Arachidonic acid metabolism in starfish oocytes

DEVELOPMENTAL

BIOLOGY

114.22-33 (1986)

Arachidonic

Acid Metabolism

in Starfish Oocytes

LAURENTMEIJER,**'JACQUESMACLOUF,~ANDROBERT *Station Biologique,

29211 Roscoff, France; +&it& Corporation, Department of Allergy Received

April

INSERM U150, H&&al Lmahidre, and InfEammution, 60 Orange Street, 22, 1985;

W. BRYANT* 75010 Paris, B&m&e&

accepted in revised fin-m October

France; and $Schering New

Jersey

Plough

07005

7, 1985

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine (I-MeAde) and, in certain species, can be mimicked, in a calcium-facilitated way by submicromolar concentrations of arachidonic acid (AA). The metabolism of AA by starfish oocytes, as related to induction of maturation, was studied by thin-layer (TLC) and high-pressure liquid (HPLC) chromatography. Although exogenous AA is very rapidly metabolized (halflife: 3 min), no difference in the global AA utilization was found between nonmaturing and maturing oocytes whether maturation was induced by AA in the presence of calcium or by l-MeAde. No difference either was found in the incorporation, by non-maturing or maturing oocytes, of AA into various lipids (triglycerides, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylcholine, lysophosphatidylethanolamine or lysophosphatidylcholine). On the other hand, a nice correlation was found between the calcium-facilitated conversion of radioactive AA into hydroxyeicosatetraenoic acids (HETE) and the calcium-facilitated induction of maturation by AA: (1) AA is more efficiently converted into HETEs in the maturing oocytes (in presence of calcium) than in nonmaturing oocytes (in the absence of calcium); (2) after separation of AA metabolites by TLC and HPLC, biological activity is only recovered in the HETE-containing fractions; (3) HETE-induced maturation occurs at much lower concentrations (about 5 X lo-’ M) and is insensitive to calcium; and (4) although AA-induced maturation is sensitive to eicosatetraynoic acid, this lipoxygenase inhibitor has no effect on HETE-induced maturation. 1-MeAde does not seem to induce an increased conversion of exogenous AA into HETE nor an increase of endogenous AA or HETE, nor an increase of lipoxygenase level. Therefore we conclude that the biological activity of AA is in fact due to HETE, as a result of the presence of calcium-stimulated lipoxygenases in the oocytes. Fatty acid-free bovine serum albumin (BSA) inhibits AA-induced but not 1-MeAdeinduced maturation. It inhibits maturation in a dose-dependent fashion and only when added during the first few minutes after AA addition to the oocytes. The plasma membrane site of action of AA is suggested by (1) the lack of stimulatory effect of microinjected AA and (2) the lack of inhibitory effects of microinjected BSA. The possible mechanism of action of AA and HETE is discussed. o 19% Academic press, he. INTRODUCTION

icle)-arrested oocytes. Upon stimulation by the hormone the oocytes undergo many biochemical, biophysical, morphological and physiological changes leading to a fertilizable cell (review in Meijer and Guerrier, 1984). Among these processes, one is particularly interesting, the appearance of an intracellular factor, the “maturation promoting factor” (MPF). This factor is able to induce maturation when injected into unstimulated oocytes (Kishimoto and Kanatani, 1976); it appears to be present in all dividing cells, whether mitotic or meiotic, and heterologous transfer experiments have shown that it is totally unspecific (Kishimoto et aL, 1982). The sequence of events leading from the binding of 1-MeAde with its plasma membrane receptor to the appearance of MPF and subsequent maturation is largely unknown. In a previous publication (Meijer et aL, 1984) it was shown that arachidonic acid (AA), as well as exogenous calcium-activated phospholipase A2 are able to induce oocyte maturation: oocytes respond to submicromolar concentrations of AA, in a calcium-facilitated fashion. The high specificity is emphasized by the fact that only 2 (AA and 5,8,11,14,17-eicosapentaenoic acid) out of 36 different fatty acids are able to mimic 1-MeAde. The

Starfish oocytes remain arrested in the first prophase stage of meiosis in the ovary. During the spawning period the follicle cells surrounding the oocytes release a hormone, 1-methyladenine (1-MeAde): which induces meiosis reinitiation or maturation (Kanatani et &, 1969). This maturation can also be induced in vitro by addition of 1-MeAde to a suspension of prophase-(germinal vesI Present address: Department of Biochemistry, University of Washington, SJ-70, Seattle, Wash. 98195. ‘Abbreviations used: AA, arachidonic acid (5-, S-,11-, lri-eicosatetraenoic acid); BSA, fatty acid-free bovine serum albumin; CaFASW, calcium-free artificial seawater; DMSO, dimethylsulfoxide; EGTA, ethylene-glycol-bis-(y-aminoethylether N,N,N’,N’-tetraacetic acid); ETYA, 5,8,11,14-eicosatetraynoic acid; FAA, free arachidonic acid; GVBD, germinal vesicle breakdown; HDP, hormone-dependent period; HETE, hydroxyeicosatetraenoic acid; HPLC, high-pressure liquid chromatography; Ia,, dose inhibiting at 5O%;LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; 1-MeAde, l-methyladenine; MPF, maturation promoting factor; NL, neutral lipids; NSW, natural seawater; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PPI, polyphosphoinositides; PS, phosphatidylserine; TCA, trichloroacetic acid; TG, triglycerides; TLC, thin-layer chromatography. 0012-1606/86 $3.00 Copyright All rights

Q 1986 by Academic Press, Inc. of reproduction in any form reserved.

22

MEIJER,

MACLOUF,

AND BRYANT

Arachidwnic

Acid

Metabolism

in Star$sh 0ocyte.s

23

pension was injected into 4 ml of ice-cold chloroformmethanol-HC1(10:20:1). The mixture was shaken, 1.2 ml of chloroform was added and then 1.2 ml of 2 N KU; after a short centrifugation most of the upper aqueous phase was removed and the lower, organic phase was collected and dried down under a stream of nitrogen and processed for TLC (see below). In another type of experiment the lOOO-~1 aliquot was injected into 4 ml ice-cold 12% TCA and the precipitate was washed three times with 12% TCA, two times with chloroform:methanol (1:3), one time with acetone, two times with 12% TCA, and finally dissolved in 1 ml 0.5 N NaOH and counted. An additional wash with chloroform: methanol:HC1(10:30:1) before the acetone wash removed most of the label (X, in Fig. 3G, presumably represents AA incorporated into proteins or other acid precipitable, chloroform:methanol-insoluble material). Thin-layer chromatography (TLC). The dry lipid resMATERIAL AND METHODS idue was dissolved in 30 ~1 of chloroform:methanol (1: 3) and a 5-~1 aliquot was then spotted (1.5 cm) on a TLC Material and oocytes processing. Asterias rubens and Silica Gel 60 plate (Merck, with concentrating zone) which had been previously activated at 110°C for 1 hr. Martha&&as glacialis were collected in the Roscoff area and kept in running seawater. The gonads were dis- The plate was then developed in chloroform:methanol: sected, dilacerated in ice-cold calcium-free artificial acetic acid:water (75:45:12:3) (System A), to separate the polar lipids, or in the upper phase of a mixture of ethseawater (CaFASW) and filtered through cheesecloth. Oocytes were collected and washed three or four times ylacetate:isooctane:acetic acid:water (80:40:16:88) (sysin CaFASW to remove the 1-MeAde-producing follicle tem B). After the run, the plates were dried and exposed cells. Oocyte maturation was recorded as the percentage to iodine vapour overnight. The spots were surrounded of oocytes showing germinal vesicle breakdown (GVBD) with a pencil, scraped into a scintillation vial, and after 30 min. Control unstimulated oocytes showed a counted with Lumagel. The individual spots were idenmaximal spontaneous maturation rate around 10% .The tified by comigration with authentic standard (5 ~1 of a hormone (or agonist)-dependent period was determined lo-mg/ml solution). by dilution of an oocyte suspension aliquot, at various To measure the biological activity of AA metabolites times after the beginning of stimulation, to an inactive after separation by TLC, a sample of lipid extract was concentration of the inducer (100 ~1 in 12 ml CaFASW). spotted (7.5 cm) on a TLC plate and developed in system The kinetics of maturation was determined by counting A or B. After the run the plate was dried and 7.0-mmthe percentage GVBD at various times after the beginwide areas were scraped into tests tubes. The silica gel ning of stimulation. in each fraction was extracted twice with 2 ml of chloIntracellular microinjections. Oocytes (internal volume roform:methanol (3:l); the dried lipid residue was reof about 2750 pl) were suspended in NSW and injected dissolved in 20 ~1 DMSO and 10 ~1 was added to 900 ~1 according to the method of Hiramoto (1974). The product of oocyte suspension to assay the biological activity; the to be injected was dissolved in ‘70 mlMNaC1 and injected percentage GVBD was recorded 30 min later. through a 3- to 6-hrn tip glass micropipette, between two High-pressure liquid chromatography (HPLC). Ten droplets of silicone oil. In a few experiments microinmicroliters of [l-14C]AA was added to various samples jections were performed without oil, and, in this case, of a 20% oocyte suspension; after 2 min incubation at to avoid contaminations, the oocyte were lying in NSW 20°C 1 ml of methanol was added, and the samples were containing fatty acid-free bovine serum albumin (5 kept at -20°C until HPLC analysis. After centrifugation mg/ml). the supernatant was recovered and acidified directly to [%$4rachidonic acid incorporation and lipid extracpH 3 with H3P04. Reversed-phase HPLC was performed tion. Twenty milliliters of a 10% (v/v) suspension of oo- on 5 X loo-mm Radial Pack Cl8 cartridges (lo-pm parcytes (in CaFASW or NSW) were incubated at 20°C with ticle size; Waters Assoc., Milford, Mass.) using gradients gentle stirring. At time 0,20 ~1 of [1-14C]arachidonic acid of methanol and acetonitrile as described by Borgeat et (Amersham, 60.1 mCi/mmole; 50 &i/ml) was added, al. (1984) except that solvent C was omitted. The meand, at various intervals, a lOOO-~1 aliquot of oocyte sus- tabolites of AA were detected by UV photometry at 280 fact that AA mimics a very proximal step in the lMeAde-induced sequence of events leading to maturation is supported (1) by identical kinetics for AA- and lMeAde-induced maturation and (2) by the coincidence between the hormone-dependent period (HDP) (during which the presence of 1-MeAde is necessary for maturation) and the period during which AA is required for maturation. In this paper we present a study of the metabolism of AA by starfish oocytes, with a special emphasis on the mechanism of action of AA inducing oocyte maturation. It clearly appears that AA acts through the conversion by lipoxygenases into active HETE and that these are likely to act at the plasma membrane level. The action of 1-MeAde does not necessarily involve an “arachidonic acid cascade.”

24

DEVELOPMENTAL

BIOLOGY

and 237 nm and the radioactivity monitored simultaneously. To measure the biological activity of AA metabolites after separation by HPLC, 200 ~1 of [14C]AA was added to 16 ml of a 15% oocyte suspension in NSW; 2 min later lipids were extracted as described above but in the presence of 1 pug/ml of butylhydroxyanisole. The organic fraction was dried under a stream of nitrogen, solubilized in ethanol, and dried again; hexane:ether (9:l) was added; the sample was centrifuged and the clear supernatant was deposited on a small column containing 0.5 g silicic acid. This column was washed with 15 ml of hexane: ether (9:l) and then eluted further with 15 ml ether: methanol (95:5). The eluate was concentrated and run on a Spherisorb SlO ODS column using methanol:distilled water:acetic acid (75:10:25) (1 ml/min). Fractions were collected every 30 set and 50 ~1 was counted for radioactivity; 200 ~1 of each fraction were dried and resuspended in 20 ~1 DMSO; 10 ~1 was added to 990 ~1 of an oocyte suspension and the percentage GVBD was recorded 30 min later. Purified 8 and 12-HETE standards were run under the same conditions and were not clearly separated in this system. Chemicals. Fatty acid-free bovine serum albumin (BSA), arachidonic acid (AA), the phospholipid standards, were obtained from Sigma Chemical Company. 5,8,11,14-Eicosatetraynoic acid (ETYA) was a generous gift of Dr. Gutmann and Dr. Weber (Hoffmann-LaRoche and Co, CH 4002 Bale, Switzerland). 8-HETE was prepared by HPLC, after singlet oxidation of AA, by one of us (R.W.B.).

VOLUME

114, 1986

Addition of radioactive AA to an oocyte suspension results in a very fast utilization of the label as shown by the amount of remaining AA measured after TLC separation on system B (Fig. 2): the half-life of AA is about 3 min and 90% of the label has disappeared within 10 min (Fig. 1). The global rate of AA metabolism is identical for maturing and nonmaturing oocytes, whether 1-MeAde (Fig. 1) or AA in the presence of calcium (data not shown) is the inducing factor. A more detailed study is thus required to correlate AA metabolism and maturation induction. Metabolism through into Other Lipids

Covalent Incorporation

Radioactive AA is rapidly incorporated into various lipids, as seen after separation by TLC: in the TLC system A, the label of the free fatty acids + neutral lipids fraction quickly decreases to a plateau level (mainly triglycerides) (Fig. 3A), whereas the label is incorporated into phosphatidylcholine mainly (about 60%) (Fig. 3B), but also into phosphatidylserine and phophatidylinositol (Fig. 3C), phosphotidylethanolamine (Fig. 3D), lysophosphatidylcholine (Fig. 3E), and a few unidentified polar lipids (Fig. 3F). The TLC system B shows the global

RESULTS -70

Arachidonic

E :: Yi -50 :;, .-c -40 ;

Acid Metabolism

The metabolism of AA, as related to the maturationinducing activity of AA, was studied in two types of experiments: (1) in the first type, an identical amount of radioactive AA (20 ~1 [14C]AA/20 ml 10% oocyte suspension, i.e., a final concentration of 0.833 PM AA) is added to two batches of oocytes (suspended in CaFASW), simultaneously treated or not with 1-MeAde; (2) in the second type, advantage is taken from the facilitating effect of calcium on AA-induced maturation (Meijer et al., 1984) (Fig. 5); indeed, two batches of oocytes, one in NSW, the other in CaFASW, are treated with an identical amount of radioactive AA (20 ~1 [14C]AA/20 ml 10% oocyte suspension, i.e., a final concentration of 0.833 pMAA), chosen in such a way that it induces complete maturation in the NSW oocyte batch and no maturation in the CaFASW oocyte batch. The metabolism of [14C]AA in the maturing and in the control oocytes is then investigated by looking at incorporation into various lipid fractions after separation by TLC (Fig. 2).

-60

-30

2 '4C-AA

4

6

8 10

Time

12 14 16

18

x

20

(min.)

FIG. 1. Arachidonic acid utilization by starfish oocytes: at time 0, 0.833 &‘[r4CJ4A (1 /,&i/16.7 nmole) was added to two batches of oocytes in CaFASW, one simultaneously treated with 1-MeAde (A) and the other untreated (A). At various intervals a l-ml aliquot of the 10% oocyte suspension was removed and injected into 4 ml of chloroform: methanohHC1 and processed as described under Material and Methods. After TLC of the samples in system B, the AA spot was scraped and counted. The percentage GVBD for each batch is indicated in the figure.

MEIJER, MACLOUF, AND BRYANT

Arachidonic

System B

System A Rf -l.O-

FAA+NL+

+TG

-0.9-

X-,

-0.6PE-,

a-AA +HETE

-0.7-0.6-

PS+PI+ LPE+ PC-,

LPC+

e

--O.‘-o--

al.

+PL

FIG. 2. Thin-layer ehromatograms of starfish oocytes lipids, after iodine vapor staining. A chloroform:methanol:HCl extract was spotted on silica gel 60 plates and developed in two different systems: system A (chloroform:methanol:acetic acidwater, 75:45:12:3) separates lysophosphatidylcholine (LPC), phosphatidylcholine (PC), lysophosphatidylethanolamine (LPE), phosphatidylserine (PS), and phosphatidylinositol (PI), phosphatidylethanolamine (PE), an unidentified polar lipid (x), and neutral lipids (NL) + free arachidonic acid (FAA); system B separates phospholipids (PL), hydroxyeicosatetraenoic acids (HETE), arachidonic acid (AA), and triglycerides (TG).

incorporation in phospholipids (Fig. 3H) and triglycerides (Fig. 31) as well as the conversion into more oxidized metabolites, as will be discussed in the next section. Finally some TCA-precipitable, but acidic-chloroform soluble phospholipids, tentatively assumed to be mainly polyphosphoinositides, are labeled and show a very fast turnover (Fig. 3G). Although most of the label is incorporated covalently into these lipids, no difference was ever observed between nonmaturing and maturing oocytes, either when the oocytes were incubated with radioactive AA in CaFASW (8% GVBD) or in NSW (100% GVBD), or when the oocytes were incubated in CaFASW with radioactive AA in the absence (4% GVBD) or in the simultaneous presence of 1-MeAde (100% GVBD). Finally, no covalent incorporation into proteins (acylation) was observed as revealed by extremely low incorporation into TCA- and chloroform:methanol-insoluble components. Furthermore, cerulenin (l-1000 p&f), a reported inhibitor of protein acylation, has no effect on maturation (data not shown). It thus appears that the covalent incorporation of AA

Acid Metabolism

in Starjsh

Oocytes

25

into these various lipids may not be part of the pathway through which AA induces starfish oocyte maturation. This idea is further supported by the long reversibility of AA action: adding the “chelating” fatty acid free bovine serum albumin (BSA) (see Fig. 7 and further in the text) or diluting AA to an inactive concentration (Meijer et ah, 1984) are still effective at a time where most of the label has already been incorporated into these lipids. Metabolism

through Oxidation

by a Lipoxygenase

On the other hand a nice correlation is found between the calcium-dependent conversion of radioactive AA into HETE and maturation. When oocytes are incubated in NSW with radioactive AA a rapid peak of radioactive HETE is observed (Fig. 4): the HETE level immediately rises after AA addition, reaches a maximum within 2 min and disappears in 6 min. Under these conditions maturation is induced. On the contrary, in CaFASW, oocytes exposed to the same amount of radioactive AA, do not undergo maturation and only a smaller HETE peak is observed (Fig. 4A). In CaFASW supplemented with the calcium chelator EGTA, the HETE peak is further reduced (Fig. 4B). HPLC analysis confirmed that exogenous radioactive AA is converted into various HETE and that this conversion is reduced when the external medium is depleted of calcium (data not shown). Since the amount of available [14C]AA is rapidly declining (Fig. 1)) the peak increase can be explained by the presence of sufficient amounts of AA for oxidation, and the peak decrease by a decrease of available AA and by the conversion of HETE to other metabolites. On the contrary, when oocytes are incubated in CaFASW with or without 1-MeAde (Fig. 4C) no difference between the radioactive HETE peaks is observed: 1-MeAde does not appear to stimulate the production of lipoxygenase metabolites from exogenous radioactive AA. In human polymorphonuclear leukocytes, formylpeptides stimulate AA metabolism through lipoxygenase when AA is presented to the cells with BSA but not when it is presented in ethanol (Clancy et al., 1983). Therefore, we repeated the experiment of Fig. 4C but AA was added with BSA 10 mg/ml, instead of ethanol. In these conditions, almost no HETE is produced (less than in the presence of CaFASW + EGTA, Fig. 4B) (data not shown); this also correlates with the negative effect of BSA on AA-induced maturation (see further in the text). Finally no increased lipoxygenase activity is revealed after 1-MeAde-induced stimulation of the oocytes, as shown by pulse experiments in which radioactive AA is added for 2 min to oocytes at various times after addition of 1-MeAde and the conversion into HETE is measured after TLC (data not shown). Furthermore no

26

DEVELOPMENTAL BIOLOGY y.

GVSD 8

o CaFASW l

100

NSW

12

16

2~ .

12

5 i

10

VOLUME 114, 1986

[G

16

4 2

E

2.5

F

8

0

12

16

2C

0

2(

% GVBD

d Control .

1 MeAde

x

Acidwash

5 98

PL

of

'

Time

' 4

'

a 8

after

8

' 12

AA

8

a 16

addition

'

' 20

(min.)

FIG. 3. Covalent incorporation of radioactive AA into various lipid fractions separated by TLC in system A (A-F), system B (H, I), or by TCA precipitation (G) (with (X) or without an acidic-chloroform wash (A, A): at time 0, [‘%]AA (20 ~1 [%]AA/20 ml, i.e., 0.833 p&f AA final concentration) was added to four batches of a 10% oocyte suspension, one in CaFASW (0), the other in NSW (0) (A-F), or one control in CaFASW (A) and the other treated with 1-MeAde (A) (G-I) at time 0. At various times after AA addition a l-ml aliquot was treated as described under Material and Methods and the incorporation into the various lipids was measured. The percentage of GVBD for each batch is indicated: 0,8%;O,lOO%;A, 5%;r, 98’%).A-F and G-I relate to two different experiments. Abbreviations as for Fig. 2; PPI, polyphosphinositides.

endogenous HETE (as measured by HPLC) seems to be produced under the influence of 1-MeAde, or their mimetics MGBG and dithiothreitol (data not shown). Correlation between Conversion into HETEs and Biological Activity Five arguments are in favor of a correlation between the conversion of AA into HETE and the biological activity of AA: (1) The biological activity of AA metabolites was studied after separation of these metabolites by TLC or by HPLC (see Material and Methods). After TLC in sys-

tem A (Fig. 2), biological activity is only observed in the free fatty acid + neutral lipid area; in system B, biological activity is detected in the AA and HETE spots only (data not shown). By HPLC the biological activity was recovered at an elution time corresponding to HETE (Fig. 5). Experiments are in progress to identify with certainty the active HETE. (2) The facilitating effect of external calcium on both the conversion of AA into HETE (Figs. 4A and B) and oocyte maturation induced by AA (Fig. 6); this effect is only observed on the conversion into HETEs and not on any other metabolism (Fig. 3).

MEIJER,

MACLOUF,

Arachidmic

AND BRYANT

Acid

Metabolism

in

Starfish

27

Oocqtes

% GVBD o CaFASW+

5mM

EGTA

1

A Control . +%MeAde

% GVBD 4 100

C t io;

2

4

6

Time(min.1

‘“C-AA

8

10

Time

12

14

16

18

2C

:

2

4

6

(min.)

:-AA

--k-L++: 8

-

10

12

14

16

18

20

Timetmin.)

%-AA

FIG. 4. Conversion of [‘%]AA into HETE: at time 0, [“C]AA was added to three couples of 10% oocyte suspension batches, one in CaFASW (0), the other in NSW (0) (4A), or one in CaFASW + 5 mM EGTA (O), the other in NSW (0) (4B), or one in CaFASW, untreated (A), the other in CaFASW but treated with 1-MeAde (1O-6 M, final concentration) (A) (4C). At various times after AA addition a l-ml aliquot was treated as described under Material and Methods, and after TLC in system B (Fig. Z), the HETE spot was scraped and counted. The percentage GVBD for each batch is indicated.

I

a c

160

-

160

-

90 -

g x 120

-

-

110

-

g 100

-

=

v ;

e F"

- If 00

140 130

i

HETE’s Y

00

7o

60

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50

0”

s s ae

70-

40

60 -

30 40 30-

20 10

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FRACTION , ,

5

6

7

8

9

20

22

24 26

28

30

32

34

36

36

40

42

44

, 10

, 11

NUMBER , , 12 13

lb

, 15

I 16

, 17

, 18

, 19

, 20

, 21

1 22

TIME

0

, 23

(MIN.)

FIG. 5. Biological activity of HETEs. Oocytes were treated with [14C]AA as described under Material and Methods and the lipids extracted after 2 min of incubation; after partial purification through a column of silicic acid, the sample was concentrated and run on a Spherisorb SlO ODS column (reversed-phase HPLC) and fractions were collected every 30 sec. 50 ~1 was removed for radioactivity counting (0) and 200 ~1 was used to monitor the maturation-inducing activity (0). The elution time of HETEs (which were not clearly separated from one another in this system) is indicated.

28

DEVELOPMENTAL BIOLOGY

VOLUME 114,1986

loogo-

OA CaFASW

8’3-0~

NSW

Agonist

concentrationCrM1

FIG. 6. Calcium dependence of AA-induced maturation and calcium independence of S-HETE-induced maturation: AA (A, A) in CaFASW or in NSW and dose-response curves for 8-HETE (0,O) in CaFASW or in NSW.

(3) Leukotrienes do not induce maturation (Meijer et ah, 1984) and among the various HETE (5-, B-, 9-, ll-, 12-, and 15-HETE) only B-HETE was found to induce maturation (Meijer et al, in prep); the biological activity we first reported for 12- and 15-HETE (Meijer et ak, 1984) could be explained by a 5% contamination with BHETE, which was not separated from these metabolites during purification (data not shown), Up to now, B-HETE thus appears as the only biologically active metabolite of AA. (4) As opposed to AA-induced maturation, B-HETEinduced maturation is not influenced by the presence of calcium in the external medium (Fig. 6). (5) Eicosatetraynoic acid (ETYA), a lipoxygenase inhibitor, completely inhibits the conversion of AA to HETE (Fig. 7A) and, correlatively, inhibits AAinduced maturation, but not HETE-induced maturation (Fig. 7B). It thus appears that the biological activity of AA plies a calcium-facilitated and ETYA-inhibited step. biological activity of B-HETE is insensitive to both tors. We believe that these results clearly correlate existence of a calcium-dependent conversion of AA HETE (lipoxygenase) and the calcium-facilitated ETYA-sensitive induction of maturation by AA.

imThe facthe into and

Site of Action of Arachidonic Acid

The site of action of AA was investigated by intracellular microinjection (Table 1): although oocytes respond to AA concentrations around 1 PM when it is

dose-response

curves

for

added externally, no maturation can be induced when AA is microinjected intracellularly, even at high final concentrations (275 PM). These injected oocytes, however, remain able to respond to externally added AA (final concentration: 25 PM). Since the microinjection technique involves injection of a definite volume entrapped between two oil droplets, it could be argued that AA is immediately trapped by the oil. However, similar results are obtained when AA is injected without oil in the microelectrode (Table 1). Apparently AA does not act in the compartment accessible by microinjection. Another insight on the site of action of AA is provided by the action of BSA (Fig. 8). Indeed, due to its ability to bind free fatty acids, and to remove them from membranes, BSA inhibits AA (but not 1-MeAde)-induced maturation (Fig. 8A) in a concentration-dependent way (Iso = 250 pg/ml for a concentration of AA of 10 PM). Addition of fatty acids to BSA lowers its inhibitory properties (data not shown). Adding BSA at various times after the addition of AA delimits a “BSA-sensitive period,” corresponding to the period during which AA is required (which itself coincides with the 1-MeAdedependent period (HDP) (Meijer et aZ., 1984) (Fig. BB). Therefore during its whole period of action, AA remains sensitive to externally added BSA. The same “BSA-sensitive period” was obtained with threshold (1 pM) or high (25 pM) concentrations of AA (data not shown). Furthermore oocytes which have been microinjected with BSA (final concentration: 3 mg/ml) remain able to respond to exogenous AA or 1-MeAde (Table 2). The experiments thus suggest that AA is acting at a site readily accessible from the external side and most

MEIJER, MACLOUF, AND BRYANT

Arachidonic

Acid Metabolism

in Starfish

Oocytes

29

0 cant rol m+ETYA

70t

60-

g

50-

x

\

.

\

.

3040-

\

. 0.25 pM 8 HETE . 1OyMAA

20-

‘\

10 0

i

2

4

Time

I

I

I

6

8

10

(min.)

04 0

I 10

I

ETY~concentration 50

II 75

100

I

I

150

250

CPM)

14C-AA FIG. ‘7. (A) Eicosatetraynoic acid (ETYA) inhibits HETE production: at time 0, [l’C]AA was added to two batches of a 10% oocyte suspension in NSW, after 30 min preincubation with (m) or without (0) 500 pM ETYA. At various times after AA addition a l-ml aliquot was treated as described under Material and Methods section, and after TLC in system B (Fig. 2), the HETE spot was scraped and counted. At the end of the experiment, the ETYA-treated oocytes were found to mature (100% GVBD) with lo-’ M 1-MeAde. (B) Eicosatetraynoic acid inhibits AA (A) but not 8-HETE (.)-induced maturation: dose-response curve. The agonist concentrations, 0.25 FMfor 8-HETE and 10 afor AA, were chosen to be equal to 4.17 times the respective dose inducing 50% maturation in this batch of oocytes (0.06 pM for 8-HETE and 2.4 pM for AA).

probably on, in, or close to the plasma membrane. They further support the role in maturation of AA conversion to HETE, which would also remain sensitive to BSA; on the other hand, they also suggest the lack of importance for maturation induction, of the covalent incorporation of AA into other lipids, which would become rapidly irreversible and insensitive to BSA.

incorporation of AA into other lipids therefore seems unlikely to be involved in the cellular response. A similar reversibility by albumin was recently demonstrated for fatty-acid-stimulated superoxide release and shape change of human neutrophils (Badwey et ak, 1984). Husebye and Flatmark (1984) further showed that BSA is able to remove up to 80% of the free unsaturated fatty acids from chromaffin granule ghosts.

DISCUSSION

Mode of Action. of Exogenous AA Site of Action of Arachidmic

Acid

Although not decisive, the lack of maturation induction by AA when microinjected intracellularly suggests a plasma membrane location of the site of action of AA inducing maturation. This is further supported by the lack of inhibitory effect of the fatty acid-chelating BSA when injected in the cytoplasm. But the best evidence for an easily and externally accessible site of action of AA in inducing maturation-most probably the external side of the plasma membrane-is provided by the inhibitory effect of BSA when added in the incubation medium from which it is unlikely to enter oocytes. The fact that BSA is still effective even 5 min after AA addition of the oocytes further points out the easily accessible site of AA action, even to a 68-kDa protein. Covalent

Although exogenous AA is rapidly taken up by the oocytes and converted into a variety of more complex lipids (phospholipids, triglycerides), this covalent binding is not likely to be important for maturation induction: indeed, (1) no difference of incorporation into these lipids was observed between nonmaturing and maturing oocytes, (2) the BSA-sensitive period corresponds to the AA-required contact time. On the other hand, the calcium-facilitated conversion of exogenous AA to HETE is clearly correlated with maturation induction: indeed, (1) biological activity is only recovered in the HETEcontaining fractions, after separation of the AA metabolites by HPLC or TLC;

30

DEVELOPMENTAL

TABLE PERCENTAGE

GVBD

AFTER

1

TREATMENT

WITH

SODIUM

(10 mM STOCK SOLUTION IN 70 mM NaCl) EITHER DITION OR BY INTRACELLULAR MICROINJECPION

Final External 0.1 0.25 1 2.5 10 25

AA concentration

BIOLOGY

(FM)

ARACHIDONATE

BY EXTERNAL.

AD-

7% GVBD

addition 7 10

47 ‘76 89 95

100

100

250

100

Internal microinjection 275 (between oil droplets) Followed by external addition 275 (no oil) Followed by external addition

0 (1000)

of 25 pM AA

100

of 25 rM

100

0 (12/12)

AA

Note. AA was injected either between two oil droplets or through an air-containing micropipet devoid of oil (in this last case, to avoid a possible external contamination by AA, the oocytes were laying in NSW containing BSA (5 mg/ml) during the microinjection, and then washed). One hour after microinjection the oocytes were checked for GVBD and then treated with 25 pM AA this time externally added, and the percentage GVBD was recorded 30 min later. The number of maturing oocytes versus the number of microinjected oocytes is indicated in parentheses.

(2) 8-HETE induces oocyte maturation and is much more efficient than AA; (3) exogenous AA is converted to HETE and this process is facilitated by external calcium, (see TLC and HPLC data);

VOLUME

114,

1986

(4) AA-induced maturation is facilitated by the presence of calcium in the external medium; (5) HETE-induced maturation is not influenced by external calcium; (6) the lipoxygenase inhibitor ETYA inhibits the conversion of exogenous AA to HETE; it blocks AAinduced maturation but not HETE-induced maturation. Lipoxygenases are known to be activated by divalent ions such as calcium (Jackschik et aZ., 1982; Narumiya and Salmon, 1982). The effect of extracellular calcium concentration on AA metabolism has been demonstrated, for example, in epithelial cells from the toad urinary bladder (Burch and Halushka, 1984). In sea urchin eggs a calcium-dependent lipoxygenase (active at lop7 Mcalcium) has been studied (Perry and Epel, 1985a); at fertilization it is activated during the period of intracellular calcium release and a transient and rapid conversion of exogenous AA into HETE occurs (Perry and Epel, 1985b). We therefore believe that most (if not all) starfish oocyte maturation-inducing activity of AA can be ascribed to HETE which is produced through the action of calcium-facilitated lipoxygenases known to be present in the oocytes (Perry and Epel, 1985a). How HETE acts to induce maturation remains unknown. The nature of the active HETE produced remains unknown, although, up to now, only 8-HETE was found to be active. Involvement of AA Metabolism Maturation?

in l-MeAde-Induced

We do not think that 1-MeAde acts by releasing AA in the external medium which then would act on plasma

. I-MeAde(*.A,m)or

AAfo.v)concentration
Time

of BSA

addition

FIG. 8. Fatty acid-free bovine serum albumin (BSA) inhibits AA- but not 1-MeAde-induced maturation: (A) l-MeAde A, 0) dose-response curves in the absence (0,O) or in the presence of 100 pgg/ml or 1000 rig/ml BSA; (B) BSA-sensitive by the effect on GVBD of BSA (1 mg/ml) added at various times after the addition of 20 pM AA at time 0.

.tmin) (0, A, n ) and AA (0, period as determined

MEIJER,

MACLOUF,

AND BRYANT

Arachidonic

TABLE 2 PERCENTAGE GVBD AFTER ADDITION OF THRESHOLD CONCENTRATIONS OF l-MeAde OR AA TO OOCYTES PRETREATED WITH FATTY ACID-FREE BSA EITHER ADDED EXTERNALLY OR MICROINJECTED INTRACELLULARLY Inducer

Treatment External addition No treatment BSA (final concentration: 1 mg/ml) Internal microinjection 70 mM NaCl BSA (final concentration: 3 mg/ml) Note. The addition or which BSA the number

0.1 jlM lMeAde

10 pM AA

100%

100%

100%

6%

100% (lO/lO)

100% (12/12)

100% (lO/lO)

90% (9/10)

respective controls were performed either by no external intracellular microinjection of the carrier 70 mMNaC1 (in was dissolved). The number of maturing oocytes versus of microinjected oocytes is indicated in parentheses.

membrane receptors after eventual conversion into other metabolites as described for induction of platelet aggregation by exogenous AA which is first converted to the active metabolite, thromboxane A2 (Feinstein and Fraser, 1975; Kinlough-Rathbone et al, 1976). This type of mechanism is unlikely to occur in the 1-MeAde-stimulated starfish oocyte since (1) BSA does not inhibit lMeAde-induced maturation, as would be expected if an AA metabolite was released, (2) no release of AA or HETE in the external medium (in the presence or absence of BSA) under the influence of 1-MeAde was observed from [14C]AA-prelabeled oocytes (data not shown), (3) calcium does not facilitate 1-MeAde-induced maturation (Meijer et al., 1984). A typical “arachidonic acid cascade” is not necessarily involved in 1-MeAde action, for the following reasons: (1) we were unable to detect any AA release from [14C]AA preloaded oocytes (prelabeled for 5 min, 30 min, or 4 hr), under the influence of l-MeAde.3 The addition of BSA to decrease reacylation did not help in revealing any release. (2) We were unable to detect any HETE appearance, under the influence of 1-MeAde, either from exogenous [14C]AA (added together with 1-MeAde: as measured by TLC (Fig. 4C) or by HPLC (data not shown) or from endogenous sources, as measured by HPLC (data not shown). The possibility remains however that a small increase was undetectable by our TLC or HPLC systems ’ Oocytes were prelabeled with [‘“C]AA for 5 min, 30 min, or 4 hr, washed to remove the external label, and treated with 1-MeAde; at various intervals, aliquots were processed for determination of free AA and HETEs by TLC as described under Material and Methods.

Acid

Metabolism

in

Starykh

Ooqtes

31

or that an increase in HETE could be masked by a change in specific activity or a rapid conversion into other metabolites. (3) No endogenous lipoxygenase activity seems to be stimulated by 1-MeAde action, as shown by pulse experiments (2min pulses with [14C]AA and analysis for conversion into [14C]HETE). (4) If AA was released and further metabolized into HETE, one would expect calcium to facilitate 1-MeAdeinduced maturation, as it does for AA-induced maturation, but this is not the case. (5) In some cells, the metabolism of phostidylinositol 4,5-diphosphate is linked to the release of free AA (Irvine, 1982, Berridge, 1984). No evidence for such a metabolism was found in the 1-MeAde-stimulated oocytes and inositol-trisphosphate has no effect on maturation (Berridge and Irvine, 1984). The possibility that AA (and HETE) acts as “second messenger” in the action of 1-MeAde on starfish oocytes remains undemonstrated, although the high specificity and efficiency of AA and HETE is highly suggestive for their physiological implication. It is also possible that AA and HETE mimic another fatty acid, yet unknown, involved in the induction of maturation by 1-MeAde. The starfish oocyte thus provides a good model for the study of the action of specific metabolites of AA although their physiological implication remain to be demonstrated. The results presented here show that the fact that AA and its metabolites are able to mimick an agonist does not necessarily mean that they are involved as internal mediators during the agonist action. This conclusion is starting to arise from a few models such as the chemotactic factors-induced aggregation and degranulation of neutrophils (review by Naccache and Shaafi, 1983). This system indeed shares many characteristics with the starfish oocyte model: (1) Neutrophil responses can be mimicked by exogenous AA (Naccache et ab, 1979) although not in all species: rabbit, but not human neutrophils are sensitive to AA; this difference may be due to different endogenous metabolic activities. Although the oocytes of all starfish species respond to 1-MeAde, some do to AA, whereas others do not (Meijer et al., 1984). (2) The response of neutrophils is enhanced by extracellular calcium. (3) The metabolites of AA, such as leukotriene B4, are much more active (Naccache and Shaafi, 1983; Bokoch and Reed, 1981). (4) Although leukotriene B4 is formed endogenously upon stimulation by chemotactic factors, the use of various of inhibitors has shown that this pathway is not necessarily central for neutrophil response (Showell et

32

DEVELOPMENTAL BIOLOGY

a~!, 1981; Smith et aL, 1981). Furthermore chemotactic factor-induced neutrophil responsiveness is inhibited by AA (Naccache et ah, 1983). (5) AA metabolism stimulated by formylpeptides in neutrophils is independent of membrane phospholipase activation (Clancy et aZ., 1983). For these reasons AA metabolites are unlikely to act as intracellular mediators of the neutrophil response but could act as intercellular messengers amplifying the recruitment of neutrophils, as long as an exogenous source of AA can be identified (Naccache and Shaafi, 1983; Clancy et ah, 1983). Great care should therefore be taken before it can be said that AA metabolites play the role of intracellular messengers in models where these AA metabolites are able to mimic the natural agonist, such as leukotriene B1-induced aggregation of marine sponge cells (Rich et al., 1984), AA-induced DNA synthesis in calcium-deprived rat liver cells (Boynton and Whitfield, 1980), 5HETE and AA-induced insulin secretion in rat pancreatic islets (Yamamoto et al, 1983), activation of lymphocytes (Parker, 1981; Wrighton et ak, 1983), platelet stimulation by thrombin (Okuma et ah, 1982; Siess et cd, 1983,1984); and the like. Work is in progress to identify the site of action of HETE during oocyte maturation and the 1-MeAde-dependent step it mimics or interacts with. We thank Mrs. Claude Guerrier for kindly preparing the figures. Many thanks to the fishermen of the “Station Biologique” for their constant disponibility to collect the starfish. The manuscript was kindly typed by Nicole Guyard. This work was supported by a grant from the “Association pour la Recherche contre le Cancer” (ARC 6268). REFERENCES BADWEY, J. A., CURN~E, J. T., ROBINSON,J. M., BERDE, C. B., KARNOVSKY, M. J., and KARNOVSKY, M. L. (1984). Effects of free fatty acids on release of superoxide and on change of shape by human neutrophils. Reversibility by albumin. J. BioL Chem 259, ‘7870-7877. BERRIDGE, M. J. (1984). Inositol trisphosphate and diacylglycerol as second messengers. Biochem. J. 220,345-360. BERRIDGE,M. J., and IRVINE, R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature (Zono!on~ 312,315321. BOKOCH, G. M., and REED, P. W. (1981). Effect of various lipoxygenase metabolites of arachidonic on degranulation of polymorphonuclear leukocytes. J. BioL Chem 256,5317-5320. BORGEAT, P., FRUTEAU DE LACLOS, B., RABINOVITCH, H., PICARDS, S., BRAQUET, P., HERBERT, J., and LAVIOLETTE, M. (1984). Generation and structures of the lipoxygenase products. Eosinophil rich human polymorphonuclear leukocyte preparations characteristically release leukotriene C, on ionophore A23187 challenge. J. Allergy Clin ZmmunoL 74.310. BOYNTON, A. L., and WHITFIELD, J. F. (1980). Possible involvement of arachidonic acid in the initiation of DNA synthesis by rat liver cells. Exp.

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VOLUME 114, 1986

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NARUMIYA, S., and SALMON, J. A. (1982). Arachidonic acid-15-lipoxygenase from rabbit peritoneal polymorphonuclear leukocytes. In “Methods in Enzymology” (W. E. M. Lands and W. L. Smith, eds.), Vol. 86, pp. 45-48. Academic Press, New York. OKUMA, M., TAKAYAMA, H., and UCHINO, M. (1982). Arachidonate peroxidation and functions of human platelets. In “Lipid Peroxides in Biology and Medicine” (K. Yagi, ed.), pp. 271-284. Academic Press, New York. PARKER, C. W. (1981). Arachidonie acid metabolism in activated lym-

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phocytes. In “Mechanism of lymphocyte activation” (K. Resch and H. Kirchner, eds.), pp. 4’7-57. Elsevier/North-Holland, Amsterdam. PERRY,G., and EPEL, D. (1985a). Characterization of a Caz+-stimulated lipid peroxidizing system in the sea urchin egg. Deu. BioL 107, 4757. PERRY, G., and EPEL, D. (1985b). Fertilization stimulates lipid peroxidation in the sea urchin egg. Dev. Biol. 107,58-65. RICH, A. M., WEISSMANN, G., ANDERSON, D., VOSSHALL, L., HAINES, K. A., HUMPHREYS, T., and DUNHAM, P. (1984). Calcium dependent aggregation of marine sponge cells is provoked by leukotriene B( and inhibited by inhibitors of arachidonic acid oxidation. B&hem. Biophgs. Res. Commun 121,863-8’70. SHOWELL, H. J.,NACCACHE, P. H., WALENGA, R. W., DALECKI, M., FEINSTEIN, M. B., SHA’AFI, R. I., and BECKER, E. L. (1981). The effects of quercetin, 1-tosylamido-2-phenylethylchloromethylketone, cytochalasin A and nordihydroguaiaretic acid on lysosommal enzyme secretion, arachidonic acid metabolism and Ca2+ fluxes in rabbit neutrophils. J. Reticdoendoth Sot. 30,167-181. SIESS, W., SIEGEL, F. L., and LAPETINA, E. G. (1983). Arachidonic acid

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stimulates the formation of 1,2-diacylglycerol and phosphatidic acid in human platelets. Degree of phospholipase C activation correlates with protein phosphorylation, platelet shape change, serotonin release and aggregation. J. Bid Chem 258,1X&36-11242. SIESS, W., WEBER, P. C., and LAPETINA, E. G. (1984). Activation of phospholipase C is dissociated from arachidonate metabolism during platelet shape change induced by thrombin or platelet-activating factor. Epinephrine does not induce phospholipase C activation or platelet shape change. J. Biol Chem 259,8286-8292. SMITH, R. J., SUN, F. F., IDEN, S. S., BOWMAN, B. J., SPRECHER,H., and MCGUIRE, J. C. (1981). An evaluation of the relationship between arachidonic acid lipoxygenation and human neutrophil degranulation. Clin. Immunol Immuwpathd 20.157-169. WRIGHTON, S. A., PAI, J. K., and MUELLER, G. C. (1983). Demonstration of two unique metabolites of arachidonic acid from phorbol esterstimulated bovine lymphocytes. Carcirwgenesis 4,1247-1251. YAMAMOTO, S., ISHI, M., NAKADATE, T., NAKAKI, T., and KATo, R. (1983). Modulation of insulin secretion by lipoxygenase products of arachidonic acid. J. Biol. Chem 258,12149-12152.