MOLECULAR REPRODUCTION AND DEVELOPMENT 25177-185 (1990)
Fertilization Envelope Assembly in Sea Urchin Eggs Inseminated in Chloride-Deficient Sea Water: 11. Biochemical Effects JEFFREY D. GREEN,' PATRICIA S. GLAS,' SOU-DE CHENG,' AND JOHN W. LYNN2 'Department of Anatomy, Louisiana State University Medical Center, New Orleans, Louisiana; 2Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana ABSTRACT Eggs of the sea urchin Strongylocentrotus purpuratus were fertilized in normal and in several chloride-deficient sea waters ((CI-1: normal > isethionate > methyl sulfonate > bromide). The fertilization envelopes (FE) were thinner and failed to harden, and the characteristic I-T transition did not occur. The permeability of the experimental FEs, as determined by release of protein from the perivitelline space, increased in the order of decreasing [CI-1. Release of the enzymes p - 1 3 glucanase and cortical granule protease were not significantly altered. O n the other hand, release of ovoperoxidase was increased three to four times in bromide sea water. Furthermore, a dose-response was observed in varying concentrations of bromide-normal sea water. With decreasing chloride (increasing bromide) concentration, more ovoperoxidase activity was observed. Cytochemical localization of ovoperoxidase activity with diaminobenzidine revealed almost a total lack of staining of FEs from bromide-substituted sea water. The results suggest that in chloride-deficient sea waters protein incorporation into the nascent FE is impaired. At least in the case of bromide, the incorporation of ovoperoxidase into the nascent FE was also inhibited. Key Words: Ovoperoxidase, Exocytosis, Extracellular macromolecular assembly, Anions
INTRODUCTION The formation of the sea urchin fertilization envelope (FE) is a striking example of regulated extracellular macromolecular assembly. First described almost 150 years ago (Derbes, 18471, it has been the subject of much investigation during the 20th century. Its functional attributes include providing a barrier against the penetration of supernumerary sperm, providing a microenvironment in which developmental processes occur, and protecting the developing embryo from mechanical insult. Mechanistically, the formation of the FE results from the addition of macromolecules secreted from cortical granules to the vitelline envelope
0 1990 WILEY-LISS, INC.
(VE) and their subsequent covalent cross linking. The VE, a n extracellular investment resting upon the oolemma, serves as a template for the developing FE. The definitive, hardened FE results from the interaction of structual proteins and enzymes. These processes have been intensively investigated during the last decade. The elevation itself appears to result from the hydration of secreted macromolecules in the perivitelline space (Schuel et al., 1974; Green and Summers, 1980). Among the investigations alluded to above, many have focused on the actions of two enzymes secreted by the egg. A protease, specifically cleaving the carboxyl terminus of arginyl residues (Fodor et al., 1975; Green, 19861, putatively functions a s a sperm receptor hydrolase and VE delaminase (Vacquier et al., 1973; Carroll and Epel, 19751, thereby preventing polyspermic fertilization (Schuel et al., 1973; Longo and Schuel, 1973; Longo et al., 1974). Ovoperoxidase is responsible for the hardening of the FE through its action of catalyzing the cross linking of tyrosyl residues in the nascent FE to form dityrosines (Foerder and Shapiro, 1977; Hall, 1978). In addition to cortical granule components, various ionic constituents of sea water have also been shown to affect the proper structuralization of the FE. Di- and monovalent cations (Ca2+,N a + , and Mg2+)have all been implicated in FE assembly (Schon and Decker, 1981; Nishioka and Cross, 1978; Schuel e t al., 1982; Kay e t al., 1982; Weidman et al., 1985; Weidman and Shapiro, 1987). Alterations in the final form of the FE can be produced by modifying the ionic composition of sea water with respect to these cations. Recently we reported that FE assembly does not occur properly in sea water deficient in chloride ions, by far the predominant anion in sea water (Lynn e t al., 1986, 1988; Green et al., 1987). By replacing C1- with four anionic
Received J u n e 8, 1989; accepted August 15, 1989 Address reprint requests to Jeffrey D. Green, Department of Anatomy, Louisiana State University Medical Center, 1901 Perdido St., New Orleans, LA 70112.
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substitutes (bromide, nitrate, isethionate, and methyl- The C1- content of the sea waters was determined as sulfonate) we demonstrated a failure of the FE to described in Lynn et al. (1988). All sea waters were harden in three species of sea urchins, Strongylocen- buffered with 10 mM TAPS (tris[hydroxymethyll trotus purpuratus, Lytechinus uariegatus, and L . pictus. methylaminopropane sulfonic acid; Sigma) and adFurthermore, the characteristic I (round) to T (an- justed to a final pH of 8.3. gular) transformation of the microvillar casts in S.purScanning Electron Microscopy (SEMI puratus (Veron e t al., 1977) did not take place. In this Eggs were fixed in 2% glutaraldehyde in 0.1 M Napaper, we extend our earlier morphological observacacodylate (pH 7.2) for 1 h r and washed overnight in tions with scanning electron microscopy and provide biochemical data regarding the secretion of cortical cacodylate. Osmication was for 1 h r in 1%OsO, in 0.1 granule protein and the activities of three major se- M cacodylate (pH 7.2). Eggs were dehydrated with ethcreted enzymes, ovoperoxidase, protease, and gluca- anol and acetone, critically point dried on a Samdri-790 (Tousimis Research Corp.), and attached to stubs with nase, in anion-substituted seawater. silver paint. Stubs were coated in a Hummer VI sputtering system (Technics). Observations were on a Jeol MATERIALS AND METHODS JSM-35CF scanning electron microscope. Gametes The purple urchin S . purpuratus was obtained from Transmission Electron Microscopy (TEM) Pacific Biomarine (Venice, CA). Gametes were colEggs were fixed as described above and dehydrated lected by introduction of 0.55 M KCl into the coelomic in ethanol, passed through propylene oxide, and emcavity, with eggs spawned into artificial sea water bedded in Embed 812 (Electron Microscopy Sciences). (MBL-SW). Sperm were collected “dry” and kept Silver sections were cut on a Reichert-Jung Ultracut E chilled until used. Eggs were dejellied by passing them with a diamond knife, stained with lead citrate and through several layers of cheese cloth and then were uranyl acetate, and viewed with a Phillips 301 transtreated with acidified sea water (0.1 N HCl, pH 5) for 3 mission electron microscope. min. The egg suspension was readjusted to pH 8.0 with Enzyme Cytochemistry 1 M Tris base added dropwise. The eggs were washed three times in sea water, followed by another two For the localization of ovoperoxidase, the method of washes in the appropriate substituted sea water (see Klebanoff et al. (1979) was used. Unfertilized eggs and below). Within 30 min eggs were suspended in a 5% eggs 15 rnin after insemination were gently settled by suspension, and “dry” sperm were added to give a 0.1% hand centrifugation, washed three times, and resusconcentration. As a n alternative to activation with pended in 0.45 M NaC1, NaBr, or Na-Ise with 5.6 mM sperm, the ionophore A23187 (free acid, molecular 3,3‘-diaminobenzidine (DAB; Sigma) buffered with 0.1 weight 523; Calbiochem) was used to activate eggs. The M Tris base to pH 8.0. DAB incubations were perionophore was dissolved in dimethylsulfoxide (DMSO; formed in equimolar NaC1, NaBr, and Na isethionate Sigma). Final concentration of ionophore was 38 pM solutions because 1)we did not want to reintroduce C1(20 pgiml) in 1%DMSO. Supernatants for the protein back into the experimental C1--deficient media and 2) and enzyme assays were collected 15 min after fertili- the DAB localization was not successful in the complex zation or ionophore activation. ionic mixture of sea water. Hydrogen peroxide (100 p1, 30%; Sigma) was added to a 5 ml volume of the above Artificial Sea Waters containing about 10% eggs. Samples were incubated Control (MBL-SW) and experimental sea waters for 10 min a t 10°C. DAB was omitted from controls. were made according to Cavanaugh (1956); bromide The reaction was stopped by the addition of 2 volumes (Br-) and isethionate (Ise) salts were substituted for of 3% glutaraldehyde for 1 hr, and the samples were chloride (C1-) salts in the substituted sea waters. washed in 0.1 M Na-cacodylate overnight and prepared Methyl Sulfonate (MeS)-substituted sea water was pre- for transmission electron microscopy as described pared a s previously described by Lynn et al. (1988). above. The compositions of the various sea waters were: 1) Total Protein MBL-NaC1, 423 mM; KC1, 9 mM; CaCl,, 9 mM; Supernatant (fertilization product; FP) over fertilMgCl,, 23 mM; MgSO,, 25 mM; NaHCO,, 2 mM; 2) Ise-Na isethionate, 422 mM; KCI, 9 mM; CaCl,, 9 ized or ionophore-activated eggs was collected and centrifuged by hand to remove any remaining whole eggs. mM; MgSO,, 48 mM; NaHCO,, 2 mM; 3) Br--NaBr, 420 mM; KBr, 10 mM; CaBr,, 10 mM; MgSO,, 48 mM; Supernatant from this was then centrifuged for 10 min NaHCO,, 2 mM; 4) MeS-substituted sea water pre- at 8,800 g in a n Eppendorf centrifuge a t 10°C to remove pared by titrating a 480 mM solution of methane sul- sperm. This final supernatant was used in the protein fonic acid with 10 N NaOH to a pH of 7.0 and by sub- and enzyme assays. sequently titrating a 10 mM solution of methane For protein determination, 1 ml of FP was precipisulfonic acid with 5 N KOH to a pH of 7.0. To this tated with 110 p1 of ice-cold 50% trichloroacetic acid solution, 65 mM MgSO, and 10 mM CaC1, were added. (TCA) on ice, centrifuged 20 min a t 8,800 g a t 10” C,
FERTILIZATION IN Cl-DEFICIENT SEA WATER drained by inversion, and air dried. The precipitate was assayed by the Lowry method (Lowry et al., 1951), with bovine serum albumin (BSA) as a standard. Preliminary experiments demonstrated no significant difference (95% confidence level) among TCA-precipitable BSA in the various sea water solutions. Control experiments utilizing unfertilized eggs incubated in the various media resulted in very small, insignificant amounts of protein in the supernatants: MBL, 1.42 pgl ml; Ise, 1.55; Br-, 1.42.
Enzyme Assays Ovoperoxidase activity was assayed using a reaction mixture of 10 mM TAPS, pH 8.0, 18 mM guaiacol, 0.3 mM H,Oz (3.3 ~1 of 30% H20, per 100 ml) in distilled H 2 0 (Deits et al., 1984). Twenty to one hundred microliters F P was placed in a 1 ml cuvette. Reaction mixture (980-900 pl) was quickly pipetted to mix rapidly with the fertilization product, and the initial reaction rate was recorded a t 436 nm (0.2 in./sec chart speed) on a Beckman ACTA spectrophotometer. Blanks were equal amounts (20-100 pl) of substituted artificial seawater treated as samples with the reaction mixture. As a control to test the effects of the various sea waters on ovoperoxidase activity, eggs were fertilized in MBL. Aliquots (20 ~ 1 of) FP were mixed with reaction mixtures (980 pl) in each of the substituted sea waters. No differences were observed in the initial reaction rates. p-1,3-Glucanase activity was assayed according to Green and Summers (1980). Aliquots (200-500 pl) of the FP were mixed with 50 p1 of the algal polysaccharide substrate laminarin (Laminaria digitata; 10 mgl ml), and the appropriate sea water was added to a total of 1 ml reaction mixture. The reaction proceeded for 2 h r a t 37°C. Two hundred microliter aliquots of the above mixtures were then removed and incubated for 10 min at 37°C with 400 pl enzymes (400 pl glucose oxidase plus 3 mg horseradish peroxidase in 50 ml of 0.25 M phosphate buffer, pH 6.0) and 400 p1 chromogen (40 mg o-dianisidine in 50 ml distilled H,O). The reaction was stopped by addition with vortexing of 800 p1 of 4 N sulfuric acid. Samples were assayed spectrophotometrically at 530 nm. Enzyme activity is measured a s pmoles of glucose formed per minute. A glucose solution served as the standard. Control incubations of glucose were assayed in the experimental sea waters and no significant differences (95% confidence level) were observed. Reagents were purchased from Sigma. Secreted protease was assayed spectrophotometrically for esterase activity using Na-benzoyl arginine ethyl ester (BAEE; Sigma; Schwert and Takenaka, 1955).One hundred microliters of a 1 mM BAEE solution (in the appropriate sea water, pH 8.3, with 10 mM TAPS) was pipetted into a quartz cuvette. The assay was initiated by adding 900 p1 of FP with mixing, and the reaction was observed a t 254 nm. Trypsin-like activity of the FP was estimated by comparing reaction rates (change in absorbance) with those produced by
179
TPCK-trypsin (Sigma). Statistical analyses were performed using Student’s t and ANOVA tests.
RESULTS SEM Fertilization in C1--deficient sea waters markedly affected the formation of the FE. Most notably, the I-T transition (from rounded to angular microvillar casts) failed to occur (Fig. 1). At 3 min postinsemination, MBL-SW FEs have completed the I-T transition, while the Br--SW eggs retained I-shaped microvillar casts. By 60 min postinsemination, the Br--SW FEs still did not complete the transition. There appears to be a graded response, presumably related to the chloride content of the media, with the extreme difference between MBL-SW (Fig. lB,C) and Br--SW (Fig. lE,F). Ise-SW, which falls between MBL- and Br--SW relative to chloride content, supported a partial I-T transition (Fig. 1D). Previously, we demonstrated with TEM that the ultrastructure of FEs in several Cl--deficient sea waters varied from the normal condition (Lynn et al., 1988). Total Protein Secreted The morphological characteristics described above suggest that the permeability of the FE might also be altered. It has been shown that various treatments of the VE before fertilization result in differences in protein permeability of the nascent FE (Veron et al., 1977; Green and Summers, 1980). Therefore, eggs were fertilized in MBL- and C1--deficient SW. After eggs and sperm were removed by gentle and rapid centrifugation, respectively, the total protein of the FP (supernatant) was assayed (Fig. 2). The MBL FEs allowed the least amount of protein (-13 pgiml FP) into the supernatant, whereas the Br--treated FE’s allowed the most (-63 pg/ml FP). Intermediate between the extremes were Ise (=18 pg1ml FP)- and MeS ( ~ 4 pgiml 6 FP)treated FEs. Since the [Cl-] increases in the order Br < MeS < Ise < MBL, the amount of protein released was inversely related to the amount of chloride present in the media. Therefore, the permeability of the FE appears to be inversely related to the chloride concentration, although the possibility of the different anionic species differentially affecting permeability cannot be ignored. Ise treatment resulted in a slightly higher protein concentration in the media, although the difference was not statistically significant (P = 0.35) from that of the control MBL SW. However, both MeS (P 5 0,001) and Br- (P 4 0.001) treatments resulted in protein release significantly higher than MBL. Enzymatic Activity Three enzymes secreted from the egg a t fertilization were assayed to determine what effects, if any, the treatments would have on their activities. We monitored the release of p-1,3-glucanase, cortical granule protease, and ovoperoxidase from the eggs following
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Fig. 1. Scanning electron microscopy of S.purpurutus vitelline and fertilization envelopes. A: VE of unfertilized egg in MBL-SW. Note the close array of rounded (I-form) microvillar casts. B: By 3 rnin postinsemination, MBL FEs have angular (T-form) casts. (Envelope collapsed during fixation and dehydration.) C: Mature MBL FE with
T-form casts a t 60 min. Note the spread array of casts compared to A. D Ise FE a t 60 min resembles 3 min MBL. I-T transition remains incomplete. E: Br- FE a t 3 min resembles unfertilized egg except that I-form casts are further separated due to expanding FE. F Br FE at 60 rnin has changed little since 3 min. x 10,000.
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fertilization or ionophore activation. The difference in release of glucanase was not statistically significant between MBL and Ise ( P = 0.071, MeS ( P = 0.571, or Br- (P = 0.29) (Fig. 3). Cortical granule trypsin-like protease activity as measured with the ester substrate BAEE corresponded to a range of 2-6 picomoles of trypsin activity per milliliter of a 5% egg suspension. Although Br- and MBL averaged higher reaction rates than Ise and MeS, these differences were not statistically significant (Ise, P = 0.07; MeS, P = 0.07; Br-, P = 0.35) a s judged using the Student's t test or one-way ANOVA (P = 0.12) (Fig. 4). In any case, protease activity was not inhibited. The only enzymatic activity measured that significantly differed from controls was that of ovoperoxidase. As determined by the guaiacol assay, eggs fertilized in Br--SW released significantly more ovoperoxidase (P 5 0.002) than eggs fertilized in MBL-SW (Fig. 5). In fact, three to four times more ovoperoxidase was detected. To ascertain whether this increase was due to increased enzyme release or increased enzyme activation, we fertilized eggs in MBL-SW, removed equal aliquots of FP, and assayed them in control and experimental SWs. There were no differences in activity, indicating that the various SWs did not affect ovoperoxidase catalysis of guaiacol. However, the results of ovoperoxidase release with MeS and Ise were enigmatic (Fig. 5). Ise released significantly lower (P 5 0.02) amounts of ovoperoxidase, and the MeS was lower than controls also, but this was not statistically significant ( P = 0.16). The number of trials in MeS was limited because with this medium it was extremely difficult to obtain 95% fertilization with S.purpuratus. When eggs were fertilized in sea waters containing varying proportions of Br--SW and MBL-SW, a dosage response was observed (Fig. 6). With decreasing con-
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Fig. 4. Spectrophotometric tracings of cortical granule protease released through FEs. Esterase activity was measured as described in Materials and Methods. Reaction rates are depicted as changes in absorbance at 254 nm. At the 95% confidence level, no significant differences were found between MBL- and experimental S W trials. Each rate is the average of two trials.
centrations of chloride, more ovoperoxidase activity appeared in the FP. Since ovoperoxidase is largely incorporated into the nascent FE, the removal of the FE precursor (VE) should result in more of the enzyme being released into the supernatant. Indeed, when eggs were pretreated with dithiothreitol (DTT) or trypsin before fertilization or ionophore activation, respectively, more ovoperoxidase was released. In the case of DTT, about two times more enzyme was released; after trypsin, four to seven times more enzyme was released than in controls. This difference is consistent with the more complete removal of the VE by trypsin than by DTT and corresponds with a four- to nine-fold increase in total protein released following trypsin treatment (data not shown).
J.D. GREEN ET AL.
182
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DAB reaction in order of decreasing chloride concentrations. That is, the MBL-treated eggs stained more darkly than the Ise-treated eggs, which stained more darkly than the Br--treated eggs. Therefore, FEs in normal SW incorporated more ovoperoxidase than FEs in the C1--deficient sea waters.
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ovoperoxidase activity (,u.mol/min/ml FP) Fig. 6. Ovoperoxidase dosage response. MBL- and Br -SWs were mixed to adjust [Cl-1, which is shown as a percentage of normal sea water (MBL). Eggs were fertilized in these concentrations, and ovoperoxidase activity was determined as described in Materials and Methods. Enzyme activity increased as [Cl I decreased (or LBr ~1 increased). Data shown are from a representative experiment.
Enzyme Cytochemistry Following the observation that more ovoperoxidase was released into the supernatant after fertilization in a chloride-deficient medium (Br--SW), and that this release appeared to be dosage dependent (Fig. 61, we hypothesized that this increase could be due to a decrease of ovoperoxidase incorporation into the FE. Therefore, we fertilized eggs in MBL-, Ise-, and Br-SWs, incubated them with DAB, and processed them for TEM. The results are shown in Figure 7. Although not quantitative, the micrographs reveal a less intense
DISCUSSION In a n attempt to analyze the physiological importance of C1- ions, the major anionic component of sea water, we previously reported that, although fertilization occurred, the elevation of the fertilization envelope of sea urchin eggs was seriously impaired (Lynn et al., 1988). Using three species of sea urchins and four different anionic substitutes (Ise, MeS, NO,-, and Br- 1, we ascertained that FEs elevated but were less birefringent than FEs in normal sea water. Furthermore, they collapsed back upon the eggs within several minutes postinsemination. Using FE collapse as a criterion for hardening, we further demonstrated that this failure to harden was related to decreasing [Cl-], especially when [Cl-] was < 50% of normal. On the other hand, when eggs were fertilized in C1--deficient SW but transferred back to normal SW within about 10 min postinsemination, normal hardening ensued. Conversely, eggs fertilized in normal SW and transferred to Cl--deficient SW earlier than 10 min postinsemination showed elevated soft FEs. This observation is consistent with earlier reports that the structuralization and cross linking of the FE require = 10-15 min (Veron et al., 1977; Schuel et al., 1982). TEM profiles of FEs revealed that the normal trilaminar morphology was absent, and, perhaps even more striking, the I-T transition of the microvillar casts ( S . purpuratus) failed to occur. This transition also appeared to be affected in a [Cl-]-dependent manner, since FEs in IseSW (24 mM C1-) and MeS-SW (17 mM C1-) had intermediate casts, whereas those of eggs fertilized in NO,--SW (
Fertilization Envelope Assembly in Sea Urchin Eggs Inseminated in Chloride-Deficient Sea Water: 11. Biochemical Effects JEFFREY D. GREEN,' PATRICIA S. GLAS,' SOU-DE CHENG,' AND JOHN W. LYNN2 'Department of Anatomy, Louisiana State University Medical Center, New Orleans, Louisiana; 2Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana ABSTRACT Eggs of the sea urchin Strongylocentrotus purpuratus were fertilized in normal and in several chloride-deficient sea waters ((CI-1: normal > isethionate > methyl sulfonate > bromide). The fertilization envelopes (FE) were thinner and failed to harden, and the characteristic I-T transition did not occur. The permeability of the experimental FEs, as determined by release of protein from the perivitelline space, increased in the order of decreasing [CI-1. Release of the enzymes p - 1 3 glucanase and cortical granule protease were not significantly altered. O n the other hand, release of ovoperoxidase was increased three to four times in bromide sea water. Furthermore, a dose-response was observed in varying concentrations of bromide-normal sea water. With decreasing chloride (increasing bromide) concentration, more ovoperoxidase activity was observed. Cytochemical localization of ovoperoxidase activity with diaminobenzidine revealed almost a total lack of staining of FEs from bromide-substituted sea water. The results suggest that in chloride-deficient sea waters protein incorporation into the nascent FE is impaired. At least in the case of bromide, the incorporation of ovoperoxidase into the nascent FE was also inhibited. Key Words: Ovoperoxidase, Exocytosis, Extracellular macromolecular assembly, Anions
INTRODUCTION The formation of the sea urchin fertilization envelope (FE) is a striking example of regulated extracellular macromolecular assembly. First described almost 150 years ago (Derbes, 18471, it has been the subject of much investigation during the 20th century. Its functional attributes include providing a barrier against the penetration of supernumerary sperm, providing a microenvironment in which developmental processes occur, and protecting the developing embryo from mechanical insult. Mechanistically, the formation of the FE results from the addition of macromolecules secreted from cortical granules to the vitelline envelope
0 1990 WILEY-LISS, INC.
(VE) and their subsequent covalent cross linking. The VE, a n extracellular investment resting upon the oolemma, serves as a template for the developing FE. The definitive, hardened FE results from the interaction of structual proteins and enzymes. These processes have been intensively investigated during the last decade. The elevation itself appears to result from the hydration of secreted macromolecules in the perivitelline space (Schuel et al., 1974; Green and Summers, 1980). Among the investigations alluded to above, many have focused on the actions of two enzymes secreted by the egg. A protease, specifically cleaving the carboxyl terminus of arginyl residues (Fodor et al., 1975; Green, 19861, putatively functions a s a sperm receptor hydrolase and VE delaminase (Vacquier et al., 1973; Carroll and Epel, 19751, thereby preventing polyspermic fertilization (Schuel et al., 1973; Longo and Schuel, 1973; Longo et al., 1974). Ovoperoxidase is responsible for the hardening of the FE through its action of catalyzing the cross linking of tyrosyl residues in the nascent FE to form dityrosines (Foerder and Shapiro, 1977; Hall, 1978). In addition to cortical granule components, various ionic constituents of sea water have also been shown to affect the proper structuralization of the FE. Di- and monovalent cations (Ca2+,N a + , and Mg2+)have all been implicated in FE assembly (Schon and Decker, 1981; Nishioka and Cross, 1978; Schuel e t al., 1982; Kay e t al., 1982; Weidman et al., 1985; Weidman and Shapiro, 1987). Alterations in the final form of the FE can be produced by modifying the ionic composition of sea water with respect to these cations. Recently we reported that FE assembly does not occur properly in sea water deficient in chloride ions, by far the predominant anion in sea water (Lynn e t al., 1986, 1988; Green et al., 1987). By replacing C1- with four anionic
Received J u n e 8, 1989; accepted August 15, 1989 Address reprint requests to Jeffrey D. Green, Department of Anatomy, Louisiana State University Medical Center, 1901 Perdido St., New Orleans, LA 70112.
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J.D. GREEN ET AL.
substitutes (bromide, nitrate, isethionate, and methyl- The C1- content of the sea waters was determined as sulfonate) we demonstrated a failure of the FE to described in Lynn et al. (1988). All sea waters were harden in three species of sea urchins, Strongylocen- buffered with 10 mM TAPS (tris[hydroxymethyll trotus purpuratus, Lytechinus uariegatus, and L . pictus. methylaminopropane sulfonic acid; Sigma) and adFurthermore, the characteristic I (round) to T (an- justed to a final pH of 8.3. gular) transformation of the microvillar casts in S.purScanning Electron Microscopy (SEMI puratus (Veron e t al., 1977) did not take place. In this Eggs were fixed in 2% glutaraldehyde in 0.1 M Napaper, we extend our earlier morphological observacacodylate (pH 7.2) for 1 h r and washed overnight in tions with scanning electron microscopy and provide biochemical data regarding the secretion of cortical cacodylate. Osmication was for 1 h r in 1%OsO, in 0.1 granule protein and the activities of three major se- M cacodylate (pH 7.2). Eggs were dehydrated with ethcreted enzymes, ovoperoxidase, protease, and gluca- anol and acetone, critically point dried on a Samdri-790 (Tousimis Research Corp.), and attached to stubs with nase, in anion-substituted seawater. silver paint. Stubs were coated in a Hummer VI sputtering system (Technics). Observations were on a Jeol MATERIALS AND METHODS JSM-35CF scanning electron microscope. Gametes The purple urchin S . purpuratus was obtained from Transmission Electron Microscopy (TEM) Pacific Biomarine (Venice, CA). Gametes were colEggs were fixed as described above and dehydrated lected by introduction of 0.55 M KCl into the coelomic in ethanol, passed through propylene oxide, and emcavity, with eggs spawned into artificial sea water bedded in Embed 812 (Electron Microscopy Sciences). (MBL-SW). Sperm were collected “dry” and kept Silver sections were cut on a Reichert-Jung Ultracut E chilled until used. Eggs were dejellied by passing them with a diamond knife, stained with lead citrate and through several layers of cheese cloth and then were uranyl acetate, and viewed with a Phillips 301 transtreated with acidified sea water (0.1 N HCl, pH 5) for 3 mission electron microscope. min. The egg suspension was readjusted to pH 8.0 with Enzyme Cytochemistry 1 M Tris base added dropwise. The eggs were washed three times in sea water, followed by another two For the localization of ovoperoxidase, the method of washes in the appropriate substituted sea water (see Klebanoff et al. (1979) was used. Unfertilized eggs and below). Within 30 min eggs were suspended in a 5% eggs 15 rnin after insemination were gently settled by suspension, and “dry” sperm were added to give a 0.1% hand centrifugation, washed three times, and resusconcentration. As a n alternative to activation with pended in 0.45 M NaC1, NaBr, or Na-Ise with 5.6 mM sperm, the ionophore A23187 (free acid, molecular 3,3‘-diaminobenzidine (DAB; Sigma) buffered with 0.1 weight 523; Calbiochem) was used to activate eggs. The M Tris base to pH 8.0. DAB incubations were perionophore was dissolved in dimethylsulfoxide (DMSO; formed in equimolar NaC1, NaBr, and Na isethionate Sigma). Final concentration of ionophore was 38 pM solutions because 1)we did not want to reintroduce C1(20 pgiml) in 1%DMSO. Supernatants for the protein back into the experimental C1--deficient media and 2) and enzyme assays were collected 15 min after fertili- the DAB localization was not successful in the complex zation or ionophore activation. ionic mixture of sea water. Hydrogen peroxide (100 p1, 30%; Sigma) was added to a 5 ml volume of the above Artificial Sea Waters containing about 10% eggs. Samples were incubated Control (MBL-SW) and experimental sea waters for 10 min a t 10°C. DAB was omitted from controls. were made according to Cavanaugh (1956); bromide The reaction was stopped by the addition of 2 volumes (Br-) and isethionate (Ise) salts were substituted for of 3% glutaraldehyde for 1 hr, and the samples were chloride (C1-) salts in the substituted sea waters. washed in 0.1 M Na-cacodylate overnight and prepared Methyl Sulfonate (MeS)-substituted sea water was pre- for transmission electron microscopy as described pared a s previously described by Lynn et al. (1988). above. The compositions of the various sea waters were: 1) Total Protein MBL-NaC1, 423 mM; KC1, 9 mM; CaCl,, 9 mM; Supernatant (fertilization product; FP) over fertilMgCl,, 23 mM; MgSO,, 25 mM; NaHCO,, 2 mM; 2) Ise-Na isethionate, 422 mM; KCI, 9 mM; CaCl,, 9 ized or ionophore-activated eggs was collected and centrifuged by hand to remove any remaining whole eggs. mM; MgSO,, 48 mM; NaHCO,, 2 mM; 3) Br--NaBr, 420 mM; KBr, 10 mM; CaBr,, 10 mM; MgSO,, 48 mM; Supernatant from this was then centrifuged for 10 min NaHCO,, 2 mM; 4) MeS-substituted sea water pre- at 8,800 g in a n Eppendorf centrifuge a t 10°C to remove pared by titrating a 480 mM solution of methane sul- sperm. This final supernatant was used in the protein fonic acid with 10 N NaOH to a pH of 7.0 and by sub- and enzyme assays. sequently titrating a 10 mM solution of methane For protein determination, 1 ml of FP was precipisulfonic acid with 5 N KOH to a pH of 7.0. To this tated with 110 p1 of ice-cold 50% trichloroacetic acid solution, 65 mM MgSO, and 10 mM CaC1, were added. (TCA) on ice, centrifuged 20 min a t 8,800 g a t 10” C,
FERTILIZATION IN Cl-DEFICIENT SEA WATER drained by inversion, and air dried. The precipitate was assayed by the Lowry method (Lowry et al., 1951), with bovine serum albumin (BSA) as a standard. Preliminary experiments demonstrated no significant difference (95% confidence level) among TCA-precipitable BSA in the various sea water solutions. Control experiments utilizing unfertilized eggs incubated in the various media resulted in very small, insignificant amounts of protein in the supernatants: MBL, 1.42 pgl ml; Ise, 1.55; Br-, 1.42.
Enzyme Assays Ovoperoxidase activity was assayed using a reaction mixture of 10 mM TAPS, pH 8.0, 18 mM guaiacol, 0.3 mM H,Oz (3.3 ~1 of 30% H20, per 100 ml) in distilled H 2 0 (Deits et al., 1984). Twenty to one hundred microliters F P was placed in a 1 ml cuvette. Reaction mixture (980-900 pl) was quickly pipetted to mix rapidly with the fertilization product, and the initial reaction rate was recorded a t 436 nm (0.2 in./sec chart speed) on a Beckman ACTA spectrophotometer. Blanks were equal amounts (20-100 pl) of substituted artificial seawater treated as samples with the reaction mixture. As a control to test the effects of the various sea waters on ovoperoxidase activity, eggs were fertilized in MBL. Aliquots (20 ~ 1 of) FP were mixed with reaction mixtures (980 pl) in each of the substituted sea waters. No differences were observed in the initial reaction rates. p-1,3-Glucanase activity was assayed according to Green and Summers (1980). Aliquots (200-500 pl) of the FP were mixed with 50 p1 of the algal polysaccharide substrate laminarin (Laminaria digitata; 10 mgl ml), and the appropriate sea water was added to a total of 1 ml reaction mixture. The reaction proceeded for 2 h r a t 37°C. Two hundred microliter aliquots of the above mixtures were then removed and incubated for 10 min at 37°C with 400 pl enzymes (400 pl glucose oxidase plus 3 mg horseradish peroxidase in 50 ml of 0.25 M phosphate buffer, pH 6.0) and 400 p1 chromogen (40 mg o-dianisidine in 50 ml distilled H,O). The reaction was stopped by addition with vortexing of 800 p1 of 4 N sulfuric acid. Samples were assayed spectrophotometrically at 530 nm. Enzyme activity is measured a s pmoles of glucose formed per minute. A glucose solution served as the standard. Control incubations of glucose were assayed in the experimental sea waters and no significant differences (95% confidence level) were observed. Reagents were purchased from Sigma. Secreted protease was assayed spectrophotometrically for esterase activity using Na-benzoyl arginine ethyl ester (BAEE; Sigma; Schwert and Takenaka, 1955).One hundred microliters of a 1 mM BAEE solution (in the appropriate sea water, pH 8.3, with 10 mM TAPS) was pipetted into a quartz cuvette. The assay was initiated by adding 900 p1 of FP with mixing, and the reaction was observed a t 254 nm. Trypsin-like activity of the FP was estimated by comparing reaction rates (change in absorbance) with those produced by
179
TPCK-trypsin (Sigma). Statistical analyses were performed using Student’s t and ANOVA tests.
RESULTS SEM Fertilization in C1--deficient sea waters markedly affected the formation of the FE. Most notably, the I-T transition (from rounded to angular microvillar casts) failed to occur (Fig. 1). At 3 min postinsemination, MBL-SW FEs have completed the I-T transition, while the Br--SW eggs retained I-shaped microvillar casts. By 60 min postinsemination, the Br--SW FEs still did not complete the transition. There appears to be a graded response, presumably related to the chloride content of the media, with the extreme difference between MBL-SW (Fig. lB,C) and Br--SW (Fig. lE,F). Ise-SW, which falls between MBL- and Br--SW relative to chloride content, supported a partial I-T transition (Fig. 1D). Previously, we demonstrated with TEM that the ultrastructure of FEs in several Cl--deficient sea waters varied from the normal condition (Lynn et al., 1988). Total Protein Secreted The morphological characteristics described above suggest that the permeability of the FE might also be altered. It has been shown that various treatments of the VE before fertilization result in differences in protein permeability of the nascent FE (Veron et al., 1977; Green and Summers, 1980). Therefore, eggs were fertilized in MBL- and C1--deficient SW. After eggs and sperm were removed by gentle and rapid centrifugation, respectively, the total protein of the FP (supernatant) was assayed (Fig. 2). The MBL FEs allowed the least amount of protein (-13 pgiml FP) into the supernatant, whereas the Br--treated FE’s allowed the most (-63 pg/ml FP). Intermediate between the extremes were Ise (=18 pg1ml FP)- and MeS ( ~ 4 pgiml 6 FP)treated FEs. Since the [Cl-] increases in the order Br < MeS < Ise < MBL, the amount of protein released was inversely related to the amount of chloride present in the media. Therefore, the permeability of the FE appears to be inversely related to the chloride concentration, although the possibility of the different anionic species differentially affecting permeability cannot be ignored. Ise treatment resulted in a slightly higher protein concentration in the media, although the difference was not statistically significant (P = 0.35) from that of the control MBL SW. However, both MeS (P 5 0,001) and Br- (P 4 0.001) treatments resulted in protein release significantly higher than MBL. Enzymatic Activity Three enzymes secreted from the egg a t fertilization were assayed to determine what effects, if any, the treatments would have on their activities. We monitored the release of p-1,3-glucanase, cortical granule protease, and ovoperoxidase from the eggs following
180
J.D. GREEN ET AL.
Fig. 1. Scanning electron microscopy of S.purpurutus vitelline and fertilization envelopes. A: VE of unfertilized egg in MBL-SW. Note the close array of rounded (I-form) microvillar casts. B: By 3 rnin postinsemination, MBL FEs have angular (T-form) casts. (Envelope collapsed during fixation and dehydration.) C: Mature MBL FE with
T-form casts a t 60 min. Note the spread array of casts compared to A. D Ise FE a t 60 min resembles 3 min MBL. I-T transition remains incomplete. E: Br- FE a t 3 min resembles unfertilized egg except that I-form casts are further separated due to expanding FE. F Br FE at 60 rnin has changed little since 3 min. x 10,000.
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Fig. 3. B-1,3-Glucanase activity released through FEs. The assay is described in Materials and Methods. Glucanase activity is reported as a mean value, with standard error bars and number (n) of trials. These data are not significantly different a t the 95% confidence level.
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fertilization or ionophore activation. The difference in release of glucanase was not statistically significant between MBL and Ise ( P = 0.071, MeS ( P = 0.571, or Br- (P = 0.29) (Fig. 3). Cortical granule trypsin-like protease activity as measured with the ester substrate BAEE corresponded to a range of 2-6 picomoles of trypsin activity per milliliter of a 5% egg suspension. Although Br- and MBL averaged higher reaction rates than Ise and MeS, these differences were not statistically significant (Ise, P = 0.07; MeS, P = 0.07; Br-, P = 0.35) a s judged using the Student's t test or one-way ANOVA (P = 0.12) (Fig. 4). In any case, protease activity was not inhibited. The only enzymatic activity measured that significantly differed from controls was that of ovoperoxidase. As determined by the guaiacol assay, eggs fertilized in Br--SW released significantly more ovoperoxidase (P 5 0.002) than eggs fertilized in MBL-SW (Fig. 5). In fact, three to four times more ovoperoxidase was detected. To ascertain whether this increase was due to increased enzyme release or increased enzyme activation, we fertilized eggs in MBL-SW, removed equal aliquots of FP, and assayed them in control and experimental SWs. There were no differences in activity, indicating that the various SWs did not affect ovoperoxidase catalysis of guaiacol. However, the results of ovoperoxidase release with MeS and Ise were enigmatic (Fig. 5). Ise released significantly lower (P 5 0.02) amounts of ovoperoxidase, and the MeS was lower than controls also, but this was not statistically significant ( P = 0.16). The number of trials in MeS was limited because with this medium it was extremely difficult to obtain 95% fertilization with S.purpuratus. When eggs were fertilized in sea waters containing varying proportions of Br--SW and MBL-SW, a dosage response was observed (Fig. 6). With decreasing con-
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Fig. 4. Spectrophotometric tracings of cortical granule protease released through FEs. Esterase activity was measured as described in Materials and Methods. Reaction rates are depicted as changes in absorbance at 254 nm. At the 95% confidence level, no significant differences were found between MBL- and experimental S W trials. Each rate is the average of two trials.
centrations of chloride, more ovoperoxidase activity appeared in the FP. Since ovoperoxidase is largely incorporated into the nascent FE, the removal of the FE precursor (VE) should result in more of the enzyme being released into the supernatant. Indeed, when eggs were pretreated with dithiothreitol (DTT) or trypsin before fertilization or ionophore activation, respectively, more ovoperoxidase was released. In the case of DTT, about two times more enzyme was released; after trypsin, four to seven times more enzyme was released than in controls. This difference is consistent with the more complete removal of the VE by trypsin than by DTT and corresponds with a four- to nine-fold increase in total protein released following trypsin treatment (data not shown).
J.D. GREEN ET AL.
182
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DAB reaction in order of decreasing chloride concentrations. That is, the MBL-treated eggs stained more darkly than the Ise-treated eggs, which stained more darkly than the Br--treated eggs. Therefore, FEs in normal SW incorporated more ovoperoxidase than FEs in the C1--deficient sea waters.
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Enzyme Cytochemistry Following the observation that more ovoperoxidase was released into the supernatant after fertilization in a chloride-deficient medium (Br--SW), and that this release appeared to be dosage dependent (Fig. 61, we hypothesized that this increase could be due to a decrease of ovoperoxidase incorporation into the FE. Therefore, we fertilized eggs in MBL-, Ise-, and Br-SWs, incubated them with DAB, and processed them for TEM. The results are shown in Figure 7. Although not quantitative, the micrographs reveal a less intense
DISCUSSION In a n attempt to analyze the physiological importance of C1- ions, the major anionic component of sea water, we previously reported that, although fertilization occurred, the elevation of the fertilization envelope of sea urchin eggs was seriously impaired (Lynn et al., 1988). Using three species of sea urchins and four different anionic substitutes (Ise, MeS, NO,-, and Br- 1, we ascertained that FEs elevated but were less birefringent than FEs in normal sea water. Furthermore, they collapsed back upon the eggs within several minutes postinsemination. Using FE collapse as a criterion for hardening, we further demonstrated that this failure to harden was related to decreasing [Cl-], especially when [Cl-] was < 50% of normal. On the other hand, when eggs were fertilized in C1--deficient SW but transferred back to normal SW within about 10 min postinsemination, normal hardening ensued. Conversely, eggs fertilized in normal SW and transferred to Cl--deficient SW earlier than 10 min postinsemination showed elevated soft FEs. This observation is consistent with earlier reports that the structuralization and cross linking of the FE require = 10-15 min (Veron et al., 1977; Schuel et al., 1982). TEM profiles of FEs revealed that the normal trilaminar morphology was absent, and, perhaps even more striking, the I-T transition of the microvillar casts ( S . purpuratus) failed to occur. This transition also appeared to be affected in a [Cl-]-dependent manner, since FEs in IseSW (24 mM C1-) and MeS-SW (17 mM C1-) had intermediate casts, whereas those of eggs fertilized in NO,--SW (
