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Arylsulfatase of sea urchin sperm
DEVELOPMENTAL
BIOLOGY
74, 343-350 (1980)
Arylsulfatase
of Sea Urchin Sperm
2. Arylsulfatase
as a Lysin of Sea Urchins’
MOTONORI Biochemical
Laboratory,
Institute
HOSHI AND TSUNEO
of Low Temperature
Received February
MORIYA
Science, Hokkaido
University,
Sapporo
060, Japan
15, 1979; accepted in revised form June 22, 1979
An arylsulfatase is defined as a lysin of sea urchin sperm from the following evidences. (1) The activity is detected in the sperm of all the sea urchins investigated, and the activity is partially liberated from the cells after the acrosome reaction (Moriya and Hoshi, 1979a). (2) Fertilization is completely inhibited in the presence of 40 n&fp-nitrophenyl sulfate, which is an artificial substrate of arylsulfatase, but is not inhibited by p-nitrophenyl phosphate at the same concentration. (3) The inhibitory effect ofp-nitrophenyl sulfate on fertilization is remarkably diminished by pretreatment of eggs with arylsulfatase before insemination. (4) Sperm arylsulfatase as well as limpet arylsulfatase appear to digest the vitelline coat and jelly coat. INTRODUCTION
Since Yamane (1935) observed dispersal of the egg investments by an extract of mammalian sperm, lysins have been found in such animals as mammals, reptiles, amphibians, tunicates, hemichordates, crustaceans, annelids, and mollusks except for cephalopods (for a review, see Dan, 1967). In echinoderms, Isaka et al. (1966) isolated a jelly-dispersing enzyme from a homogenate of sea urchin sperm, but it is not known whether this enzyme is actually involved in the penetration of sperm through the jelly coat. Levine et al. (1978) have recently found an acrosin-like enzyme in the sperm of sea urchin, which seems to be involved in the fertilization process. The investments of sea urchin eggs, namely, the vitelline coat and jelly coat, comprise protein, carbohydrate, and sulfate (Glabe and ’ This paper is dedicated to Professor Juro Ishida on the occasion of his 70th birthday. A preliminary report of this work was presented at the 8th International Congress of the International Society of Developmental Biologists at Tokyo, 1977. This work was supported in part by grants from the Ministry of Education of Japan and from the Itoh Science Foundation.
Vacquier, 1977; Tayler, 1956). Fucose-4-sulfate (Ishihara et al., 1973) and sialic acids (Isaka et al., 1970) have been reported as main sugar components of the jelly. We expected, therefore, that sulfatase, sialidase, other glycosidases, and protease are possible candidates for the lysin(s) of sea urchins. We have recently reported that an arylsulfatase exists in both the spermatozoa and the seminal plasma of the sea urchins examined (Moriya and Hoshi, 1979a, b) and that about 20% of the total activity is released from the spermatozoa when the sperm undergo acrosome reaction. These observations have led us to assume that arylsulfatase is the lysin, or at least one of the lysins, in sea urchins. In order to define arylsulfatase as a lysin, it is essential to examine the following points: (i) if the enzyme digests the vitelline coat and/or jelly coat; and (ii) if fertilization is blocked when the enzyme of sperm is inhibited in some way, for example, by the addition of an artificial substrate such asp-nitrophenyl sulfate. We show here that the sperm arylsulfatase fulfills these requirements for the lysin. 343 0012.1600/80/020343-08$02.00/0 (‘opyright All rights
0 1980 by Academic Press. of reproduction in any form
11~ reserved.
344
DEVELOPMENTAL BIOLOGY MATERIALS
AND
METHODS
VOLUME 74,198O
egg suspension to make the jelly coat visible, if necessary. Electron microscopy. The eggs were fixed for 60 min in a solution of 2.5% glutaraldehyde in ASW at 0°C. They were washed twice with ASW and were postfixed for 40 min in 2% osmium tetroxide in ASW. After dehydration through a graded series of ethanol, the eggs were infiltrated and embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate (Venable and Coggeshall, 1965) and examined with an electron microscope, JEM1OOL(JEOL, LTD).
Animals. The sea urchins Hemicentrotus pulcherrimus and Strongylocentrotus intermedius were used. Fertilization assay. Experiments were performed at 18-20°C unless otherwise specified. Eggs and sperm were collected by introducing 0.5 M KC1 into the coelom. Dry sperm were passed through a nylon mesh and diluted to 104-fold with seawater at 0°C just before insemination. Eggs were washed three times with filtered seawater. For insemination, 50 ~1 of the diluted sperm suspension was added to 0.5 ml of an egg RESULTS suspension containing about 200 eggs in the Effects of p-nitrophenyl esters on fertilipresence or absence of test reagents. Prior to insemination, the sperm concentration zation. In order to provide evidence that was adjusted by measuring the turbidity to sperm arylsulfatase is responsible for fergive a critical concentration which is re- tilization in sea urchins, p-nitrophenyl sulquired to fertilize 100% of the eggs. The fate, an artificial substrate of arylsulfatase critical concentration was usually between (Roy, 1971), was added to the test egg susOD 0.05 and 0.10 at 360 run. Eggs were pension before insemination. An artificial washed twice with fresh seawater 15 min substrate such as p-nitrophenyl sulfate is after insemination and were raised in sea- naturally assumed to inhibit the enzyme water. The fertilization ratios were deter- action on a native substrate in a competimined on the basis of cleavage as well as tive fashion. As demonstrated in Figs. 1 and 2, no eggs were fertilized in the presence of elevation of the fertilization envelope. Surface modification of eggs with aryl- potassium p-nitrophenyl sulfate (Boehrinsulfatase. The following preparations of ar- ger Mannheim) at 40 milf, but spermatozoa ylsulfatase were used: limpet arylsulfatase (Type IV, Sigma Chemical), partially purified arylsulfatase from the sperm of Strongylocentrotus intermedius (Moriya and Hoshi, 1979b), and a crude preparation from Hemicentrotus pulcherrimus (supernatant at 105,OOOg of the sperm wash for 1 hr). Egg suspensions (about 250 eggs, 0.5 ml) in ASW2 containing various concentrations of the enzyme were incubated at 20°C for 40 min with occasional shaking. The lo@ 10-5 10-4 10-l 10-2 IU’ IO0 eggs were washed twice with ASW and p- Nitrophenyl sulfate CM) most of them were used for fertilization FIG. 1. Effect of p-nitrophenyl sulfate on the ferassay and some for inspection with both a light microscope and an electron micro- tilization of the sea urchin, H. pulcherrimus. An aliquot (50 ~1) of sperm suspension was added to 0.5 ml scope. A bit of India ink was added to the of an egg suspension containing about 200 eggs in the ’ Abbreviations used: ASW, artificial 8.2; p-NPS, p-nitrophenyl sulfate.
seawater, pH
presence of various concentrations of p-nitrophenyl sulfate. Fertility was expressed in the ratio of the number of cleaved eggs to the total.
HOSHI
AND
MORIYA
AT-$. sulfatase Is a Sea Urchin Lysin
sulfate (40 FIG. 2. Eggs of H. pulcherrimus inseminated in the absence (A) or presence (B) of p-nitrophenyl m&f). The photographs were taken 5 min after inset nination. x 140. Inset shows at a higher magnification (X 220) that sperm remain in jelly coat apart from the cell even 15 min after insemination.
appeared to swim actively around the eggs. In the presence of this compound, a few spermatozoa appeared to reach the vitelline coat within 15 min after insemination, but most spermatozoa still stayed apart from the egg surface (Fig. 2). To the contrary, fertilization was not affected by p-nitrophenyl phosphate (Nakarai Chemicals) at 40 mit4. In the presence of p-nitrophenol (Nakarai Chemicals) at 1.5 n-J4 or more, the motility of spermatozoa was considerably reduced and thus failed to fertilize eggs, but the fertility was not affected under 0.15 n-&f. Because of such toxicity of p-nitrophenol on sperm and possible contamination ofp-nitrophenol in commercialp-nitrophenyl sulfate, we determined spectrophotometrically (at 400 nm) the concentration of p-nitrophenol in 40 mit4 of p-nitrophenyl sulfate. The result indicated that the concentration was lower than 0.125 m&f. When eggs were treated with the arylsul-
fatase prior to insemination, the inhibitory effect of p-nitrophenyl sulfate on fertilization was remarkably diminished. As a typical example, the fertilization ratios of the eggs of H. pulcherrimus pretreated with limpet enzyme are summarized in Table 1. Surface modification of eggs by arylsulfatase. Since we observed, as described in the previous section, that fertilization was not retarded by p-nitrophenyl sulfate when the eggs were pretreated with arylsulfatase, we examined morphologically what happened in the jelly and vitelline coat after treatment with arylsulfatase. As shown in Fig. 3, the jelly coat was completely dissolved by limpet arylsulfatase and this digestion was considerably delayed by pnitrophenyl sulfate. The vitelline coat was also seriously affected by this enzyme (Fig. 4). Figure 4 shows electron micrographs of unfertilized eggs after the treatment with the arylsulfatase preparations from limpet and sea
346
DEVELOPMENTAL BIOLOGY TABLE
I
FERTILIZATION IN THE PRESENCE OF pNITROPHENYL SULFATE OF EGGS PRETREATED WITH ARYLSULFATASE”
Concentra- Concentra- Elevation of tion of en- tion of p- fertilization NPS membrane zyme (uni&/ml) (mM) (%) 0 0 100 100 0.4 0 100 0.8 0 0 4.0 0 0 8.0 0 7 0 40 4 0.4 40 23 0.8 40 0 4.0 40 0 8.0 40
Cleaa;ge
VOLUME 74,198O
projections. The eggs pretreated with sulfatase developed quite normally and embryos “hatched” out of the capsule at the same time when normal embryos leave the fertilization envelopes. DISCUSSION
100 loo 100 100 100 7
The present study has clearly revealed that the fertilization of sea urchin eggs is completely blocked by p-nitrophenyl sulfate, an artificial substrate of arylsulfatase (Roy, 1971; Moriya and Ho&i, 1979b), 8 28 while the motility of sperm is not affected 38 appreciably by this compound, and that the 38 pretreatment of eggswith arylsulfatase prea Eggs (H. pulcherrimus) were pretreated with limvents the eggs from such a blocking of pet arylsulfatase at the various concentrations listed fertilization (Figs. 1 and 2 and Table 1). at 20°C for 40 min. After being washed twice with This inhibition of fertilization by p-nitroASW, the eggs were inseminated in the presence or phenyl sulfate is not due to contamination absence of p-nitrophenyl sulfate. ’ One unit of the enzyme hydrolyzed 1.0pmole ofpof p-nitrophenol, because p-nitrophenol exnitrophenyl sulfate per hour at 37°C in a reaction hibited its toxicity on sperm only when the mixture consisting of 0.5 ml of 0.2 M sodium acetate concentration was higher than 1.5 miV, buffer, pH 5.0,0.4 ml of 6.25 mdfp-nitrophenyl sulfate, which was far higher than the actual conand 0.1 ml of enzyme solution in 0.2% sodium chloride. centration (0.125 miU) of this compound contaminated in the p-nitrophenyl sulfate urchin sperm. After the treatment with solution used. In addition, the acrosome either preparation, microvilli on the egg reaction induced by jelly water was not surface decreased in number and the vitel- affected by the addition of p-nitrophenyl line coat disappeared from microvilli, but sulfate (unpublished observation), revealstill remained in the open spaces among ing that the block of fertilization by this microvilli. Except for these morphological chemical is not due to blocking of the acchanges, no appreciable effects were ob- rosome reaction. served in the egg surface after the treatIn the absence of p-nitrophenyl sulfate, ment. needless to say, most spermatozoa reached The eggs which had been treated with the egg cells promptly. When this comthe enzyme at higher concentrations before pound was present, to the contrary, most insemination were monospermically fertil- spermatozoa appeared to remain in the jelly ized, but they were unable to elevate the coat (Fig. 2). We also clearly demonstrated normal fertilization envelopes; instead, only that the jelly coat was considerably divery thin capsules were formed at a close gested by an arylsulfatase (Fig. 3). These distance from the egg surface (Fig. 5 and observations strongly indicate that arylsulTable 1). The capsule was hardly visible fatase promotes the penetration of sperwith the light microscope, but it was de- matozoa across the jelly coat, and agree tected under the electron microscope. As with reports that the jelly coat of sea urshown in Fig. 6, the capsule exhibits a chins consists of sulfated glycoproteins (Israther
fluffy
structure,
whereas
the normal
fertilization envelope has a structure in consistent thickness with uniformly distributed
aka, 1970; Ishihara,
1973). The vitelline
coat
as well as the jelly coat is generally assumed to be comprised of sulfated glycoproteins
Fro 3. Digestion of the egg jelly with limpet arylsulfatase. Eggs of S. intermedius were allowed to stand at room temperature for various periods as indicated in ASW (A) or ASW containing 4 units/ml arylsulfatase (B) or ASW containing both 4 units/ml arylsulfatase and 40 tip-nitrophenyl sulfate (C). In the control experiment (A), the egg cells were separated from each other by the jelly layer surrounding each egg for 20 hr or more. In the presence of sulfatase (B), the eggs were dispersed at the beginning, and the eggs came gradually into contact with each other, and arranged in hexagonal array after 20 hr, revealing the complete digestion of the jelly by the sulfatase. In the presence of both sulfa&se and p-nitrophenyl sulfate (C), the eggs were separated from each other, even after 5 hr. After 20 hr, the eggs became closer to each other, but the typical hexagonal array was not formed.
(Glabe and Vacquier, 1977). The observation that arylsulfatases prepared from limpet and sperm of two sea urchins (Strongylocentrotus and Hemicentrotus) had a capacity to digest the vitelline coats (Fig. 4) provides additional evidence to support the above assumption, and coincides with our previous finding that sulfate is also present in the fertilization envelope (Hoshi and Nagai, 1967). In this connection, it is interesting that the microvilli of the egg surface disappear after digestion of the vitelline layer with arylsulfatase (Fig. 4), implying that microvilli need the vitelline coat
to retain their structure. Since the vitelline coat is easily digested by proteases, it might be possible to attribute the digestion of the vitelline coat by the arylsulfatase preparations to the action of a contaminating protease (but not to that of arylsulfatase itself). However, we could not detect any activity of chymotrypsin-like or trypsin-like proteases in the preparation of sperm arylsulfatase which digested the vitelline coat as well as limpet arylsulfatase (unpublished data). A thin capsule was formed in the fertilized eggs, instead of the normal fertilization
FIG. 4. Electron micrographs of sea urchin egg surface (H. pulcherrimus). (A) Intact egg; microvilli are seen on the egg surface, and surrounded by the vitelline coat. (B and C) The eggs were treated with 4 units/ml of limpet sulfatase and 18.8 units/ml of sperm sulfatase for 40 min at 2O”C, respectively. The number of microviHi on the egg surface remarkably decreases. The vitelline coat disappears from the microvilli, but still remains in the open space between microvilli. VC, vitelline coat. PM, plasma membrane. A-l, B-l, C-l, x 9300. A-2, B-Z, c-z, x 35,000.
FIG. 5. Fertilized eggs of H. pulcherrimus with or without sulfatase pretreatment. (A) Control. (B) Eggs were treated with 4 units/ml of limpet sulfatase for 40 min at 20°C before insemination. x 270. 348
HOSHI AND MORIYA
Arylsulfatase
Is a Sea Urchin Lysin
FIG. 6. Electron micrographs of the surface of fertilized eggs of H. pulcherrimus. (A) Normal fertilization envelope (FE). The fertilization envelope at a close distance from the egg surface is due to an artifact by fixation. (B) The egg was treated with limpet sulfatase (4 units/ml) for 40 min at 20°C before insemination. The egg elevated no fertilization envelope; instead, a capsule (Cap) was formed (see Fig. 5). X 11,ooO.
envelope, when the eggs were pretreated with sulfatase before insemination (Figs. 5 and 6). The normal fertilization envelope is believed to be made from two sources, the contents of cortical granules and the vitelline coat (Endo, 1961). It is therefore assumed that the capsule is formed of the partially digested vitelline coat and the contents of cortical granules. It should be pointed out, however, that the digestion of the vitelline coat by sperm in the natural fertilization process must be strictly local, whereas the whole surface of the vitelline coat undergoes digestion when the eggs are exposed to sulfatase action, and this may explain the formation of a capsule which is an artificial structure. Monospermical fertilization of the sulfatase-treated eggs (Fig. 5) reveals that the system preventing the eggs from poly-
spermy was not affected so seriously by this enzyme, and implies that this system, if present, is not in the vitelline coat itself but probably in the plasma membrane. The data presented in this study reveal that sperm arylsulfatase of sea urchins promotes the penetration of sperm across the jelly and vitelline coat, and lead us to conclude that this enzyme plays a role as a lysin in the natural fertilization process in these animals. The authors wish to express their gratitude to the Director and the staff of the Akkeshi Marine Biological Station, Hokkaido University, where part of the study was carried out. They also would like to give thanks to the staff of the Laboratory of Developmental Biology, Waseda University, for their generous supply of animals. REFERENCES
DAN, J. C. (1967). Acrosome reaction and lysins. In
350
DEVELOPMENTALBIOL~3GY
“Fertilization, Comparative Morphology, Biochemistry, and Immunology” (C. B. Metz and A. Monroy, eds.), Vol. 1, pp. 237-293, Academic Press, New York. ENDO, Y. (1961). Changes in the cortical layer of sea urchin eggs at fertilization as studied with the electron microscope. Exp. Cell Res. 25, 383-397. GLABE, C. G., and VACQUIER,V. D. (1977). Isolation and characterization of the vitelline layer of sea urchin eggs. J. Cell Biol. 76,410-421. HOSHI, M., and NAGAI, K. (1967). Abstracts of 7th Int. Congr. Biochem., Vol. V, p. 965. ISAKA, W., KANATANI, H., and SUZUKI,N. (1966). Jelly dispersing enzyme obtained from spermatozoa of sea urchin, Anthocidaris crassispina. Exp. Cell Res. 44,66-72. ISAKA, S., HO~TA, K., and KUROKAWA,M. (1970). Jelly coat substances of sea urchin eggs. 1. Sperm isoagglutination and sialopolysaccharide in the jelly. Exp. Cell Res. 56,37-42. ISHIHARA, K., OGURI, K., and TANIGUCHI, H. (1973). Isolation and characterization of fucose sulfate from jelly coat glycoprotein of sea urchin egg. Biochim. Biophys. Acta 320,628-634. LAVINE, A. E., WALSH, K. A., and FODOR,E. J. B.
VOLUME ?4,1980
(1978). Evidence of an acrocin-like enzyme in sea urchin sperm. Develop. Biol. 63,299-306. MORIYA, T., and HOSHI, M. (1979a). Arylsulfatase of sea urchin sperm. 1. Distribution of arylsulfatase in the gonads and gametes of echinoderms. Comp. Biochem. Physiol. (in press). MORIYA, T., and HOSHI, M. (1979b). Characterization and partial purification of arylsulfatase from the sperm of sea urchin, Strongylocentrotus intermedius (submitted for publication). ROY, A. B. (1971). The hydrolysis of sulfate esters. In “The Enzyme” (P. D. Boyer, ed.), Vol. 5, pp. 21-41. Academic Press, New York. TAYLER, A. (1956). Physico-chemical properties of the fertilizins of the sea urchin, Arbaciapunctulata and the sand dollar, Echinarachnius parma. Exp. Cell Res. 10,377-383. VENABLE, J. H., and COGGESHAL,R. (1965). A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25,407-408. YAMANE, J. (1935). Kausal-analytishe studien iiber die befruchtung des Kanincheneies. 1. Die dispersion der follikelzeller und die Ablosung der zeller der Corona radiata des eies durch spermatozoen. Cytologia 6.233-255.
BIOLOGY
74, 343-350 (1980)
Arylsulfatase
of Sea Urchin Sperm
2. Arylsulfatase
as a Lysin of Sea Urchins’
MOTONORI Biochemical
Laboratory,
Institute
HOSHI AND TSUNEO
of Low Temperature
Received February
MORIYA
Science, Hokkaido
University,
Sapporo
060, Japan
15, 1979; accepted in revised form June 22, 1979
An arylsulfatase is defined as a lysin of sea urchin sperm from the following evidences. (1) The activity is detected in the sperm of all the sea urchins investigated, and the activity is partially liberated from the cells after the acrosome reaction (Moriya and Hoshi, 1979a). (2) Fertilization is completely inhibited in the presence of 40 n&fp-nitrophenyl sulfate, which is an artificial substrate of arylsulfatase, but is not inhibited by p-nitrophenyl phosphate at the same concentration. (3) The inhibitory effect ofp-nitrophenyl sulfate on fertilization is remarkably diminished by pretreatment of eggs with arylsulfatase before insemination. (4) Sperm arylsulfatase as well as limpet arylsulfatase appear to digest the vitelline coat and jelly coat. INTRODUCTION
Since Yamane (1935) observed dispersal of the egg investments by an extract of mammalian sperm, lysins have been found in such animals as mammals, reptiles, amphibians, tunicates, hemichordates, crustaceans, annelids, and mollusks except for cephalopods (for a review, see Dan, 1967). In echinoderms, Isaka et al. (1966) isolated a jelly-dispersing enzyme from a homogenate of sea urchin sperm, but it is not known whether this enzyme is actually involved in the penetration of sperm through the jelly coat. Levine et al. (1978) have recently found an acrosin-like enzyme in the sperm of sea urchin, which seems to be involved in the fertilization process. The investments of sea urchin eggs, namely, the vitelline coat and jelly coat, comprise protein, carbohydrate, and sulfate (Glabe and ’ This paper is dedicated to Professor Juro Ishida on the occasion of his 70th birthday. A preliminary report of this work was presented at the 8th International Congress of the International Society of Developmental Biologists at Tokyo, 1977. This work was supported in part by grants from the Ministry of Education of Japan and from the Itoh Science Foundation.
Vacquier, 1977; Tayler, 1956). Fucose-4-sulfate (Ishihara et al., 1973) and sialic acids (Isaka et al., 1970) have been reported as main sugar components of the jelly. We expected, therefore, that sulfatase, sialidase, other glycosidases, and protease are possible candidates for the lysin(s) of sea urchins. We have recently reported that an arylsulfatase exists in both the spermatozoa and the seminal plasma of the sea urchins examined (Moriya and Hoshi, 1979a, b) and that about 20% of the total activity is released from the spermatozoa when the sperm undergo acrosome reaction. These observations have led us to assume that arylsulfatase is the lysin, or at least one of the lysins, in sea urchins. In order to define arylsulfatase as a lysin, it is essential to examine the following points: (i) if the enzyme digests the vitelline coat and/or jelly coat; and (ii) if fertilization is blocked when the enzyme of sperm is inhibited in some way, for example, by the addition of an artificial substrate such asp-nitrophenyl sulfate. We show here that the sperm arylsulfatase fulfills these requirements for the lysin. 343 0012.1600/80/020343-08$02.00/0 (‘opyright All rights
0 1980 by Academic Press. of reproduction in any form
11~ reserved.
344
DEVELOPMENTAL BIOLOGY MATERIALS
AND
METHODS
VOLUME 74,198O
egg suspension to make the jelly coat visible, if necessary. Electron microscopy. The eggs were fixed for 60 min in a solution of 2.5% glutaraldehyde in ASW at 0°C. They were washed twice with ASW and were postfixed for 40 min in 2% osmium tetroxide in ASW. After dehydration through a graded series of ethanol, the eggs were infiltrated and embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate (Venable and Coggeshall, 1965) and examined with an electron microscope, JEM1OOL(JEOL, LTD).
Animals. The sea urchins Hemicentrotus pulcherrimus and Strongylocentrotus intermedius were used. Fertilization assay. Experiments were performed at 18-20°C unless otherwise specified. Eggs and sperm were collected by introducing 0.5 M KC1 into the coelom. Dry sperm were passed through a nylon mesh and diluted to 104-fold with seawater at 0°C just before insemination. Eggs were washed three times with filtered seawater. For insemination, 50 ~1 of the diluted sperm suspension was added to 0.5 ml of an egg RESULTS suspension containing about 200 eggs in the Effects of p-nitrophenyl esters on fertilipresence or absence of test reagents. Prior to insemination, the sperm concentration zation. In order to provide evidence that was adjusted by measuring the turbidity to sperm arylsulfatase is responsible for fergive a critical concentration which is re- tilization in sea urchins, p-nitrophenyl sulquired to fertilize 100% of the eggs. The fate, an artificial substrate of arylsulfatase critical concentration was usually between (Roy, 1971), was added to the test egg susOD 0.05 and 0.10 at 360 run. Eggs were pension before insemination. An artificial washed twice with fresh seawater 15 min substrate such as p-nitrophenyl sulfate is after insemination and were raised in sea- naturally assumed to inhibit the enzyme water. The fertilization ratios were deter- action on a native substrate in a competimined on the basis of cleavage as well as tive fashion. As demonstrated in Figs. 1 and 2, no eggs were fertilized in the presence of elevation of the fertilization envelope. Surface modification of eggs with aryl- potassium p-nitrophenyl sulfate (Boehrinsulfatase. The following preparations of ar- ger Mannheim) at 40 milf, but spermatozoa ylsulfatase were used: limpet arylsulfatase (Type IV, Sigma Chemical), partially purified arylsulfatase from the sperm of Strongylocentrotus intermedius (Moriya and Hoshi, 1979b), and a crude preparation from Hemicentrotus pulcherrimus (supernatant at 105,OOOg of the sperm wash for 1 hr). Egg suspensions (about 250 eggs, 0.5 ml) in ASW2 containing various concentrations of the enzyme were incubated at 20°C for 40 min with occasional shaking. The lo@ 10-5 10-4 10-l 10-2 IU’ IO0 eggs were washed twice with ASW and p- Nitrophenyl sulfate CM) most of them were used for fertilization FIG. 1. Effect of p-nitrophenyl sulfate on the ferassay and some for inspection with both a light microscope and an electron micro- tilization of the sea urchin, H. pulcherrimus. An aliquot (50 ~1) of sperm suspension was added to 0.5 ml scope. A bit of India ink was added to the of an egg suspension containing about 200 eggs in the ’ Abbreviations used: ASW, artificial 8.2; p-NPS, p-nitrophenyl sulfate.
seawater, pH
presence of various concentrations of p-nitrophenyl sulfate. Fertility was expressed in the ratio of the number of cleaved eggs to the total.
HOSHI
AND
MORIYA
AT-$. sulfatase Is a Sea Urchin Lysin
sulfate (40 FIG. 2. Eggs of H. pulcherrimus inseminated in the absence (A) or presence (B) of p-nitrophenyl m&f). The photographs were taken 5 min after inset nination. x 140. Inset shows at a higher magnification (X 220) that sperm remain in jelly coat apart from the cell even 15 min after insemination.
appeared to swim actively around the eggs. In the presence of this compound, a few spermatozoa appeared to reach the vitelline coat within 15 min after insemination, but most spermatozoa still stayed apart from the egg surface (Fig. 2). To the contrary, fertilization was not affected by p-nitrophenyl phosphate (Nakarai Chemicals) at 40 mit4. In the presence of p-nitrophenol (Nakarai Chemicals) at 1.5 n-J4 or more, the motility of spermatozoa was considerably reduced and thus failed to fertilize eggs, but the fertility was not affected under 0.15 n-&f. Because of such toxicity of p-nitrophenol on sperm and possible contamination ofp-nitrophenol in commercialp-nitrophenyl sulfate, we determined spectrophotometrically (at 400 nm) the concentration of p-nitrophenol in 40 mit4 of p-nitrophenyl sulfate. The result indicated that the concentration was lower than 0.125 m&f. When eggs were treated with the arylsul-
fatase prior to insemination, the inhibitory effect of p-nitrophenyl sulfate on fertilization was remarkably diminished. As a typical example, the fertilization ratios of the eggs of H. pulcherrimus pretreated with limpet enzyme are summarized in Table 1. Surface modification of eggs by arylsulfatase. Since we observed, as described in the previous section, that fertilization was not retarded by p-nitrophenyl sulfate when the eggs were pretreated with arylsulfatase, we examined morphologically what happened in the jelly and vitelline coat after treatment with arylsulfatase. As shown in Fig. 3, the jelly coat was completely dissolved by limpet arylsulfatase and this digestion was considerably delayed by pnitrophenyl sulfate. The vitelline coat was also seriously affected by this enzyme (Fig. 4). Figure 4 shows electron micrographs of unfertilized eggs after the treatment with the arylsulfatase preparations from limpet and sea
346
DEVELOPMENTAL BIOLOGY TABLE
I
FERTILIZATION IN THE PRESENCE OF pNITROPHENYL SULFATE OF EGGS PRETREATED WITH ARYLSULFATASE”
Concentra- Concentra- Elevation of tion of en- tion of p- fertilization NPS membrane zyme (uni&/ml) (mM) (%) 0 0 100 100 0.4 0 100 0.8 0 0 4.0 0 0 8.0 0 7 0 40 4 0.4 40 23 0.8 40 0 4.0 40 0 8.0 40
Cleaa;ge
VOLUME 74,198O
projections. The eggs pretreated with sulfatase developed quite normally and embryos “hatched” out of the capsule at the same time when normal embryos leave the fertilization envelopes. DISCUSSION
100 loo 100 100 100 7
The present study has clearly revealed that the fertilization of sea urchin eggs is completely blocked by p-nitrophenyl sulfate, an artificial substrate of arylsulfatase (Roy, 1971; Moriya and Ho&i, 1979b), 8 28 while the motility of sperm is not affected 38 appreciably by this compound, and that the 38 pretreatment of eggswith arylsulfatase prea Eggs (H. pulcherrimus) were pretreated with limvents the eggs from such a blocking of pet arylsulfatase at the various concentrations listed fertilization (Figs. 1 and 2 and Table 1). at 20°C for 40 min. After being washed twice with This inhibition of fertilization by p-nitroASW, the eggs were inseminated in the presence or phenyl sulfate is not due to contamination absence of p-nitrophenyl sulfate. ’ One unit of the enzyme hydrolyzed 1.0pmole ofpof p-nitrophenol, because p-nitrophenol exnitrophenyl sulfate per hour at 37°C in a reaction hibited its toxicity on sperm only when the mixture consisting of 0.5 ml of 0.2 M sodium acetate concentration was higher than 1.5 miV, buffer, pH 5.0,0.4 ml of 6.25 mdfp-nitrophenyl sulfate, which was far higher than the actual conand 0.1 ml of enzyme solution in 0.2% sodium chloride. centration (0.125 miU) of this compound contaminated in the p-nitrophenyl sulfate urchin sperm. After the treatment with solution used. In addition, the acrosome either preparation, microvilli on the egg reaction induced by jelly water was not surface decreased in number and the vitel- affected by the addition of p-nitrophenyl line coat disappeared from microvilli, but sulfate (unpublished observation), revealstill remained in the open spaces among ing that the block of fertilization by this microvilli. Except for these morphological chemical is not due to blocking of the acchanges, no appreciable effects were ob- rosome reaction. served in the egg surface after the treatIn the absence of p-nitrophenyl sulfate, ment. needless to say, most spermatozoa reached The eggs which had been treated with the egg cells promptly. When this comthe enzyme at higher concentrations before pound was present, to the contrary, most insemination were monospermically fertil- spermatozoa appeared to remain in the jelly ized, but they were unable to elevate the coat (Fig. 2). We also clearly demonstrated normal fertilization envelopes; instead, only that the jelly coat was considerably divery thin capsules were formed at a close gested by an arylsulfatase (Fig. 3). These distance from the egg surface (Fig. 5 and observations strongly indicate that arylsulTable 1). The capsule was hardly visible fatase promotes the penetration of sperwith the light microscope, but it was de- matozoa across the jelly coat, and agree tected under the electron microscope. As with reports that the jelly coat of sea urshown in Fig. 6, the capsule exhibits a chins consists of sulfated glycoproteins (Israther
fluffy
structure,
whereas
the normal
fertilization envelope has a structure in consistent thickness with uniformly distributed
aka, 1970; Ishihara,
1973). The vitelline
coat
as well as the jelly coat is generally assumed to be comprised of sulfated glycoproteins
Fro 3. Digestion of the egg jelly with limpet arylsulfatase. Eggs of S. intermedius were allowed to stand at room temperature for various periods as indicated in ASW (A) or ASW containing 4 units/ml arylsulfatase (B) or ASW containing both 4 units/ml arylsulfatase and 40 tip-nitrophenyl sulfate (C). In the control experiment (A), the egg cells were separated from each other by the jelly layer surrounding each egg for 20 hr or more. In the presence of sulfatase (B), the eggs were dispersed at the beginning, and the eggs came gradually into contact with each other, and arranged in hexagonal array after 20 hr, revealing the complete digestion of the jelly by the sulfatase. In the presence of both sulfa&se and p-nitrophenyl sulfate (C), the eggs were separated from each other, even after 5 hr. After 20 hr, the eggs became closer to each other, but the typical hexagonal array was not formed.
(Glabe and Vacquier, 1977). The observation that arylsulfatases prepared from limpet and sperm of two sea urchins (Strongylocentrotus and Hemicentrotus) had a capacity to digest the vitelline coats (Fig. 4) provides additional evidence to support the above assumption, and coincides with our previous finding that sulfate is also present in the fertilization envelope (Hoshi and Nagai, 1967). In this connection, it is interesting that the microvilli of the egg surface disappear after digestion of the vitelline layer with arylsulfatase (Fig. 4), implying that microvilli need the vitelline coat
to retain their structure. Since the vitelline coat is easily digested by proteases, it might be possible to attribute the digestion of the vitelline coat by the arylsulfatase preparations to the action of a contaminating protease (but not to that of arylsulfatase itself). However, we could not detect any activity of chymotrypsin-like or trypsin-like proteases in the preparation of sperm arylsulfatase which digested the vitelline coat as well as limpet arylsulfatase (unpublished data). A thin capsule was formed in the fertilized eggs, instead of the normal fertilization
FIG. 4. Electron micrographs of sea urchin egg surface (H. pulcherrimus). (A) Intact egg; microvilli are seen on the egg surface, and surrounded by the vitelline coat. (B and C) The eggs were treated with 4 units/ml of limpet sulfatase and 18.8 units/ml of sperm sulfatase for 40 min at 2O”C, respectively. The number of microviHi on the egg surface remarkably decreases. The vitelline coat disappears from the microvilli, but still remains in the open space between microvilli. VC, vitelline coat. PM, plasma membrane. A-l, B-l, C-l, x 9300. A-2, B-Z, c-z, x 35,000.
FIG. 5. Fertilized eggs of H. pulcherrimus with or without sulfatase pretreatment. (A) Control. (B) Eggs were treated with 4 units/ml of limpet sulfatase for 40 min at 20°C before insemination. x 270. 348
HOSHI AND MORIYA
Arylsulfatase
Is a Sea Urchin Lysin
FIG. 6. Electron micrographs of the surface of fertilized eggs of H. pulcherrimus. (A) Normal fertilization envelope (FE). The fertilization envelope at a close distance from the egg surface is due to an artifact by fixation. (B) The egg was treated with limpet sulfatase (4 units/ml) for 40 min at 20°C before insemination. The egg elevated no fertilization envelope; instead, a capsule (Cap) was formed (see Fig. 5). X 11,ooO.
envelope, when the eggs were pretreated with sulfatase before insemination (Figs. 5 and 6). The normal fertilization envelope is believed to be made from two sources, the contents of cortical granules and the vitelline coat (Endo, 1961). It is therefore assumed that the capsule is formed of the partially digested vitelline coat and the contents of cortical granules. It should be pointed out, however, that the digestion of the vitelline coat by sperm in the natural fertilization process must be strictly local, whereas the whole surface of the vitelline coat undergoes digestion when the eggs are exposed to sulfatase action, and this may explain the formation of a capsule which is an artificial structure. Monospermical fertilization of the sulfatase-treated eggs (Fig. 5) reveals that the system preventing the eggs from poly-
spermy was not affected so seriously by this enzyme, and implies that this system, if present, is not in the vitelline coat itself but probably in the plasma membrane. The data presented in this study reveal that sperm arylsulfatase of sea urchins promotes the penetration of sperm across the jelly and vitelline coat, and lead us to conclude that this enzyme plays a role as a lysin in the natural fertilization process in these animals. The authors wish to express their gratitude to the Director and the staff of the Akkeshi Marine Biological Station, Hokkaido University, where part of the study was carried out. They also would like to give thanks to the staff of the Laboratory of Developmental Biology, Waseda University, for their generous supply of animals. REFERENCES
DAN, J. C. (1967). Acrosome reaction and lysins. In
350
DEVELOPMENTALBIOL~3GY
“Fertilization, Comparative Morphology, Biochemistry, and Immunology” (C. B. Metz and A. Monroy, eds.), Vol. 1, pp. 237-293, Academic Press, New York. ENDO, Y. (1961). Changes in the cortical layer of sea urchin eggs at fertilization as studied with the electron microscope. Exp. Cell Res. 25, 383-397. GLABE, C. G., and VACQUIER,V. D. (1977). Isolation and characterization of the vitelline layer of sea urchin eggs. J. Cell Biol. 76,410-421. HOSHI, M., and NAGAI, K. (1967). Abstracts of 7th Int. Congr. Biochem., Vol. V, p. 965. ISAKA, W., KANATANI, H., and SUZUKI,N. (1966). Jelly dispersing enzyme obtained from spermatozoa of sea urchin, Anthocidaris crassispina. Exp. Cell Res. 44,66-72. ISAKA, S., HO~TA, K., and KUROKAWA,M. (1970). Jelly coat substances of sea urchin eggs. 1. Sperm isoagglutination and sialopolysaccharide in the jelly. Exp. Cell Res. 56,37-42. ISHIHARA, K., OGURI, K., and TANIGUCHI, H. (1973). Isolation and characterization of fucose sulfate from jelly coat glycoprotein of sea urchin egg. Biochim. Biophys. Acta 320,628-634. LAVINE, A. E., WALSH, K. A., and FODOR,E. J. B.
VOLUME ?4,1980
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