THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Soeiety of Biological Chemists, Inc. Vol. 261, No. 36, Issue of December 25, pp. 17040-17047 1986

Printed in h.S.A.

Stereospecific Induction of Starfish Oocyte Maturation by (8R)-Hydroxyeicosatetraenoic Acid*

(Received for publication, July 15, 1986)

Laurent MeijerSi?, Alan R. Brash% Robert W. Bryant 11, Kwokei Ngll , Jacques MaclouP*, and Howard SprecherSS From the $Department of Biochemistry, University of Washington, Seattle, Washington 98195, the BDepartment of Phnrmobgy, Vanderbilt University School of Medicine, Nashville, Tennessee 372.32, the JIDepartWnt of Allergy and Infkzmmation, Schering Plough Corporation, Bloomfield, New Jersey 07003, the **Unite Insern U150, Hopital kriboisjere, 75010 Paris, France, and the $$Department of Physiological Chemistry, Ohio State University, Columbus, Ohio 43210

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine. This induction of meiotic divisions can be triggered also by four fatty acids: 5,8,11-20:3; 5,8,11,14-20:4 ( arachidonic acid); 6,9,12,15-20:4; 5,8,11,14,17- 20:5, all other fatty acids being completely inactive. This maturation triggered by eicosanoids occurs in the micromolar range and is facilitated by the presence of calcium. A variety of arachidonic acid derivatives (es- ters, epoxides, etc.) and metabolites (cyclooxygenase and lipoxygenase products) has been tested; the biolog- ical activity is restricted to 8-hydroxyeicosatetraenoic acid (8-HETE), other mono- and poly-HETEs being completely inactive. Maturation triggered by 8-HETE occurs around 10 nM and is insensitive to the presence of calcium. 8-HETE methyl ester and 8-hydroperox- yeicosatetraenoic acid are able to induce maturation at higher concentrations. Both (8s) and (8R) stereoiso- mers have been tested; the biological activity is strictly restricted to the (8R) isomer. 8-HETE triggers a com- plete maturation, i.e. maturation-promoting factor ap- pearance, germinal vesicle breakdown, emission of the polar bodies, and formation of a female pronucleus. (8R)-HETE, but not (8S)-HETE, triggers the typ- ical decrease in cyclic AMP concentration induced by 1-methyladenine and the burst of protein phosphoryl- ation associated with maturation. Starfish oocytes ox- idize exogenous arachidonic acid into 8-HETE and other HETEs. 8-HETE was identified, after high pres- sure liquid chromatography purification, by gas chro- matography mass spectrometry. Furthermore, it was found that the starfish oocytes only produce the (8R)- HETE isomer. This highly stereospecific induction of oocyte maturation by (8R)-HETE suggests that this fatty acid, or a very closely related fatty acid, may play a role in the transduction of the 1-methyladenine message at the plasma membrane level.

* This work was supported by Grant ARC 6268 from the Associa- tion pour la Recherche contre le Cancer, by a grant from the PIRMED/Centre National de la Recherche Scientifique (“Lipides pharmacologiquement actifs”) (L. M.), by National Institutes of

tutes of Health Grants AM 35275 and HL 05797 (A. R. B.), and by Health Grants AM 18844 and AM 20387 (H. S.), by National Insti-

National Institutes of Health Grant GM 23810 (to B. Shapiro). It was partly performed during a sabbatical leave in Seattle, supported by a Centre National de la Recherche Scientifique/National Science Foundation exchange program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 To whom reprint requests and correspondence should be ad- dressed: Station Biologique, 29211 Roscoff, France.

Two major pathways of arachidonic acid (AA)’ oxidation have been described the cyclooxygenase pathway leading to prostaglandins and thromboxanes; and the lipoxygenase path- way leading to hydroxyeicosatetraenoic acids (HETEs) and leukotrienes. These metabolites have been demonstrated to play fundamental regulatory roles in a number of cellular events; indeed numerous agonists seem to activate cells through an “arachidonic acid cascade,” i.e. the transient re- lease of arachidonic acid from membrane phospholipids or diglycerol and its rapid oxidation to biologically active media- tors (see reviews in Pace-Asciak and Smith, 1983; Borgeat, 1985). During the last 3 years, we have been investigating the mediator role of AA in starfish oocyte maturation (Meijer et al., 1984, 1986a).

Starfish oocytes are naturally arrested in the first prophase stage of meiosis until the spawning period. At this time, the follicle cells surrounding the oocytes release a hormone, 1- methyladenine (MeAde), which triggers meiosis reinitiation or maturation (Kanatani et al., 1969). The simple addition of MeAde to isolated prophase-arrested oocytes results in the synchronous induction of maturation in uitro, as first visual- ized by the rupture of the nuclear envelope (or germinal vesicle breakdown, GVBD), followed by the emission of the two polar bodies. As a consequence of the interaction of MeAde with its plasma membrane receptors, the oocytes undergo many biochemical, biophysical, morphological, and physiological changes (review in Meijer and Guerrier, 1984). Among these is the appearance of a cytoplasmic “maturation-promoting factor” (MPF), which is able to induce maturation when injected into unstimulated oocytes (Kishimoto and Kanatani, 1976); this MPF appears transiently in all dividing cells whether mitotic or meiotic, and heterologous transfer exper- iments have shown that it is not species specific (Kishimoto et al., 1982, 1984).

In a previous paper (Meijer et al., 1984) we showed that AA, eicosapentaenoic acid (EPA), as well as exogenous cal- cium-activated phospholipase AP, are able to induce oocyte maturation. This fatty acid-triggered maturation occurs in the micromolar range, is facilitated by the presence of calcium in the external medium, and is inhibited by lipoxygenase inhibitors. Starfish oocytes metabolize exogenous AA into

The abbreviations used are: AA, arachidonic acid (5,8,11,14- eicosatetraenoic acid); CaFASW, calcium-free artificial seawater; EPA, eicosapentaenoic acid; GVBD, germinal vesicle breakdown; HETE, hydroxyeicosatetraenoic acid; 8-HETE, 8-hydroxy-5,9,11,14- eicosatetraenoic acid; HPLC, high pressure liquid chromatography; MeAde, 1-methyladenine; MPF, maturation-promoting factor; NSW, natural seawater.

17040

(8R)-HETE Triggers Starfish Oocyte Maturation 17041

phospholipids and triglycerides in a calcium-independent way and into HETE-like compounds in a calcium-dependent and lipoxygenase inhibitor-sensitive way (Meijer et al., 1986a). Biological activity is restricted to the HETE-containing frac- tions, and HETE-induced maturation is independent of cal- cium and insensitive to lipoxygenase inhibitors.

In this paper we have studied the high specificity of fatty acids inducing oocyte maturation; the active fatty acids share three double bonds in positions 5,8, and 11. Among oxidized metabolites, only (8R)-HETE, but not its stereoisomer (8s)- HETE, triggers maturation (concentrations around 10 nM are required for 50% maturation). (8R)-HETE, but not (8s)- HETE, triggers a decrease of the cyclic AMP concentration and a burst of protein phosphorylation, two events typically associated with maturation. Starfish oocytes are able to syn- thesize (8R)-HETE from exogenous arachidonic acid. The possibilities that (8R)-HETE might be the intracellular me- diator of MeAde-induced maturation or that (8R)-HETE might mimic a step in the transduction of the hormone message at the plasma membrane level are discussed.

MATERIALS AND METHODS

Preparation and Handling of the Oocytes-Asterias rubens and Marthasterias glacialis were collected in the Roscoff area (France) and kept in running seawater. Orthasterias koehleri and Evasterins troschelii were collected in the Seattle, WA area and kept in closed- circuit aquaria. The gonads were dissected out of the animals and gently torn open in ice-cold calcium-free artificial seawater (Ca- FASW). The oocytes were filtered through cheesecloth and washed three or four times in CaFASW to remove the MeAde-producing follicle cells. Oocyte maturation was triggered by the active fatty acids in 2-ml multiwell plates, in a final volume of 1 ml of CaFASW or natural seawater (NSW). Oocyte maturation was recorded as the percentage of oocytes showing GVBD after 30 min, and the 50% maturation dose (MD,) was obtained from dose-response curves (for methodology on starfish oocytes, see Meijer et al., 1985).

Arachidonic Acid Metabolism-A 20% oocyte suspension (Euaster- ias) was treated at time 0 with radioactive AA (10 p l of ["CIAA (59.6 mCi/mmol; 50 mCi/ml) per ml of oocyte suspension). At different times after AA addition a 1-ml aliquot of the oocyte suspension was injected into 1 ml of methanol containing 2 pg of butylhydroxy- toluene/ml. The zero time point was done by injection of the oocyte suspension aliquot into methanol + butylhydroxytoluene before ad- dition of 10 pl of [I4C]AA. The samples were stored at -80 "C, under nitrogen, until further analysis. After thawing, they were treated with 0.1 mg of NaEiH, to minimize peroxide-induced auto-oxidation during sample workup. Mixed cold HETEs were added as carriers to each sample. Samples were acidified, extracted with chloroform, dried with Na2S04 and redissolved in hexane:2-propanol, and chromatographed on a DuPont Zorbax Sil/HPLC column (4 X 250 mm). The solvent used was hexane:2-propanol:acetic acid (980:ZO:l) a t 1 ml/min. The cold carrier HETEs were detected with a Waters 480 UV monitor at 235 mm. The radioactive AA metabolites were detected with a Ra- mona detector after mixing of the HPLC effluent with 2.5 ml of Scintisol mixture/min.

In another type of experiment performed with Orthosterias, oocytes were treated with [14C]AA and extracted with chloroform: methano1:HCl (1O:ZOl); after centrifugation the supernatants were injected on a Waters MB-C,, column (8 X 100 mm). The AA metab- olites were eluted with a gradient of (A) methan0kwater:acetic acidEDTA (6634:0.08:0.01) adjusted to pH 6.0 with NH,OH and (B) methanol. The elution scheme was: 0-15 min, 100% A; 15-20 min, from 100% A to 100% B; 25-27 min, 100% B. The flow rate was 2 ml/min. The peak of HETEs (which are not separated from one another by this procedure), eluting at 23 min, was collected and injected on a DuPont Zorbax Si1 Column (4.8 X 250 mm); the various HETEs were separated after elution with hexane:2-propanokacetic acid (990:201). Gas chromatography-mass spectrometry was carried on methyl ester, trimethylsilyl ether derivatives as previously de- scribed (Anthes et al., 1986).

Preparative Separation of (8R)- and (BS)-HETEs-Milligram quan- tities of (8R,8S)-HETE were prepared via controlled auto-oxidation of arachidonic acid in the presence of a-tocopherol (Peers and Coxon, 1983) followed by reduction of the resulting hydroperoxides and

HPLC separation of the individual HETEs. Eight milligrams of (8R,8S)-HETE was converted to the methyl ester and reacted with the isocyanate of dehydroabiethylamine (Corey and Hashimoto, 1981) to form the diastereomeric urethane derivatives of the (8R) and (8s) enantiomers. The diastereomers were completely resolved by semi- preparative HPLC using an Alltech 10-pm 25 X 1-cm silica column and a solvent system of hexane:isopropyl alcohol (1W0.2, v/v) run at 4 ml/min. The urethanes eluted at 37 and 46 min; unreacted 8- HETE methyl ester was recovered in later fractions. A 500-pg aliquot of each urethane was cleaved to the corresponding (8R) or (85') alcohol by treatment with 7.5 mg of triethylamine and 5.5 mg of trichlorosilane in 200 p1 of benzene (Corey and Hashimoto, 1981); after stirring overnight at room temperature under argon, water (0.3 ml) was added and the benzene evaporated under nitrogen. Hydrolysis of the methyl ester was effected by addition of methanolic 1 N KOH (0.5 ml) and a further 0.3 ml of water. After stirring for 1 h at room temperature, the 8-HETE was recovered by acidification and extrac- tion with dichloromethane. Each 8-HETE enantiomer was then re- purified by normal phase HPLC using a solvent system of hex- ane:isopropyl alcohokglacial acetic acid (100:1.60.1, by volume) and quantified by UV spectroscopy (Amm 236 nm, c = 30,000).

Assignment of Chirality of 8-HETE Enantiomers-The absolute configuration of the 8-HETE enantiomers was established using the method of Hamberg (1971) as previously described (Maas et al., 1981). This method is based on conversion of the HETE alcohol to a diastereomeric menthoxycarbonyl derivative, followed by oxidative ozonolysis, methylation, and gas chromatographic comparison of the resulting derivative of malic acid to derivatives prepared from au- thentic R and S malic acids. Using this approach, the 8-HETE enantiomer which chromatographed as the earlier eluting urethane derivative on normal phase HPLC could be assigned as the (8R) configuration, and the enantiomer corresponding to the second ure- thane peak as (8s). The diastereomeric menthoxycarbonyl deriva- tives chromatograph in the same order on normal phase HPLC (Brash et al., 1985). An aliquot of each enantiomer was converted to the urethane derivative of the methyl ester and reanalyzed for chiral purity by HPLC. This showed that the (8R)- and (8S)-HETEs were, respectively, 5 and 3% contaminated with the opposite enantiomer, evidence of the slight degree of epimerization associated with cleavage of the urethane derivative.

A new method was developed in order to confirm the (8R) and (8s) assignments. It was reasoned that the soybean lipoxygenase could be used to convert (8R)- and (8S)-HETE to (8RJ5S)- and (8S,15S)-diols, respectively (after reduction of the (15S)-hydroper- oxide products) and that the latter diastereomer should correspond to the well characterized (8S,15S)-diol obtained via reaction of ara- chidonic acid with high concentrations of the same enzyme (Van Os et al., 1981). This was indeed found to occur. Racemic 8-HETE (10 pg/ml) reacted smoothly with soybean lipoxygenase (1 pg/ml Sigma Type IV, pH 8.5) to form equal amounts of two products with indistinguishable triene chromophores. After reduction of the (15s)- hydroperoxide the resulting diols were easily separated by normal phase HPLC (as shown in Fig. 7). The earlier eluting 8,15-diol had identical UV and HPLC properties to the authentic (8s,15S)-diol formed from arachidonic acid. The individual 8-HETE enantiomers were each converted to a single major product; the enantiomer des- ignated as (8s) gave a product which co-chromatographed with the authentic (85',15S)-diol, and the product from (8R)-HETE corre- sponded to the second of the two HPLC peaks obtained from racemic 8-HETE. This method was also found suitable for measurement of the degree of contamination of the (8R)- and (8S)-HETEs with the opposite enantiomer. The HPLC profiles contained minor peaks formed from the contaminating enantiomer. The (8S,15S) contami- nant in the (8R)-derived sample was 5% the area of the main peak; the (8S)-HETE gave a minor (8R,15S)-diol of 3% abundance, both results being in agreement with the chiral purity determined by chemical methods (vide supra).

Steric Analysis of 8-HETE from Starfish Oocytes-The use of the soybean lipoxygenase reaction in the assignment of 8-HETE chirality proved to be very suitable for analysis of small amounts of sample. Accordingly, the method was applied to 8-HETE obtained from E. troschelii. Oocytes were incubated for 2 min with [l-"Clarachidonic acid, and further reaction was stopped by addition of 4 volumes of methanol. The sample was subsequently subjected to Bligh and Dyer extraction (Bligh and Dyer, 1959), followed by isolation of the radio- labeled peak of 8-HETE by reversed phase HPLC (Altex 5-pm ODS Ultrasphere column, 25 X 0.4 cm, solvent system methanol: water:acetic acid (75:25:0.01, by volume), retention volume of 8-

17042 (8R)-HETE Triggers Starfish Oocyte Maturation HETE = 40.5 ml). One microgram of unlabeled (8R,8S)-HETE was then added and the sample further purified by normal phase HPLC (Alltech 5-pm silica column, 25 X 0.4 cm, solvent system hex-

volume of 8-HETE = 11 ml). The sample was then reacted with 1 ane:isopropyl alcoho1:acetic acid (100:3:0.1, by volume); retention

pg/ml soybean lipoxygenase (in 0.5 ml of phosphate buffer, pH 8.5) for 5 min at room temperature, followed by acidification (pH 4) and extraction into dichloromethane. After treatment with sodium boro- hydride in methanol and re-extraction, the sample was analyzed on normal phase HPLC (Alltech 5-pm silica, solvent system hexane: isopropyl alcoho1:acetic acid (10050.1, by volume).

Measurement of Cyclic AMP Concentrations-Oocytes were prein- cubated for 60 min with 100 pM forskolin. They were then treated with 5 PM (final concentration) (8R)-HETE or (8S)-HETE or with a corresponding amount of dimethyl sulfoxide (1-5 pi of dimethyl sulfoxide/ml of oocyte suspension; control). At various intervals, 1- ml aliquots were rapidly centrifuged (5 s in an Eppendorf centrifuge at full speed). The supernatant was removed, and the oocyte pellet was rapidly resuspended in 1 ml of ice-cold 70% (v/v) ethanol. After 30 min on ice, the samples were sonicated for 30 s (Braun Sonic 2000, needle probe, setting 60); after another 30 min on ice the extracts were centrifuged for 15 min at 4 "C in an Eppendorf centrifuge. The pellet was preserved for protein determination according to Bradford (1976), after solubilization in 1 ml of 0.5 N NaOH. The ethanolic supernatant was recovered and either kept a t -20 "C or directly dried at 40-50 "C under a stream of air. The dried residue was dissolved in 950 pl of 50 mM sodium acetate, pH 6.2,0.01% sodium azide (buffer A) and stored at -20 "C until further use.

Cyclic AMP levels were determined in duplicates by a radioim- munoassay method (Biomedical Technologies Inc., 378 Page St., Stoughton, MA 02072) 80 pl of buffer A were added to 20 p1 of extract, 100 pl of '=I-succinyl cAMP tyrosine methyl ester along with normal rabbit IgG were added, followed by 100 pl of cyclic AMP antiserum (containing sheep antirabbit IgG). After incubation at 4 "C for 18-20 h, 1 ml of cold buffer A was added, and the tubes were centrifuged for 30 min at 4 "C at 2000 X g. The supernatant was removed by aspiration and the pellet counted for 3 min or 10,000 cpm in a y spectrometer. Cyclic AMP levels were obtained by comparison with standards processed similarly. The concentration of cAMP was then calculated as pmol of cAMP/mg of protein and plotted as a percentage of the initial cAMP concentration.

Protein Phosphorylation-In vivo phosphorylation was measured by the following method. Oocytes (40 ml of a 10% suspension) were preincubated for 4 h in the presence of 20 pCi (40 pI at 5 mCi/ml) of [32P]phosphate (New England Nuclear). After 4 h the oocytes were washed four times with NSW and redistributed into 3 batches of 6 ml (10% suspension). At time 0, (8R)-HETE or ( W - H E T E (10 pM final concentration) were added; control oocytes were treated with a corresponding volume of dimethyl sulfoxide (final concentration 1%). At various intervals a 500-pl aliquot of the oocyte suspensions was injected into 4 ml of ice-cold 10% trichloroacetic acid. The trichlo- roacetic acid-precipitated oocytes were washed with 5 ml of 10% trichloroacetic acid, dissolved in 0.5 ml of 0.5 N NaOH, reprecipitated with 10% trichloroacetic acid washed one more time with 10% tri- chloroacetic acid, and dissolved in 1 ml of 0.5 N NaOH. An aliquot was preserved for protein determination according to Bradford (19761, while 500 pl were counted with 8 ml of Aquasol. Protein phosphoryl- ation was calculated as (32P]phosphate cpm incorporated/mg of pro- tein and plotted as percent of the initial phosphorylation level.

Intracellular Microinjections-Oocytes (internal volume: M. glaci- al&, 2750 pl) were suspended in NSW and injected according to the method of Hiramoto (1974). To detect the presence of MPF, cyto- plasm was withdrawn from oocytes undergoing GVBD, under the influence of 8-HETE, and transferred, through a 3-6-pm tip glass micropipette, into immature nonstimulated oocytes. These oocytes were scored for GVBD 30 min later.

Chemicals-MeAde, forskolin, AA, EPA, eicosane, eicosanoic acid, 11-20:1, 11,14-202, 8,11,14-20:3, 11,14,17-203, AA-alcohol, AA methyl ester, AA ethyl ester, AA acetate, AA-coenzyme A, prosta- glandins E2, Fla, 12, and Dz, and thromboxane BP were obtained from Sigma. 8-Eicosanoic acid was obtained from Nu-check-Prep, Elysian,

204, and 8,11,14,17-204 were prepared by one of us (H. S.); 12- hydroxy-5,8,10-heptadecatrenoic acid was prepared by J. M.; the mono-HETEs were prepared by singlet oxidation of AA by one of us (R. W. B.) who also prepared the epoxides and the diHETEs. The hydroxy epoxides were a generous gift of Dr. Pace-Asciak. (Hospital for Sick Children, Toronto). 5,8,11,14-Eicosatetraynoic acid was

MN. 5,8,11-20:3, 5,11,14-203, 5,8,14-20:3, 4,7,10,13-20~4, 6,9,12,15-

kindly offered by Dr. Guttmann and Dr. Weber (Hoffmann-La Roche and Co., CM-4002 Basel, Switzerland). All fatty acids were stored at -60 "C, under nitrogen and in methanol solutions. Just before use, they were dried under nitrogen and dissolved in dimethyl sulfoxide which was added to the oocytes at concentrations lower than 1% (v/ v). This concentration of dimethyl sulfoxide had no effect on the oocytes.

RESULTS

Induction of Oocyte Maturation by Fatty Acids-In a pre- vious study (Meijer et al., 1984) we tested 36 fatty acids ranging from 4- to 24-carbon length and from 0 to 6 double bonds. Active fatty acids were restricted to eicosanoids. We further extended this study with other eicosanoids (Table I). Only four fatty acids were found to trigger oocyte matura- tion: 5,8,11-20:3; 5,8,11,14-20:4 (AA); 6,9,12,15-20:4; and 5,8,11,14,17-20:5 (EPA) (Fig. 1). In each case, oocyte matura- tion is triggered in the micromolar range and found to be stimulated by the presence of calcium in the external medium, as revealed by the dose-response curve in NSW and CaFASW (Fig. 1). Other fatty acids remain completely inactive, even at the highest concentration tested.

Although AA is active at micromolar doses, modification of the acid function (by esterification, for example) completely abolishes the biological activity. Modification of the double bonds, such as epoxidation at one double bond, also eliminates the biological activity (Table I).

Although all cyclooxygenase metabolites are inactive, we found some biological activity in 12-hydroxy-5,8,10-hepta- decatrienoic acid; this activity occurs in the high micromolar range and is also sensitive to calcium (Fig. 1E).

Induction of Oocyte Maturation by (8R)-HETE"Among all lipoxygenase metabolites tested, ie . leukotrienes, mono- and polyhydroxyeicosatetraenoic acids, and lipoxins, only 8- HETE is able to trigger maturation. This 8-HETE-induced maturation occurs, however, at much lower concentrations (50% maturation at 12 nM in Marthasterim, at 130 nM in Euasterias) and is insensitive to the presence of calcium in the external medium (Fig. 2A). Modification of the acid function by esterification decreases the activity by an order of magnitude; 8-HETE-methyl ester-induced maturation is also insensitive to calcium (Fig. 2A). The corresponding per- oxide 8-hydroperoxyeicosatetraenoic acid (8-HPETE) was also found to be 10 times less potent than 8-HETE (Fig. 2 B ) . Addition of another function to 8-HETE, such as another hydroxy radical in 8,15-diHETE or an epoxide in 8-hydroxy- 11,12-epoxy eicosatetraenoic acid, completely abolishes the biological activity (Table I).

Since the 8-HETE we first used was obtained by singlet oxidation of AA it contains a 1:l racemic mixture of both (88)- and (8R)-HETE stereoisomers. Using isolated isomers it was found that the biological activity is restricted to the (8R) isomer (Fig. 3); the 50% maturation dose of (8R)-HETE is half the 50% maturation dose of the racemic mixture (in Fig. 3, MDS0 for (8R)-HETE and (8R,BS)-HETE are 0.071 and 0.141 p ~ , respectively). The little activity contained in (8S)-HETE (in Fig. 3, MD, for (8s)-HETE is 1.450 phi) can be completely accounted for by a 3% contamination by the other stereoisomer (1.450 p M (8S)-HETE contains about 0.044 NM (8R)-HETE).

8-HETE triggers a complete maturation, ie. GVBD, emis- sion of the two polar bodies, and formation of the female pronucleus. MeAde triggers a decrease of CAMP concentration which is made even more obvious when oocytes are pretreated with forskolin, an agent that, by activating adenylate cyclase, increases the basal cAMP level.' 8-HETE triggers a rapid

__ L. Meijer and P. Zarutskie, manuscript submitted.

(8R)-HETE Triggers Starfish Oocyte Maturation 17043 TABLE I

Effects of several eicosanoids and their metabolites on starfish oocyte maturation The fatty acids dissolved in dimethyl sulfoxide were added to oocyte suspensions in NSW and CaFASW; -, no

effect on oocytes; +, induction of maturation (50% maturation doses (MDW) in NSW and CaFASW are indicated in parentheses).

Agonists Range of

concentrations tested

Biological activity (MD, in NSW/MDm in CaFASW)

Arachidonic acid and congeners Eicosane Eicosanoic 8-Eicosenoic 11-Eicosenoic 11,14-Eicosadienoic 5,8,11-Eicosatrienoic 5,8,14-Eicosatrienoic 5,11,14-Eicosatrienoic 8,11,14-Eicosatrienoic 11,14,17-Eicosatrienoic 4,7,10,13-Eicosatetraenoic 5,8,11,14-Eicosatetraenoic 6,9,12,15-Eicosatetraenoic 8,11,14,17-Eicosatetraenoic 5,8,11,14,1?-Eicosapentaenoic

Arachidonic acid esters Arachidonoyl alcohol Arachidonic methyl ester Arachidonic ethyl ester Arachidonyl acetate Arachidonyl coenzyme A

5,8,11,I4-Eicosatetraynoic 5,6-Epoxy-8,11,14-eicosatrienoic 8,9-Epoxy-5,11,14-eicosatrienoic 11,12-Epoxy-5,8,14-eicosatrienoic + 14,15-epoxy-5,8,11-eicosatrienoic

Arachidonic acid with modified double bonds

Cyclooxygenase metabolites Prostaglandin E, Prostaglandin F2- Prostaglandin I2 Prostaglandin D2 Thromboxane B2 12-Hydroxy-5,8,10-heptadecatrienoic

5-Hydroxy-6,8,11,14-eicosatetraenoic 8-Hydroxy-5,9,11,14-eicosatetraenoic 8-Hydroxy-5,9,11,14-eicosatetraenoic methyl ester 8-Hydroperoxy-5,9,11,14-eicosatetraenoic 9-Hydroxy-5,7,11,14-eicosatetraenoic ll-Hydroxy-5,8,12,14-eicosatetraenoic 12-Hydroxy-5,8,12,14-eicosatetraenoic 15-Hydroxy-5,8,11,13-eicosatetraenoic Leukotriene A,-methyl ester Leukotriene B, Leukotriene C, Leukotriene D4 Leukotriene E, 5,15-Dihydroxy-6,8,11,13-eicosatetraenoic 8,15-Dihydroxy-5,9,11,13-eicosatetraenoic 8-Hydroxy-l1,12-epoxy-5,9,14-eicosatrienoic 1O-Hydroxy-l1,12-epoxy-5,8,14-eicosatrienoic 8-Hydroxy-11,12-epoxy-5,9,14,17-eicosatetraenoic 10-Hydroxy-11,12-epoxy-5,8,14,17-eicosatc

Lipoxygenase metabolites

0.1-1000 0.01-1000 0.01-1000 0.01-1000 0.01-1000

0.01-100 0.01-100 0.01-1000 0.01-1000 0.01-100

0.01-100

0.01-1000 0.1-1000

0.01-1000 0.01-100 0.01-100

10-250 0.01-10 0.01-20 0.01-20

0.2-200 0.1-500 0.1-500 0.1-500 0.1-500

0.01-50

0.01-20 0.01-20 0.01-20 0.01-20 0.1-500 0.1-1000 0.1-1000 0.1-1000 0.1-1000 0.1-10

2-10 0.01-10 0.01-10 0.01-10 0.01-10

- + (0.2/0.85) + (0.72/2.6)

f (1.1/6.3) -

- + (1.8/12)

- + (0.012) + (0.1) +

decrease of the CAMP concentration, both in untreated and in forskolin-pretreated oocytes (data not shown). Fig. 4 shows that only (8R)-HETE triggers this cyclic AMP concentration decrease. Like AA (Meijer et al., 1984), 8-HETE triggers the typical protein phosphorylation burst associated with the release from prophase block (Meijer et al., 1986b). Here also, only the (8R)-HETE isomer triggers the biochemical response (Fig. 5). The slight increase of protein phosphorylation in- duced by (8S)-HETE could be accounted for by the 3% contamination with (8R)-HETE. Using intracellularmicroin-

jection techniques we verified that 8-HETE, like MeAde, triggers the appearance of cytoplasmic MPF (8 oocytes out of 8 underwent GVBD upon microinjection of cytoplasm from 8-HETE-treated oocytes).

Synthesis of (8R)-HETE by Starfish Oocytes-To determine if starfish oocytes were able to produce 8-HETE from exoge- nous AA they were exposed to radioactive AA, and at various intervals, the products of oxidation were extracted and run on a straight-phase HPLC, resolving the different HETEs (Fig. 6, upper trace). In other experiments the HETEs were

17044 (8R)-HETE Triggers Starfish Oocyte Maturation

FIG. 1. Fatty acids inducing star- fish oocyte maturation: dose-re- sponse curves. Oocytes were incubated, in the presence (closed symbols) or in the absence (open symbols) of calcium, with various concentrations of fatty acids. The percentage of GVBD was scored 30 min later. A , B, C, E, M. glacialiq D, A. rubens.

FIG. 2. Induction of starfish oo- cyte maturation by 8-HETE. A, dose- response curves for 8-HETE and 8- HETE methyl ester in the presence (closed symbols) or absence (open sym- bols) of calcium ( M . glacialis). B, dose- response curves for 8-HETE and 8- HPETE (E . troschelii).

100 -

80-

40-

0.05 0.1 0.5 1 5

8HETEcoNcENTRATK)Nw

r = f

8 IR+SI - HETE

40-

20

0 a01 io5 0.1 0.5 1 5

8HETEcoNcENTRATK)Nw FIG. 3. Stereospecific induction of starfish oocyte matura-

tion by (8R)-HETE. Oocytes were incubated with various concen- trations of (8R)-HETE, (8S)-HETE, or the racemic mixture; the percentage of GVBD was scored 30 min later (E. trosctielii).

100-

- 80-

- 60 -

- 40-

- 20-

- 0

B I I 1 1 I I I I I I 1

0.1 1 10

AGONIST CoNcEMRATloN

first purified on reverse-phase HPLC before being separated by straight-phase HPLC. In both cases, the data clearly show that the oocytes are able to produce 8-HETE, in addition to other HETEs (Fig. 6, middle truce). 8-HETE was produced by the oocytes of the two species investigated, Evasterias and Orthasterias. Other HETEs produced included 9-, 12-, and 15-HETEs; their appearance depended on the species and on the time elapsed after AA addition (data not shown). When the purified 8-HETE was treated with soybean lipoxygenase, it was converted into a product comigrating with 8,15-di- HETE (see further in the text). When the 8-HETE produced by the oocytes was treated with diazomethane, it comigrated on HPLC, with standard 8-HETE-methyl ester (data not shown). Control samples, where oocytes were extracted before addition of the radioactive AA did not show any 8-HETE (Fig. 6, lower truce); this eliminates the possibility of 8-HETE production by nonenzymatic oxidation during processing of the samples. Finally, 8-HETE from oocytes was positively identified by gas chromatography-mass spectrometry as the methyl ester trimethylsilyl ether derivative (data not shown).

To investigate the stereochemistry of the 8-HETE produced by starfish oocytes, these cells were incubated with radioactive AA, and the radioactive 8-HETE produced was purified, after mixing with a racemic mixture of cold 8-HETE, by reverse- phase and straight-phase HPLC, consecutively. The purified

(8R)-HETE Triggers Starfish Oocyte Maturation -. 6 c

17045

TlME (MlN3 FIG. 4. Effects of (8R)-HETE and (8S)-HETE on the con-

centration of CAMP in forskolin-pretreated oocytes. Oocytes were preincubated for 60 min with 100 pM forskolin. They were then divided in 3 batches and treated, at time 0, with either 5 FM (8R)- HETE, 5 PM (SS)-HETE, or dimethyl sulfoxide (5 Fl/ml oocyte suspension) (control). At various times aliquots of the oocyte suspen- sion were removed and processed for cAMP determination. The initial concentrations of cAMP were 24.2 (8R)-HETE, 20.79 (8S)-HETE, and 15.31 (control) pmol/mg of protein.

3 I I

(u 0 1 1 1 1 1 1 1 1 1 1

0 1 0 2 0 3 0 4 0

TIME WlWTES) FIG. 5. Effect of (8R)-HETE and (8S)-HETE on protein

phosphorylation. Oocytes were preloaded with [32P]phosphate for 4 h. They were then divided in 3 batches, one was treated with dimethyl sulfoxide (control), the two others with 10 PM (final concen- tration) (8R)-HETE or (8S)-HETE. At various times aliquots of the oocyte suspension were removed and processed for measurement of phosphate incorporation in proteins as described under “Materials and Methods.” The phosphorylation was calculated as 32P cpm incor- porated/mg of protein and plotted as a percent of the initial levels which were 2011 (control), 2031 ((8s)-HETE), and 1910 ((8R)- HETE) cpm/mg of protein (E. troschelii).

8-HETE was then treated with soybean lipoxygenase which produced the mixture of (8S,15S)-diHETE and (8R,15S)- diHETE which were resolved by HPLC (Fig. 7). The fact that the radioactivity is only recovered in the (8R,15S)-diHETE demonstrates that the starfish oocytes only synthesize the (8R)-HETE isomer (Fig. 7).

DISCUSSION

Eicosanoids have been discovered in many invertebrates (review in Bundy, 1985). Starfish contain a large variety of polyunsaturated fatty acids, which include several eicosanoids

1440 1 ; ’:u 240 480 0

8 HETE

7

FIG. 6. Production of 8-HETE by starfish oocytes. Oocytes were treated with [“Clarachidonic acid and, after 3 min, the oxidation products were extracted, mixed with nonradiolabeled HETEs, and resolved by straight-phase HPLC. Both the absorbance at 235 mm and the radioactive profiles were monitored simultaneously. The upper truce shows the absorbance profile of the various HETEs; the middle trace shows the radioactive profile and the radioactive 8- HETE peak. The tower truce shows the radioactive profile of a control sample where oocytes were extracted before addition of [14C]AA (E. troschelii).

and prostaglandins (Rodegker and Nevenzel, 1964; Allen, 1968; Ferguson, 1976; Nomura and Ogata, 1976; Korotchenko et al., 1983; Sargent et al., 1983; Isay and Busarova, 1984). We have detected a large variety of polyunsaturated eicosa- noids in starfish oocyte^.^ The discovery of 15-lipoxygenase (Doerge and Corbett, 1982) and 8-lipoxygenase (Bundy et al., 1986) in corals, the powerful effects of leukotrienes on sponge aggregation (Rich et al., 1984), and the recent demonstration of 10,11,12-trihydroxy-5,8,14,17-eicosatetraenoic acid as the barnacle hatching factor (Holland et al., 1985) show that lipoxygenase metabolites of eicosanoids may play as impor- tant roles in invertebrates as they do in vertebrates.

The induction of starfish oocyte maturation by a few fatty acids may represent another example of the numerous phys- iological roles of eicosanoids in cellular activation. A struc- tural analysis of the active and inactive fatty acids provides some insight on biologically active sites of the molecule. A double bond is required in position 5 (8,11,14-20:3 and 8,11,14,17-204 are ineffective); the double bond in position 8 is also absolutely necessary for biological activity (5,11,14- 20:3 is ineffective); the double bond on carbon 11 is also required (5,8,14-20:3 is ineffective). However, the double

J. C. Marty and L. Meijer, unpublished data.

17046 (8R)-HETE Triggers Starfish Oocyte Maturation

800-

P "I f z.

")-

Z0 " 200-

100 -

I

dl HETE dlHETE as,1ss 8R,15S

n . . . 1 2 3 4 5 6 7 6 9 10 11 12 13 I4 15 16 17 18 18 20

FRACTION NUMBER FIG. 7. Demonstration that the starfish oocytes produce

(8R)-HETE. Oocytes were treated with ["Clarachidonic acid and, after 2 min, the oxidation products were extracted, mixed with non- radiolabeled (8R,BS)-HETE, and purified by reverse-phase and straight-phase HPLC. The purified 8-HETE was then treated with soybean lipoxygenase which after NaBH, reduction of the (154)- hydroperoxide gave a mixture of @S,lSS)-diHETE and (8RJ5.S)- diHETE which was resolved by HPLC. Both the absorbance at 270 nm and the radioactivity were recorded. The upper trace shows the absorbance profile of the HPLC run, showing the position of the two isomers produced by soybean lipoxygenase; downward spikes indicate the beginning of a new fraction being collected. The lower trace shows the amount of radioactivity in each fraction (E . troschelii).

bonds in position 14 and 17 are not necessary for maturation inducing activity (5,8,11-20:3 and 5,8,11,14-204 trigger mat- uration). A single carbon shift of all double bonds of AA (leading to 6,9,12,15-204) maintains some biological activity. A similar shift but toward the carboxylic end of the molecule leads to an inactive fatty acid (4,7,10,13-204). The acid func- tion of AA seems to be important (esters are devoid of biolog- ical activity). The common features shared by the active fatty acids are thus 1) three double bonds (5,8,11) and 2) a free acid function a t 3) a well-defined distance. The metabolism of AA (Meijer et al., 1986a) showed that the biological activity was related to the metabolism by lipoxygenase. The results pre- sented here clearly show that the maturation-inducing activ- ity can be attributed to the 8-hydroxy derivative of these fatty acids.

8-HETE has been produced chemically from arachidonic acid by regio-random oxidation by Cu2+\H202 (Boeynaems et al., 1980), by air (Porter et al., 1979b), by singlet oxidation (Porter et al., 1979a; Terao and Matsushita, 1981; Brash et al., 1985), or by total chemical synthesis (Adams and Rokach, 1984). 8-Hydroxyeicosapentaenoic acid has been obtained by autoxidation or photosensitized oxidation of EPA (Yamauchi et al., 1983).

The enzymatic production of 8-HETE by cells has only been briefly reported in a few systems: human neutrophils (Goetzl and Sun, 1979), mouse peritoneal macrophages (Ra- binovitch et al., 1981a, 1981b), mouse liver (Hughes et d., 1983), mouse and guinea pig peritoneal monocytes (McGuire and Sun, 19801, rat kidney glomerulii (Jim et al., 1982), psoriatic skin (Camp et al., 1983), epithelial cells from human trachea (Hunter et al., 1985), human primary squamous car- cinomas of head and neck (El Attar et al., 1985), cultured aortic smooth muscle cells (Larrue et al., 1983), rat hepatic microsomes (Falck et al., 19841, the lipoxygenase of cherry tomato fruit described by Matthew et al. (1977) (Goetzl et al,, 1980), and wheat caryopses lipoxygenase (Kuhn et al., 1985). Recently two thorough studies have reported the specific production of 8-HETE by biological systems: extracts of phorbol ester-stimulated mouse skin (Gschwendt et al., 1986) and extracts of the gorgonian coral Pseudoplexaura (Bundy et al., 1986). The stereochemistry has only been determined in the coral which produces the (8R) isomer (Bundy et al., 1986). In contrast, when another eicosanoid, bis-homo-y- linolenic acid (8,11,14-20:3), was oxidized at position 8 by lipoxygenase of rabbit peritoneal neutrophils (Borgeat et al., 1976) or potato tuber homogenates (Shimizu et al., 1984,1985) only the (8s) isomer was obtained. The present study clearly establishes that the starfish oocyte is able to metabolize exogenous arachidonic acid into 8-HETE. Furthermore, only the (8R) isomer is produced.

The study of the biological activity of lipoxygenase metab- olites has been almost completely restricted to 5-, 12-, and 15-HETEs and the leukotrienes. The cellular effects of hy- droxy fatty acids have been reviewed recently (Vanderhoek, 1985). The effects of 8-HETE have only been tested in a few cases: its chemotactic effect on human neutrophils is less effective than 5-HETE (Goetzl and Sun, 1979; Goetzl et al., 1979; Goetzl et al., 1980); mouse lymphocyte mitogenesis induced by concanavalin A or phytohemagglutinin A is inhib- ited by all HPETEs including 8-HPETE (Gualde et al., 1983); spleen lymphocyte mitogenesis is inhibited by all HETEs (Low et al., 1984); 8-HETE, like other HETEs, triggers an increase of mucous release by human cultured airways (Ma- rom et al., 1983); the proinflammatory effects of 8-HETE are intermediate between that of 5- and 12-HETE (Cunningham et al.., 1985). In all these cases, the biological activity is not limited to 8-HETE, other HETEs being more or less efficient; also the biological activity is observed in the micromolar range and above (except in the report of Marom et al., 1983). Oocyte maturation induction in starfish is strictly limited to 8-HETE; all other HETEs are completely inactive. Furthermore, it is strictly restricted to the naturally produced stereoisomer (8R)-HETE. Finally, it is biologically active at concentrations as low as 10 nM. Esterification of 8-HETE reduces its biolog- ical activity by 10-fold; the biological activity of 8-HETE methyl ester can be either attributed to the molecule itself or to 8-HETE after conversion of the ester by esterases present in the oocytes. Interestingly, the chemotactic activity of 12- and 5-HETE methyl esters are 10 times lower than their corresponding free acids (Goetzl et al., 1979, 1980). The very high specificity and efficiency of (8R)-HETE strongly suggest that it may be physiologically involved in MeAde-induced oocyte maturation.

In a previous paper (Meijer et al.., 1986a) we have shown that a classical "AA cascade" is not demonstrated to be involved in MeAde-induced maturation; no release of AA was observed from [14C]AA prelabeled oocytes. The possibility remains, however, that 8-HETE might be directly released from plasma membrane phospholipids in which it had been

(8RAHETE Triggers Starfish Oocyte Maturation 17047

previously incorporated covalently. Covalent incorporation of HETEs into phospholipids has already been described in a few cases. An alternative possibility is that 8-HETE might mimic a part of a larger molecule (glycolipid?) involved in the transduction mechanism activated by the hormone MeAde. Whatever the case, it shows that a rarely studied lipoxygenase metabolite may be involved in the control of oocyte matura- tion. Recent experiments in mammals have shown that lipox- ygenase may be involved in a related event, ovulation (Reich et al., 1983, 1985).

Acknowledgments-We would like to thank Prof. B. Shapiro for reading this manuscript, for many discussions, and for continuous enthusiastic support during a sabbatical year (L. M.) spent in Seattle. Many thanks to the fishermen of the "Station Biologique" for col- lecting Marthasterius and Asterias; we also wish to thank Roland Anderson, biologist at the Seattle Aquarium, for kindly providing us with Euasterins and Orthasterius. We thank Carol McCaulley for typing the manuscript.

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47,2897-2902