To ensure normal development, most animals have evolved a number of mechanisms to block polyspermy including prevention of binding to surface coats as well as sperm-egg fusion. Ascidian sperm bind to vitelline coat (VC) glycosides. In the genus Ascidia, N-acetylglu-cosamine (GlcNAc) is the ligand to which sperm bind. The number of sperm bound to the VC is biphasic following fertilization; sperm binding increases through the first minute or so, then abruptly declines. At fertilization, the eggs of Ascidia callosa, A. ceratodes, A. méntula, A. nigra and Phallusia mammillata release N- acetylglucosaminidase into the sea water (SW). This has been shown to inactivate VC GlcNAc groups, blocking the binding of supernumerary sperm and polyspermy in A. nigra. This block to polyspermy is inactivated by GlcNAc (2mm) or 150mm-Na+ (choline substituted) SW. These treatments are not additive and therefore probably affect the same process. In A. callosa, fertilization in low Na+ SW causes a 60 % decline in enzyme release and a similar increase in the number of sperm remaining on the VC at 4 min as well as a great increase in polyspermy. Thus the principal block to polyspermy in ascidian eggs involves the release of N-acetylglucosa-minidase which appears to be Na+ dependent. Enzyme activity is found in the supernatant SW by 15 s after fertilization, suggesting that it is stored very near the egg surface. Histochemical staining of whole eggs and embryos shows loss of surface-associated enzyme activity following fertilization. Like other lysosomal enzymes this N-acetylglucosaniinidase is mannosylated and has an acidic pH optimum.

For most eggs, fertilization by more than a single sperm results in chaotic cleavages and death of the embryo. Polyspermy is prevented by a number of different mechanisms, some of which prevent supernumerary sperm from access to the egg surface and others that block the fusion of sperm and egg plasma membranes. Many eggs have more than a single block (for review see Jaffe & Gould, 1985). Ascidian eggs are surrounded by a vitelline coat (VC) with follicle cells on its outer surface and a generous perivitelline space within which are found the test cells overlying the egg surface. Sperm bind to VC surface glycosides exposed between the follicle cells (Rosati & De Santis, 1980) then penetrate through the VC and perivitelline space to reach the surface of the egg. In the genera Ascidia (Lambert, 1986) and Phallusia (Honegger, 1982, 1986), sperm bind to VC N-acetylglucosamine (GlcNAc) groups while in Ciona fucose is the ligand (Rosati & De Santis, 1980; Hoshi et al. 1985).

Ascidians have evolved effective strategies for avoiding polyspermy. Seconds after the first sperm enters the egg it becomes immune to the entrance of additional sperm. The block to polyspermy involves Na+ in its formation and is progressive in nature, the eggs becoming able to withstand greater densities of challenging sperm with increasing time, finally becoming completely non-fertilizable after several minutes and first polar body production (Lambert & Lambert, 1981; Lambert, 1986). Part of this block involves a change in the VC sperm receptor GlcNAc groups so that they no longer bind sperm. This is the result of the egg releasing a protein after fertilization that is responsible for this modification (Lambert, 1986). In the previous work, although we had demonstrated that incubation of A. nigra eggs in fertilization supernatant inhibits their ability to bind sperm and trypsin reverses this, we had not identified the nature of the protein or the relationship of its release to Na+. Here I report that the eggs of several ascidians release the enzyme N-acetylglucos-aminidase in response to fertilization and that release is Na+-dependent. The enzyme is released from near the egg surface and modifies the VC so that supernumerary sperm fail to bind. This serves as a principal block to polyspermy in ascidian eggs.

Portions of this research were done in several laboratories including the Kewalo laboratory of the University of Hawaii, Friday Harbor Laboratory of the University of Washington, and the Roscoff and Kristineberg marine laboratories. Several different species from the genus Ascidia and closely related Phallusia mammillata were used. Most of the experiments were done with more than a single species with very similar results. The reported experiments are those that demonstrated the point most advantageously. Eggs were surgically removed from the oviduct and soaked for lh in filtered sea water (SW) made pH5·8–6·5 by the addition of HC1 followed by several washes in pH8·2 SW before use. This treatment was necessary because eggs from the genus Ascidia do not undergo synchronous fertilization without removal of oviducal jelly by either low pH or treatment with a proteolytic enzyme (Lambert & Epel, 1979). Sperm were pelleted in a microcentrifuge for 1min at 13000 g and stored on ice until used. Eggs of Ascidia ceratodes, A. nigra and Phallusia mammillata were used at room temperature while those of A. callosa, A. paratropa and A. mentula were used at sea water temperature of Friday Harbor Washington or Kristineberg Sweden 12–15°C. Sperm were activated in SW raised to pH 9·2 with 1 N-Tris (hydroxymethyl) aminomethane for 4 min before fertilization (Lambert, 1986). Sperm binding was studied as before (Lambert, 1986) by exposing the eggs (2000-3000 ml-1) to a rather dense sperm suspension (3μl ‘dry’ sperm added to 1·0ml pH9·2 SW diluted to 10 ml of egg suspension) followed by fixation in 0·5 % glutaraldehyde SW and gentle pipetting three times to remove sperm loosely associated but not firmly bound to the VC (Bleil & Wassar-man, 1980). Sperm attached through a fixed range of focus at the egg equator were determined with a 20× objective using differential interference contrast illumination. The extent of polyspermy was determined by counting the number of cells which either divided into more than 2 cells at first cleavage or that had multiple clear zones in the cytoplasm. This method may underestimate polyspermy as some polyspermic eggs fail to cleave. However, the presence of follicle and test cell nuclei makes impractical methods for polyspermy assessment based upon counting the number of sperm nuclei per egg by staining the DNA by fluorescent or histochemical methods (Lambert & Lambert, 1981; Lambert, 1986). Low Na+ SW contained 150mm-NaCl, 9mm-KCl, 9mm-CaCl2, 23mm-MgCl2, 26mm-MgSO4, 2 mm-NaHC03, and 273 mm either choline chloride or N-methylglucamine to make up the osmotic deficit of the low Na+ levels (Lambert, 1986).

N-acetylglucosaminidase was assayed using 4-methyl-umbelliferyl-GlcNAc (Sigma) as a substrate (Hoshi et al. 1985). To 0·5 ml citrate buffer at pH4·2 or at the specified pH was added 2 μl of a 25mm-stock solution of the substrate in dimethylsulphoxide (DMSO) following which was added 100 μl of the fertilization supernatant. Tris buffer was used for pHs above 7. After 30min, 3ml of 0·2M-glycine buffer pH 10·4 was added and the fluorescence determined in a Turner or Perkin Elmer fluorometer using 365 nm excitation and 395 nm emission wavelengths. The concanavalin A (ConA)-binding characteristics of the enzyme were determined as before (Lambert, 1988). ConA Sepharose (Sigma, 100 μl) was washed with 1 ml of SW in a centrifuge tube and 0·5 ml of the fertilization supernatant added and allowed to bind for 30 min. The ConA Sepharose was then sedimented by centrifugation, the supernatant saved and the pellet extracted for 15 min with 1 ml Sepharose equilibrium buffer (SEB) (0·02M-citrate, 0·75m-NaCl, 0·001 M-MnCl2, 0·001M-CaCl2 and 0·1 % Triton ×100 at pH5·2). Following this two more extractions with 0·5ml portions of SEB containing 0·5M-methyl mannoside were carried out. Portions of the original supernatant and SEB extracts were then assayed for enzyme activity as described above.

The histochemical investigation of enzyme release involved unfertilized and 2-cell embryos fixed briefly in 1 % formaldehyde SW and stained (Pearse, 1972) with a mixture containing 3mg of naphthol AS-BL-V-acetylglucosaminide (Polysciences; other brands did not give consistent results) dissolved in 0·3ml DMSO and added to 5·0ml citrate buffer pH5·2. To the substrate was added 0·6 ml of freshly prepared hexazotized pararosanaline (0·3ml each of 0 ·04g ml-1 of pararosanaline (Sigma) in 2N-HC1 and 0 ·04g ml-1 NaNO2 in H2O). The eggs were incubated in this mixture for 20 min, washed and mounted in SW for viewing. Omission of the substrate resulted in no staining.

Kinetics of glycosidase release, sperm binding and the block to polyspermy

It is likely that the block to polyspermy includes several processes. Kinetics for formation of the block to polyspermy used the method of primary insemination in a minimal sperm concentration for complete fertilization followed by challenging these eggs with much higher sperm densities at various times after the initial fertilization (Ziomeck & Epel, 1975; Lambert & Lambert, 1981; Lambert, 1986). The overall block forms at a rate which is dependent upon sperm density (Lambert & Lambert, 1981; Lambert, 1986). Previous studies used A. nigra’, increasing numbers of sperm bind through the first minute, then the number of sperm bound decreases rapidly. Sperm binding (Fig. 1) and the block to polyspermy (Fig. 2) follow nearly the same kinetics in the cold-water ascidian A. callosa. Here the peak of sperm binding occurs at l·5min followed by a sharp decline. The block to polyspermy follows nearly the same time course. Enzyme release was followed by withdrawing samples through 102 μm Nytex mesh at the indicated time intervals followed by centrifugation to remove sperm. The earliest time point at which a sample could be collected was 15 s by which time there was substantial enzyme activity in the external medium (Fig. 3). A maximum was reached by 1min, following which activity fluctuated around a plateau level. The experiment depicted in Fig. 3 represents the mean values from 3 independent experiments done in a single day. All other replicates of this experiment also included the fairly wide fluctuations around the plateau value shown here.

Fig. 1.

Kinetics of sperm binding to Ascidia callosa eggs. The number of sperm per egg profile is plotted against the time of fixation after insemination. Each data point represents the mean and standard errors from three independent experiments in which 10 eggs were evaluated.

Fig. 1.

Kinetics of sperm binding to Ascidia callosa eggs. The number of sperm per egg profile is plotted against the time of fixation after insemination. Each data point represents the mean and standard errors from three independent experiments in which 10 eggs were evaluated.

Fig. 2.

Time course of formation of the block to polyspermy in Ascidia callosa eggs. The per cent polyspermy is plotted against the time of challenge refertilization after initial insemination. Each data point represents the mean and standard errors from four independent experiments in which 100 eggs were evaluated.

Fig. 2.

Time course of formation of the block to polyspermy in Ascidia callosa eggs. The per cent polyspermy is plotted against the time of challenge refertilization after initial insemination. Each data point represents the mean and standard errors from four independent experiments in which 100 eggs were evaluated.

Fig. 3.

Release of N-acetylglucosaminidase following the fertilization of Ascidia callosa eggs. Fertilization supernatant was collected at the indicated times after insemination and assayed for N-acetylglucosaminidase activity by formation of a fluorescent product. Enzyme activity is in arbitrary fluorescence units. Data points indicate the means and standard deviations of three independent experiments.

Fig. 3.

Release of N-acetylglucosaminidase following the fertilization of Ascidia callosa eggs. Fertilization supernatant was collected at the indicated times after insemination and assayed for N-acetylglucosaminidase activity by formation of a fluorescent product. Enzyme activity is in arbitrary fluorescence units. Data points indicate the means and standard deviations of three independent experiments.

Egg glycosidase

Sperm binding by Ascidia nigra eggs is drastically curtailed after incubation in fertilization supernatant. This effect can be blocked by treatment of the supernatant with trypsin, suggesting that these eggs release a protein at fertilization that decreases the number of VC surface GlcNAc groups available to bind sperm (Lambert, 1986). Although the effect of the factor could be destroyed by trypsin, no further characterization of the factor was attempted in the previous report (Lambert, 1986). Since Xenopus eggs release N-acetyl-glucosaminidase at fertilization (Greve et al. 1985; Prody et al. 1985), and VC GlcNAc groups are modified in the block to sperm binding, it was important to assay for the presence of N-acetylglucosaminidase in the fertilization supernatant. Using a fluorometric assay, N- acetylglucosaminidase activity was in fact detected in the fertilization supernatants of A. nigra, A. ceratodes, A. callosa, A. paratropa, A. mentula and Phallusia mammillata.

As is common with lysosomal enzymes, this enzyme has a pH optimum close to 4·2 with very little activity at pH 8, the pH of SW. In addition to having an acid pH optimum, lysosomal enzymes are also extensively man-nosylated (Farooqui & Srivastava, 1980). To determine the presence of mannose groups on the egg glycosidase, binding to the mannose-glucose-specific lectin, ConA was studied in a simple batch procedure in a microcentrifuge tube. In this experiment, egg supernatant from A. callosa was incubated with ConA immobilized on Sepharose beads and the fraction bound determined. The beads were then washed in buffer, followed by washing in buffer containing a mannoside and the elution pattern of enzyme activity determined. Here 100 μl of a 1:5 dilution of the fertilization supernatant contained sufficient enzyme to_give a fluorescence value of 58·7 ± 9·4 arbitrary units (X ± standard error of the mean from 3 independent experiments). To 0·5ml of the supernatant was added 100 μl of ConA Sepharose and 30 min later the ConA Sepharose removed by centrifugation. No enzyme activity was found in 100 μl of this supernatant. The ConA Sepharose was then extracted with two 0·5 ml washes of SEB buffer containing 0·5mm-methyl mannoside. The total enzyme activity contained in 100 μl samples of these washes was 55·8±4·8. This shows that the glycosidase bound quantitatively to ConA in a manner which is completely reversible with a mannoside and suggests that the enzyme contains mannose groups.

Na+ dependency of glycosidase release, sperm binding and the block to polyspermy

Sperm of Ascidia nigra bind to VC surface GlcNAc groups before penetration. Sperm binding increases through the first minute, then sharply declines. The decline can be blocked by the presence of 2 mm-GlcNAc in the SW, which also increases polyspermy (Lambert, 1986). In addition, the block requires Na+ for its formation (Lambert & Lambert, 1981). To investigate the relationship between the modification of VC GlcNAc and Na+, sperm binding and release was studied in reduced Na+ SW. Fertilization does not occur in the absence of Na+ (Lambert & Lambert, 1981), therefore it was necessary to determine the lowest Na+ concentration that would reliably support fertilization. This was determined to be around 150 mm-Na+ using either choline chloride or N-methylglucamine as Na+ substitutes. Sperm binding is slightly lower in the low-Na+ SW but the normal decline completed by 2 min is completely suppressed (Table 1). The polyspermy block also requires Na+ for its formation (Lambert & Lambert, 1981). Since sperm detachment and the block to polyspermy could both be modified by either lowering the Na+ levels or adding GlcNAc to the SW, the question arises as to whether these two treatments are operating on two different polyspermy blocks or the same one. Accordingly the two treatments were combined in a single experiment, the expectation being that if GlcNAc and low-Na+ SW affected different mechanisms their effects should be additive. In these experiments, the protocols followed in the kinetic studies were modified to utilize a single fertilization with sperm densities high enough to cause a low but measurable level of polyspermy in the control cultures. As can be seen from Table 2, the two treatments are not additive. This implies that both treatments affect the same process and that glycosidase release requires external Na+.

Table 1.

Effect of Na+ replacement on sperm detachment by A. nigra eggs

Effect of Na+ replacement on sperm detachment by A. nigra eggs
Effect of Na+ replacement on sperm detachment by A. nigra eggs
Table 2.

Inhibition of the block to polyspermy in A. nigra eggs by low-Nc+ STV and N-acetylglucosamine

Inhibition of the block to polyspermy in A. nigra eggs by low-Nc+ STV and N-acetylglucosamine
Inhibition of the block to polyspermy in A. nigra eggs by low-Nc+ STV and N-acetylglucosamine

This would explain the requirement for Na+ in the postfertilization decline in sperm binding and the block to polyspermy. To test this hypothesis, eggs of A. callosa were fertilized in 150mm-Na+ SW and enzyme release, sperm binding and resistance to polyspermic fertilization evaluated. Lowering of extracellular Na+ caused a nearly 60% decline in enzyme release (Table 3). Sperm detachment was inhibited by a similar amount and polyspermy greatly increased. These last two findings confirm the results with A. nigra and point out the interdependence of enzyme release, sperm binding and polyspermy. P. mammillata also possesses the same requirement for Na+ in enzyme release and the block to polyspermy.

Table 3.

The effect of low-Na+ SW on enzyme release, sperm binding and the block to polyspermy in A. callosa eggs

The effect of low-Na+ SW on enzyme release, sperm binding and the block to polyspermy in A. callosa eggs
The effect of low-Na+ SW on enzyme release, sperm binding and the block to polyspermy in A. callosa eggs

Histochemical demonstration of fertilization-dependent enzyme release

Ascidian eggs lack the cortical granules which are prominent features of echinoderm and mammalian eggs (Rosati et al. 1977). Nevertheless they clearly release glycosidase in response to fertilization. To study enzyme release, cultures of A. callosa and P. mammillata eggs were divided in two, and one half was fertilized. When the fertilized sample reached the 2-cell stage, both samples were fixed briefly in 1% formaldehyde, washed and mixed together for staining in the same tube as explained in the Methods section. The test cells, follicle cells and VC do not stain. The entire surface of the unfertilized eggs becomes intensely pigmented by 20 min while the 2-cell embryo is only slightly stained (Fig. 4). This is another demonstration of enzyme release at fertilization and suggests that the enzyme is close enough to the cell surface to react with the stain without any special permeabilization being necessary.

Fig. 4.

Histochemical staining of unfertilized eggs and 2-cell embryos of Ascidia callosa for N-acetyiglucosaminidasc activity. Unfertilized eggs and 2-cell embryos were separately fixed, then mixed for staining in the same tube. Arrow marks the egg surface. Notice that the test cells, follicle cells and VC are unstained. The unfertilized egg is intensely stained over the entire surface and the 2-cell embryo is only slightly stained.

Fig. 4.

Histochemical staining of unfertilized eggs and 2-cell embryos of Ascidia callosa for N-acetyiglucosaminidasc activity. Unfertilized eggs and 2-cell embryos were separately fixed, then mixed for staining in the same tube. Arrow marks the egg surface. Notice that the test cells, follicle cells and VC are unstained. The unfertilized egg is intensely stained over the entire surface and the 2-cell embryo is only slightly stained.

A few minutes in 1 % formaldehyde would not be expected to result in free permeability to both substrate and coupler.

Ascidian fertilization involves a biphasic sperm-binding curve with increasing numbers of sperm bound during the first minute followed by a rapid decline. Sperm cease binding to the VC surface because the GlcNAc groups needed for binding are no longer available. GlcNAc inhibits sperm release and the block to polyspermy. The egg releases a trypsin-sensitive factor which changes the ability of the VC to bind sperm or wheat germ agglutinin, a GlcNAc-binding lectin (Lambert, 1986). This factor has now been identified as N-acetylglucosaminidase through the use of a fluorometric assay of enzyme activity. The enzyme has been identified in the fertilization supernatants of A. callosa, A. ceratodes, A. mentula, A. nigra, A. paratropa and Phallusia mammillata. Thus far only A. callosa, A. nigra, A. ceratodes and A. paratropa have been found to have biphasic sperm-binding curves, but the presence of the enzyme activity in the fertilization supernatant of all 6 species suggests that they may all share the same polyspermy-preventing mechanism. Sperm of Ascidia (Lambert, 1988) and Phallusia (Hoshi et al. 1985) have membrane-associated N-acetylglucosaminidase activity which is not released in a SW wash. It is unlikely that this activity is a significant contribution to the enzyme release reported here, as adding the quantity of sperm used to fertilize the cultures of A. callosa eggs used in these experiments gave no detectable enzyme activity when added to SW without eggs. In addition, the Ca2+/H+ ionophore A 23187 which can increase intracellular pH causes N-acetylglucosaminidase release without sperm from the eggs of A. nigra (Lambert, unpublished) and A. ceratodes (Kochumian, unpublished).

In our original report (Lambert & Lambert, 1981), we determined that Na+ was required for fertilization. We also showed that it was needed for formation of the block to polyspermy in A. nigra by fertilizing in complete SW followed by rapid transfer to Na+-free (choline substituted) SW. After the control cultures had formed their polyspermy block these eggs were refertilized in complete SW and became highly polyspermic. Removal of other ions did not increase polyspermy (Lambert & Lambert, 1981). In the following work, it was demonstrated that these eggs release a factor at fertilization that modifies the VC sperm-binding sites (Lambert, 1986). Since release of this egg factor might involve Na+, experiments were performed to see if fertilization could be achieved in low-Na+ rather than Na+-free SW. Na+ concentrations below 100 mm inhibited fertilization but 150mm-Na+ gave satisfactory fertilization with A. nigra, A. callosa and Phallusia mammillata using either choline chloride or /V-methyl glucamine to substitute for the osmotic contribution of Na+. This allowed the design of other experiments involving fertilization in the presence of GlcNAc in low-Na+ SW to see if the effects of the two polyspermyenhancing agents were additive. Low Na+ and GlcNAc are not additive in their effect on polyspermy. This is circumstantial evidence that they affect the same system. To investigate further the connection between these processes, eggs of A. callosa were fertilized in low-Na+ SW and enzyme release, sperm binding and degree of polyspermy determined. Fertilization in low-Na+ SW inhibits enzyme release, sperm detachment and the block to polyspermy. Similar experiments with P. mammillata yielded the same findings with enzyme release and the block to polyspermy. These experiments support a role for Na+ in the release of the glycosidase which is involved in the polyspermy block but additional experiments will be required to delineate the exact role of Na+. The possibility also exists that fertilization in reduced Na+ SW has a nonspecific deleterious effect on the eggs not specifically related to fertilization. This is a difficult problem to resolve; however, monospermic eggs fertilized in low-Na+ SW undergo normal cleavage in this medium and, if transferred to complete SW, subsequently form normal larvae which are capable of metamorphosis. The role suggested for Na+ in glycosidase release may not be the only role played by this ion in the overall block; other organisms such as sea urchins (Jaffe, 1976), Urechis (Gould-Somero et al. 1979) and Mytilus (Dufresne-Dube et al. 1983) have polyspermy blocks involving Na+ but without glycosidase release.

The finding of significant amounts of the enzyme in the supernatant SW by 15 s after fertilization demonstrates that release is rapid enough to be operational as an effective polyspermy block. The release of cortical granule glucanase and protease from sea urchin eggs begins at a slower rate and reaches a maximum at nearly the time (1 · 5min) of ascidian eggs (Vacquier et al. 1973). After fertilization, Xenopus eggs release N-acetylglucosaminidase from their cortical granules which decreases sperm binding and polyspermy. This release follows the same kinetics as the cortical reaction, with a midpoint in the release curve at 4 · 3min (Greve et al. 1985; Prody et al. 1985). In Xenopus the slow polyspermy block is completed by 6 min (Grey et al. 1982) in good agreement with the enzyme release data. However, there are other events occurring including the reaction of egg lectin with extracellular coats (Greve & Hedrick, 1978) which also contribute to the block.

Ascidian eggs lack cortical granules but do have subcortical granules that seem to be released at the egg surface after fertilization (Rosati et al. 1977). This release in Ciona begins several minutes after fertilization, which is much too late to be involved in N-acetylglucosaminidase release. N-acetylglucos-aminidase appears to be located either very close to the inner surface of the plasma membrane or on the outer surface prior to fertilization, as whole unfertilized eggs can be readily stained for enzyme activity in the absence of special permeabilization steps. The rapid transit through the perivitelline space and VC also argue for storage near the egg surface. The exact localization of the enzyme awaits further ultrastructural histochemistry.

Egg N-acetylglucosaminidase is the second glycosidase involved in ascidian fertilization. Sperm binding to the VC is dependent upon sperm surface glycosidase forming an enzyme-substrate complex with glycosides of the VC surface (Hoshi et al. 1985). Ciona sperm show fucosidase activity (Hoshi et al. 1985) while Phallusia (Hoshi et al. 1985) and Ascidia (Lambert, 1988) have a membrane N-acetylglucosaminidase. Thus it would appear that Ascidia spp. use the same type of enzyme to bind sperm to the VC and to alter the VC after fertilization so that supernumerary sperm fail to bind. The sperm enzyme is an integral membrane protein that can be removed with Triton × 100, while the egg enzyme is released free into the SW. Both enzymes are manno-sylated as shown by binding to ConA, and both have the acidic pH optima typical of lysosomal enzymes. Further comparison awaits biochemical characterization of the enzymes.

I am grateful to G. Lambert for help with the manuscript and several experiments as well as for drawing Figs 1 – 3. R. Koch, M. Kochumian and M. Hoshi gave helpful discussions of the work in progress. The staffs of the Friday Harbor, Kewalo, Kristineburg, and Roscoff marine laboratories have my thanks for making space and equipment available and help in collecting animals. The early work was supported by NSF Grant PCM 83 – 08571 recent work by a CSUF faculty research grant.

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