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Studies on the interactions of sperm with the surface of the sea urchin egg
DEVELOPMENTAL
BIOLOGY
84,397-406
(1981)
Studies on the Interactions of Sperm with the Surface of the Sea Urchin Egg1 CHARLES Department
of Physiological
GLABE,~,~
Chemistry,
The Johns Received
June
MAURY Hopkins
BUCHALTER, University
11, 1980; accepted
School
J. LENNARZ
AND WILLIAM of Medicine,
in revised form
725 N. Wolfe
December
Street,
Baltimore,
Maryland
21205
5, 1980
We have examined the relationship between sperm adhesion and fertilization in the cross species insemination of Arbaeggs by Strongylocentrotus purpuratus sperm. As previously reported (Kinsey et al., 1980) the addition of S. puqwuratus egg jelly results in induction of the acrosome reaction in sperm and significant numbers of S. purpuratus sperm adhere to A. punctulata eggs. However, in the absence ofS. purpuratus egg jelly, S. purpuratus sperm fail to bind to A. punctulata eggs. Although at least 200 S. pzqwwatus sperm bind to an A. punctulata egg in the presence of S. purpuraGtus jelly, less than 8% of the eggs are fertilized. The adhesion of S. purpuratus sperm meets the same functional criteria as homologous A. punctulata sperm-egg adhesion. Electron microscopy shows that S. purpuratus sperm that have undergone the acrosome reaction adhere to A. pun&la& eggs by their bindin-coated acrosomal process in a manner that is morphologically identical to that observed with homologous A. punctulata sperm. We have also compared the ability of S. purpuratus and A. punctulatu sperm to fuse and fertilize with A. punctulatu eggs after removal of the vitelline layer. Using high levels of sperm of either species, heterologous as well as homologous fertilization is readily detectable. Under these conditions, where stable binding is not demonstrable, there is no difference in the ability of S. purpumtus and A. punctulata sperm to fertilize A. punctulata eggs. These observations suggest that the failure of S. puqwumtus sperm to fertilize A. punctulata eggs under normal conditions may be due to their inability to penetrate the vitelline layer so that they can fuse with the egg plasma membrane. In relation to the possible mechanism of vitelline layer penetration, we have also investigated the mode of action of chymostatin, an inhibitor of chymotrypsin that has been reported to inhibit fertilization of sea urchin eggs (Hoshi et al., 1979). Our findings suggest that the fertilization inhibitory activity of chymostatin is not related to its antichymotrypsin activity. Rather, it appears that this inhibition is due to the induction of an abnormal acrosome reaction in sperm that precludes formation of the acrosome process. cia punctulata
INTRODUCTION
19’77; Glabe and Vacquier, 1978). Both the induction of the acrosome reaction and sperm adhesion have been Fertilization in sea urchins can be described as a series shown to contribute to the overall specificity of fertilizaof cell-cell interactions which take place in a matter of tion, but the extent of species specificity may vary conseconds upon mixing of gametes. This series of interacsiderably between species (Summers and Hylander, tions can be conveniently divided into four sequential 1975; SeGall and Lennarz, 1979; Kato and Sugiyama, events, the tist of which is the acrosome reaction in 1978; Kinsey et al., 1980, reviewed by Vacquier, 1979b). sperm (Dan, 1954a,b). This consists of the exocytosis of The two subsequent events consist of the penetration the acrosome vesicle and extension of the acrosomal proof the vitelline layer by the acrosomal process of the cess (Summers et al., 1975). The acrosome reaction is insperm, and the fusion of the plasma membranes of the duced by a component of the egg jelly which surrounds sperm and the egg. In contrast to the first two events, the egg and has recently been identified as a sulfated futhe penetration of the vitelline layer and the fusion of case polymer (SeGall and Lennarz, 1979). The second sperm and egg plasma membranes are poorly underevent is the adhesion of reacted sperm to the vitelline stood. This, in fact, may be due to the difficulty in seplayer covering the egg plasma membrane. This adhesion arating these later processes from the earlier steps. is mediated by the interaction of the sperm adhesive proIt has previously been reported that under conditions tein, bindin, which covers the acrosomal process, with where Strongylocentrotus purpuratus sperm are incomponents of the vitelline layer (Vacquier and Moy, duced to undergo the acrosome reaction, significant numbers of these sperm adhere to the surface of Arbacia 1 This work was supported by a grant from the National Institutes of punctulata eggs (Kinsey et al., 1980). We observed that Health (HD 08357) to W. J. L. although many heterologous sperm adhere, very few of * Present Address: Department of Anatomy, University of California the eggs subsequently fertilize. More extensive studies Medical Center, San Francisco, California 94143. of this phenomenon have provided us the opportunity to 3 C. G. was the recipient of a Special Postdoctoral Fellowship in Reproductive Biology from The Rockefeller Foundation. gain some insight into the processes of penetration of the 397
0012-1606/81/080397-10$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
DEVELOPMENTAL BIOLOGY
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Sperm added/Egg
FIG. 1. The adhesion of S. purpuratus sperm to A. punctulato eggs. The egg suspension was either supplemented with S. pqmratus egg jelly at a concentration of 80 nmoles hexose equivalents per ml (04); or inseminated in the absence of homologous jelly (O-----O). Five minutes after insemination, samples were fixed with formaldehyde and nonadherent sperm removed by repeated washing.
vitelline layer and fusion of plasma membranes and their relationship to the overall process of species specific fertilization. With regard to penetration of the vitelline layer, Hoshi et al. (19’79) have studied the inhibitory effect of chymostatin, a potent inhibitor of chymotrypsin, on fertilization. Based on these studies they suggested that a chymotrypsin-like activity associated with sperm is involved in penetration of the vitelline layer. Chymotrypsin-like activity has been detected in sea urchin sperm (Harris et al., 1977). In this report we present evidence which suggests that the inhibitory effect of chymostatin is not related to its antichymotrypsin activity. Our findings suggest that the inhibition of fertilization is due to the induction of an abnormal acrosome reaction in sperm. These findings are discussed in the context of models for the penetration of the vitelline layer by sperm.
VOLUME 84. 1981
density of 0.5 corresponds to a concentration of 2.3 x lo7 sperm/ml. Crude egg jelly was prepared as previously described (SeGall and Lennarz, 1980). To assay for sperm adhesion varying numbers of sperm (6 x 105-6 x 107) in a total volume of 0.3 ml were added to 0.3 ml of an egg suspension containing 5 X 103 eggs in 10 mM Tris-buffered sea water, pH 7.8. The egg suspension contained egg jelly homologous to the sperm at a concentration of 80 nmoles hexose equivalents per ml. Hexose was determined by the method of DuBois et al. (1958). Sperm adhesion was stopped by the addition of 0.6 ml of 6% formaldehyde in sea water (Vacquier and Payne, 1973). After fixation, the unbound sperm were removed by repeated washing in sea water, and the average number of sperm bound per egg was determined by the perimeter counting method described previously (Kinsey et aZ., 1980). Fertilization was determined by counting 200 eggs and scoring for the presence of a fertilization envelope or, in the case of eggs devoid of the vitelline layer, scoring cleavage after incubation for 90 min at 14°C. The vitelline layer was removed by limited trypsin digestion (Epel, 1970). A 10% v/v suspension of eggs was treated with 50 pg/ml trypsin (3 x crystallized Worthington, Freehold, N. J.) for 4-8 min at 4°C. Trypsin digestion was terminated by adding an excess of soy bean trypsin inhibitor (SBTI) (Sigma, St. Louis, MO.) and the eggs washed 3x in 100 vol of cold sea water. Removal of the vitelline layer was determined by the absence of a fertilization envelope upon activation of the eggs by ionophore A23187 as previously described (Glabe and Vacquier, 1978). Removal of the vitelline layer by the “vitel-
800
MATERIALS
- ;p-” -__------
1200
l
AND METHODS
A. punctulata were obtained from Florida Marine Biological Specimen Co., Panama City, Fl. or Marine Biological Laboratories, Woods Hole, Ma. and S. purpuratus from Pacific Biomarine, Venice, Ca. and maintained in artificial sea water (Instant Ocean, Eastlake, Oh.) at 18 and 6”C, respectively. Gametes were obtained from both species by electric shock or intracoelomic injection of 0.5 M KCl. Sperm were stored on ice until use. A. punctulata eggs were mechanically dejellied by repeated passage through 90 pm Nitex mesh (Tetko, Inc., Elmsford, N.Y.) as previously described (Glabe and Lennarz, 1979). Sperm concentration was estimated by measuring the absorbance of a diluted sample at 660 nm, a slight modification of the method reported by Vacquier and Payne (1973), ‘or by counting with a Coulter electronic particle counter (Coulter Electronics, HiaIeah, FI.). An optical
:
c-,P
(00
n
80 60
31
FIG. 2. Kinetics of sperm adhesion and fertilization of A. punetulata and S. pquurutus sperm at a ratio of 6660 sperm per egg. To an egg suspension containing 5 x 103 eggs sperm were added as described in Materials and Methods. Sperm adhesion and fertilization were assessed as described in Materials and Methods. Samples were fixed with formaldehyde at various times after insemination, and the percentage of fertilized eggs (O-----O) and the number of bound sperm determined (A) A. punetulata inseminated with A. p&&data sperm. (0 -0). (I3 A. punctulata eggs inseminated with S. purpuratus sperm.
GLABE,BUCHALTER,AND
LENNARZ
line delaminase-DTT” procedure was done as previously described (Carroll et al., 1978). Chymostatin was obtained from Vega-Fox (Tucson, AZ.) and dissolved in dimethyl sulfoxide (DMSO) to a concentration of 81 mM. A reduced derivative of chymostatin was prepared as follows: Ten milligrams of chymostatin was dissolved in 2 ml of N, N’-dimethyl formamide (DMF). One milliliter of a solution of sodium borohydride (3 mg/ml) in DMF was added and the solution incubated on ice overnight. The sample was taken to dryness under a stream of nitrogen and dissolved in 1 ml of 4 M acetic acid. The reduced chymostatin was desalted on a 40 ml column of Biogel P-2 (BioRad, Richmond, Ca.), lyophilized, and dissolved in DMSO. The concentration of chymostatin was determined using the extinction coefficient at 255 nm of 5.45 x lo5 mole-l. The amount of free aldehyde groups remaining was estimated by reactivity with the Schiff reagent. Antichymotrypsin activity was determined using 3x crystallized chymotrypsin and the synthetic substrate benzoyl-tyrosine ethyl ester (Sigma, St. Louis, MO.) (Worthington, 1972). Samples for electron microscopy were fixed for 1 hr in 3% glutaraldehyde in sea water and postfixed in 0.1% osmium tetroxide in 50 mM NaH,PO, buffer, pH 6, on ice for 30 min, optimum conditions for the fixation of actin filaments (Maupin-Szamier and Pollard, 1978). For transmission electron microscopy, samples were embedded in Epon and ultrathin sections examined in a Zeiss EM 10 electron microscope. For scanning electron microscopy, samples were spread on polylysine-coated coverslips, critical point dried, and rotary shadowed with gold palladium. Samples were examined in an ETEC scanning electron microscope. RESULTS
In an earlier study (Schmell et al., 1977), using a sensitive bioassay to measure fertilization as a function of sperm concentration, it was established that even at very high concentrations of sperm, A. pun&data eggs could not be fertilized by S. purpuratus sperm. Since it is known that S. purpuratus sperm do not undergo the acrosomal reaction in the presence of A. punctulata jelly coat (SeGall and Lennarz, 1979), we investigated the effect of the jelly coat of S. purpuratus eggs on the adhesion of S. purpuratus sperm to A. pun&data eggs (Kinsey et al., 1980). This study revealed that there was no binding of S. purpuratus sperm to A. punctulata eggs in the absence of S. purpuratus jelly coat; however, when jelly coat homologous to the sperm was present heterologous gamete binding could readily be detected. In the current study we have further examined this phenomenon using a wider concentration range of heterologous sperm. It is clear from the results shown in Fig. 1 that
Fertilization
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heterologous sperm adhesion is readily detected in the presence, but not in the absence of S. purpuratus egg jelly. This binding, as well as fertilization (as judged by fertilization envelope elevation, pronuclear activation, or cytokinesis), was examined as a function of time. As expected, using A. punctulata sperm and A. punctulata eggs, an extensive, transient binding of sperm occurred and 100% fertilization was observed (Fig. 2A). When an equal number of S. purpuratus sperm were mixed with A. punctulata eggs, the adhesion of heterologous sperm was readily detected, but only a very low level of fertilization was observed (Fig. 2B). It should be noted that although only approximately 10 sperm per egg are necessary for 100% fertilization among homologous gametes (Schmell et al., 1977), in this experiment 6000 sperm were added per egg. Thus although a relatively large number of heterologous sperm bind (200), only a very low number of eggs are fertilized, and in many experiments no heterologous fertilization was observed. This adhesion of S. purpuratus sperm to A. punctulata eggs is morphologically indistinguishable from the adhesion of A. pun&data sperm. Scanning electron micrographs, shown in Fig. 3, reveal that apparently all of the sperm associated with the egg surface have undergone the acrosome reaction, with the acrosome process in close association with the egg surface. Transmission electron microscopy confirmed this observation. As shown in Fig. 4, the bindin coated acrosomal process of S. pwpuratus sperm (Moy and Vacquier, 1979) is in intimate contact with the vitelline layer of A. pun&data eggs. Is the failure of adherent S. purpuratus sperm to fertilize A. punctulata eggs due to the existence of a species-specific recognition event at the level of the fusion of the sperm and egg plasma membranes, or due to the inability of the sperm to penetrate the vitelline layer? To examine the first possibility, we have investigated the sperm-egg fusion event per se by comparing the fertilization of A. pun&data eggs by A. punctulata and S. pwpuratus sperm after removal or disruption of the vitelline layer. The methods we have chosen to remove the vitelline layer also destroy the ability of the egg surface to bind sperm, thereby removing sperm adhesion as a factor in determining the fertilizability of the eggs. The first method we explored for removal of the vitelline layer was exposure of eggs to trypsin (50 pg/ml) at 4°C for 4 min. We have previously determined that these are the minimal conditions required to prevent the formation of a fertilization envelope upon parthenogenetic activation of the eggs and to completely abolish the ability of the eggs to agglutinate with isolated bindin particles from sperm (Glabe and Lennarz, 1979). Eggs treated in this fashion were then incubated with sperm in the presence of egg jelly homologous to the
DEVELOPMENTAL
BIOLOGY
VOLUME
84, 1981
FIG. 3. Scanning eggs. Samples were fixed with glutaraldehyde 5 min electron micrograph of S. purpuratus sperm bound to A. punctulata and prepared for microscopy as described in Materials and Methods. All of the adherent sperm have undergone ! the after ins emination X7500. acrosomt f reaction. FIG. 4. Transmission electron micrograph of S. purpuratus sperm bound to A. punctulata eggs. Samples were fixed with glutaraldeh yde 5 min afte r insemination and prepared for microscopy as described in Materials and Methods. The bindin coated acrosomal process is in contact with the egg vitelline layer. The intact cortical granules are characteristic of unfertilized eggs. X48,000. intimate
GLABE,BUCHALTER,ANDLENNARZ
sperm to induce the acrosome reaction. As shown in Fig. 5A, with the vitelline layer intact, A. punctulata sperm are at least 100-500 times more effective than S. purpuratus sperm in fertilizing A. punctulata eggs (Fig. 5A). However, when the vitelline layer is removed, A. punctulata and S. purpuratus sperm are equally effective (Fig. 5B). This equality results because the efficiency of A. punctulata sperm to fertilize A. punctulata decreases while the efficiency of S. purpuratus sperm increases. It could be argued that trypsin treatment might also destroy a putative specific receptor for membrane fusion. To investigate this possibility we have utilized another method for removing the vitelline layer which is reported to be more specific than the trypsin procedure (Carroll et al., 197’7). The specificity of this procedure depends on the specificity of the cortical granule protease for the vitelline layer as a substrate. When the vitelline layer is removed by this procedure the results were identical to those obtained using trypsin; S. purpuratus sperm were nearly as effective as A. punctulata sperm in fertilizing A. punctulata eggs. The results of these experiments suggest that there is no species specific recognition event at the level of the fusion of sperm and egg plasma membranes per se and that an intact vitelline layer is required to maintain the species-specific selection of sperm for fertilization. Therefore, it appears likely that the failure of adherent S. purpuratus sperm to fertilize A. punctulata
60 i’ f
2000 Sperm
FIG. 5. Fertilization of A. removal of the egg vitelline pun&data sperm (O-----O) fixed after a 5min incubation termined. Fertilization was Methods.
4000 added /Egg
6000
punctulatu eggs before (A) and after layer. Eggs were inseminated with or S. purpuratus sperm (04) period and the extent of fertilization assessed as described in Materials
(B) A. and deand
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looor 9
Sperm
added
i0
per egg X IO+
FIG. 6. Sperm adhesion (O-----O) and fertilization (04) of S. purpuratus eggs inseminated with S. purpuratus sperm. Following insemination samples were fixed at 30 set for determination of sperm adhesion and at 5 min for determination of the extent of fertilization as described in Materials and Methods. (A) Eggs were inseminated in the presence of 0.325 m&f chymostatin. (B) Eggs were inseminated in the absence of chymostatin.
eggs is due to a failure of the sperm to penetrate the vitelline layer. Levine et al. (1978) have described a serine protease activity in S. purpuratus sperm, that is detectable only after the acrosome reaction has occurred. Furthermore, a trypsin-like protease has been ultrastructurally localized to the reacted acrosomal process in sperm of the same species (Green and Summers, 1980). However, the role of sperm proteases in sea urchin fertilization is not clear (see discussion Green and Summers, 1980). Recently, it has been reported that chymostatin, a noncompetitive inhibitor of chymotrypsin (Umezawa, 1977), inhibits the fertilization of several species of sea urchins (Hoshi et al., 1979). Based on these observations, it was suggested that a chymotrypsin-like enzyme is involved in the penetration of the vitelline layer. If a chymotrypsinlike activity does promote penetration then, in the presence of chymostatin, large numbers of homologous sperm should adhere to the egg surface, but no fertilization would result. We have tested this prediction with S. purpuratus gametes. In Fig. 6, the effect of 0.325 mM (200 pg/ml) chymostatin (Hoshi et al., 1979) on sperm adhesion and fertilization is shown as a function of sperm concentration. In the presence (Fig. 6A) or absence (Fig. 6B) of chymostatin, equivalent binding of sperm to eggs is observed, but in the presence of chymostatin very few of the eggs fertilize. Similar results were obtained with A punctulata gametes (data not shown). Chymostatin exerts its effect on sperm; pretreatment
402
DEVELOPMENTALBIOLOGY
.-‘0 ‘0N
60
-
40
-
:z h a-”
-m -2
0 Chymosiatin Addition (SKI
FIG. 7. Kinetics of chymostatin inhibition and the chymostatin sensitive period of gamete interaction. Chymostatin (0.325 rnk? was added at various times in relation to the time of sperm addition (to) and the extent of fertilization determined after 5 min.
of the sperm with chymostatin followed by a loo-fold dilution of the chymostatin reduced the fertilizability of the sperm by 99%. In contrast, pretreatment of the eggs with chymostatin, followed by its removal by washing, had no effect on their fertilizability. In addition we have investigated the effect of prior induction of the acrosome reaction on the ability of sperm to fertilize eggs in the presence of chymostatin. No fertilization was observed at any time after the induction of the acrosome reaction including the simultaneous addition of chymostatin and jelly to sperm. The kinetics of the inhibitory effect of chymostatin was determined by adding chymostatin to gametes at various times relative to the addition of sperm. If chymostatin is added at any time up to 2 see before sperm addition, fertilization is completely inhibited (Fig. 7). However, if chymostatin is added 2 set after sperm addition most of the eggs are fertilized. These results demonstrate that the inhibitory effect of chymostatin on sperm is rapid and that the chymostatin-sensitive step of sperm-egg interaction is rapidly completed upon mixing of gametes. Chymostatin contains an aldehyde group that is critical for its chymotrypsin inhibitory activity (Umezawa, 19’77). After reduction of this aldehyde group with NaBH, , less than 1% of the aldehyde group remained, as determined by reactivity with the Schiff reagent. The re-
VOLUME 84, 1981
duced derivative of chymostatin was found to be two orders of magnitude less potent as an inhibitor of chymotrypsin, with a Ki of 9 X IOe7 as compared to 8 X lops M for chymostatin. The effect of chymostatin on sperm does not appear to be related to its activity as an irreversible inhibitor of chymotrypsin. As shown in Fig. 8, the reduced derivative of chymostatin is nearly as effective as chymostatin in inhibiting fertilization. Parenthetically, it should be noted that the concentration of chymostatin required to inhibit fertilization is extremely high. Chymostatin at a concentration of 0.325 mM would be sufficient to completely inhibit the proteolytic activity of chymotrypsin present at the very high concentration of 1 mg/ml. In view of our finding that the inhibitory effect of chymostatin could not be ascribed to its activity as an inhibitor of chymotrypsin, we investigated the morphological effect of chymostatin on sperm treated with 0.325 m&Z chymostatin by transmission electron microscopy. Observations of thin sections revealed that chymostatin induces an abnormal acrosome reaction in greater than 90% of the sperm (Fig. 9A). This abnormal acrosome reaction induced by chymostatin consists of vesiculation of material at the apex of the sperm accompanied by a failure of the acIO0 -
so -
60
-
-4 Chymoslatin
Concentration LOQ M
FIG. 8. Concentration dependence of the inhibition of S. purpuratus fertilization by chymostatin (04) or reduced chymostatin (O----O). We have observed in several experiments that the concentration required for 50% inhibition of fertilization with either chymostatin or reduced chymostatin varies by a factor of approximately 2 (from 0.1 to 0.2 mMJ with different samples of gametes.
GLABE, BUCHALTER, AND LENNARZ
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FIG. 3. Transmission electron micrographs of chymostatin-treated and control S. pqnwatus sperm. (A) Sperm treated with 0.325 mM chymostatin for 30 set before fixation. (B) Control sperm treated with DMSO. (C) Sperm treated with egg jelly for 30 see to induce the acrosome reaction and then treated with 0.325 mM chymostatin. (D) Egg jelly treated sperm subsequently treated with DMSO. All figures X39,400.
rosome process to fully extend, as compared to the normal jelly induced acrosome reaction (Fig. 9D). DMSO, the solvent used for chymostatin, had no morphological effect on sperm or on the ability of sperm to undergo the acrosome reaction (Figs. 9B and D). The reduced derivative of chymostatin was found to have the same morphological effect on sperm as chymostatin (data not shown). Since as mentioned above, not only unreacted sperm but also sperm prereacted with egg jelly were inactivated by chymostatin, we examined the morphology of the reacted sperm by transmission electron microscopy. As shown in Fig. 9C, these sperm display an abnormal acrosomal region similar to that observed in nonreacted sperm that were treated with chymostatin. Chymostatin-treated sperm were also readily distinguishable from normal acrosome reacted sperm by electron microscopy of whole sperm mounted on Formvar-coated grids (Decker et al., 1976). Using this technique to evaluate
the sperm, it was found that 90% of the sperm in a population underwent the acrosome reaction upon jelly treatment. Treating these acrosome-reacted sperm with 0.325 mlM chymostatin caused more than 60% of them to exhibit an abnormal vesiculated acrosome process. These observations clearly establish that even the already extended acrosome process is disordered upon chymostatin treatment. As shown earlier (Fig. S), sperm-egg adhesion occurs efficiently in the presence of chymostatin, although the eggs fail to fertilize. It is apparent from the electron micrograph of thin section specimens of eggs inseminated in the presence of chymostatin (Fig. 10) that these abnormally reacted sperm adhere to the egg surface, even though they fail to fertilize the eggs. DISCUSSION
Based on a number of recent studies, it seems reasonable that the high degree of species specificity observed
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FIG. 10. Transmission electron micrograph of chymostatin-treated S. pwpurutus sperm adhering to the surface of a homologous egg. The vesiculated material covering the acrosomal process appears to be located at the site of adhesion between sperm and egg. x 39,400.
in sea urchin fertilization is the cumulative result of limited specificity in at least two of the sequential events of sperm-egg interaction (Summers and Hylander, 19’75; Glabe and Vacquier, 1977; SeGall and Lennarz, 1979; Glabe and Lennam, 1979; Kinsey et al., 1980). The early events of sperm-egg interaction, consisting of the induction of the acrosome reaction and subsequent sperm adhesion, have been shown to exhibit a range of specificities in different species of urchins (see Introduction). In contrast, very little is known about the later events of penetration of the vitelline layer and the fusion of sperm and egg plasma membranes or the possible contribution of these events to species specificity. In the few cases where a significant degree of heterologous sperm adhesion was observed, a high percentage of the eggs subsequently fertilized (Summers and Hylander, 1975; Kato and Sugiyama, 1978; Aketa and Ohto, 1979; Glabe, unpublished). This investigation is the first report of a case where significant numbers of heterologous sperm bind and yet the majority of eggs fail to fertilize. By analyzing the mechanism of this block to fertilization, we have explored the possible contribution of the later events of penetration of the vitelline layer and plasma membrane fusion to the overall species specificity of fertilization. We have found that in the absence of the vitelline layer and therefore sperm adhesion, A. punctulata and S. purpuratus sperm are equally effective in
fertilizing A. punctulata eggs. This suggests that there is no specificity in the fusion of sperm-egg plasma membrane . Earlier work can now be interpreted in the context of a modern model for sperm-egg interaction. Hultin (1948) originally showed that trypsin treatment of eggs enhances the capacity for cross fertilization. Hagstrom (1959) measured the kinetics of fertilization of trypsintreated eggs and found that the rates of fertilization for homologous and heterologous species were nearly equal. The rate of homologous fertilization decreased and heterologous fertilization increased when compared to the rate with the vitelline layer intact. These results and our observations present an apparent paradox in that the vitelline layer appears to act as both an effector of homologous fertilization and a barrier to heterologous fertilization. The one explanation for this is that the vitelline layer increases the efficiency of homologous fertilization by virtue of its ability to bind sperm. For heterologous sperm that do not adhere, the vitelline layer acts as a barrier by covering the egg plasma membrane and therefore preventing the less efficient process of direct plasma membrane fusion. From this work, it appears that there is a more important function for sperm adhesion than merely immobilizing the apex of the sperm head at the vitelline layer and that penetration of the vitelline layer plays a role in de-
GLABE,
BUCHALTER,
AND LENNARZ
termining species specificity. Perhaps the function of sperm adhesion is to anchor the apex of the sperm in such a manner as to provide support for the mechanical penetration of the vitelline layer by the extending acrosome process as proposed by Dan (1960) for star fish fertilization. Evidence for this type of mechanism has been presented for the penetration of the vitelline envelope of Lim&us eggs by sperm (Brown, 1976). In terms of a mechanical penetration model, the failure of S. purpuratus to fertilize A. punctulata eggs may be explained by postulating that the strength of adhesion between S. puqnwatus sperm and A. punctulata eggs is below the threshold required for penetration of the vitelline layer, but sufficient to immobilize the sperm. We have previously presented evidence which suggests that such species specific differences exist in the strength of the adhesive interaction between isolated bindin and eggs of these two species (Glabe and Lennarz, 1979). Another possible mechanism for the penetration of the vitelline layer is by the action of hydrolytic enzymes, as suggested for annelids (Colwin and Colwin, 1960), mollusks (Heller and Raftery, 19’76), and mammals (Austin and Bishop, 1959). In this regard, a trypsin-like protease associated with the acrosome reaction has been recently purified and characterized (Levine and Walsh, 1980). The preparation of sea urchin sperm protease was shown to possess both chymotrypsin and trypsin-like activity and to be inhibited by chymostatin. Hoshi et al. (1979) have observed that chymostatin inhibits fertilization in several species of sea urchins. In this report we have further investigated the effect of chymostatin on fertilization. Two lines of evidence suggest that the inhibitory effect of chymostatin on fertilization is not related to its activity as an inhibitor of chymotrypsin. A reduced analog of chymotrypsin, which is lOO-fold less potent as an inhibitor of chymotrypsin, is nearly as effective as chymostatin in inhibiting fertilization. Second, chymostatin is only an effective inhibitor of fertilization at extremely high concentrations approaching or exceeding its solubility in sea water. Our observations demonstrate that the primary effect of chymostatin is to induce an abnormal acrosome reaction in sperm consisting of the vesiculation of material at the apex of the sperm and an incomplete extension of the acrosome process. Chymostatin is an amphipathic peptide containing hydrophobic amino acid residues. Since it is an effective inhibitor only at high concentrations (0.325 mM>, this effect may be due to a detergent-like activity. We have previously observed that A. punctulata bindin forms vesicles upon treatment of sperm with the detergent Triton X-100 (Glabe and Lennarz, 1979), Perhaps, S. purpuratus bindin forms vesicles upon treatment with chymotrypsin. We have also demonstrated that the effect of chymostatin on sperm is rapid and that the chymostatin-sensi-
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tive events in sperm-egg interaction are completed rapidly upon mixing of gametes. There is no fertilization when chymostatin is added any time before the addition of sperm, but if chymostatin is added 2 set after insemination, most of the eggs subsequently fertilize. Since the primary effect of chymotrypsin appears to be inhibition of a normal acrosome reaction, we investigated the effect of preinducing the acrosome on the ability of the sperm to fertilize eggs in the presence of chymostatin. We found that these preinduced sperm still fail to fertilize eggs in the presence of chymostatin. Electron microscopic examination of such sperm revealed that the acrosomal process had been altered by chymostatin so that it was no longer fully extended. Thus chymostatin induces an abnormal reaction in unreacted sperm, as well as altering the morphology of the process in prereacted sperm. The fact that preinduced sperm do not fertilize eggs in the presence of chymostatin, along with the observation that the chymostatin sensitive interactions are rapidly completed upon insemination, suggest that the precise sequence and timing of the multiple events of sperm-egg interaction may be critical for successful fertilization. Our observations raise the possibility that penetration of the vitelline layer is accomplished by the extending acrosome process, although they certainly do not rule out the participation of hydrolytic enzymes. Mechanical penetration of the vitelline layer by the extending acrosome process may explain why the fertilizing capacity of sperm decreases rapidly after induction of the acrosome reaction, as reported previously by a number of investigators (Takahashi and Sugiyama, 1973; Vacquier, 1979a; Kinsey et al., 1980). Once the acrosome process has fully extended, sperm should no longer be able to mechanically penetrate the vitelline layer. If this model is correct, one implication is that the familiar electron microscopic image of sperm adhering to the vitelline layer by the extended acrosome process may not represent an intermediate step in the process of gamete interaction, but rather the nonproductive interaction of a typical unsuccessful sperm. However, it is clear that further work will be necessary to test this idea. The assistance of Ms. Ann Fuhr in the preparation is gratefully acknowledged.
of this manuscript
REFERENCES K., and OHTA, T. (1979). Possible causal relation between the acrosome reaction and cross-fertilization in the sea urchins Hemicentrotus pdcherrimus and Pseuabcentrotus depressus. Gamete Res. 2, 15-23. AUSTIN, C. R., and BISHOP, M. W. H. (1958). Some features of the acrosome and perforatorium in mammalian spermatozoa. Proc. ROY. Sot. London B 149.234-240. BROWN, G. G. (1976). Scanning electron microscopical and other obserAKETA,
DEVELOPMENTAL BIOLOGY
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reactions in Limulus
polyphemus.
J.
Sci. 22, 547-562.
CARROLL, E. J., JR., BYRD, E. W., JR., and EPEL, D. (1977). A novel procedure for obtaining denuded sea urchin eggs and observations of the role of the vitelline layer in sperm reception and egg activation. Exp.
Cell
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108, 365-374.
COLWIN, A. L., and COLWIN, L. H. (1960). Egg membrane lytic activity of sperm extract and its significance in relation to sperm entry in Hydroidec hexagonus (Annelida). J. Biophys. Biochem. Cytol. 7, 321-328. DAN J. C. (1954a). Studies on the acrosome: II. Acrosome reaction in starfish spermatozoa. Biol. Bull. 107, 203-217. DAN, J. C. (195413). Studies on the acrosome: III. Effect of calcium deficiency. Biol. Bull. 107, 335-349. DAN, J. C. (1960). Studies on the acrosome: VI. Fine structure of the starfish acrosome. Exp. Cell Res. 19, 13-28. DECKER, G. L., JOSEPH, D. B., and LENNARZ, W. J. (1976). A study of factors involved in induction of the acrosomal reaction in sperm of the sea urchin, Arbacia punctulata. Develop. Biol. 53, 115-125. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., and SMITH, F. (1956). Calorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350-356. EPEL, D. (1970). Methods for removal of the vitelline membrane of sea urchin eggs. Exp. Cell Res. 61, 69-70. GLABE, C. G., and VACQUIER, V. D. (1978). Glycoprotein receptor for bindin isolated from the surface of sea urchin eggs. Proc. Nat. Acad. Sci. USA 75, 881-885. GLABE, C. G., and LENNARZ, W. J. (1979). Species-specific sperm adhesion in sea urchins: A quantitative investigation of bindin-mediated egg agglutination. J. Cell Biol. 83, 595-604. HAGSTROM, B. E. (1959). Experiments on hybridization of sea urchins. Arkiv. Zool. 12, 127-135. HARRIS, S., CSERNANSKY, J., ZIMMERMAN, M., and TROL, W. (1977). A chymotrypsin-like enzyme present in Arbacia punctulata spermatozoa. Biot. Bull. 153, 428-429. HELLER, E., and RAFTERY, M. A. (1976). The vitelline envelope of eggs from the giant keyhole limpet Megathura crenulata. Biochemistry 15, 1194-1198. HOSHI, M., MORIYA, T., AOYAGI, T., UMEZAWA, H., MOHRI, H., and NAGAI, Y. (1979). Effects of hydrolase inhibitors on fertilization of sea urchins: I. Protease inhibitors. Gamete Res. 2, 107-119. HULTIN, T. (1948). Species specificity in fertilization reaction. The role of the vitelline membrane of sea urchin eggs in species specificity. Arkiv.
Zoot.
A40,
E-14.
KATO, K. H., and SUGIYAMA, M. (1978). Species-specific adhesion of spermatozoa to the surface of fixed eggs in sea urchins. Develop., Growth
and Differ.
20, 337-347.
KINSEY, W. H., SEGALL, G. K., and LENNARZ, W. J. (1979). The ef-
VOLUME 84, 1981
fect of the acrosome reaction on the respiratory activity and fertilizing capacity of echinoid sperm. Develop. Biol. 71, 49-59. KINSEY, W. H., RUBIN, J. A., and LENNARZ, W. J. (1980). Studies on the specificity of sperm binding in echinoderm fertilization. Develop. Biol.
74, 245-250.
LEVINE, A. E., WALSH, K. A., and FOWR, F. J. B. (1978). Evidence of an acrosome-like enzyme in sea urchin sperm. Develop. Biol. 63, 299-306.
LEVINE, A. E., and WALSH, K. A. (1980). Purification of an Acrosinlike enzyme from sea urchin sperm. J. Biol. Chem. 255, 4814-4820. MAUPIN-SZAMIER, P., and POLLARD, T. D. (1978). Actin filament destruction by osmium tetroxide. J. Cell. Biol. 77, 837-852. MOY, G. W., and VACQUIER, V. A. (1979). Immunoperoxidase localization of bindin during the adhesion of sperm to sea urchin eggs. Current
Topics
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13, 31-44.
SCHMELL, E., EARLES, B. J., BREAUX, C., and LENNARZ, W. J. (1977). Identification of a sperm receptor on the surface of the eggs of the sea urchin Arbacia punctulata. J. Cell Biol. 72, 35-46. SEGALL, G. K., and LENNARZ, W. J. (1979). Chemical characterization of the component of the jelly coat from sea urchin eggs responsible for induction of the acrosome reaction. Develop. Biol. 71, 33-48. SUMMERS, R. G., and HYLANDER, B. L. (1975). Species specificity of aerosome reaction and primary gamete binding in echinoids. Exp. Cell
Res. 96, 63-68.
SUMMERS, R. G., HYLANDER, B. L., COLWIN, L. H., and COLWIN, A. L. (1975). The functional anatomy of the echinoderm spermatozoa and its interaction with the egg at fertilization. Amer. Zool. 15, 523551.
TAKAHASHI, Y. M., and SUGIYAMA, M. (1973). Relation between the acrosome reaction and fertilization in the sea urchin. I. Fertilization in Ca++ free sea water with egg-water treated spermatozoa. Develop.,
Growth
Difter.
15, 261-267.
UMEZAWA, H. (1976). Protease inhibitors of microbial origin. Methods Enzymol. 45, 678-695. VACQUIER, V. D. (1979a). The fertilizing capacity of sea urchin sperm rapidly decreases after induction of the acrosome reaction. Develop., Growth Differ. 21, 61-69. VACQUIER, V. D. (197913). The interactions of sea urchin gametes during fertilization. Amer. Zool. 19, 838-849. VACQUIER, V. D., and PAYNE, J. E. (1973). Methods for quantitating sea urchin sperm-egg binding. Exp. Cell Res. 82, 227-235. VACQUIER, V. D. and MOY, G. W. (1977). Isolation of bindin. The protein responsible for adhesion of sperm to sea urchin eggs. Proc. Nat. Acad.
Sci.
USA
74, 2456-2460.
WORTHINGTON (1972). “Worthington Enzyme Reagents and Related Biochemicals.” Worthington Biochemical Corporation, Freehold, N.J.
BIOLOGY
84,397-406
(1981)
Studies on the Interactions of Sperm with the Surface of the Sea Urchin Egg1 CHARLES Department
of Physiological
GLABE,~,~
Chemistry,
The Johns Received
June
MAURY Hopkins
BUCHALTER, University
11, 1980; accepted
School
J. LENNARZ
AND WILLIAM of Medicine,
in revised form
725 N. Wolfe
December
Street,
Baltimore,
Maryland
21205
5, 1980
We have examined the relationship between sperm adhesion and fertilization in the cross species insemination of Arbaeggs by Strongylocentrotus purpuratus sperm. As previously reported (Kinsey et al., 1980) the addition of S. puqwuratus egg jelly results in induction of the acrosome reaction in sperm and significant numbers of S. purpuratus sperm adhere to A. punctulata eggs. However, in the absence ofS. purpuratus egg jelly, S. purpuratus sperm fail to bind to A. punctulata eggs. Although at least 200 S. pzqwwatus sperm bind to an A. punctulata egg in the presence of S. purpuraGtus jelly, less than 8% of the eggs are fertilized. The adhesion of S. purpuratus sperm meets the same functional criteria as homologous A. punctulata sperm-egg adhesion. Electron microscopy shows that S. purpuratus sperm that have undergone the acrosome reaction adhere to A. pun&la& eggs by their bindin-coated acrosomal process in a manner that is morphologically identical to that observed with homologous A. punctulata sperm. We have also compared the ability of S. purpuratus and A. punctulatu sperm to fuse and fertilize with A. punctulatu eggs after removal of the vitelline layer. Using high levels of sperm of either species, heterologous as well as homologous fertilization is readily detectable. Under these conditions, where stable binding is not demonstrable, there is no difference in the ability of S. purpumtus and A. punctulata sperm to fertilize A. punctulata eggs. These observations suggest that the failure of S. puqwumtus sperm to fertilize A. punctulata eggs under normal conditions may be due to their inability to penetrate the vitelline layer so that they can fuse with the egg plasma membrane. In relation to the possible mechanism of vitelline layer penetration, we have also investigated the mode of action of chymostatin, an inhibitor of chymotrypsin that has been reported to inhibit fertilization of sea urchin eggs (Hoshi et al., 1979). Our findings suggest that the fertilization inhibitory activity of chymostatin is not related to its antichymotrypsin activity. Rather, it appears that this inhibition is due to the induction of an abnormal acrosome reaction in sperm that precludes formation of the acrosome process. cia punctulata
INTRODUCTION
19’77; Glabe and Vacquier, 1978). Both the induction of the acrosome reaction and sperm adhesion have been Fertilization in sea urchins can be described as a series shown to contribute to the overall specificity of fertilizaof cell-cell interactions which take place in a matter of tion, but the extent of species specificity may vary conseconds upon mixing of gametes. This series of interacsiderably between species (Summers and Hylander, tions can be conveniently divided into four sequential 1975; SeGall and Lennarz, 1979; Kato and Sugiyama, events, the tist of which is the acrosome reaction in 1978; Kinsey et al., 1980, reviewed by Vacquier, 1979b). sperm (Dan, 1954a,b). This consists of the exocytosis of The two subsequent events consist of the penetration the acrosome vesicle and extension of the acrosomal proof the vitelline layer by the acrosomal process of the cess (Summers et al., 1975). The acrosome reaction is insperm, and the fusion of the plasma membranes of the duced by a component of the egg jelly which surrounds sperm and the egg. In contrast to the first two events, the egg and has recently been identified as a sulfated futhe penetration of the vitelline layer and the fusion of case polymer (SeGall and Lennarz, 1979). The second sperm and egg plasma membranes are poorly underevent is the adhesion of reacted sperm to the vitelline stood. This, in fact, may be due to the difficulty in seplayer covering the egg plasma membrane. This adhesion arating these later processes from the earlier steps. is mediated by the interaction of the sperm adhesive proIt has previously been reported that under conditions tein, bindin, which covers the acrosomal process, with where Strongylocentrotus purpuratus sperm are incomponents of the vitelline layer (Vacquier and Moy, duced to undergo the acrosome reaction, significant numbers of these sperm adhere to the surface of Arbacia 1 This work was supported by a grant from the National Institutes of punctulata eggs (Kinsey et al., 1980). We observed that Health (HD 08357) to W. J. L. although many heterologous sperm adhere, very few of * Present Address: Department of Anatomy, University of California the eggs subsequently fertilize. More extensive studies Medical Center, San Francisco, California 94143. of this phenomenon have provided us the opportunity to 3 C. G. was the recipient of a Special Postdoctoral Fellowship in Reproductive Biology from The Rockefeller Foundation. gain some insight into the processes of penetration of the 397
0012-1606/81/080397-10$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
DEVELOPMENTAL BIOLOGY
398
Sperm added/Egg
FIG. 1. The adhesion of S. purpuratus sperm to A. punctulato eggs. The egg suspension was either supplemented with S. pqmratus egg jelly at a concentration of 80 nmoles hexose equivalents per ml (04); or inseminated in the absence of homologous jelly (O-----O). Five minutes after insemination, samples were fixed with formaldehyde and nonadherent sperm removed by repeated washing.
vitelline layer and fusion of plasma membranes and their relationship to the overall process of species specific fertilization. With regard to penetration of the vitelline layer, Hoshi et al. (19’79) have studied the inhibitory effect of chymostatin, a potent inhibitor of chymotrypsin, on fertilization. Based on these studies they suggested that a chymotrypsin-like activity associated with sperm is involved in penetration of the vitelline layer. Chymotrypsin-like activity has been detected in sea urchin sperm (Harris et al., 1977). In this report we present evidence which suggests that the inhibitory effect of chymostatin is not related to its antichymotrypsin activity. Our findings suggest that the inhibition of fertilization is due to the induction of an abnormal acrosome reaction in sperm. These findings are discussed in the context of models for the penetration of the vitelline layer by sperm.
VOLUME 84. 1981
density of 0.5 corresponds to a concentration of 2.3 x lo7 sperm/ml. Crude egg jelly was prepared as previously described (SeGall and Lennarz, 1980). To assay for sperm adhesion varying numbers of sperm (6 x 105-6 x 107) in a total volume of 0.3 ml were added to 0.3 ml of an egg suspension containing 5 X 103 eggs in 10 mM Tris-buffered sea water, pH 7.8. The egg suspension contained egg jelly homologous to the sperm at a concentration of 80 nmoles hexose equivalents per ml. Hexose was determined by the method of DuBois et al. (1958). Sperm adhesion was stopped by the addition of 0.6 ml of 6% formaldehyde in sea water (Vacquier and Payne, 1973). After fixation, the unbound sperm were removed by repeated washing in sea water, and the average number of sperm bound per egg was determined by the perimeter counting method described previously (Kinsey et aZ., 1980). Fertilization was determined by counting 200 eggs and scoring for the presence of a fertilization envelope or, in the case of eggs devoid of the vitelline layer, scoring cleavage after incubation for 90 min at 14°C. The vitelline layer was removed by limited trypsin digestion (Epel, 1970). A 10% v/v suspension of eggs was treated with 50 pg/ml trypsin (3 x crystallized Worthington, Freehold, N. J.) for 4-8 min at 4°C. Trypsin digestion was terminated by adding an excess of soy bean trypsin inhibitor (SBTI) (Sigma, St. Louis, MO.) and the eggs washed 3x in 100 vol of cold sea water. Removal of the vitelline layer was determined by the absence of a fertilization envelope upon activation of the eggs by ionophore A23187 as previously described (Glabe and Vacquier, 1978). Removal of the vitelline layer by the “vitel-
800
MATERIALS
- ;p-” -__------
1200
l
AND METHODS
A. punctulata were obtained from Florida Marine Biological Specimen Co., Panama City, Fl. or Marine Biological Laboratories, Woods Hole, Ma. and S. purpuratus from Pacific Biomarine, Venice, Ca. and maintained in artificial sea water (Instant Ocean, Eastlake, Oh.) at 18 and 6”C, respectively. Gametes were obtained from both species by electric shock or intracoelomic injection of 0.5 M KCl. Sperm were stored on ice until use. A. punctulata eggs were mechanically dejellied by repeated passage through 90 pm Nitex mesh (Tetko, Inc., Elmsford, N.Y.) as previously described (Glabe and Lennarz, 1979). Sperm concentration was estimated by measuring the absorbance of a diluted sample at 660 nm, a slight modification of the method reported by Vacquier and Payne (1973), ‘or by counting with a Coulter electronic particle counter (Coulter Electronics, HiaIeah, FI.). An optical
:
c-,P
(00
n
80 60
31
FIG. 2. Kinetics of sperm adhesion and fertilization of A. punetulata and S. pquurutus sperm at a ratio of 6660 sperm per egg. To an egg suspension containing 5 x 103 eggs sperm were added as described in Materials and Methods. Sperm adhesion and fertilization were assessed as described in Materials and Methods. Samples were fixed with formaldehyde at various times after insemination, and the percentage of fertilized eggs (O-----O) and the number of bound sperm determined (A) A. punetulata inseminated with A. p&&data sperm. (0 -0). (I3 A. punctulata eggs inseminated with S. purpuratus sperm.
GLABE,BUCHALTER,AND
LENNARZ
line delaminase-DTT” procedure was done as previously described (Carroll et al., 1978). Chymostatin was obtained from Vega-Fox (Tucson, AZ.) and dissolved in dimethyl sulfoxide (DMSO) to a concentration of 81 mM. A reduced derivative of chymostatin was prepared as follows: Ten milligrams of chymostatin was dissolved in 2 ml of N, N’-dimethyl formamide (DMF). One milliliter of a solution of sodium borohydride (3 mg/ml) in DMF was added and the solution incubated on ice overnight. The sample was taken to dryness under a stream of nitrogen and dissolved in 1 ml of 4 M acetic acid. The reduced chymostatin was desalted on a 40 ml column of Biogel P-2 (BioRad, Richmond, Ca.), lyophilized, and dissolved in DMSO. The concentration of chymostatin was determined using the extinction coefficient at 255 nm of 5.45 x lo5 mole-l. The amount of free aldehyde groups remaining was estimated by reactivity with the Schiff reagent. Antichymotrypsin activity was determined using 3x crystallized chymotrypsin and the synthetic substrate benzoyl-tyrosine ethyl ester (Sigma, St. Louis, MO.) (Worthington, 1972). Samples for electron microscopy were fixed for 1 hr in 3% glutaraldehyde in sea water and postfixed in 0.1% osmium tetroxide in 50 mM NaH,PO, buffer, pH 6, on ice for 30 min, optimum conditions for the fixation of actin filaments (Maupin-Szamier and Pollard, 1978). For transmission electron microscopy, samples were embedded in Epon and ultrathin sections examined in a Zeiss EM 10 electron microscope. For scanning electron microscopy, samples were spread on polylysine-coated coverslips, critical point dried, and rotary shadowed with gold palladium. Samples were examined in an ETEC scanning electron microscope. RESULTS
In an earlier study (Schmell et al., 1977), using a sensitive bioassay to measure fertilization as a function of sperm concentration, it was established that even at very high concentrations of sperm, A. pun&data eggs could not be fertilized by S. purpuratus sperm. Since it is known that S. purpuratus sperm do not undergo the acrosomal reaction in the presence of A. punctulata jelly coat (SeGall and Lennarz, 1979), we investigated the effect of the jelly coat of S. purpuratus eggs on the adhesion of S. purpuratus sperm to A. pun&data eggs (Kinsey et al., 1980). This study revealed that there was no binding of S. purpuratus sperm to A. punctulata eggs in the absence of S. purpuratus jelly coat; however, when jelly coat homologous to the sperm was present heterologous gamete binding could readily be detected. In the current study we have further examined this phenomenon using a wider concentration range of heterologous sperm. It is clear from the results shown in Fig. 1 that
Fertilization
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heterologous sperm adhesion is readily detected in the presence, but not in the absence of S. purpuratus egg jelly. This binding, as well as fertilization (as judged by fertilization envelope elevation, pronuclear activation, or cytokinesis), was examined as a function of time. As expected, using A. punctulata sperm and A. punctulata eggs, an extensive, transient binding of sperm occurred and 100% fertilization was observed (Fig. 2A). When an equal number of S. purpuratus sperm were mixed with A. punctulata eggs, the adhesion of heterologous sperm was readily detected, but only a very low level of fertilization was observed (Fig. 2B). It should be noted that although only approximately 10 sperm per egg are necessary for 100% fertilization among homologous gametes (Schmell et al., 1977), in this experiment 6000 sperm were added per egg. Thus although a relatively large number of heterologous sperm bind (200), only a very low number of eggs are fertilized, and in many experiments no heterologous fertilization was observed. This adhesion of S. purpuratus sperm to A. punctulata eggs is morphologically indistinguishable from the adhesion of A. pun&data sperm. Scanning electron micrographs, shown in Fig. 3, reveal that apparently all of the sperm associated with the egg surface have undergone the acrosome reaction, with the acrosome process in close association with the egg surface. Transmission electron microscopy confirmed this observation. As shown in Fig. 4, the bindin coated acrosomal process of S. pwpuratus sperm (Moy and Vacquier, 1979) is in intimate contact with the vitelline layer of A. pun&data eggs. Is the failure of adherent S. purpuratus sperm to fertilize A. punctulata eggs due to the existence of a species-specific recognition event at the level of the fusion of the sperm and egg plasma membranes, or due to the inability of the sperm to penetrate the vitelline layer? To examine the first possibility, we have investigated the sperm-egg fusion event per se by comparing the fertilization of A. pun&data eggs by A. punctulata and S. pwpuratus sperm after removal or disruption of the vitelline layer. The methods we have chosen to remove the vitelline layer also destroy the ability of the egg surface to bind sperm, thereby removing sperm adhesion as a factor in determining the fertilizability of the eggs. The first method we explored for removal of the vitelline layer was exposure of eggs to trypsin (50 pg/ml) at 4°C for 4 min. We have previously determined that these are the minimal conditions required to prevent the formation of a fertilization envelope upon parthenogenetic activation of the eggs and to completely abolish the ability of the eggs to agglutinate with isolated bindin particles from sperm (Glabe and Lennarz, 1979). Eggs treated in this fashion were then incubated with sperm in the presence of egg jelly homologous to the
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FIG. 3. Scanning eggs. Samples were fixed with glutaraldehyde 5 min electron micrograph of S. purpuratus sperm bound to A. punctulata and prepared for microscopy as described in Materials and Methods. All of the adherent sperm have undergone ! the after ins emination X7500. acrosomt f reaction. FIG. 4. Transmission electron micrograph of S. purpuratus sperm bound to A. punctulata eggs. Samples were fixed with glutaraldeh yde 5 min afte r insemination and prepared for microscopy as described in Materials and Methods. The bindin coated acrosomal process is in contact with the egg vitelline layer. The intact cortical granules are characteristic of unfertilized eggs. X48,000. intimate
GLABE,BUCHALTER,ANDLENNARZ
sperm to induce the acrosome reaction. As shown in Fig. 5A, with the vitelline layer intact, A. punctulata sperm are at least 100-500 times more effective than S. purpuratus sperm in fertilizing A. punctulata eggs (Fig. 5A). However, when the vitelline layer is removed, A. punctulata and S. purpuratus sperm are equally effective (Fig. 5B). This equality results because the efficiency of A. punctulata sperm to fertilize A. punctulata decreases while the efficiency of S. purpuratus sperm increases. It could be argued that trypsin treatment might also destroy a putative specific receptor for membrane fusion. To investigate this possibility we have utilized another method for removing the vitelline layer which is reported to be more specific than the trypsin procedure (Carroll et al., 197’7). The specificity of this procedure depends on the specificity of the cortical granule protease for the vitelline layer as a substrate. When the vitelline layer is removed by this procedure the results were identical to those obtained using trypsin; S. purpuratus sperm were nearly as effective as A. punctulata sperm in fertilizing A. punctulata eggs. The results of these experiments suggest that there is no species specific recognition event at the level of the fusion of sperm and egg plasma membranes per se and that an intact vitelline layer is required to maintain the species-specific selection of sperm for fertilization. Therefore, it appears likely that the failure of adherent S. purpuratus sperm to fertilize A. punctulata
60 i’ f
2000 Sperm
FIG. 5. Fertilization of A. removal of the egg vitelline pun&data sperm (O-----O) fixed after a 5min incubation termined. Fertilization was Methods.
4000 added /Egg
6000
punctulatu eggs before (A) and after layer. Eggs were inseminated with or S. purpuratus sperm (04) period and the extent of fertilization assessed as described in Materials
(B) A. and deand
Fertilization
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401
looor 9
Sperm
added
i0
per egg X IO+
FIG. 6. Sperm adhesion (O-----O) and fertilization (04) of S. purpuratus eggs inseminated with S. purpuratus sperm. Following insemination samples were fixed at 30 set for determination of sperm adhesion and at 5 min for determination of the extent of fertilization as described in Materials and Methods. (A) Eggs were inseminated in the presence of 0.325 m&f chymostatin. (B) Eggs were inseminated in the absence of chymostatin.
eggs is due to a failure of the sperm to penetrate the vitelline layer. Levine et al. (1978) have described a serine protease activity in S. purpuratus sperm, that is detectable only after the acrosome reaction has occurred. Furthermore, a trypsin-like protease has been ultrastructurally localized to the reacted acrosomal process in sperm of the same species (Green and Summers, 1980). However, the role of sperm proteases in sea urchin fertilization is not clear (see discussion Green and Summers, 1980). Recently, it has been reported that chymostatin, a noncompetitive inhibitor of chymotrypsin (Umezawa, 1977), inhibits the fertilization of several species of sea urchins (Hoshi et al., 1979). Based on these observations, it was suggested that a chymotrypsin-like enzyme is involved in the penetration of the vitelline layer. If a chymotrypsinlike activity does promote penetration then, in the presence of chymostatin, large numbers of homologous sperm should adhere to the egg surface, but no fertilization would result. We have tested this prediction with S. purpuratus gametes. In Fig. 6, the effect of 0.325 mM (200 pg/ml) chymostatin (Hoshi et al., 1979) on sperm adhesion and fertilization is shown as a function of sperm concentration. In the presence (Fig. 6A) or absence (Fig. 6B) of chymostatin, equivalent binding of sperm to eggs is observed, but in the presence of chymostatin very few of the eggs fertilize. Similar results were obtained with A punctulata gametes (data not shown). Chymostatin exerts its effect on sperm; pretreatment
402
DEVELOPMENTALBIOLOGY
.-‘0 ‘0N
60
-
40
-
:z h a-”
-m -2
0 Chymosiatin Addition (SKI
FIG. 7. Kinetics of chymostatin inhibition and the chymostatin sensitive period of gamete interaction. Chymostatin (0.325 rnk? was added at various times in relation to the time of sperm addition (to) and the extent of fertilization determined after 5 min.
of the sperm with chymostatin followed by a loo-fold dilution of the chymostatin reduced the fertilizability of the sperm by 99%. In contrast, pretreatment of the eggs with chymostatin, followed by its removal by washing, had no effect on their fertilizability. In addition we have investigated the effect of prior induction of the acrosome reaction on the ability of sperm to fertilize eggs in the presence of chymostatin. No fertilization was observed at any time after the induction of the acrosome reaction including the simultaneous addition of chymostatin and jelly to sperm. The kinetics of the inhibitory effect of chymostatin was determined by adding chymostatin to gametes at various times relative to the addition of sperm. If chymostatin is added at any time up to 2 see before sperm addition, fertilization is completely inhibited (Fig. 7). However, if chymostatin is added 2 set after sperm addition most of the eggs are fertilized. These results demonstrate that the inhibitory effect of chymostatin on sperm is rapid and that the chymostatin-sensitive step of sperm-egg interaction is rapidly completed upon mixing of gametes. Chymostatin contains an aldehyde group that is critical for its chymotrypsin inhibitory activity (Umezawa, 19’77). After reduction of this aldehyde group with NaBH, , less than 1% of the aldehyde group remained, as determined by reactivity with the Schiff reagent. The re-
VOLUME 84, 1981
duced derivative of chymostatin was found to be two orders of magnitude less potent as an inhibitor of chymotrypsin, with a Ki of 9 X IOe7 as compared to 8 X lops M for chymostatin. The effect of chymostatin on sperm does not appear to be related to its activity as an irreversible inhibitor of chymotrypsin. As shown in Fig. 8, the reduced derivative of chymostatin is nearly as effective as chymostatin in inhibiting fertilization. Parenthetically, it should be noted that the concentration of chymostatin required to inhibit fertilization is extremely high. Chymostatin at a concentration of 0.325 mM would be sufficient to completely inhibit the proteolytic activity of chymotrypsin present at the very high concentration of 1 mg/ml. In view of our finding that the inhibitory effect of chymostatin could not be ascribed to its activity as an inhibitor of chymotrypsin, we investigated the morphological effect of chymostatin on sperm treated with 0.325 m&Z chymostatin by transmission electron microscopy. Observations of thin sections revealed that chymostatin induces an abnormal acrosome reaction in greater than 90% of the sperm (Fig. 9A). This abnormal acrosome reaction induced by chymostatin consists of vesiculation of material at the apex of the sperm accompanied by a failure of the acIO0 -
so -
60
-
-4 Chymoslatin
Concentration LOQ M
FIG. 8. Concentration dependence of the inhibition of S. purpuratus fertilization by chymostatin (04) or reduced chymostatin (O----O). We have observed in several experiments that the concentration required for 50% inhibition of fertilization with either chymostatin or reduced chymostatin varies by a factor of approximately 2 (from 0.1 to 0.2 mMJ with different samples of gametes.
GLABE, BUCHALTER, AND LENNARZ
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FIG. 3. Transmission electron micrographs of chymostatin-treated and control S. pqnwatus sperm. (A) Sperm treated with 0.325 mM chymostatin for 30 set before fixation. (B) Control sperm treated with DMSO. (C) Sperm treated with egg jelly for 30 see to induce the acrosome reaction and then treated with 0.325 mM chymostatin. (D) Egg jelly treated sperm subsequently treated with DMSO. All figures X39,400.
rosome process to fully extend, as compared to the normal jelly induced acrosome reaction (Fig. 9D). DMSO, the solvent used for chymostatin, had no morphological effect on sperm or on the ability of sperm to undergo the acrosome reaction (Figs. 9B and D). The reduced derivative of chymostatin was found to have the same morphological effect on sperm as chymostatin (data not shown). Since as mentioned above, not only unreacted sperm but also sperm prereacted with egg jelly were inactivated by chymostatin, we examined the morphology of the reacted sperm by transmission electron microscopy. As shown in Fig. 9C, these sperm display an abnormal acrosomal region similar to that observed in nonreacted sperm that were treated with chymostatin. Chymostatin-treated sperm were also readily distinguishable from normal acrosome reacted sperm by electron microscopy of whole sperm mounted on Formvar-coated grids (Decker et al., 1976). Using this technique to evaluate
the sperm, it was found that 90% of the sperm in a population underwent the acrosome reaction upon jelly treatment. Treating these acrosome-reacted sperm with 0.325 mlM chymostatin caused more than 60% of them to exhibit an abnormal vesiculated acrosome process. These observations clearly establish that even the already extended acrosome process is disordered upon chymostatin treatment. As shown earlier (Fig. S), sperm-egg adhesion occurs efficiently in the presence of chymostatin, although the eggs fail to fertilize. It is apparent from the electron micrograph of thin section specimens of eggs inseminated in the presence of chymostatin (Fig. 10) that these abnormally reacted sperm adhere to the egg surface, even though they fail to fertilize the eggs. DISCUSSION
Based on a number of recent studies, it seems reasonable that the high degree of species specificity observed
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FIG. 10. Transmission electron micrograph of chymostatin-treated S. pwpurutus sperm adhering to the surface of a homologous egg. The vesiculated material covering the acrosomal process appears to be located at the site of adhesion between sperm and egg. x 39,400.
in sea urchin fertilization is the cumulative result of limited specificity in at least two of the sequential events of sperm-egg interaction (Summers and Hylander, 19’75; Glabe and Vacquier, 1977; SeGall and Lennarz, 1979; Glabe and Lennam, 1979; Kinsey et al., 1980). The early events of sperm-egg interaction, consisting of the induction of the acrosome reaction and subsequent sperm adhesion, have been shown to exhibit a range of specificities in different species of urchins (see Introduction). In contrast, very little is known about the later events of penetration of the vitelline layer and the fusion of sperm and egg plasma membranes or the possible contribution of these events to species specificity. In the few cases where a significant degree of heterologous sperm adhesion was observed, a high percentage of the eggs subsequently fertilized (Summers and Hylander, 1975; Kato and Sugiyama, 1978; Aketa and Ohto, 1979; Glabe, unpublished). This investigation is the first report of a case where significant numbers of heterologous sperm bind and yet the majority of eggs fail to fertilize. By analyzing the mechanism of this block to fertilization, we have explored the possible contribution of the later events of penetration of the vitelline layer and plasma membrane fusion to the overall species specificity of fertilization. We have found that in the absence of the vitelline layer and therefore sperm adhesion, A. punctulata and S. purpuratus sperm are equally effective in
fertilizing A. punctulata eggs. This suggests that there is no specificity in the fusion of sperm-egg plasma membrane . Earlier work can now be interpreted in the context of a modern model for sperm-egg interaction. Hultin (1948) originally showed that trypsin treatment of eggs enhances the capacity for cross fertilization. Hagstrom (1959) measured the kinetics of fertilization of trypsintreated eggs and found that the rates of fertilization for homologous and heterologous species were nearly equal. The rate of homologous fertilization decreased and heterologous fertilization increased when compared to the rate with the vitelline layer intact. These results and our observations present an apparent paradox in that the vitelline layer appears to act as both an effector of homologous fertilization and a barrier to heterologous fertilization. The one explanation for this is that the vitelline layer increases the efficiency of homologous fertilization by virtue of its ability to bind sperm. For heterologous sperm that do not adhere, the vitelline layer acts as a barrier by covering the egg plasma membrane and therefore preventing the less efficient process of direct plasma membrane fusion. From this work, it appears that there is a more important function for sperm adhesion than merely immobilizing the apex of the sperm head at the vitelline layer and that penetration of the vitelline layer plays a role in de-
GLABE,
BUCHALTER,
AND LENNARZ
termining species specificity. Perhaps the function of sperm adhesion is to anchor the apex of the sperm in such a manner as to provide support for the mechanical penetration of the vitelline layer by the extending acrosome process as proposed by Dan (1960) for star fish fertilization. Evidence for this type of mechanism has been presented for the penetration of the vitelline envelope of Lim&us eggs by sperm (Brown, 1976). In terms of a mechanical penetration model, the failure of S. purpuratus to fertilize A. punctulata eggs may be explained by postulating that the strength of adhesion between S. puqnwatus sperm and A. punctulata eggs is below the threshold required for penetration of the vitelline layer, but sufficient to immobilize the sperm. We have previously presented evidence which suggests that such species specific differences exist in the strength of the adhesive interaction between isolated bindin and eggs of these two species (Glabe and Lennarz, 1979). Another possible mechanism for the penetration of the vitelline layer is by the action of hydrolytic enzymes, as suggested for annelids (Colwin and Colwin, 1960), mollusks (Heller and Raftery, 19’76), and mammals (Austin and Bishop, 1959). In this regard, a trypsin-like protease associated with the acrosome reaction has been recently purified and characterized (Levine and Walsh, 1980). The preparation of sea urchin sperm protease was shown to possess both chymotrypsin and trypsin-like activity and to be inhibited by chymostatin. Hoshi et al. (1979) have observed that chymostatin inhibits fertilization in several species of sea urchins. In this report we have further investigated the effect of chymostatin on fertilization. Two lines of evidence suggest that the inhibitory effect of chymostatin on fertilization is not related to its activity as an inhibitor of chymotrypsin. A reduced analog of chymotrypsin, which is lOO-fold less potent as an inhibitor of chymotrypsin, is nearly as effective as chymostatin in inhibiting fertilization. Second, chymostatin is only an effective inhibitor of fertilization at extremely high concentrations approaching or exceeding its solubility in sea water. Our observations demonstrate that the primary effect of chymostatin is to induce an abnormal acrosome reaction in sperm consisting of the vesiculation of material at the apex of the sperm and an incomplete extension of the acrosome process. Chymostatin is an amphipathic peptide containing hydrophobic amino acid residues. Since it is an effective inhibitor only at high concentrations (0.325 mM>, this effect may be due to a detergent-like activity. We have previously observed that A. punctulata bindin forms vesicles upon treatment of sperm with the detergent Triton X-100 (Glabe and Lennarz, 1979), Perhaps, S. purpuratus bindin forms vesicles upon treatment with chymotrypsin. We have also demonstrated that the effect of chymostatin on sperm is rapid and that the chymostatin-sensi-
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tive events in sperm-egg interaction are completed rapidly upon mixing of gametes. There is no fertilization when chymostatin is added any time before the addition of sperm, but if chymostatin is added 2 set after insemination, most of the eggs subsequently fertilize. Since the primary effect of chymotrypsin appears to be inhibition of a normal acrosome reaction, we investigated the effect of preinducing the acrosome on the ability of the sperm to fertilize eggs in the presence of chymostatin. We found that these preinduced sperm still fail to fertilize eggs in the presence of chymostatin. Electron microscopic examination of such sperm revealed that the acrosomal process had been altered by chymostatin so that it was no longer fully extended. Thus chymostatin induces an abnormal reaction in unreacted sperm, as well as altering the morphology of the process in prereacted sperm. The fact that preinduced sperm do not fertilize eggs in the presence of chymostatin, along with the observation that the chymostatin sensitive interactions are rapidly completed upon insemination, suggest that the precise sequence and timing of the multiple events of sperm-egg interaction may be critical for successful fertilization. Our observations raise the possibility that penetration of the vitelline layer is accomplished by the extending acrosome process, although they certainly do not rule out the participation of hydrolytic enzymes. Mechanical penetration of the vitelline layer by the extending acrosome process may explain why the fertilizing capacity of sperm decreases rapidly after induction of the acrosome reaction, as reported previously by a number of investigators (Takahashi and Sugiyama, 1973; Vacquier, 1979a; Kinsey et al., 1980). Once the acrosome process has fully extended, sperm should no longer be able to mechanically penetrate the vitelline layer. If this model is correct, one implication is that the familiar electron microscopic image of sperm adhering to the vitelline layer by the extended acrosome process may not represent an intermediate step in the process of gamete interaction, but rather the nonproductive interaction of a typical unsuccessful sperm. However, it is clear that further work will be necessary to test this idea. The assistance of Ms. Ann Fuhr in the preparation is gratefully acknowledged.
of this manuscript
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