THE JOURNAL OF EXPERIMENTAL ZOOLOGY 25483-87 (1990)

Trypsin-likeHatching Protease From Mouse Embryos: Evidence for the Presence in Culture Medium and Its Enzymatic Properties HITOSHI SAWADA, KIMIE YAMAZAKI, AND MOTONORI HOSHI Department o f Life Science, Faculty of Science, Tokyo Institute of Technology, O-okayama, Meguro, Tokyo 152 (H.S.,M.H.); Mitsubishi-Kasei Institute of Life Science, Minamioya, Machida, Tokyo 194 (K.Y.), Japan ABSTRACT Enzymatic properties of a protease involved in hatching of mouse embryos were examined. A trypsin-like protease, which most efficiently hydrolyzed t-butoxycarbonyl-Leu-SerThr-Arg-4-methylcoumaryl-7-amide, was demonstrated in culture medium of mouse hatching embryos. The enzyme was strongly inhibited by diisopropyl fluorophosphate, phenylmethanesulsoybean trypsin inhibitor, and fonyl fluoride, leupeptin, antipain, Ncu-tosyl-L-lysyl-chloromethane, Trasylol, but not or weakly inhibited by p-chloromercuribenzoic acid, EDTA, E-64,pepstatin, chymostatin, and bestatin, suggesting a trypsin-like serine proteinase. The protease activity in the medium gradually elevated during the course of hatching, whereas the embryo-associated activity showed no significant change. Furthermore, pyroglutamyl-Leu-argininal, the strongest inhibitor for the enzyme among peptidyl argininals, all of which are potent trypsin inhibitors, showed the strongest inhibition toward hatching. Thus, a trypsin-like protease secreted from hatching embryos into the culture medium may participate in mouse hatching, probably as a hatching enzyme.

In many animals including echinoderms (Yasumasu, '61; Edwards et al., '77; Takeuchi et al., '79), prochordates (Hoshi and Numakunai, ,811, amphibia (Carroll and Hedrick, '74; Katagiri, '75), and teleosts (Yamagami, '72; Hagenmaier, '741, it is well known that the embryo secretes a hatching enzyme, mostly a protease, in order to hatch out of the surrounding investment. In mammals, however, little information is available on the properties of the hatching enzyme (Denker and Fritz, '79; Perona and Wassarman, '86). Mammalian ovum is covered with a noncellular investment called the zona pellucida, and cellular investments called corona radiata and cumulus oophorus. After fertilization, the embryos develop to the stage of blastocyst, in which the blastomeres are classified into two groups called inner cell mass (ICM) and trophectoderm. The former cells give rise to the fetus, and the latter form the extraembryonic membranes and embryonic placenta. The blastocysts thus develOped expand and make a in the 'Ona pellucida, through which the embryos pass. Since only the hatched embryo can implant to the uterus, the hatching process is essential for successful pregnancy. The mouse zona pellucida is composed of three u

0 1990 WILEY-LISS, INC.

glycoproteins called ZP1-3 (Wassarman, '88). Therefore, it seems likely that a protease released from o r associated with the hatching embryo may be one of the hatching enzymes. Actually, possible involvement of a trypsin-like protease in mouse hatching has already been suggested by using protease inhibitors (Dabich, '81; Y amazaki et al., '85; Perona and Wassarman, '86). In order t o elucidate the properties and the mode of action of mammalian hatching enzyme, we attempted to identify and characterize the trypsin-like protease participating in mouse blastocyst hatch. In this paper, we present evidence that a t-butoxycarbonyl (Boc)-Leu-Ser-Thr-Arg-4methylcoumaryl-7-amide (MCA)-hydrolyzingenzyme detected in hatching medium plays a key role in mouse blastocyst hatch, most probably as a hatching enzyme.

MATERIALS AND METHODS Collection and culture of embryos ~~~~l~ mice (ICR:slc; Shizuoka Laboratory Animal Center, Shizuoka, Japan) at 7 weeks of age were injected with 7 IU pregnant mare's Received March 23, 1989; revision accepted July 20, 1989. This paper is dedicated to the late Dr. Yoshihiro Kato of MitsubishiKasei Institute of Life Science.

84

H. SAWADA ET AL.

serum gonadotropin (PMSG; Teikoku Zoki, Tokyo, Japan). Forty-eight hours later, 7 IU human chorionic gonadotropin (hCG; Teikoku Zoki) was intraperitoneally administrated t o the animals, followed by mating overnight. At 88 h after hCG injection, embryos were collected by flushing dissected oviducts and uteri of plugged female mice using a-MEM (GIBCO, No. 4101900) supplemented with 3 mg/ml bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, MO). Approximately 20-40 embryos were cultured in vitro in a micro-droplet (0.5 pl/embryo) of the above medium under paraffin oil at 37°C in a humidified atmosphere of 5% COz in air.

cals Ltd., Cambridge, England) were purchased from the Peptide Institute.

Inhibition toward the enzyme activity Inhibitory potencies of various protease inhibitors toward the enzyme were determined by measuring the residual protease activity of the incubation with each inhibitor. The inhibitors used are diisopropyl fluorophosphate (DFP; Sigma), phenylmethanesulfonyl fluoride (PMSF; Sigma), p-chloromercuribenzoic acid (Sigma), E-64 (Peptide Institute), EDTA (Dojin Kagaku Co., Kumamoto, Japan), pepstatin (Peptide Institute), leupeptin (gift from Dr. T. Aoyagi of Institute of Microbial Chemistry), antipain (Dr. T. Aoyagi), Leupeptin analogs chymostatin (Dr. T. Aoyagi), bestatin (Dr. T. AoLeupeptin analogs (peptidyl-argininals), semi- yagi), Na-tosyl-L-lysyl-chloromethane (TLCK, synthesized as previously described (Someno and Sigma), soybean trypsin inhibitor (Type I-s, Ishii, '86; Saino et al., ,881, were provided by Dr. Sigma), and Trasylol (Bayer AG, WuppertalT. Someno of Nippon Kayaku Co. The analogs Elberfeld, FRG). used are pyroglutamyl-Leu-m-argininal, acetylInhibition of mouse blastocyst hatch by Phe-Leu-argininal, and benzyloxycarbonyl-Proleupeptin analogs argininal. Unless otherwise noted, all the amino Early blastocysts collected at 88 h after hCG acid residues and argininal moiety of the analogs were L-form. In the case of pyroghtamyl-Leu-DL- injection were cultured in vitro in a-MEM conargininal, its concentration was determined by taining 3 mgiml BSA in the presence or absence assuming that only L-form is inhibitory. All the of leupeptin analogs. To count the percentage of above analogs are known to be potent trypsin in- hatching, the embryos were observed under a mihibitors capable of binding covalently to the ac- croscope at 95,111, 115,119, and 135 h after hCG tive-site serine residue of trypsin-like protease at injection. The embryos, at least a part of which emerged from zona pellucida, were counted as their C-terminal aldehyde group. hatched embryos. Enzyme preparation RESULTS The culture medium of the embryos, in which the hatching ratio was approximately 50%, was Properties of trypsin-like protease in culture medium of mouse hatching embryos frozen at - 80 or - 30°C until use. The frozen medium was thawed and used as a crude enzyme It is known that embryos from various animals secrete the hatching enzyme and that mouse preparation. hatching is blocked by trypsin inhibitors such as Enzyme assay leupeptin, antipain, and TLCK (Yamazaki et al., The protease activity was fluorophotometrically '85; Perona and Wassarman, '86). Thus, we atdetermined according to the method described tempted t o examine whether a trypsin-like propreviously (Sawada et al., '82). One-half milliliter tease is present in the culture medium of hatchof 0.1 mM Boc-Leu-Ser-Thr-Arg-MCA(Peptide In- ing embryos by using peptidyl-Arg (or Lys)-MCA stitute, Inc., Osaka, Japan) in 50 mM TrisiHCl as substrates. As shown in Table 1, the activity (pH 9.0) was preincubated at 37°C. The enzymatic toward Boc-Leu-Ser-Thr-Arg-MCA was highest, reaction was started by adding 3-5 pl of the en- and those toward Boc-Leu-Gly-Arg-MCA, Boczyme solution, and the initial velocity was deter- Phe-Ser-Arg-MCA, and glutaryl-Gly-Arg-MCA mined at 37°C. Under the conditions tested, the followed in this order, suggesting the presence of activity was linearly increased by increasing the a trypsin-like protease. On the substrate specificamount of the enzyme, and, also, the amount of ity, the enzyme preferred Thr, Ser, or Gly residue the reaction product, AMC, was linearly in- at the Pz position (for designation of subsite, see creased. The other substrates except tosyl-Gly- Schechter and Berger, '67), though the specificity Pro-Lys-MCA (Cambridge Research Biochemi- was not so narrow. At the PI position, strong pref-

85

HATCHING ENZYME OF MOUSE EMBRYOS TABLE 1. Substrate specificity of trypsin-like protease in culture medium of mouse hatching embryos Substrate

p5

p4

p3

p2

Activity (%)

p1

Boc-Leu- Ser-Thr-Arg- MCA Boc-Leu-Gly- Arg- MCA Boc-Phe- Ser-Arg- MCA Glt-Gly-Arg-MCA Pro-Phe- Arg- MCA Tos- Gly- Pro- Lys- MCA Boc- Val- Pro- Arg- MCA Boc-Glu- LYS-LYS-MCA Z-Phe- Arg- MCA Bz- Arg- MCA

100 96

87 77 72 60 44 43 40 24

Alteration of trypsin-like protease activity associated to embryos and released in medium during the hatching process The activities of trypsin-like enzymes both associated t o and liberated from embryos were determined during the course of hatching. As depicted in Figure 1,the activity in the medium was gradually elevated during this process, whereas that of the embryo was not significantly changed. These results suggest that the trypsin-like protease may be gradually secreted from embryos into the culture medium during the hatching process.

The activity was determined a t 37"C, pH 9.0, by using each fluorogenic substrate a t 0.1 mM, and expressed as % activity by defining the maximum value as 100%. Abbreviations: Boc, t-butoxycarbonyl; MCA, methylcoumaryl-7-amide; Glt, glutaryl; Tos, tosyl; -Z, benzyloxycarbonyl; Bz, Benzyol.

Involvement of the trypsin-like protease in hatching In order t o confirm the participation of Boc-LeuSer-Thr-Arg-MCA-hydrolyzing enzyme in the erence of Arg over Lys residue, which was re- hatching process, we compared the inhibitory ported in rabbit blastocyst trypsin-like proteinase abilities of leupeptin and its analogs toward the (Denker and Fritz, '79), was not found in the protease activity (Table 3) with those toward mouse enzyme. hatching (Fig. 2). Pyroglutamyl-Leu-argininal Inhibitor spectrum of the protease was then ex- was found to be the strongest inhibitor for the amined by using the best substrate, Boc-Leu-Ser- protease activity. Acetyl-Phe-Leu-argininal, leuThr-Arg-MCA. The enzyme was strongly in- peptin (acetyl-Leu-Leu-argininal), and benzylhibited by DFP, PMSF, leupeptin, antipain, oxycarbonyl-Pro-argininal showed much weaker TLCK, soybean trypsin inhibitor, and Trasylol, inhibition against the activity. Similarly, in the but not or weakly inhibited by p-chloromercuri- hatching process, pyroglutamyl-Leu-argininal benzoic acid, E-64, EDTA, pepstatin, chymo- showed the most powerful inhibition at 2 mM (see statin, and bestatin (Table 2). This suggests that Fig. 2). The other argininal derivatives including the protease concerned is indeed a serine protein- leupeptin exhibited no or only weak inhibition at ase, rather than cysteine, aspartic, or metallo- the same concentration. Furthermore, all the proteinase. The activity was potently inhibited by in- above analogs did not affect normal development hibitors toward hatching, leupeptin, antipain, and at the concentrations used, suggesting that the TLCK (Yamazaki et al., '851, suggesting that this inhibition of hatching by the analogs was not due protease participates in the mouse blastocyst hatch. t o the possible side effects of the analogs. These TABLE 2 . Effects of protease inhibitors on trypsin-like protease in culture medium of mouse hatching embryos Inhibitor None DFP PMSF p-Chloromercuribenzoic acid E-64 EDTA Pepstatin Leupeptin Antipain Chymostatin Bestatin TLCK Soybean trypsin inhibitor Trasylol "A slight activation was observed.

Concentration

Inhibition (%)

-

0 75 50 Oa 11 0" 0" 100 100 0 0 52 61 100

1 mM 1 mM 1mM 50 p.g/ml 1 mM 50 Fgiml 50 Fgirnl 50 Fg/ml 50 Fgirnl 50 Fg/rnl 100 FM 100 Fg/ml 10 pg/ml

H. SAWADA ET AL.

86

-

100

I

T

&

/

s

'

/-

.

L

u)

b

50

E w

E 0

c.

2

0

105 117 -110 -120

93 -96 I

I

I

129 -135 I

J

Y

90 100 110 120 130 140 Time after hCG Injection, h Fig. 1. Alteration of activities of Boc-Leu-Ser-Thr-ArgMCA-hydrolyzing protease associated with or secreted from embryos during the course of mouse blastocyst hatch. Embryos cultured in vitro were withdrawn at 93-96, 105-110, 117-120, and 129-135 h after hCG injection and suspended in the same volume of the fresh medium. After freezing and thawing, the embryo suspension was homogenated with a glass micro-homogenizer and used as an enzyme preparation. The activity in the corresponding hatching medium was assayed after freezing and thawing. Embryo-associated activity (open column), activity in the medium (dotted column), % hatching (dashed line). The value in each column was obtained from four to six experimental data by using around 100 embryos as a total and expressed as a mean standard error.

*

TABLE 3. Effects of leupeptin and its analogs on trypsin-like protease i n culture medium of mouse hatchine embryos

" lo0 110 120 130 140 Time after hCG Injection, h Fig. 2. Effects of leupeptin and its analogs on mouse blastocysts hatch. Mouse embryos obtained at 88 h after hCG injection were cultured in vitro in the presence or absence of inhibitor at 2 mM. At an appropriate time, hatching ratio was determined by counting the shedding or shedded blastocysts under a photomicroscope. The embryos with at least a part of themselves emerged were counted as hatched. Control (C--+J), bezyloxycarbonyl-Pro-arginind(A.......A), acetyl-PheLeu-argininal (&..-A), acetyl-Leu-leu-argininal (leupeptin, t.-o), pyroglutamyl-Leu-argininal (G---0).

associated protease activity (Wassarman et al., '84; Perona and Wassarman, '86). However, since several intracellular proteinases susceptible to Leupeptin analog" 8 Activity the hatching inhibitors would be present in embryos, whether the embryo-associated proteinNone 100 ase(s) participates in hatching remains t o be de1 F'yroglutamyl-Leu-argininal bated. In contrast, our protease is most probably a Acetyl-Phe-Leu-argininal 48 Acetyl-Leu-Leu-argininal (leupeptin) 63 hatching protease, because the protease(s) seBenzvloxvcarbonvl-Pro-areininal 72 creted from embryos during the course of hatching could directly attack the zona pellucida. Fur'Concentration of leupeptin or its analog was 1 PM, thermore, a good correlation between inhibitory results indicate that this trypsin-like protease abilities of argininal derivatives toward the enplays an important role in the mouse blastocyst zyme activity and hatching strongly indicates the involvement of our protease in the mouse hatchhatch, most probably as a hatching enzyme. ing process. DISCUSSION Inhibitory potencies of leupeptin analogs toThe present paper demonstrates the presence of ward hatching was much weaker than those toa trypsin-like hatching protease in the hatching ward the protease activity. However, these reliquid (culture medium) of mouse embryos. It has sults are not at variance with our assumption of already been suggested that the embryo-associ- the involvement of the protease in hatching, beated trypsin-like protease (strypsin) may partici- cause their inhibitory abilities against hatching pate in hatching of mouse blastocysts, because are greatly affected by the concentration and K, hatching inhibitors such as leupeptin, antipain, of the physiological substrate (Sawada et al., '84). The level of protease activity in the culture meand soybean trypsin inhibitor block the embryo-

HATCHING ENZYME OF MOUSE EMBRYOS

dium was relatively high (2.9 pmol/min/ml) even before the hatching, and the increase in activity after hatching was only about two-fold. These results are not at variance with our preliminary findings that the hatching protease functions just before the shedding of the embryos as revealed by the pulse treatment experiment of TLCK during the course of hatching (to be published). In contrast t o the secreted protease activity, the embryo-associated activity was not significantly changed during hatching. This result does not necessarily suggest that a level of the hatching protease in embryo is constant, since Boc-LeuSer-Thr-Arg-MCA could be digested by several intracellular proteinases. Mechanical force or movement may also be necessary for successful hatching, since the embryos shed the zona pellucida from a single small hole. These observations together with our present findings lead us t o propose that a trypsin-like protease secreted from or associated with the embryos or both does not completely dissolve but partially digests or weakens the zona pellucida at a stage before hatching and thus enables the embryos to shed the covering. Recently, the amino acid sequence of Z P 3 was estimated from the data of its cDNA sequence (Ringuette et al., ’88). Interestingly, about 26% of the amino acid sequence around Arg residue showed Thr(or Ser)-Arg-X sequence, suggesting that ZP3 may be very susceptible to the presently found protease. Purification of the enzyme, identification of its physiological substrate, and its localization in blastocysts are currently under investigation in our laboratory.

ACKNOWLEDGMENTS We are grateful t o the late Dr. Yoshihiro Kato of the Mitsubishi-Kasei Institute of Life Science for his helpful discussion and encouragement throughout this work. We are also indebted t o Miss Eriko Hojo for her skillful technical assistance. We thank Dr. Tetsuya Someno of the Nippon Kayaku Co. and Dr. Takaaki Aoyagi of the Institute of Microbial Chemistry for generous gifts of protease inhibitors. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan to H.S. and M.H. and from the Uehara Memorial Foundation to H.S. LITERATURE CITED Carroll, E.J., and J.L. Hedrick (1974) Hatching in the toad Xenopus laeuis: Morphological events and evidence for a hatching enzyme. Dev. Biol., 38:l-13.

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Dabich, D. (1981) Impairment of mouse blastocyst hatching by naturally occurring serine proteinase inhibitors. Fed. Proc., 40:1808. Denker, H.-W., and H. Fritz (1979) Enzymatic characterization of rabbit blastocyst proteinase with synthetic substrates of trypsin-like enzymes. Hoppe-Seyler’s Z. Physiol. Chem., 360: 107-1 13. Edwards, B.F., W.R. Allen, and D. Barrett (1977) Purification and partial characterization of hatching protease of the sea urchin, Strongylocentrotus purpuratus. Arch. Biochem. Biophys., 182:696-704. Hagenmaier, H.E. (1974) The hatching process in fish embryos. V. Characterization of hatching protease (chorionase) from the perivitelline fluid of the rainbow trout, Salmo gaidneri Rich as a metalloenzyme. Wilhelm Roux’ Arch., 175:157-162. Hoshi, M., and T. Numakunai (1981) Hatching enzyme of the solitary ascidian, Hulocynthia roretzi. Acta Embryol. Morphol. Exp., New Series, 2:163-169. Katagiri, Ch. (1975) Properties of the hatching enzyme from frog embryos. J. Exp. Zool., 193:109-118. Perona, R.M., and P.M. Wassarman (1986) Mouse blastocysts hatch i n uitro by using a trypsin-like proteinase associated with cells of mural trophectoderm. Dev. Biol., 114:42-52. Ringuette, M.J., M.E. Chamberlin, A.W. Baur, D.A. Sobieski, and J . Dean (1988) Molecular analysis of cDNA coding for ZP3, a sperm binding protein of the mouse zona pellucida. Dev. Biol., 127:287-295. Saino, T., T. Someno, S.Ishii, T. Aoyagi, and H. Umezawa (1988) Protease-inhibitory activities of leupeptin analogues. J . Antibiot. (Tokyo), 41 :220-225. Sawada, H., H. Yokosawa, M. Hoshi, and S. Ishii (1982) Evidence for acrosin-like enzyme in sperm extract and its involvement in fertilization of the ascidian, Hulocynthiu roretzi. Gamete Res., 5:291-301. Sawada, H., H. Yokosawa, T. Someno, T. Saino, and S.Ishii (1984) Evidence for the participation of two sperm proteases, spermosin and acrosin, in fertilization of the ascidian, Halocynthia roretzi: Inhibitory effects of leupeptin analogs on enzyme activities and fertilization. Dev. Biol., 105:246-249. Schechter, I., and A. Berger (1967) On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun., 27:157-162. Someno, T., and S. Ishii (1986) A simple method for semisynthesis of peptidyl argininals as potent inhibitors of trypsinlike proteases. Chem. Pharm. Bull., 34:1748-1754. Takeuchi, K., H. Yokosawa, and M. Hoshi (1979) Purification and characterization of hatching enzyme of StrongyZocentrotus intermedius. Eur. J. Biochem., 100:257-265. Wassarman, P.M. (1988) Zona pellucida glycoproteins. Annu. Rev. Biochem., 57:415-442. Wassarman, P.M., J.M. Greve, R.M. Perona, R.J . Roller, and G.S. Salzmann (1984) How mouse eggs put on and take off their extracellular coat. In: Molecular Biology of Development. E.H. Davidson and R.A. Firtel, eds. Alan R. Liss, Inc., New York, pp. 213-225. Yamagami, K. (1972) Isolation of choriolytic enzyme (hatching enzyme) of the teleost, Oryzias latipes. Dev. Biol., 29:343-348. Yamazaki, K., Y. Kato, and M. Hoshi (1985) Protease inhibitors block the zona shedding of mouse embryos in uitro. Dev. Growth Differ., 27:491. Yasumasu, I. (1961) Crystallization of hatching enzyme of the sea urchin, Anthocidaris crussispina. Sci. Papers Coll. Gen. Educ. Univ. Tokyo, 11:275-280.