Proc. Nati. Acad. Sci. USAVol. 75, No. 7, pp. 3183-3187, July 1978Biochemistry

Hydrogen peroxide production, chemiluminescence, and therespiratory burst of fertilization: Interrelated eventsin early sea urchin development

(peroxidase/oxygen/respiration/polyspermy)

CHARLES A. FOERDER*, SEYMOUR J. KLEBANOFFt, AND BENNETT M. SHAPIRO*Departments of * Biochemistry and t Medicine, University of Washington, Seattle, Washington 98195

Communicated by E. R. Stadtman, May 2,1978

ABSTRACT After fertilization of the sea urchin, Strongyl-ocentrotus purpuratus, a crosslinked fertilization membraneis formed; the crosslinks (dityrosine residues) are synthesizedin a reaction catalyzed by an ovoperoxidase that is released fromthe cortical granules during fertilization. The substrate forovoperoxidase activity, hydrogen peroxide, is generated by theegg coincident with the "respiratory burst" that follows par-thenogenetic activation by the divalent ionophore A23187 orfertilization. This burst of oxygen consumption may be almostquantitatively accounted for by hydrogen peroxide evolution,as measured by the peroxidase-catalyzed quenching of scopo-letin fluorescence. Neither the burst of oxygen consumption norhydrogen peroxide production occurs when the inhibitor ofcortical granule discharge, procaine, is present at fertiliza-tion.

Fertilization or parthenogenetic activation with A23187 alsois associated with a burst of light emission. This chemilumi-nescence is inhibited in vivo by inhibitors of the ovoperoxidase,such as 3-amino-1,2,4-triazole, phenylhydrazine, sulfite, or azide.A crude ovoperoxidase preparation catalyzes hydrogen perox-ide-dependent chemiluminescence that is similarly inhibited.Thus, the bursts of oxygen uptake, peroxide production, andchemiluminescence appear to be several manifestations of theperoxidative system released at fertilization. This system mayadditionally be responsible for spermicidal activity and thusmay act as a component of the block to polyspermy.

One of the early biochemical observations on the activation ofegg metabolism at fertilization was that of Otto Warburg (1),who noted a marked increase in oxygen consumption on fer-tilization of sea urchin eggs. This observation has been con-firmed with eggs of several different species (2). Oxygen con-sumption incb eases in an initial burst to reach a maximum atabout 2 min post-fertilization and then falls to a plateau levelslightly higher than that present before fertilization (3, 4). Themetabolic basis for this respiratory burst is not known.

In a study of fertilization-induced alterations in the egg ofthe sea urchin, Strongylocentrotus purpuratus, we found (5)that the glycoprotein coat on the egg surface is converted intoa rigid, relatively impermeable "fertilization membrane" bythe formation of crosslinks between tyrosine residues. Theformation of the di- and trityrosine structures is catalyzed byan ovoperoxidase that is released from cortical granules in amassive exocytosis which occurs within seconds of fertilization(6). The occurrence of such a peroxidase-mediated reaction inthe vicinity of the fertilized egg implies that the substrate forthat enzyme, hydrogen peroxide (H202), is either formed orliberated by the egg with kinetics appropriate to its involvementin the crosslinking reaction. In this paper, we show that the eggdoes produce H202 at the appropriate time, and, additionally,

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked"advertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

3183

H202 production accounts almost quantitatively for the res-piratory burst at fertilization. Additionally, an emission of light(chemiluminescence) occurs immediately after fertilization andappears to be, at least in part, a consequence of the ovoperox-idase-H202 reaction. An abstract describing portions of thiswork has appeared (7).

MATERIALS AND METHODSMaterials. S. purpuratus eggs, sperm, and sea water were

collected and prepared as reported previously (5). The sea waterwas filtered through a 0.45 ,um Millipore filter (MSW) beforeuse. Measurements were made at 90 unless otherwise noted. Inall experiments, the frequency of fertilization on exposure ofeggs to sperm was at least 90%. Ionophore A23187 was a giftfrom R. Hamill (Eli Lilly Co., Indianapolis, IN). a-N-ben-zoyl-L-arginine [3H]ethyl ester was a generous gift from AlanLevine. Scopoletin, horseradish peroxidase (HRP) (Type II),and 3-amino-1,2,4-triazole were from Sigma Chemical Co.Catalase was from Worthington Biochemical. Sodium [14C]-formate was from New England Nuclear. All other chemicalswere of the highest quality available.Measurement of H202 Production. Formate oxidation was

measured as described by Klebanoff and Smith (8) (see legendto Table 1 for components of the reaction mixture) and scopo-letin oxidation, aq described by Root et al. (9), with the followingmodifications. For the measurement of the kinetics of H202production, fertilization was initiated as described in Fig. 1Awith HRP added to a final concentration of 1.7 ng/ml at 10 spost-fertilization and scopoletin added at 10 tM final concen-tration 10 s later. Scopoletin was dissolved in dimethylsulfoxide,resulting in a final dimethylsulfoxide concentration in the assaymixture of 7 mM. Incubation mixtures were swirled to keep theeggs suspended, and 2.7-ml aliquots were removed at intervalsand immediately mixed with 0.3 ml of 100mM NaCN in MSW,pH 8.0, to stop scopoletin oxidation. After centrifugation at 1000X g for 10 min at 40, the supernatant solutions were decantedand fluorescence was measured by excitation at 350 nm andmonitoring at 460 nm in a Perkin-Elmer MPF-44A fluores-cence spectrophotometer. Scopoletin oxidation was plotted asa function of time, and rates were calculated from theslopes.

For the quantitation of H202 production, fertilization wasinitiated in the presence of aminotriazole, and H202 was addedas described in Fig. 2. At 15 min post-fertilizaton, the suspen-sions were centrifuged at 1500 X g for 10 min and the super-nates decanted. To a 5-ml portion was added scopoletin at 40AM (final concentration) and HRP at 3.9 ng/ml (final con-centration). Control experiments demonstrated that 250,M

Abbreviations: MSW, sea water filtered through 0.45 ,m Milliporefilters; HRP, horseradish peroxidase.

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FIG. 1. Bursts of H202 production (A) and oxygen uptake (B)after fertilization. (A) Sperm, at a final concentration of 1-5 106/ml

was added at zero time to eggs (40,000/ml) in MSW (3). Procaine (5mM) was present in the reaction mixture where indicated (U). Sco-poletin oxidation was determined as described in Materials andMethods. Values are the means of two experiments. (B) Sperm (1-5X 106/ml) were added at zero time to either 120,000 eggs in 3.0 ml ofMSW (0) or MSW alone (0) and the rate of oxygen uptake deter-mined as a function of time post-fertilization. Values are the meansof three experiments. In A and B experimental variation was < 10%of mean.

(final concentration) aminotriazole did not inhibit the oxidationof scopoletin by HRP and H202 under the conditions employed.Fluorescence was measured as described above after another15 min incubation.Oxygen Uptake. For the measurement of the kinetics of

oxygen consumption, fertilization was initiated as describedin Fig. 1B, and oxygen uptake was continually monitored witha Clark-type oxygen electrode (Yellow Springs Instrument Co.)connected to a Linear Instruments 2550 recorder. The eggsamples were equilibrated with air and initial measurementswere made for several minutes before beginning the experimentby addition of sperm; samples were stirred gently to avoid egglysis.

Cumulative oxygen consumption in the first 15 min afterfertilization was measured in a reaction mixture containing180,000 eggs, sperm at a final concentration of 1-5 X 107/ml,and 250 1.M (final concentration) 3-amino-1,2,4-triazole in 3.0ml of MSW. Uptake of oxygen by sperm was measured as de-scribed above with eggs omitted. Aminotriazole has no effecton the kinetics and extent of oxygen uptake.

Chemiluminescence. Eggs (1-2%, vol/vol; settled eggs/seawater), suspended in 2.0 ml of MSW saturated with 02 (unlessotherwise noted), were incubated with sperm at a final dilutionof 1:1000 in scintillation vials maintained at 15° in a shakingwater bath. At intervals, the vials were placed in a liquid scin-tillation counter (Beckman Instrument Co.) for measurementof chemiluminescence as described by Rosen and Klebanoff(10).

Preparation of Fertilization Product. "Fertilization prod-uct" was prepared by treating unfertilized eggs with trypsin

(11) and collecting the material released after fertilization. Atwo-step centrifugation method, as described for the prepara-tion of a crude ovoperoxidase fraction (6), was used.Treatment with Procaine. Eggs were treated with procaine,

as described by Vacquier (12). The method involves preincu-bation of eggs with 10 mM procaine for 10 min, fertilizationin 5mM procaine, and, at 16 min post-fertilization, dilution into2 mM procaine, all at final concentrations and in MSW, pH 8.0.In all cases, cleavage of procaine-treated eggs occurred onschedule and at a frequency equal to the untreated, fertilizedcontrols, but fertilization membrane elevation was inhibitedas anticipated. This technique was developed to inhibit corticalgranule exocytosis; we have found that cortical granule proteaserelease is 27% of that seen with untreated controls (data notshown). Cortical granule-protease release was measured bycollecting fertilization product as described, without priortrypsinization, and assaying activity by a modification of themicroassay technique of Anderson et al. (13). We used 0.2 MTris-HCl buffer, pH 8.0, and 1.25 mM a-N-benzoyl-L-arginine[3H]ethyl ester (50 ,Ci/mmol) for our assays.We were unable to measure the inhibition of ovoperoxidase

release by procaine treatment because procaine, an aromaticamine, completely inhibits the ovoperoxidase assay (6) underthe conditions used.

RESULTSH202 Production by Eggs after Fertilization. The oxidation

of formate by catalase and of scopoletin by HRP were em-ployed as measures of H202 formation. H202 measurement byformate oxidation is based on the observation of Chance (14)that formate is oxidized to CO2 by catalase in the presence oflow, steady-state concentrations of H202. This technique hasbeen used to measure H202 formation by leukocytes (15),bacteria, and bovine spermatozoa (8). Table 1 shows the resultsof such a study with sea urchin eggs and sperm. Formate oxi-dation after fertilization or parthenogenetic activation withionophore A23187 (5, 6, 16) is dramatically increased over thatseen with eggs or sperm alone. Omission of catalase does not

Table 1. Formate oxidation after fertilization orparthenogenetic activation

nmol 14CO2produced

Fertilization, complete system* 5.1 (4)tMinus sperm 0.9 (5)Minus eggs 0.1 (5)Minus catalase 2.6 (5)

Plus denatured catalaset 1.2 (5)Plus 1 mM NaN3 0.5 (5)Plus 1 mM NaCN 0.4 (5)

Parthenogenesis, complete system§ 9.1 (5)Minus eggs 0.1 (3)Minus A23187 plus 7 mM Me2SO 0.4 (5)

Formate oxidation was measured -as described (8), except that in-cubation was for 60 min at 90 or 15°; data from the two temperatureswere nearly identical. Reactions were initiated by addition of spermor ionophore.* Incubation volume of 0.5 ml, containing 60% (vol/vol) egg suspen-sion, 130 nmol sodium [14C]formate, 0.1 pg catalase (60 unit/pg), and1-5 X 107 sperm in oxygen-saturated MSW.

t Mean of (n) experiments minus the background in the absence ofboth sperm and eggs (0.6 nmol).

I Catalase heated at 100° for 10 min.§ Incubation as in *, in 5 AM A23187 and 7 mM dimethylsulfoxide(Me2SO) and without sperm.

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completely eliminate formate oxidation, perhaps because ofthe presence of endogenous ovoperoxidase (6) or catalase. Ad-dition of sodium azide or sodium cyanide inhibits catalase andovoperoxidase activities, as well as formate oxidation, but doesnot inhibit fertilization under the conditions employed in Table1. We conclude that fertilization or parthenogenetic activationby ionophore A23187 leads to release of H202 from eggs.The second technique used to monitor H202 production was

that of scopoletin oxidation. Scopoletin (7-hydroxy-6-methoxy-coumarin) loses its fluorescence when oxidized by HRP andH202 (17-19), providing a sensitive technique for measuringsmall quantities of H202 in biological materials (9). We havemodified this procedure for estimation of H202 at fertilizationto avoid interference of the fluorescence signal by turbid eggsuspensions. At different times after fertilization, scopoletinoxidation was stopped by adding sodium cyanide (see Materialsand Methods). The eggs and sperm were removed by centrif-ugation, and the supernatant solutions were monitored forfluorescence. Results of typical experiments are shown in Fig.1A. There was clearly a burst of scopoletin oxidation (and,therefore, 11202 production) between 2 and 14 min post-fer-tilization under these conditions with a maximum at 7-8 min.Control experiments (data not shown), in which 20 MM (finalconcentration) scopoletin was added instead of 10 MM, showedsimilar kinetics and extent of scopoletin oxidation, indicatingthat scopoletin was not limiting. Note that procaine treatmentof eggs, which blocks exocytosis of cortical granules at leastpartially (ref. 12; see Materials and Methods), completelyabolished scopoletin oxidation. In control experiments with 35MM (final concentration) H202 added instead of eggs andsperm, 5 mM procaine inhibited scopoletin oxidation by 24%after a 15-min incubation with HRP. Thus, the complete in-hibition of scopoletin oxidation after fertilization in the presenceof procaine cannot be accounted for entirely by interferencewith the assay. This suggests that H202 production is associatedwith the release of granular components during the corticalreaction of fertlization, although additional effects of procainecannot be excluded.

Quantitation of H202 production was performed by usinginternal H202 standards, as shown in Fig. 2. Obtaining a correctvalue for H202 generation is difficult due to competing reac-tions [in this case the ovoperoxidase-catalyzed crosslinking ofthe fertilization membrane (6)] which do not permit stoichio-metric capture of the peroxide by the assay system. In an at-tempt to control for this, we have added small quantities ofH202, in the range of that evolved, to eggs immediately afterfertilization, and then we tested for the amount of H202 re-maining after 15 min. 3-Amino-1,2,4-triazole was added toinhibit the ovoperoxidase during this period (6). This establishedan internal standard curve, based on the assumption that en-dogenously evolved H202 and added H202 undergo similarmetabolic fates as a common pool. A reasonably linear responsewas found on the addition of H202, and from this we estimatethe concentration of the generated H202 at 15 min to be 321AMand thus the H202 production per embryo during the 15 minafter fertilization to be 520 fmol.Oxygen Uptake by Eggs after Fertilization. Because H202

is usually generated by a reduction of molecular oxygen, weanticipated that a burst of oxygen consumption would ac-company the peroxide evolution. This has been described withfertilization of S. purpuratus eggs (4), but the incubationtemperature was significantly different from that used in ourstudies. To compare oxygen consumption to 11202 production,measurements were made under conditions similar to thoseused for estimating scopoletin oxidation. A burst of oxygenconsumption was seen (Fig. 1B), which reached a maximum

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FIG. 2. Quantitation of H202 production after fertilization. Eggs(60,000/ml) were fertilized with 1-5 X 107 sperm/ml in MSW con-taining 250 IM (final concentration) aminotriazole. Known concen-trations of H202 were added 30 s post-fertilization as internal stan-dards, and scopoletin oxidation was determined after 15 min. Thearrow indicates the point on the standard curve at which is found anincrement in scopoletin oxidation equivalent to that found in theabsence of added H202. The values shown are means of three exper-iments, and experimental variation was <5% of mean.

at about 4 min post-fertilization and then fell. The maximal,instantaneous, steady-state rate of oxygen consumption agreeswith that previously reported (4) and was about 4 times that forscopoletin oxidation. When oxygen consumption was measuredcumulatively for the first 15 min after fertilization (see Mate-rals and Methods), we obtained the value of 770 fmol of 02consumed per embryo (data not shown). Comparison of thisvalue to the optimal H202 production per embryo estimatedfrom the data in Fig. 2, suggests that at least two-thirds of the02 taken up in the 15 min after fertilization was converted toH202. The fertilization-induced burst of oxygen consumptionwas suppressed when eggs were treated with procaine (Fig. 3),suggesting that both H202 production and oxygen uptake re-quire the release of cortical granule components.Chemiluminescence at Fertilization. Allen et al. (20) have

shown that phagocytosis by human polymorphonuclear leu-kocytes is associated with the generation of light (chemilumi-nescence). Evidence that myeloperoxidase activity is requiredfor optimum chemiluminescence by the leukocytes was sub-sequently provided (10, 21, 22). Because a peroxidase-mediated

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FIG. 3. Effect of inhibition of cortical granule exocytosis on theburst of oxygen consumption. Eggs were fertilized as described in Fig.1 except that in (0) procaine was present before, during, and afterfertilization (see Materials and Methods). Values are the means oftwo experiments, and experimentalvariation was S15% of mean.

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FIG. 4. Effect of egg concentration on the burst of chemilumi-nescence. Light emission was measured for egg concentrations of 0.5%(-); 1% (@); 5% (o); and 10% (0).

40

reaction occurs after fertilization (6), we examined the sea ur-

chin system for a similar chemiluminescence. Fig. 4 shows a

dramatic burst of chemiluminescence at fertilization, the ki-netics and total amount of which were dependent upon the eggconcentration. The kinetics of the chemiluminescence gener-

ation were similar at egg concentrations below 1%. Lightemission began at about 2 min and reached a maximum at 4-5min, and the total amount of light emission was proportionalto the egg concentration. At higher egg concentrations, the peakchemiluminescence was shifted to a later time, and the yieldof light per egg dropped. For these reasons, the remainder ofour chemiluminescence studies were carried out at egg con-

centrations of 1-2%. A similar decrease in oxygen consumptionand H202 production occurred at high egg concentrations, eventhough essentially all eggs were fertilized (data not shown). Wehave not explored this phenomenon in detail, but we have beencareful to use similar egg concentrations when making com-

parisons between the several phenomena.To test the role of the ovoperoxidase in generating chemi-

luminescence, we added inhibitors of the enzyme to eggs at thetime of fertilization and then measured light emission. Fig. 5Ashows the effect of 10 ,uM phenylhydrazine; similar results wereseen at 250 ,uM 3-amino-1,2,4-triazole, 1 mM Na2SO3, or 20mMNaN3 (data not shown). All of these inhibitors completely in-hibit ovoperoxidase activity in Witro and fertilization membranehardening in vvo (6). This suggests that ovoperoxidase activityis required for the fertilization-associated chemiluminescenceburst, an hypothesis that is supported by studies with fertil-ization product. Fertilization product contains the ovoperox-idase (6), as well as other enzymes and structural proteins (cf.ref. 5). Table 2 shows that the fertilization product was capableof generating chemiluminescence in the presence of H202, andthat this luminescence was inhibited by ovoperoxidase inhibi-tors.We reported that hardening of the fertilization membrane

occurred at a slower rate after parthenogenetic activation ofeggs with A23187 than after sperm addition, as measured byseveral criteria (5). Ionophore-activated eggs generate chemi-luminescence with significantly delayed kinetics compared tofertilized eggs (Fig. 5B) in agreement with the delay seen in therespiratory burst (16) and in other aspects of the hardeningreaction (5).

0

Time, min post-activationFIG. 5. (A) Effect of phenylhydrazine on the chemiluminescence

burst at fertilization. Eggs were fertilized at zero time, and at 75 sphenylhydrazine was added to a final concentration of 10MM to onebatch of eggs (@), and the chemiluminescence was compared to thatof a control preparation (0). (B) Comparison of the chemilumines-cence burst after ionophore activation to that produced by fertiliza-tion. Eggs were either fertilized (o) or activated by 10MM A23187 (-)at time zero. The delay in chemiluminescence seen with this con-centration of A23187 was also seen at 25 MAM ionophore (data notshown).

DISCUSSIONFertilization of sea urchin eggs is associated with a burst ofoxygen consumption. The studies reported here indicate thata substantial portion (two-thirds) of the oxygen consumedduring the 15 min after fertilization is converted to a substancethat can act as a substrate for both catalase and HRP. Thissubstance is presumed to be H202, although other hydroper-oxides also can act as substrates for these enzymes. The amountof H202 detected is a minimum value because the added cat-alase or HRP must compete with cellular systems for the H202formed, raising the possibility that essentially all of the extraoxygen consumed is converted to H202.The finding of peroxide production soon after fertilization

is in agreement with our previous hypothesis (6) that hardening

Table 2. Ovoperoxidase-dependent chemiluminescenceof fertilization product

Chemiluminescence,cpm

Complete system 163,202Minus H202 6,209Minus fertilization product 5,189Plus 250MgM 3-amino-1,2,4-triazole 12,100Plus 10,uM phenylhydrazine 9,506Plus 1 mM Na2SO3 32,415

The complete system contained 1.0 ml fertilization product (pre-pared as in Materials and Methods) and 50MAM H202 in 5 ml MSW.The scintillation vial was mixed after H202 addition, and chemi-luminescence was monitored 2 min after incubation at 250.

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of the fertilization membrane is dependent upon the release ofan ovoperoxidase that catalyzes the crosslinking of tyrosylresidues in the "soft" fertilization membrane (5). This perox-ide-dependent system may have additional physiological roles.One obvious possibility, as suggested (6), is that H202 and theovoperoxidase participate in a spermicidal system that killsexcess sperm in the vicinity of the egg, thus serving as an ad-ditional block to polyspermy. H202 has a toxic effect on echi-noderm (23) and mammalian sperm (24), lactoperoxidase ac-tivity is spermicidal for S. purpuratus (B. Shapiro, unpublisheddata), and uterine fluid peroxidase, lactoperoxidase, and my-eloperoxidase, when combined with H202 and iodide (orthiocyanate), are toxic to bovine sperm (25). Although neitherperoxidase release nor H202 production by mammalian eggshas yet been described, the presence of a peroxidase in theuterine fluid of certain species (8) raises the possibility thatH202 generated by the fertilized egg may limit the survival ofadjacent spermatozoa through its interaction with the uterineperoxidase. Since mammalian sperm are rich in polyunsatu-rated, long chain fatty acids (26), peroxidase-catalyzed reactionsmay cause their oxidation to fatty acid hydroperoxides (27, 28)that are spermicidal (26, 29-31). Thus sperm, because of theirunusual fatty acid composition, may be particularly susceptibleto attack by peroxidase supplied either by the egg or the uterus.The H202 required for this system may be generated either byegg or by sperm (8) metabolism.

Fertilization of sea urchin eggs is also associated with theemission of light (Figs. 4 and 5; refs. 7 and 32; G. Perry and D.Epel, personal communication). This chemiluminescence istemporally related to the increased oxygen consumption andH202 production and appears to be dependent, at least in part,on ovoperoxidase activity; it is inhibited by agents previouslyshown to inhibit this enzyme. Chemiluminescence by peroxi-dase catalyzed reactions was found over 60 years ago by E. N.Harvey (33), who noted the emission of light on the additionof pyrogallol to turnip extracts in the presence of H202. Recentexperiments have shown that the luciferase of Balanoglossusbiminiensis is peroxidatic in nature (34, 35) and that myelo-peroxidase activity accounts for a portion of the chemilumi-nescence seen with phagocytosing polymorphonuclear leuko-cytes; the remainder is dependent upon the superoxide anion(10). In this regard, there are many interesting similarities be-tween the system activated in the leukocyte by phagocytosisand that in the sea urchin egg by fertilization; polymorpho-nuclear leukocytes dramatically increase oxygen consumption(36), generate hydrogen peroxide (9, 15) and emit light (10, 20)after phagocytosis. Because these cells also generate the su-peroxide anion (37) and perhaps singlet molecular oxygen (38,39), we wonder whether these other oxygen species also aregenerated after fertilization, where they may play some rolein either chemiluminescence or spermicidal activity.

We are grateful to Alan Levine for performing the protease assays.We are grateful to Joanne Fluvog and Ann Waltersdorph for theirexcellent technical assistance. We are grateful to Christopher Gabeland James Thomson for suggestions and careful review and criticismof the manuscript. This research was supported in part by NationalInstitutes of Health Training Grants GM00052 and HD00266 (C.F.),National Institutes of Health Research Grants GM23910 (B.M.S.) andHD02266 (S.J.K.), and National Science Foundation Grant 77-20472(B.M.S.).

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