MOLECULAR REPRODUCTION AND DEVELOPMENT 26:319-323 (1990)
Chymotrypsin-Like Enzymes Are Involved in Sperm Penetration Through the Vitelline Coat of Ciona intestinalis Egg MARIA ROSARIA PINTO,'*2 MOTONORI HOSHI," RITA MARINO,'*' ALESSANDRO AMOROS0,2 ROSARIA DE SANTIS' 'Institute of Protein Biochemistry and Enzymology, CNR, Arco Felice, Napoli, Italy; 2Stazione Zoologica di Napoli, Villa Comunale, Napoli, Italy; 3Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo, Japan ABSTRACT In Ciona intestinalis, sperm penetration through the egg vitelline coat is an essential event of fertilization. We investigated whether trypsin- and chymotrypsin-like enzymes are involved in this event. Inhibitors and peptide substrates for chymotrypsin-like enzymes blocked the overall process of fertilization in a concentration-dependent manner. The inhibitory activity was specifically exerted on the step of sperm penetration. Chymotrypsin-like protease activity was identified in spermatozoa with the fluorogenic synthetic substrate Suc-Ala-Ala-Phe-AMC, which was the most effective substrate in blocking sperm penetration. These data indicate that a chymotrypsin-like protease activity is a sperm lysin of Ciona intestinalis.
AND
Two trypsin-like proteases-spermosin and acrosin-have been purified from spermatozoa of the ascidian Halocynthia roretzi, and it has been suggested that these enzymes, together with chymotrypsin-like enzyrne(s1, are involved in a lysin system (Hoshi et al., 1981; Sawada et al., 1984). The timing of action of these enzymes in sperm penetration has also been described (Sawada et al., 1986). Preliminary screening to detect protease activity involved in sperm penetration produced evidence that in the Pleurogona ascidians both chymotrypsin- and trypsin-like activities are necessary, whereas in Enterogona only the chymotrypsinlike activity seems required (Hoshi, 1985; Yokosawa et al., 1987). The involvement of chymotrypsin-like enzymes in sperm penetration has been suggested also in the Enterogona Ciona intestinalis (Hoshi, 19851,but no experimental data are available in this species. Key Words: Spermatozoa, Lysins, Fertilization, Hence we used inhibitors and specific substrates to Ascidians determine the participation of lysins in sperm penetration through the VC in Ciona intestinalis and found that chymotrypsin-like proteases participate in this INTRODUCTION event. Lastly, chymotrypsin-like activity was detected In Ciona intestinalis the binding of spermatozoa to on the spermatozoa, which suggests that sperm chymothe egg vitelline coat (VC) is a prerequisite for fertili- trypsin-like enzymes are involved in the penetration zation (Rosati and De Santis, 1978). Much is known through the VC. These results not only provide further about the mechanisms and molecules involved in this information on the early events of sperm-egg interacearly step (De Santis et al., 1983; Hoshi et al., 1985; De tion in Ciona intestinalis, but also add weight to the Santis and Pinto, 1987; Casazza et al., 1988). However, hypothesis of the distribution of sperm proteases reinformation on the acrosome reaction, sperm penetra- quired for penetration through the VC in Pleurogona tion through the VC, and sperm-egg fusion, is still frag- and Enterogona ascidians. mentary. MATERIALS AND METHODS The sperm head is known to be endowed with vesiChemicals cles (Cotelli et al., 1980; Rosati et al., 1985; Fukumoto, The protease inhibitors used were soybean trypsin 1988). These vesicles, interpreted as an acrosome, uninhibitor (SBTI; type I-s, Sigma Chemical Co., St. dergo morphological changes described as an acrosome Louis, MO), chymostatin (Sigma),bestatin (Sigma),anreaction (De Santis et al., 1980; Rosati et al., 1985; tipain (Peptide Institute, Osaka), N-tosyl-L-Phe-CH2C1 Usui et al., 1987; Fukumoto, 1988). It is also known that the spermatozoon bores a hole through the VC to reach the perivitelline space (De Santis et al., 1980). It has been inferred that the acrosomal vesicles contain Received November 21, 1989; accepted January 15, 1990. lysins which are required for penetration of the sper- Address reprint requests to Maria Rosaria Pinto, Stazione Zoologica, Villa Comunale, 80121 Napoli, Italy. matozoon through the VC (Rosati et al., 1985).
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(TPCK; Seikagaku Kogyo Co., Tokyo), and leupeptin (AMC) (Peptide Institute); 7-amino-4-methylcoumarin was from Sigma. The peptide substrates were succinyl (Suc)-Ala-Ala-Phe-AMC, Suc-Ala-Ala-Ala-AMC, carbobenzoxy(CBZ)-Gly-Phe-NH,,CBZ-Gly-Gly-NH2(Sigma) and Suc-Ala-Ala-Pro-Phe-p-nitroanilide(pNA) (Bachem, Switzerland).
Fertilization and Binding Assays Cionu intestinalis eggs and spermatozoa were collected from the gonoducts with a Pasteur pipette and used within a few hours in binding and fertilization assays. Fertilization assay was carried out routinely with 100 living eggs inseminated with 2.5 x lo5 spermatozoa in a final volume of 1 ml of artificial seawater buffered with 20 mM Tris-HC1, pH 8.0 (ASW). Naked eggs were fertilized as follows: 20 eggs, manually deprived of their investments by needles, were inseminated with 2.5 x lo5 spermatozoa in a final volume of 1 ml of buffered ASW in agar-coated dishes. After 45 min, the percentage of fertilization of both intact and naked eggs was determined by counting the eggs that had undergone the first cleavage. Binding assay was as previously described (De Santis et al., 1983). When fertilization and binding assays were carried out with inhibitors and substrates, the ASW pH was adjusted to 8.0 by increasing the molarity of Tris-HC1 up to 100 mM, according to the pH changes induced by these chemicals. All samples contained a final concentration of 1%DMSO. In the experiments with inhibitors, eggs were preincubated for 5 min before insemination.
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Fig. 1. Effects of protease inhibitors on fertilization of intact eggs. Eggs were preincubated in seawater with increasing concentrationsof the inhibitor for 5 min and then inseminated. Chymostatin ( 0 ) ;TPCK (A);SBTI (0); antipain (0); leupeptin (m).
sample for 5 min. The appearance of fluorescence was monitored with excitation at 380 nm and emission at 460 nm in a Turner 430 spectrofluorometer.
RESULTS Effects of Inhibitors and Substrates The involvement of trypsin- and chymotrypsin-like enzymes in sperm penetration through the VC was assessed according to the capability of inhibitors of these Enzyme Preparation enzymes to prevent fertilization. The effects of the inSpermatozoa, collected from gonoducts with a Pas- hibitors on fertilization of intact eggs are illustrated in teur pipette, were prepared and stored at -20°C as Figure 1. Fertilization was inhibited by chymostatin previously described (Sawada et al., 1982). The thawed and TPCK. Total inhibition was obtained with 400 p M sperm suspension (2.5 x 10" spermatozoa/ml) was chymostatin and 250 FM TPCK. At the concentrations mixed with 2.5 volumes of 0.46 M NaC1,lO mM CaCl,, used, the other inhibitors tested did not affect fertili50 mM MgC12, and 10 mM KC1 buffered with 50 mM zation. None of the inhibitors against trypsin-like enTris-HC1, pH 8.0, and homogenized with a Teflon ho- zymes interfered with fertilization. mogenizer a t 4". After centrifugation (18,000 x g,60 Because only inhibitors of chymotrypsin-like enm i d , the resulting supernatant was used as an enzyme zymes affected fertilization, we focused on the effect extract. exerted by protease substrates specific for chymotrypChymotrypsin-like activity was assayed using the sin-like enzymes on the overall process of fertilization fluorogenic substrate Suc-Ala-Ala-Phe-AMC (Zim- (Fig. 2). The fluorogenic protease substrate Suc-Alamerman et al., 1977).Because a crude extract was used Ala-Phe-AMC was the most effective in preventing feras an enzyme preparation, AMC may be liberated as a tilization, followed by Suc-Ala-Ala-Pro-Phe-pNA and result of successive digestion by a nonchymotryptic en- CBZ-Gly-Phe-NH,. As shown in Figure 2, much higher dopeptidase and an aminopeptidase. Therefore, a po- concentrations of the last two substrates than of Suctent aminopeptidase inhibitor, bestatin, was added to Ala-Ala-Phe-AMC were required to obtain total inhithe assay medium. Aliquots of 100 ~1of enzyme extract bition. Suc-Ala-Ala-Ala-AMC and CBZ-Gly-Gly-NH2, with 2.1 mg/ml of proteins (Lowry et al., 1951) were which are not chymotrypsin substrates, did not inhibit incubated in a final volume of 2 ml containing 20 mM fertilization a t all (Fig. 2). Fertilization was not afCaCl,, 50 mM Tris-HC1,pH 8.0,lO pg/ml bestatin, and fected in control experiments with free AMC up to 4 0.1 mM substrate. After incubation at 37"C, the reac- nM, thus demonstrating that the effect of the substrate tion was stopped a t different intervals by boiling the is not due to the toxicity of AMC.
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Concentration (mM Fig. 2. Effects of protease substrates on fertilization of intact eggs. Eggs were inseminated in the presence of increasing concentrations of substrates. Suc-Ala-Ala-Phe-AMC (A); Suc-Ala-Ala-Pro-Phe-pNA( 0 ) ; CBZ-Gly-Phe-NH, (W; Suc-Ala-Ala-Ala-AMC (0); CBZ-Gly-Gly- NH, (0).
The substrates tested could exert their inhibitory action on one or more of the individual steps of the fertilization process, i.e., binding of the spermatozoa to the VC, penetration through the coat, and sperm-egg fusion. In Ciona intestinalis, binding and sperm-egg fusion can be studied separately: the binding step can be isolated using a binding assay between spermatozoa and glycerol-treated eggs, and the fertilization of naked eggs can be studied by removing the egg coats with needles (Rosati and De Santis, 1978). If a chymotrypsin-like enzyme is required for sperm binding and sperm-egg fusion, inhibitors and substrates of chymotrypsin should prevent these events by competing with the natural protein substrate. Hence we examined the effects induced by the inhibitor chymostatin and by the most effective substrate Suc-Ala-Ala-Phe-AMC on sperm binding to the VC of the egg and on fertilization of naked eggs. Chymostatin (400 pM) prevented both binding and fertilization of eggs deprived of their coats whereas Suc-Ala-Ala-Phe-AMC (4 mM) did not affect either sperm binding to the VC of glycerol-treated eggs or fertilization of naked eggs. These observations demonstrate that in Ciona intestinalis, fertilization is inhibited by compounds that interact with chymotrypsin-like enzymes and that chymotrypsin-like enzymes are involved at least in the penetration of spermatozoa through the VC.
Demonstration of Chymotrypsin-LikeEnzymes in Spermatozoa Because protease inhibitors and specific substrates for chymotrypsin-like enzymes seem to interfere with
o
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t i m e (h) Fig. 3. Hydrolysis of chymotrypsin substrate Suc-Ala-Ala-PheAMC by enzyme extract. The enzyme extract was incubated with 0.1 mM Suc-Ala-Ala-Phe-AMC ( 0 )or with 0.1 mM Suc-Ala-Ala-Ala-AMC ( 0 ) for the times indicated. The amount of free AMC released was spectrofluorometricaIly assayed.
sperm penetration through the VC, a chymotrypsinlike activity should be detectable in the spermatozoa. We tested this postulate using an enzyme preparation from spermatozoa obtained as described under Materials and Methods. The chymotrypsin-like activity was examined with the fluorogenic protease substrate SucAla-Ala-Phe-AMC, which was the most effective in inhibiting sperm penetration. Chymotrypsin-like proteases hydrolyze AMC from the substrate to produce free AMC that can be fluorometrically quantitated. As shown in Figure 3, the enzyme preparation contains a protease that hydrolyzes Suc-Ala-Ala-Phe-AMC,thus producing free AMC. Hydrolysis was linear for at least 5 hours and was temperature dependent. The rate of the reaction was almost doubled by increasing the temperature from 25°C to 37°C. Hydrolysis of the fluorogenic substrate Suc-Ala-Ala-Phe-AMC was significantly reduced (>go%) with 80 pM of chymostatin. Suc-Ala-Ala-Ala-AMC, which is not a chymotrypsin substrate, was hydrolyzed minimally.
DISCUSSION Fertilization is the result of an ordered series of events starting with specific recognition and terminating in gamete fusion. Hence penetration of the spermatozoon through the egg coats is chronologically the central event in the overall process of fertilization (De Santis and Pinto, 1988). In all the systems studied so far, both in invertebrates and vertebrates, spermatozoa are endowed with lytic agents, called Zysins, that allow them to cross the
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egg investments (Hoshi, 1985). In Ciona intestinalis, information about sperm penetration is limited to morphological data showing spermatozoa crossing the VC (De Santis et al., 1980; Fukumoto, 1988). However, in analogy with other Enterogona, chymotrypsin-like enzymes are probably involved in sperm penetration through the VC (Hoshi, 1985). Preliminary screening with protease inhibitors in fertilization assays demonstrated that, of the inhibitors tested, only the inhibitors of chymotrypsin-like enzymes, namely chymostatin and TPCK, inhibited fertilization. In line with these results, synthetic substrates for chymotrypsin-like proteases inhibited fertilization of intact eggs. Suc-Ala-Ala-Phe-AMC was the most effective substrate in preventing fertilization, whereas the peptide analogue of this substrate, in which Ala replaces Phe a t the PI site (Suc-Ala-AlaAla-AMC) did not affect this event. The ability of inhibitors and substrates for chymotrypsin-like proteases to interfere specifically with fertilization supports the hypothesis that chymotrypsin-like activity is required for fertilization to occur. To demonstrate, albeit indirectly, the involvement of chymotrypsin-like activity in sperm penetration, we tested the effects of chymostatin and Suc-Ala-Ala-PheAMC on sperm binding and sperm-egg fusion. Chymostatin inhibited both of these events, thus suggesting the involvement of chymotrypsin-like enzymes in these two steps a t least. The fluorogenic substrate Suc-AlaAla-Phe-AMC did not affect either binding of the spermatozoa to the VC of glycerol-treated eggs or fertilization of naked eggs. Hence the inhibitory activity of the substrate is exerted specifically on the intermediate step of sperm penetration as a result of the interaction with chymotrypsin-like enzymes. In this context, inhibition of binding and fusion by chymostatin may suggest either the involvement of chymotrypsin-like enzyme(s) with a different substrate specificity or a secondary effect exerted on the sites of the spermatozoon involved in recognition. The finding that a chymotrypsin-like activity is functional in sperm penetration through the VC prompted us to look for chymotrypsin-like enzyme(s), possibly acting as a lysin, on the spermatozoon. Indeed, a chymotrypsin-like activity was detected by the sensitive fluorogenic substrate Suc-Ala-Ala-Phe-AMC in a n enzyme preparation from spermatozoa. Because chymostatin almost totally inhibited hydrolysis of this substrate, all activity of the enzyme extract on the substrate must be ascribed to chymotrypsin-like enzyme(s). These data indicate that this chymotrypsinlike protease is a sperm lysin of Ciona intestinalis. In ascidians most of the information about lysins has been obtained in Halocynthia roretzi. Two trypsin-like enzymes, ascidian acrosin and spermosin, and a chymotrypsin-like enzyme are involved in a lysin system that regulates sperm penetration (Sawada et al., 1984). Only the chymotrypsin-like enzyme exerts a lytic ac-
tivity on the VC, whereas the role of ascidian acrosin and spermosin and their functional relationship with the chymotrypsin-like enzyme has yet to be elucidated. Thus, i t is evident that sperm penetration through the egg coats is mediated by a very complicated mechanism. This is supported by studies on sea urchins and mammals (Hoshi, 1985): many enzymes, mostly acrosomal, presumed to be functional in sperm penetration have been identified and in some cases purified. Hence it seems likely that also in these species spermatozoa cross the egg coats by means of several lysins acting cooperatively and sequentially a s a system. In Ciona intestinalis, data about the molecules participating in sperm penetration are too preliminary to allow any conclusion a s to the presence of a lysin system in this species. It is noteworthy that, in all the ascidians-both Pleurogona and Enterogona-examined so far, chymotrypsin-like activity has been always detected in spermatozoa and related to sperm penetration through the VC. Only in Pleurogona has trypsin-like activity been found to be associated with chymotrypsin-like activity as lysins. Therefore it has been suggested that distribution of trypsin- and chymotrypsin-like enzymes as lysins in Pleurogona and Enterogona may have a n evolutionary meaning (Hoshi, 1985). The results obtained in the Enterogona Ciona intestinalis support the hypothesis of the biological and evolutionary significance of sperm lysins in ascidians. However, only when the enzymes have been obtained in a homogeneous form and their functional characterization carried out will it be possible to gain insight into the precise role of trypsin- and chymotrypsin-like enzymes in sperm penetration through the VC.
REFERENCES Casazza G, De Santis R, Pinto MR (1988): Plasma membrane glycoproteins of Cmna intestinalis spermatozoa that interact with the egg. Dev Growth Differ 30:147-158. Cotelli F, De Santis R, Rosati F, Monroy A (1980): Acrosome differentiation in the spermatogenesis of Ciona rntestrnalis. Dev Growth Differ 22:561-569. De Santis R, Jamunno G, Rosati F (1980): A study of the chorion and the follicle cells in relation to sperm-egg interaction in the ascidian Ciona i n t e s t i d i s . Dev Biol 74:490-499. De Santis R, Pinto MR, Cotelli F, Rosati F, Monroy A, DAlessio G (1983): A fucosyl-glycoprotein component with sperm receptor and sperm activating activites from the vitelline coat of Czona tntestrnalis eggs. Exp Cell Res 148:508-513. De Santis R, Pinto MR (1987): Isolation and partial characterization of a glycoprotein complex with sperm receptor activity from Ciona intestinalis ovary. Dev Growth Differ 29:617-625. De Santis R, Pinto MR (1988):The pathway of sperm-egg interaction in ascidians: Biology and chemistry. Zoo1 Sci 5:919-924. Fukumoto M (1988): Fertilization in ascidians: Apical processes and gamete fusion in Ciona intestinalis spermatozoa. J Cell Sci 89:189196. Hoshi M, Numakunai T, Sawada H (1981): Evidence for participation of sperm proteinases in fertilization of the solitary ascidian, Halocynthia roretzt: Effects of protease inhibitors. Dev Biol 86:117-121. Hoshi M (1985): Lysins. In CB Metz, A Monroy (eds): “Biology of Fertilization.” Orlando, FL: Academic Press, Vol 2, pp 431-462.
CHYMOTRYPSIN-LIKEENZYMES OF CIONA SPERM Hoshi M, De Santis R, Pinto MR, Cotelli F, Rosati F (1985):Sperm glycosidases as mediators of sperm-egg binding in the ascidians. Zoo1 Sci 265-69. Lowry OH, Rosebrough NJ, Farr AL, Randall R J (1951):Protein measurement with Folin phenol reagent. J Biol Chem 193265-275. Rosati F, De Santis R (1978):Studies on fertilization in ascidians. I Self-sterility and specific recognition between gametes of C i o m intestinalis. Exp Cell Res 112:lll-119. Rosati F, Pinto MR, Casazza G (1985):The acrosomal region of the spermatozoon of Ciona intestinalis: Its relationship with the binding to the vitelline coat of the egg. Gamete Res 11:379-389. Sawada H, Yokosawa H, Hoshi M, Ishii S (1982):Evidence for acrosinlike enzyme in sperm extract and its involvement in fertilization of the ascidian, Halocynthia roretzi. Gamete Res 5291-301. Sawada, H, Yokosawa H, Ishii S (1984):Purification and characterization of two types of trypsin-like enzymes from sperm of the as-
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cidian (Protochordata) HaloCynthia roretzi. J Biol Chem 25929002904. Sawada H, Yokosawa H, Numakunai T, Ishii S (1986):Timing of action of sperm proteases in ascidian fertilization. Experientia 42: 74-75. Usui N, Takahashi I, Hoshi M (1987):Membrane differentiation in ascidian spermatozoa.In H Mohri (ed):“New Horizon in Sperm Cell Research.” Tokyo: Japan Sci SOCPress/New York: Gordon and Breach Science Publishers, pp 265-274. Yokosawa H, Numakunai T, Murao S, Ishii S (1987):Sperm chymotrypsin-like enzymes of different inhibitor-susceptibility as lysins in ascidians. Experientia 43:925-927. Zimmerman M, Ashe B, Yurewicz EC, Pate1 G (1977):Sensitive assays for trypsin, elastase, and chymotripsin using new fluorogenic substrates. Anal Biochem 78:47-51.
Chymotrypsin-Like Enzymes Are Involved in Sperm Penetration Through the Vitelline Coat of Ciona intestinalis Egg MARIA ROSARIA PINTO,'*2 MOTONORI HOSHI," RITA MARINO,'*' ALESSANDRO AMOROS0,2 ROSARIA DE SANTIS' 'Institute of Protein Biochemistry and Enzymology, CNR, Arco Felice, Napoli, Italy; 2Stazione Zoologica di Napoli, Villa Comunale, Napoli, Italy; 3Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo, Japan ABSTRACT In Ciona intestinalis, sperm penetration through the egg vitelline coat is an essential event of fertilization. We investigated whether trypsin- and chymotrypsin-like enzymes are involved in this event. Inhibitors and peptide substrates for chymotrypsin-like enzymes blocked the overall process of fertilization in a concentration-dependent manner. The inhibitory activity was specifically exerted on the step of sperm penetration. Chymotrypsin-like protease activity was identified in spermatozoa with the fluorogenic synthetic substrate Suc-Ala-Ala-Phe-AMC, which was the most effective substrate in blocking sperm penetration. These data indicate that a chymotrypsin-like protease activity is a sperm lysin of Ciona intestinalis.
AND
Two trypsin-like proteases-spermosin and acrosin-have been purified from spermatozoa of the ascidian Halocynthia roretzi, and it has been suggested that these enzymes, together with chymotrypsin-like enzyrne(s1, are involved in a lysin system (Hoshi et al., 1981; Sawada et al., 1984). The timing of action of these enzymes in sperm penetration has also been described (Sawada et al., 1986). Preliminary screening to detect protease activity involved in sperm penetration produced evidence that in the Pleurogona ascidians both chymotrypsin- and trypsin-like activities are necessary, whereas in Enterogona only the chymotrypsinlike activity seems required (Hoshi, 1985; Yokosawa et al., 1987). The involvement of chymotrypsin-like enzymes in sperm penetration has been suggested also in the Enterogona Ciona intestinalis (Hoshi, 19851,but no experimental data are available in this species. Key Words: Spermatozoa, Lysins, Fertilization, Hence we used inhibitors and specific substrates to Ascidians determine the participation of lysins in sperm penetration through the VC in Ciona intestinalis and found that chymotrypsin-like proteases participate in this INTRODUCTION event. Lastly, chymotrypsin-like activity was detected In Ciona intestinalis the binding of spermatozoa to on the spermatozoa, which suggests that sperm chymothe egg vitelline coat (VC) is a prerequisite for fertili- trypsin-like enzymes are involved in the penetration zation (Rosati and De Santis, 1978). Much is known through the VC. These results not only provide further about the mechanisms and molecules involved in this information on the early events of sperm-egg interacearly step (De Santis et al., 1983; Hoshi et al., 1985; De tion in Ciona intestinalis, but also add weight to the Santis and Pinto, 1987; Casazza et al., 1988). However, hypothesis of the distribution of sperm proteases reinformation on the acrosome reaction, sperm penetra- quired for penetration through the VC in Pleurogona tion through the VC, and sperm-egg fusion, is still frag- and Enterogona ascidians. mentary. MATERIALS AND METHODS The sperm head is known to be endowed with vesiChemicals cles (Cotelli et al., 1980; Rosati et al., 1985; Fukumoto, The protease inhibitors used were soybean trypsin 1988). These vesicles, interpreted as an acrosome, uninhibitor (SBTI; type I-s, Sigma Chemical Co., St. dergo morphological changes described as an acrosome Louis, MO), chymostatin (Sigma),bestatin (Sigma),anreaction (De Santis et al., 1980; Rosati et al., 1985; tipain (Peptide Institute, Osaka), N-tosyl-L-Phe-CH2C1 Usui et al., 1987; Fukumoto, 1988). It is also known that the spermatozoon bores a hole through the VC to reach the perivitelline space (De Santis et al., 1980). It has been inferred that the acrosomal vesicles contain Received November 21, 1989; accepted January 15, 1990. lysins which are required for penetration of the sper- Address reprint requests to Maria Rosaria Pinto, Stazione Zoologica, Villa Comunale, 80121 Napoli, Italy. matozoon through the VC (Rosati et al., 1985).
0 1990 WILEY-LISS, INC.
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(TPCK; Seikagaku Kogyo Co., Tokyo), and leupeptin (AMC) (Peptide Institute); 7-amino-4-methylcoumarin was from Sigma. The peptide substrates were succinyl (Suc)-Ala-Ala-Phe-AMC, Suc-Ala-Ala-Ala-AMC, carbobenzoxy(CBZ)-Gly-Phe-NH,,CBZ-Gly-Gly-NH2(Sigma) and Suc-Ala-Ala-Pro-Phe-p-nitroanilide(pNA) (Bachem, Switzerland).
Fertilization and Binding Assays Cionu intestinalis eggs and spermatozoa were collected from the gonoducts with a Pasteur pipette and used within a few hours in binding and fertilization assays. Fertilization assay was carried out routinely with 100 living eggs inseminated with 2.5 x lo5 spermatozoa in a final volume of 1 ml of artificial seawater buffered with 20 mM Tris-HC1, pH 8.0 (ASW). Naked eggs were fertilized as follows: 20 eggs, manually deprived of their investments by needles, were inseminated with 2.5 x lo5 spermatozoa in a final volume of 1 ml of buffered ASW in agar-coated dishes. After 45 min, the percentage of fertilization of both intact and naked eggs was determined by counting the eggs that had undergone the first cleavage. Binding assay was as previously described (De Santis et al., 1983). When fertilization and binding assays were carried out with inhibitors and substrates, the ASW pH was adjusted to 8.0 by increasing the molarity of Tris-HC1 up to 100 mM, according to the pH changes induced by these chemicals. All samples contained a final concentration of 1%DMSO. In the experiments with inhibitors, eggs were preincubated for 5 min before insemination.
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Fig. 1. Effects of protease inhibitors on fertilization of intact eggs. Eggs were preincubated in seawater with increasing concentrationsof the inhibitor for 5 min and then inseminated. Chymostatin ( 0 ) ;TPCK (A);SBTI (0); antipain (0); leupeptin (m).
sample for 5 min. The appearance of fluorescence was monitored with excitation at 380 nm and emission at 460 nm in a Turner 430 spectrofluorometer.
RESULTS Effects of Inhibitors and Substrates The involvement of trypsin- and chymotrypsin-like enzymes in sperm penetration through the VC was assessed according to the capability of inhibitors of these Enzyme Preparation enzymes to prevent fertilization. The effects of the inSpermatozoa, collected from gonoducts with a Pas- hibitors on fertilization of intact eggs are illustrated in teur pipette, were prepared and stored at -20°C as Figure 1. Fertilization was inhibited by chymostatin previously described (Sawada et al., 1982). The thawed and TPCK. Total inhibition was obtained with 400 p M sperm suspension (2.5 x 10" spermatozoa/ml) was chymostatin and 250 FM TPCK. At the concentrations mixed with 2.5 volumes of 0.46 M NaC1,lO mM CaCl,, used, the other inhibitors tested did not affect fertili50 mM MgC12, and 10 mM KC1 buffered with 50 mM zation. None of the inhibitors against trypsin-like enTris-HC1, pH 8.0, and homogenized with a Teflon ho- zymes interfered with fertilization. mogenizer a t 4". After centrifugation (18,000 x g,60 Because only inhibitors of chymotrypsin-like enm i d , the resulting supernatant was used as an enzyme zymes affected fertilization, we focused on the effect extract. exerted by protease substrates specific for chymotrypChymotrypsin-like activity was assayed using the sin-like enzymes on the overall process of fertilization fluorogenic substrate Suc-Ala-Ala-Phe-AMC (Zim- (Fig. 2). The fluorogenic protease substrate Suc-Alamerman et al., 1977).Because a crude extract was used Ala-Phe-AMC was the most effective in preventing feras an enzyme preparation, AMC may be liberated as a tilization, followed by Suc-Ala-Ala-Pro-Phe-pNA and result of successive digestion by a nonchymotryptic en- CBZ-Gly-Phe-NH,. As shown in Figure 2, much higher dopeptidase and an aminopeptidase. Therefore, a po- concentrations of the last two substrates than of Suctent aminopeptidase inhibitor, bestatin, was added to Ala-Ala-Phe-AMC were required to obtain total inhithe assay medium. Aliquots of 100 ~1of enzyme extract bition. Suc-Ala-Ala-Ala-AMC and CBZ-Gly-Gly-NH2, with 2.1 mg/ml of proteins (Lowry et al., 1951) were which are not chymotrypsin substrates, did not inhibit incubated in a final volume of 2 ml containing 20 mM fertilization a t all (Fig. 2). Fertilization was not afCaCl,, 50 mM Tris-HC1,pH 8.0,lO pg/ml bestatin, and fected in control experiments with free AMC up to 4 0.1 mM substrate. After incubation at 37"C, the reac- nM, thus demonstrating that the effect of the substrate tion was stopped a t different intervals by boiling the is not due to the toxicity of AMC.
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Concentration (mM Fig. 2. Effects of protease substrates on fertilization of intact eggs. Eggs were inseminated in the presence of increasing concentrations of substrates. Suc-Ala-Ala-Phe-AMC (A); Suc-Ala-Ala-Pro-Phe-pNA( 0 ) ; CBZ-Gly-Phe-NH, (W; Suc-Ala-Ala-Ala-AMC (0); CBZ-Gly-Gly- NH, (0).
The substrates tested could exert their inhibitory action on one or more of the individual steps of the fertilization process, i.e., binding of the spermatozoa to the VC, penetration through the coat, and sperm-egg fusion. In Ciona intestinalis, binding and sperm-egg fusion can be studied separately: the binding step can be isolated using a binding assay between spermatozoa and glycerol-treated eggs, and the fertilization of naked eggs can be studied by removing the egg coats with needles (Rosati and De Santis, 1978). If a chymotrypsin-like enzyme is required for sperm binding and sperm-egg fusion, inhibitors and substrates of chymotrypsin should prevent these events by competing with the natural protein substrate. Hence we examined the effects induced by the inhibitor chymostatin and by the most effective substrate Suc-Ala-Ala-Phe-AMC on sperm binding to the VC of the egg and on fertilization of naked eggs. Chymostatin (400 pM) prevented both binding and fertilization of eggs deprived of their coats whereas Suc-Ala-Ala-Phe-AMC (4 mM) did not affect either sperm binding to the VC of glycerol-treated eggs or fertilization of naked eggs. These observations demonstrate that in Ciona intestinalis, fertilization is inhibited by compounds that interact with chymotrypsin-like enzymes and that chymotrypsin-like enzymes are involved at least in the penetration of spermatozoa through the VC.
Demonstration of Chymotrypsin-LikeEnzymes in Spermatozoa Because protease inhibitors and specific substrates for chymotrypsin-like enzymes seem to interfere with
o
i
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3
4
5
t i m e (h) Fig. 3. Hydrolysis of chymotrypsin substrate Suc-Ala-Ala-PheAMC by enzyme extract. The enzyme extract was incubated with 0.1 mM Suc-Ala-Ala-Phe-AMC ( 0 )or with 0.1 mM Suc-Ala-Ala-Ala-AMC ( 0 ) for the times indicated. The amount of free AMC released was spectrofluorometricaIly assayed.
sperm penetration through the VC, a chymotrypsinlike activity should be detectable in the spermatozoa. We tested this postulate using an enzyme preparation from spermatozoa obtained as described under Materials and Methods. The chymotrypsin-like activity was examined with the fluorogenic protease substrate SucAla-Ala-Phe-AMC, which was the most effective in inhibiting sperm penetration. Chymotrypsin-like proteases hydrolyze AMC from the substrate to produce free AMC that can be fluorometrically quantitated. As shown in Figure 3, the enzyme preparation contains a protease that hydrolyzes Suc-Ala-Ala-Phe-AMC,thus producing free AMC. Hydrolysis was linear for at least 5 hours and was temperature dependent. The rate of the reaction was almost doubled by increasing the temperature from 25°C to 37°C. Hydrolysis of the fluorogenic substrate Suc-Ala-Ala-Phe-AMC was significantly reduced (>go%) with 80 pM of chymostatin. Suc-Ala-Ala-Ala-AMC, which is not a chymotrypsin substrate, was hydrolyzed minimally.
DISCUSSION Fertilization is the result of an ordered series of events starting with specific recognition and terminating in gamete fusion. Hence penetration of the spermatozoon through the egg coats is chronologically the central event in the overall process of fertilization (De Santis and Pinto, 1988). In all the systems studied so far, both in invertebrates and vertebrates, spermatozoa are endowed with lytic agents, called Zysins, that allow them to cross the
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egg investments (Hoshi, 1985). In Ciona intestinalis, information about sperm penetration is limited to morphological data showing spermatozoa crossing the VC (De Santis et al., 1980; Fukumoto, 1988). However, in analogy with other Enterogona, chymotrypsin-like enzymes are probably involved in sperm penetration through the VC (Hoshi, 1985). Preliminary screening with protease inhibitors in fertilization assays demonstrated that, of the inhibitors tested, only the inhibitors of chymotrypsin-like enzymes, namely chymostatin and TPCK, inhibited fertilization. In line with these results, synthetic substrates for chymotrypsin-like proteases inhibited fertilization of intact eggs. Suc-Ala-Ala-Phe-AMC was the most effective substrate in preventing fertilization, whereas the peptide analogue of this substrate, in which Ala replaces Phe a t the PI site (Suc-Ala-AlaAla-AMC) did not affect this event. The ability of inhibitors and substrates for chymotrypsin-like proteases to interfere specifically with fertilization supports the hypothesis that chymotrypsin-like activity is required for fertilization to occur. To demonstrate, albeit indirectly, the involvement of chymotrypsin-like activity in sperm penetration, we tested the effects of chymostatin and Suc-Ala-Ala-PheAMC on sperm binding and sperm-egg fusion. Chymostatin inhibited both of these events, thus suggesting the involvement of chymotrypsin-like enzymes in these two steps a t least. The fluorogenic substrate Suc-AlaAla-Phe-AMC did not affect either binding of the spermatozoa to the VC of glycerol-treated eggs or fertilization of naked eggs. Hence the inhibitory activity of the substrate is exerted specifically on the intermediate step of sperm penetration as a result of the interaction with chymotrypsin-like enzymes. In this context, inhibition of binding and fusion by chymostatin may suggest either the involvement of chymotrypsin-like enzyme(s) with a different substrate specificity or a secondary effect exerted on the sites of the spermatozoon involved in recognition. The finding that a chymotrypsin-like activity is functional in sperm penetration through the VC prompted us to look for chymotrypsin-like enzyme(s), possibly acting as a lysin, on the spermatozoon. Indeed, a chymotrypsin-like activity was detected by the sensitive fluorogenic substrate Suc-Ala-Ala-Phe-AMC in a n enzyme preparation from spermatozoa. Because chymostatin almost totally inhibited hydrolysis of this substrate, all activity of the enzyme extract on the substrate must be ascribed to chymotrypsin-like enzyme(s). These data indicate that this chymotrypsinlike protease is a sperm lysin of Ciona intestinalis. In ascidians most of the information about lysins has been obtained in Halocynthia roretzi. Two trypsin-like enzymes, ascidian acrosin and spermosin, and a chymotrypsin-like enzyme are involved in a lysin system that regulates sperm penetration (Sawada et al., 1984). Only the chymotrypsin-like enzyme exerts a lytic ac-
tivity on the VC, whereas the role of ascidian acrosin and spermosin and their functional relationship with the chymotrypsin-like enzyme has yet to be elucidated. Thus, i t is evident that sperm penetration through the egg coats is mediated by a very complicated mechanism. This is supported by studies on sea urchins and mammals (Hoshi, 1985): many enzymes, mostly acrosomal, presumed to be functional in sperm penetration have been identified and in some cases purified. Hence it seems likely that also in these species spermatozoa cross the egg coats by means of several lysins acting cooperatively and sequentially a s a system. In Ciona intestinalis, data about the molecules participating in sperm penetration are too preliminary to allow any conclusion a s to the presence of a lysin system in this species. It is noteworthy that, in all the ascidians-both Pleurogona and Enterogona-examined so far, chymotrypsin-like activity has been always detected in spermatozoa and related to sperm penetration through the VC. Only in Pleurogona has trypsin-like activity been found to be associated with chymotrypsin-like activity as lysins. Therefore it has been suggested that distribution of trypsin- and chymotrypsin-like enzymes as lysins in Pleurogona and Enterogona may have a n evolutionary meaning (Hoshi, 1985). The results obtained in the Enterogona Ciona intestinalis support the hypothesis of the biological and evolutionary significance of sperm lysins in ascidians. However, only when the enzymes have been obtained in a homogeneous form and their functional characterization carried out will it be possible to gain insight into the precise role of trypsin- and chymotrypsin-like enzymes in sperm penetration through the VC.
REFERENCES Casazza G, De Santis R, Pinto MR (1988): Plasma membrane glycoproteins of Cmna intestinalis spermatozoa that interact with the egg. Dev Growth Differ 30:147-158. Cotelli F, De Santis R, Rosati F, Monroy A (1980): Acrosome differentiation in the spermatogenesis of Ciona rntestrnalis. Dev Growth Differ 22:561-569. De Santis R, Jamunno G, Rosati F (1980): A study of the chorion and the follicle cells in relation to sperm-egg interaction in the ascidian Ciona i n t e s t i d i s . Dev Biol 74:490-499. De Santis R, Pinto MR, Cotelli F, Rosati F, Monroy A, DAlessio G (1983): A fucosyl-glycoprotein component with sperm receptor and sperm activating activites from the vitelline coat of Czona tntestrnalis eggs. Exp Cell Res 148:508-513. De Santis R, Pinto MR (1987): Isolation and partial characterization of a glycoprotein complex with sperm receptor activity from Ciona intestinalis ovary. Dev Growth Differ 29:617-625. De Santis R, Pinto MR (1988):The pathway of sperm-egg interaction in ascidians: Biology and chemistry. Zoo1 Sci 5:919-924. Fukumoto M (1988): Fertilization in ascidians: Apical processes and gamete fusion in Ciona intestinalis spermatozoa. J Cell Sci 89:189196. Hoshi M, Numakunai T, Sawada H (1981): Evidence for participation of sperm proteinases in fertilization of the solitary ascidian, Halocynthia roretzt: Effects of protease inhibitors. Dev Biol 86:117-121. Hoshi M (1985): Lysins. In CB Metz, A Monroy (eds): “Biology of Fertilization.” Orlando, FL: Academic Press, Vol 2, pp 431-462.
CHYMOTRYPSIN-LIKEENZYMES OF CIONA SPERM Hoshi M, De Santis R, Pinto MR, Cotelli F, Rosati F (1985):Sperm glycosidases as mediators of sperm-egg binding in the ascidians. Zoo1 Sci 265-69. Lowry OH, Rosebrough NJ, Farr AL, Randall R J (1951):Protein measurement with Folin phenol reagent. J Biol Chem 193265-275. Rosati F, De Santis R (1978):Studies on fertilization in ascidians. I Self-sterility and specific recognition between gametes of C i o m intestinalis. Exp Cell Res 112:lll-119. Rosati F, Pinto MR, Casazza G (1985):The acrosomal region of the spermatozoon of Ciona intestinalis: Its relationship with the binding to the vitelline coat of the egg. Gamete Res 11:379-389. Sawada H, Yokosawa H, Hoshi M, Ishii S (1982):Evidence for acrosinlike enzyme in sperm extract and its involvement in fertilization of the ascidian, Halocynthia roretzi. Gamete Res 5291-301. Sawada, H, Yokosawa H, Ishii S (1984):Purification and characterization of two types of trypsin-like enzymes from sperm of the as-
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cidian (Protochordata) HaloCynthia roretzi. J Biol Chem 25929002904. Sawada H, Yokosawa H, Numakunai T, Ishii S (1986):Timing of action of sperm proteases in ascidian fertilization. Experientia 42: 74-75. Usui N, Takahashi I, Hoshi M (1987):Membrane differentiation in ascidian spermatozoa.In H Mohri (ed):“New Horizon in Sperm Cell Research.” Tokyo: Japan Sci SOCPress/New York: Gordon and Breach Science Publishers, pp 265-274. Yokosawa H, Numakunai T, Murao S, Ishii S (1987):Sperm chymotrypsin-like enzymes of different inhibitor-susceptibility as lysins in ascidians. Experientia 43:925-927. Zimmerman M, Ashe B, Yurewicz EC, Pate1 G (1977):Sensitive assays for trypsin, elastase, and chymotripsin using new fluorogenic substrates. Anal Biochem 78:47-51.
