [15] and the penaeoid shrimp, S. ingentis [7, 14]. However, the mechanisms of these enzymes in the AR response in the two shrimp species, S. ingentis and P. monodon, are dissimilar. The proteolytic activity in S. ingentis specifically mediates the acrosomal filament formation [7, 14]. This mechanism does not apply to P. monodon sperm, since such a morphological change does not occur in sperm undergoing the AR in this species. EW trypsin-like proteases of S. ingentis is involved in the AR induction, whereas EW trypsin-like enzymes of P. monodon appear to be unimportant for this event. This observation stems from the fact that denatured EW (HI-EW) or APMSF-EW was still capable of inducing AR responses to a similar extent to that of native EW (Fig. 3). Therefore, trypsin-like proteases essential for the AR induction in P. monodon likely belonged to sperm. We provide the results here indicating that trypsin-like enzymes existed both on the sperm surface and in the acrosome, the latter of which was revealed as a 45-kDa band in the zymogram (Fig. 6).
Our results also revealed that sperm AR was inhibited when SBTI was present throughout sperm-EW coincubates (Figs. 2 and 5), while the AR was still inducible with 5-min exposure of SBTI-treated sperm with HI-EW (Fig. 5). Since the surface of SBTI-pretreated sperm contained only minimal levels of trypsin-like activity within 5–10 min after SBTI removal (Table 2), our AR results shown in Figure 5 (condition IV) suggest that the sperm surface trypsin activity might not be essential for AR induction (Fig. 5). Rather, the trypsin-like activity in the acrosomal vesicles/content may be more pertinent for sperm AR in P. monodon shrimp. In fact, the presence of a 45-kDa trypsin-like enzyme was observed in this study (Fig. 6). In the cases in which PMSF and APMSF were used for incubation with sperm, these cell-permeant inhibitors could readily reach the acrosome and inhibit the trypsin-like activity in this organelle. However, for the sperm incubation with SBTI, it would reach the trypsin-like enzyme(s) in the acrosome once pores on the sperm surface membranes were formed via fusion between the sperm plasma membrane and the outer acrosomal membrane at the onset of AR. Our results also suggest that the participation of these acrosomal trypsin enzymes in AR events was downstream of the initial interaction between the EW and the sperm head plasma membrane, which may lead to the fusion between the sperm plasma membrane and outer acrosomal membrane. This would allow the release of the acrosomal trypsin-like enzyme, which might be responsible for the digestion of the sperm anterior spike. Our ongoing work involves the purification and proteomic analyses of this acrosomal 45-kDa trypsin-like protease. We intend to discern its peptide sequence which will then be comapred with that of acrosomal trypsin-like enzymes of invertebrate animals (ascidians [16] and abalones [25]), known to be involved in enzymatic and nonenzymatic egg coat digestion, respectively. In addition, comparison will be made with acrosin and testis serine protease 5, two well-characterized serine proteases in the mammalian sperm acrosome that are believed to engage in sperm acrosomal matrix dispersion and zona pellucida digestion, respectively [26, 27].
Recently, sperm proteasomes have been shown to be involved in egg jelly-induced AR [28, 29] and vitelline layer hydrolysis [29] in sea urchins. Proteasomes have also been found in mammalian sperm, and play similar roles in AR induction, as observed in sea urchin gametes and in sperm-zona pellucida interaction [30, 31]. The 26S proteasome is a large protein complex, consisting of a 19S regulatory particle and a 20S core particle. Based on their substrate specificity, three threonine proteases, present in the latter particle, are categorized to be trypsin-like, chymotrypsin-like, and peptidyl-glutamyl peptide hydrolyzing enzymes. The confinement of these three different enzymes within the 20S particle may accelerate the hydrolysis of their common substrates, which are usually ubiquitinated [32]. While it is tempting to speculate that the trypsin-like enzyme found in the acrosome of P. monodon sperm may be a component of proteosomes, evidence available so far argues against this possibility. First, the molecular masses of proteases in the 20S proteasome particle are usually in the range of 20–30 kDa [33]. Second, our unpublished results also revealed relatively low chymotrypsin-like activity, both in the acrosome and on the sperm surface of this shrimp species. Experiments are ongoing in our laboratory to determine whether the 20S 
subunits of the proteasome exist in the sperm lysate of P. monodon.
Despite the deficit in proteolytic activity, HI-EW still possessed high competency in inducing AR (Fig. 3). The initial step of the AR induction was likely dependent on the direct interaction of EW components with sperm (Fig. 4). These EW constituents were derived from TF and egg components, including the egg jelly (originated from the CR precursor upon spawning [34, 35]) and the egg VE. Both the egg jelly in echinoderms [36–38] and VE in shrimp [39, 40] have been known to engage in AR induction, presumably through their binding to the sperm surface. Since P. monodon sperm that were stored in the thelycum and continuously exposed to TF (T-sperm) only had a minimal level of spontaneous AR (as described previously here), EW components from the CR and VE may be of greater importance in the sperm binding that leads to AR induction.
The significance of EW components and sperm acrosomal trypsin-like proteases in the acrosomal exocytosis in P. monodon is summarized pictorially in Figure 7. Initially, EW components bind specifically to the sperm membrane receptors (Fig. 7A). This ligand-receptor binding would likely induce the downstream signaling events that lead to the fusion of the sperm plasma membrane and the acrosomal membrane. The acrosomal trypsin-like enzymes that are then released through the membrane pores may be responsible for dissociating the sperm anterior spike (Fig. 7B). Subsequently, the acrosome is completely ruptured, leaving a dense spherical mass lying in place of the anterior spike against the nuclear pole of the AR sperm (Fig. 7C). Whether the dense spherical material is analogous to the acrosomal filament of S. ingentis, or a protease-resistant remnant of the depolymerized spike, is currently under investigation.
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ACKNOWLEDGMENTS
The authors would like to thank Dr. Athula Wikramanayake for his valuable comments and proof reading of this manuscript, and Dr. Boonyarath Pratoomchat, Department of Aquatic Science, Faculty of Science, Buraphar University, for kindly providing shrimp samples.
FOOTNOTES
1Supported by funding from the National Science and Technology Development Agency of Thailand to B.W., the Thailand Research Fund to W.W. and P.S., Thailand Research Fund-Royal Golden Jubilee Ph.D. Program grant PHD/0275/2545 to R.V., and from the Commission on Higher Education to H.K. and S.I. 
Correspondence: 2Wattana Weerachatyanukul, Department of Anatomy, Faculty of Science, Mahidol University, Rama 6 Rd., Payathai, Bangkok 10400, Thailand. FAX: 66 2 354 7168; e-mail: scwwy{at}mahidol.ac.th
3These authors contributed equally to this work.
Received: 25 October 2007.
First decision: 21 November 2007.
Accepted: 27 March 2008.
REFERENCES
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