THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl. 268, No. 11. Iasue of April 15, pp. 7983-7989,1993 Printed in U. S. A.

Meiosis Reinitiation in Surf Clam Oocytes Is Mediated via a 5- HydroxytryptamineB Serotonin Membrane Receptor and a Vitelline Envelope-associated High Affinity Binding Site*

(Received for publication, July 20, 1992)

Slavica Kranticzg, Pierre GuerrierS, and FranCois Dubell From the 4Luboratoire de Biologie MoEculaire et Cellulaire, UMR49, Centre National de la Recherche Scientifique et Ecole Normale Sugrieure de Lyon, 46 AlEe d’ltalie, 69364 Lyon Cedex 07, France and the TDepartement d’oceanographie, Uniuersite du Quibec a Rimouski, 310, AlEe des Ursulines, Rimouski, Qutbec G5L 3A1, Canada

The neurohormone serotonin (5-hydroxytrypta- mine, 5HT) triggers meiosis reinitiation in prophase- arrested oocytes of Spisula solidissima. The original pharmacological profile of this response prompted us to examine whether it involved a novel type of 5HT receptor. In order to characterize these putative recep- tors, we performed [’H]5HT binding assays. Given the complexity of oocyte surface architecture, [‘H]5HT- specific binding was measured in both plasma mem- brane and vitelline envelope fractions.

Our data reveal the existence of a single class of BHT-specific binding sites in each fraction. These bind- ing sites appear distinct by their kinetic properties: 1) plasma membrane binding sites are of lower affinity but are more numerous than vitelline envelope sites; 2) [‘H]5HT binding is more rapid in membrane fraction.

The pharmacological profiles of both binding sites were defined in competition experiments by using six- teen BHT-related and unrelated compounds. Both membrane and vitelline envelope sites displayed novel profiles. However, only the pharmacological profile determined for the plasma membrane sites corresponds to that observed for BHT-induced meiosis reinitiation. These new 5HT6 binding sites are therefore the phys- iological receptors that mediate the effects of 5HT in Spisula oocytes. The putative role of vitelline envelope sites is discussed.

The biogenic amine serotonin, or 5-hydroxytryptamine (5HT),’ is widely distributed throughout animal phyla. I t elicits and/or modulates a wide range of complex behaviors in both vertebrates and invertebrates. For example, in mam- mals, it is involved in the control of sexual and feeding behavior, sleep and arousal, pain perception, learning, and memory (1-3). In invertebrates, various functions including

* This work was supported by Association pour la Recherche sur le Cancer Grant 6145, Institut National de la Sante et de la Recherche M&dicale Grant 910708, Programme de Coop6ration Franco-QuBbe- coise Grant 20128890, Fondation pour la Recherche Medicale, and NSERC-Canada and FCAR-Quebec grants (to F. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer-

this fact. tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate

5 To whom correspondence and reprint requests should be ad- dressed Laboratoire de Biologie Moleculaire et Cellulaire, Ecole Normale Superieure de Lyon, 46 Allie d’Italie, 69364 Lyon Cedex 07, France. Tel.: 33-72-72-81-97; Fax: 33-72-72-80-80.

The abbreviations used are: 5HT, 5-hydroxytryptamine; GVBD, germinal vesicle breakdown; 8-OH DPAT, 8-hydroxy-2-(di-N-pro- py1amino)tetralin hydrobromide; TFMPP, 1-/3-trifluoromethyl- phenyl piperazine; PEI, polyethyleneimine.

circadian rhythmicity, aggressive posture, and escape behav- iors are regulated by 5HT (4-6). In addition, in some molluscs, 5HT plays a role in the control of reproduction. 5HT thus induces spawning in a number of bivalve species such as Crassostrea, Pecten, Spisula, and Ruditapes (7-10). Moreover, 5HT is apparently the natural maturating neurohormone for some of these animals. Indeed, in Crassostrea and Ruditapes, during the spawning process, 5HT induces meiosis reinitiation of the prophase-arrested oocytes, thus ensuring germinal ves- icle breakdown (GVBD) and transition toward the metaphase stage, i.e. the physiological stage at which fertilization occurs (10).

During the last decade, it became clear that 5HT elicited such a variety of biological effects via a panoply of specific binding sites. In mammals, these binding sites have been classified into four major pharmacological types: 5HT1 (fur- ther subdivided into A, B, C, and D), 5HT2,5HT3, and 5HT4 (11, 12). Most of these binding sites have been successfully correlated with behavioral and biochemical effects of 5HT and, therefore, assigned as the physiological receptors for 5HT action (1 1).

In contrast, the physiological receptors involved in trans- duction of the biological effects of 5HT in invertebrates are still unknown. Only very recently have 5HT binding sites positively (5HT-droJ or negatively coupled to adenylate cy- clase (5HT-drozA and 5HT-droZ~), been cloned from a Dro- sophila head cDNA library (13, 14). Based on the distribution pattern of the 5HT-drozB receptor, it has been suggested that this receptor might play a role in the 5HT-dependent regu- lation of motor control (14); the physiological functions of 5HT-drol and 5HT-dro2~ binding sites remain unknown. These invertebrate binding sites, although pharmacologically distinct, display a high homology with the mammalian 5HTlA receptors. In contrast, they are genetically unrelated to 5HTz receptors (14).

We recently reported that, in the surf clam Spisula solidis- sima, 5HT triggers meiosis reinitiation with a specific phar- macological profile (15), which was consistent with involve- ment of a novel type of 5HT binding sites in this oocyte response. These sites appeared sensitive to both 8-OH DPAT and ketanserin, i.e. to compounds defined as selective, respec- tively, for the 5 H T I ~ and 5HT2 subtypes, on mammalian 5HT receptors (15). These results suggested that putative Spisula oocyte 5HT receptors might be different and phylogenetically older than those from Drosophila brain.

In the present work, we assessed these possibilities by performing detailed kinetic and pharmacological characteriza- tions of [3H]5HT binding with the putative receptor sites localized on the oocyte surface. Our results reveal the exist- ence of 5HT-specific binding sites in both plasma membrane and vitelline coat fractions. The properties of these binding

7983

7984 Serotonin Receptors and Meiosis Reinitiation in Clam Oocytes

sites are discussed in comparison with well characterized mammalian and Drosophila 5HT receptors. Finally, to assess the physiological involvement of Spisula oocyte 5HT binding sites, we compared their pharmacological profiles with that observed for the 5HT-induced meiosis reinitiation response.

EXPERIMENTAL PROCEDURES

Materials-[3H]5HT creatinine sulfate (30 Ci/mmol; 1 Ci = 3.7 GBq) was purchased from New England Nuclear (Dupont de Nem- ours, France). 3-Tropanyl-3,5-dichlorobenzoate (MDL 72222) was obtained from Merrel Dow Research Institute (Strasbourg, France). 3-Tropanyl-indole-3-carboxylate (ICs 205930) was a generous gift from Sandoz Pharma Ltd. (Rueil-Malmaison, France). Other 5HT analogs were ordered from RBI (Natik, MA). All other chemicals and drugs were obtained from standard commercial sources.

Handling of Oocytes and Quantification of Their Response to Sero- tonin-Surf clams were collected at Iles de la Madeleine (Quhbec, Canada) and kept in running sea water tanks until used. Oocytes were dissected out from ovaries of mature females, filtered, and washed three times in artificial sea water as previously described (15).

Each batch of oocytes was divided into two experimental groups. One was kept intact (control group) while the other was treated with 1 M glycerol in 0.2 M phosphate buffer (pH 8) for 3 min in order to remove the vitelline envelope. The oocyte suspension was then sedi- mented (2 min in Tabletop International centrifuge, setting No. 2) and washed in 15 volumes of artificial sea water. The treated group displayed normal morphology and remained in the prophase-arrested stage when examined by light microscopy.

The oocyte suspensions from both groups were adjusted to a 0.5% (v/v) concentration in artificial sea water. One-ml samples of the oocyte suspensions were then incubated for 30 min at 18 “C with different concentrations of 5HT (final concentration ranging from 1 nM to 10 pM). After 30 min, the oocytes were fixed for 15 min in 5% formaldehyde. The fixative was then rinsed out, and GVBD was scored by random counting of 100-200 oocytes per condition.

Oocyte Membrane and/or Vitelline Coat Preparations-Plasma membranes of oocytes were isolated together with (“E”) or without (“Mb”) vitelline coats by differential centrifugation. The vitelline layer of the oocytes was removed by incubating 1 volume of oocytes with 10 volumes of 1 M glycerol in 20 mM phosphate buffer (KH,P04/ Na2HP04), pH 8, for 3 min at 25 “C (16). The oocyte suspension was then sedimented in a Tabletop International centrifuge (2 min at setting No. 2). The pellet consisted of vitelline coat-free oocytes, and was used for Mb fraction preparation (see below). The supernatant used for vitelline envelope (“VE”) fraction preparation was centri- fuged (45,000 X g, 15 min). The resulting pellet was washed with 50 mM Tris-HC1 buffer, pH 7.4 (Tris-HC1 buffer), centrifuged (15 min, 45,000 X g), suspended in a small volume of Tris-HC1 buffer, frozen in liquid nitrogen, and kept at -80 “C until used.

Intact oocytes and oocytes devoid of vitelline coat were homoge- nized in 15 volumes of Tris-HC1 buffer with a glass-glass homogenizer by 10 hand strokes. Crude membrane fractions prepared from entire (E) and from vitelline envelope-free oocytes (Mb) were centrifuged (45,000 X g, 15 min), washed in Tris-HC1 buffer (1:15, v/v), and centrifuged (15 min, 45,000 X g) again. Crude membrane pellets were suspended in a small volume of Tris-HC1 buffer, frozen in liquid nitrogen, and kept at -80 “C until used.

Na/K-ATPase activity was assayed as previously described (17) to estimate a quantitative index of plasma membrane content (18) in oocyte homogenates and both VE and Mb fractions.

Ultrastructure of each fraction was checked by transmission elec- tron microscopy on ultrathin (0.08-pm-thick) sections of Epon-em- bedded Mb and VE pellets initially fixed with 4% glutaraldehyde (16).

Binding Assays-Binding assays were performed by a modification of the method of Fillion and colleagues (19). Different oocyte fractions

at 25 ‘C with 100 p1 of [3H]5HT (30 Ci/mmol) in 1.5 ml of Tris-HC1 (protein equivalent: 40-80 pg per 100 pl) were incubated for 30 min

buffer. The nonspecific component of total binding was determined in the presence of 100 p~ nonradioactive 5HT.

In competition experiments, 100 p1 of various serotoninergic (8- hydroxy-2-(di-N-propylamino)tetralin hydrobromide, 8-OH DPAT, 1-/3-trifluoromethylphenyl/piperazine, TFMPP; MDL 72222; 3-tro- panyl-indole-3-carboxylate, ICs 205930; metoclopramide; ritanserin, mianserin, propranolol) and nonserotoninergic (acetylcholine, car- bachol, haloperidol, pyrilamine, isoproterenol, and prazosin) com- pounds were incubated with oocyte membranes and [3H]5HT. All drugs were freshly prepared as 10 mM solutions in either methanol

(ritanserin, carbachol, haloperidol, ICs 205930, and prazosin) or Tris- HC1 buffer (all others). They were then diluted in the same buffer and tested at final concentrations ranging from 1 nM to 100 p ~ .

Incubations were stopped by rapid filtration over Whatman GF/B glass fiber filters (presoaked in 0.1% polyethyleneimine for 2 h) at reduced pressure. Filters were rinsed three times with 5 ml of ice-cold Tris-HC1 buffer in less than 15 s, dried overnight at room tempera- ture, dropped into 7 ml of Aquasol (New England Nuclear, Dupont de Nemours, France), and shaken vigorously. A few hours later, radioactivity was assayed by liquid scintillation counting with 60% efficiency.

Binding data were expressed in picomoles of [3H]5HT bound per mg of protein. Protein concentration was measured according to Lowry and colleagues (20).

Data Analysis-Equilibrium saturation and drug competition bind- ing data were analyzed by iterative curve-fitting using the Mc Pherson modified ligand program (21). Differences between data were statis- tically assessed using one-way analysis of variance (ANOVA) and considered significant at p < 0.05.

RESULTS

Oocyte Response to Serotonin in Vivo: Role of the Vitelline Enuelope-Both intact and vitelline coat-free oocytes re- sponded to 5HT in a dose-dependent manner (Fig. 1). How- ever, the neurohormone triggered GVBD almost 10 times more efficiently in intact oocytes: ECso concentration deter- mined for intact oocytes was 0.18 f 0.06 pM, whereas it was 1.31 k 0.29 p~ when determined for the same batches of oocytes after removal of the vitelline envelope. In addition, only about 75% of vitelline coat-free oocytes responded by GVBD to 10 PM 5HT, whereas, in the same experimental conditions, all intact oocytes responded.

Binding of [‘HISHT: Choice of Experimental Coditions- To choose among several types of incubation buffers, intact oocytes were homogenized, suspended, and incubated in the following media: 2 mM Hepes in artificial sea water, pH 8; Ca2+-free artificial sea water, pH 8; 50 mM Hepes, pH 8; 25 mM Tris-HC1, pH 7.4; 50 mM Tris-HC1 with or without 1 mM EDTA, pH 7.4 or 8; 150 mM Tris-HC1, pH 7.4. The best specific-to-nonspecific binding ratio was obtained when 50 mM Tris-HC1, pH 7.4 was used. This buffer was therefore chosen for further experiments.

The incubation of [3H]5HT with crude oocyte membrane preparations (E) was performed at two different temperatures: 25 “C and 30 “C. After 30 min of incubation, the resulting specific binding was not significantly different in these two

D

> W

m

s

5 - H T c o n c e n t r a t i o n (-log [MI)

FIG. 1. Vitelline envelope is necessary for full oocyte re- sponse to 6HT. Each batch of oocytes was separated into two halves: one half was used intact (+uitelline), while the other was treated with 1 M glycerol in 0.2 M phosphate buffer, pH 8, for vitelline envelope removal (-uitelline). In both experimental conditions, the 0.5% oo- cyte suspensions were incubated for 30 min at 18 “C with different concentrations of 5HT. Values represent the mean 2 S.E. for the responses of oocytes dissected out from four different females, ob- tained in two independent experiments.

Serotonin Receptors and Meiosis Reinitiation in Clam Oocytes 7985

experimental conditions. In subsequent experiments, the in- cubation was carried out at 25 “C.

Two different types of glass fiber filters (Whatman GF/C and GF/B) were presoaked for 2 h at room temperature in: (a) Tris-HC1 buffer; ( b ) 0.1% polyethyleneimine (PEI); (c) 0.5% PEI; or (d) 1% PEI. They were then tested for their capacity to separate E oocyte preparation-complexed [3H] - 5HT from free [3H]5HT. GF/B filters presoaked in PEI retained less free [3H]5HT than GF/B filters presoaked in Tris-HC1 buffer or GF/C filters presoaked in PEI. Moreover, GF/B filters presoaked in 0.1% PEI did not “bind” [3H]5HT “specifically.” In other words, in the absence of crude mem- brane preparations, the amount of [3H]5HT retained by filters was not different after incubation of samples containing either [3H]5HT alone or [3H]5HT in the presence of 100 p~ non- radioactive 5HT. We therefore chose to use GF/B filters presoaked for 2 h in 0.1% PEI in further experiments.

The [3H]5HT-specific binding represented only 20-30% of the total binding, when measured on crude membrane frac- tions prepared from entire (E) oocytes (Fig. 2). By contrast, after removal of vitelline envelope, the specific binding signal displayed by the crude membrane preparations (Mb) doubled (Fig. 2). [3H]5HT-specific binding could also be measured in the VE fractions, where it represented up to 99% of the total binding (Fig. 2). Biochemical analysis (Table I) showed the enrichment of plasma membrane- associated Na/K-ATPase activity in Mb fractions (2.6- to 4.9-fold range). However, VE fractions displayed a weak contamination (1.7- to 1.8-fold range) by this plasma membrane marker.

Transmission electron microscopic analysis further docu- mented the plasma membrane enrichment of Mb and weak

FIG. 2. Signal-to-noise ratio obtained for [%]5HT binding in different oocyte preparations. Each batch of oocytes was separated into two halves: one half was used to prepare crude mem- branes from entire oocytes ( E ) , while the other was processed as described under “Experimental Procedures” in order to obtain the membrane (Mb) and the vitelline envelope fractions (VE) . Three different batches of oocytes were processed in the same experiment (the [3H]5HT concentration used was 0.5 p ~ ) . The specific binding calculated as described under “Experimental Procedures” was ex- pressed as percentage of total binding. The reference 100% values for total binding were: for E, 5.76, 4.48, and 5.41 pmol/mg of protein; for Mb, 4.63, 3.01, and 3.76 pmol/mg of protein; for VE, 0.07, 0.09, and 0.08 pmol/mg of protein.

TABLE I Na/K-ATPase activity in Spisula oocyte preparations

Total Total ATPase Na/K-ATPase . ”

ATPase (A) + ouabain (B) (A - B) pmol PJmg @mol PJmg rmol PJmg proteinlh proteinlh proteinlh

Homogenate 2.60 f 0.20b 2.34 f 0.20 0.26 f 0.01 1 Mb fraction 7.77 f 0.98 6.71 f 0.77 1.37 f 0.96 3.96 f 0.65 VE fraction 2.74 f 0.06 2.27 f 0.04 0.47 -+ 0.02 1.75 f 0.02

F = enrichment factor, Represents mean f S.E. of three independent determinations,

contamination of VE fractions (Fig. 3, A and B). The kinetic and the pharmacological characterizations of

[3H]5HT binding were therefore performed on both the Mb and VE fractions. [3H]5HT-specific binding was dependent on protein content of oocyte preparations in both fractions. Assays were carried out under linear conditions, i.e. 40- to 80- pg protein equivalent in 100-p1 samples.

Kinetic Characteristics of 5HT Binding Sites-Binding of [3H]5HT to Mb sites was slightly more rapid than that to VE binding sites (Fig. 4, A and B) . The time for half-maximal association (tl/z) and KO, constant for Mb binding sites was, respectively, 7.4 f 0.2 min and 0.095 f 0.003 rnin”. For VE binding sites, t1I2 was 11.9 f 0.9 min with a corresponding KO, of 0.061 f 0.005 min”. A 20- and 30-min period was therefore chosen for incubation of the Mb and VE fractions, respec- tively.

The specific component of the [3H]5HT binding was satu- rable in both VE (Fig. 5A) and Mb fractions (Fig. 5B). Scatchard plots of direct binding data were linear and yielded Kd of 15 f 2 nM for VE preparations and of 0.23 k 0.08 p M for Mb preparations. Corresponding Bmax values for, respec- tively, the VE and the Mb preparations were estimated at 0.093 f 0.024 pmol/mg of protein and 2.5 f 0.9 pmol/mg of protein. The Hill coefficient was close to unity for both oocyte preparations: it was 0.95 k 0.01 and 1.02 f 0.02 for VE and Mb fractions, respectively.

Pharmacological Profile of 5HT Binding Sites-In both VE and Mb preparations, only 5HT and related compounds could inhibit the [3H]5HT-specific binding (Fig. 6, A, A’, B, and B’). All nonrelated monoaminergic compounds tested in a concentration range extending from 1 nM to 100 p M (acetyl- choline, haloperidol, carbachol, pyrilamine, isoproterenol, and prazosin) had no effect on [3H]5HT-specific binding.

The inhibition curves obtained for all efficient analogs (except mianserin in VE fractions, Fig. 6A) were monophasic and did not extend over more than 2 orders of magnitude. However, in both VE and Mb preparations, only 5HT was able to completely inhibit [3H]5HT-specific binding (Fig. 6, A and B). In contrast, one-third to one-half of the [3H]5HT- specific binding remained uninhibited in competition experi- ments performed with all other active analogs (Fig. 6, A, A’, B, and B’).

The rank order of potency of active analogs for inhibition of [3H]5HT-specific binding was determined by analysis of IC6o values (Table 11). In VE preparations, 5HT was the sole active agonist. In Mb preparations among agonists it was 8-

The rank order of potency for the antagonists was : MDL 72222 2 ICs 205930 = mianserin > imipramine > metoclo- pramide for the VE fractions, while it was metoclopramide 2 MDL 72222 2 ritanserin 2 propranolol 2 ICs 205930 z imipramine for the Mb preparations.

Comparisons between the efficiencies of active analogs to inhibit [3H]5HT-specific binding with their efficiencies to either trigger GVBD (for the agonists) or to inhibit 5HT- induced GVBD (for the antagonists), revealed the existence of a correlation (r = 0.574) only for the Mb fractions.

OH DPAT > 5HT > TFMPP.

DISCUSSION

In the present study we have described two novel types of 5HT-specific binding sites present in the plasma membrane and the vitelline coat of S. solidissima oocytes. The prepara- tion of oocytes for the present [3H]5HT assays was adapted from the protocols commonly used for the study of 5HT binding sites in mammalian tissues (22). In particular, oocyte homogenization was gentle enough to preserve membrane interactions of binding molecules with transduction systems

7986 Serotonin Receptors and Meiosis Reinitiation in Clam Oocytes

FIG. 3. Transmission electron mi- crographs of ultrathin sections of vitelline envelope ( A ) and plasma membrane ( B ) pellets from oocyte homogenates at the initial magnifi- cation of 8000. A , vitelline envelope fractions with glycoprotein matrix ( g ) , microvilli membrane vesicles (m), and yolk granules (y). B, typical plasma membrane vesicles. Note the homoge- neity of this fraction.

(22), and homogenates were centrifuged and decanted as for standard membrane preparations previously described for mammalian (22) and Drosophila (23) tissues. However, char- acterization of [3H]5HT binding sites became possible only after processing the oocyte surface and separating plasma membranes from the vitelline envelopes using the method of Maul (16). As previously reported by this author, the hyper- tonic glycerol caused a swelling of the fibrous layers of vitel- line envelope and its subsequent fragmentation.

[3H]5HT-specific signal-to-noise ratio was much higher in either the vitelline envelope or the plasma membrane frac- tions than in the entire oocyte surface fraction. In the last case, the specific binding ratio was too low to permit a valuable kinetic and pharmacological analysis. [3H]5HT binding meas- ured in Mb fractions was due to the presence of specific binding sites on oocyte plasma membranes as confirmed by our biochemical and ultrastructural data. In contrast, the precise ultrastructural location of VE fraction binding sites remains unknown. However, a weak Na/K-ATPase activity was apparently present in VE fractions, thus suggesting its contamination by plasma membrane fragments. Indeed, the hypertonic glycerol treatment triggers a pinching-off of the plasma membrane microvilli (which are an intrinsic part of vitelline envelope) at their bases (18). VE binding sites de-

scribed here might therefore be located in the microvilli subfraction of vitelline envelope; alternatively, they could be situated in its glycoprotein fibrous layer.

The kinetic analysis of the vitelline envelope and plasma membrane binding sites, based on monolinear Scatchard plots and Hill coefficients close to unity, revealed that both frac- tions contain a single class of putative 5HT receptors. How- ever, binding sites from the Mb and VE fractions appeared different according to their respective affinity and number: VE binding sites had a t least 10-fold better affinity but were about 25 times less numerous than Mb binding sites. Also, binding of [3H]5HT was slightly more rapid for the Mb than for the VE fraction sites.

VE and Mb binding sites share some pharmacological sim- ilarities. First, specificity of both VE and Mb fraction binding sites was restricted to 5HT and related compounds. Second, the inhibition curves obtained for the competition between active analogs and [3H]5HT were monophasic and did not extend over more than 2 orders of magnitude for both binding sites. However, all active analogs were at least 10 times more potent on VE fraction binding sites. Even more striking was the complete inefficiency of both 8-OH DPAT and metoclo- pramide on the latter binding sites. These different pharma- cological properties reveal the existence of two different bind-

Serotonin Receptors and Meiosis Reinitiation in Clam Oocytes

0.3 r 7987

0.3 r

A

1 0.1 1 m

t ,*'* f

a

T I M E ( m i n )

FIG. 4. Association kinetics of ['H]5HT to the VE fraction ( A ) and Mb fraction (B) binding sites. 0.5 PM ['H]5HT was incubated with either 54-pg (A) or 75-pg ( B ) protein equivalent of oocyte preparations for different time periods. Insets represent linear In (1 - B/Bmu) uersus time plots used for the determination of rate constants. Values represent the means of triplicate determinations (S.E. were less than 5%) obtained in one out of three different experiments in which similar results were obtained.

ing sites in VE and Mb fractions. The pharmacological profiles defined for both vitelline en-

velope and plasma membrane binding sites are qualitatively different from all those so far reported for mammalian and Drosophila 5HT receptors. For example, 5 H T 1 ~ receptors are sensitive to 5HT and 8-OH DPAT in nanomolar concentra- tion (8-OH DPAT itself is at least 10 times more potent than 5HT); they are also relatively sensitive to TFMPP. In our study, the vitelline envelope binding site was completely in- sensitive to 8-OH DPAT, while both the vitelline envelope and the plasma membrane binding sites were completely insensitive to TFMPP. In addition, binding sites from both fractions were sensitive to 5HT only in micromolar concen- tration. Therefore, neither the VE nor the Mb fraction bind- ing sites belong to the 5HT1* type. These binding sites cannot be classified as: ( a ) 5HT1~, given that, according to this profile, 5HT and TFMPP are equipotent; ( b ) 5HTIc because these sites are equally sensitive to prazosin and haloperidol; (c) 5HTlo as this receptor subtype interacts with 5HT in nanomolar concentration. The binding sites that we charac- terized in VE and Mb fractions did not display any of these properties.

Similarly, the binding sites studied are pharmacologically distinct from 5HTz, 5HT3, and 5HT4 receptors. For example, high and low affinity forms of the 5HTz receptor are equally sensitive to either TFMPP or 8-OH DPAT. In our study, 8- OH DPAT had no effect on [3H]5HT binding to VE sites, while TFMPP was inefficient on both VE and Mb binding sites. 5HT3 receptors are at least 10 times more sensitive to 8-OH DPAT than to metoclopramide. We found that 8-OH

/. /' 0 0.2 0.4 0.6 0.0 I .o

3 H - 5 H T c o n c e n t r a t i o n ( p M )

FIG. 5. Saturation of ['H]5HT-specific binding by increas- ing concentration of free ['HISHT in VE ( A ) and Mb ( B ) preparations. Insets represent the Scatchard linearization of spe- cific binding data. Each value is the mean of triplicate determinations (S.E. were less than 5%) from one representative experiment.

DPAT and metoclopramide were inefficient on VE fraction binding sites, while they were equipotent on Mb fraction binding sites. Finally, 5HT4 receptors are insensitive to MDL 72222. In our model system, both VE and Mb binding sites were extremely sensitive to this antagonist. So, the pharma- cological profiles described for either the vitelline envelope or the plasma membrane binding sites do not match any of those described for the mammalian 5HT receptors.

The pharmacological profiles of either of the Spisula 5HT binding sites do not match any of those described for Dro- sophila. For example, according to the 5HT-drol, 5HT-drozA, and 5HT-droz~ profiles, prazosin should be relatively efficient. In addition, 8-OH DPAT is a weak agonist on the Drosophila 5HT receptors: the IC5o values reported for the subtypes of 5HT-droz receptors ranged from 10 to 100 p ~ , while it was of 200 pM for the 5HT-drol receptor (14). In our experimental conditions, both prazosin and 8-OH DPAT were inefficient on the vitelline envelope binding sites; on plasma membrane binding sites, prazosin was inefficient, while 8-OH DPAT was the most potent competitor.

Taken altogether, our data demonstrate the presence of two novel types of 5HT binding sites in, respectively, vitelline envelope and plasma membrane fractions prepared from S. solidissima oocytes. Moreover, for the reasons discussed above, none of these two Spisula oocyte binding sites can belong either to the 5 H T I ~ or the 5HT3 types, in contrast to a previous statement (24).

The divergence between the 5HT binding sites described herein and the 5HT-dro receptors involves, in addition to the

Serotonin Receptors and Meiosis Reinitiation in Clam Oocytes

FIG. 6. Competition of various BHT-related compounds for ['HI SHT-specific binding in VE (A and A') and Mb ( B and B') oocyte frac- tions. [3H]5HT concentration was 20 f 4 nM and 0.21 f 0.03 p~ for A and B, respectively. Each value corresponds to the mean f S.E. of three independent experiments, each done in triplicate.

m I I 100 -

10 c 0

$ 60 -

20 -

B

Amianser in o m e l o c l o p r o m i d e

0 9 6 7 6 5 4

D r u g concentration (-log [MI)

I A'

1, o p r o p r a n o l o l * M D L 7 2 2 2 2

1 I

0 9 0 7 6 5 4

D r u g concentration (-log [MI)

TABLE I1 Comparison between the efficiencies of various 5HT-related compounds to induce (for agonists) or inhibit (for anti-agonists)

the meiosis reinitiation and their efficiencies to inhibit r H I 5 H T specific binding Meiosis reinitiation response was quantified by random scoring of GVBD; these data are taken from our previous paper (15).

Compound

5HT

Ritanserin Mianserin Metoclopramide ICs 205930

TFMPP Propranolol

MDL 72222 Imimamine

8-OH DPAT

GVBD

ECwIICm

P M

0.55 f 0.18 111-6

0.41 f 0.10 3.60 f 0.28

12 f 1 1.63 f 0.22 3.33 f 0.72

>loo >loo ND

E - I L %

100 91 f 5 95 f 5 97 f 2 77 f 11 77 f 11 79 f 13 9 f 3 ND ND

VE fractions

ICW" I,:

P M % 0.91 f 0.06 100

>loo 9 f 1 ND' ND

0.07 f 0.01 71 1- 6 >loo 16 f 1

0.05 f 0.01 60 f 2 ND ND >loo ND

0.02 f 0.001 56 f 1 0.45 f 0.24 68 f 10

Mb fractions

ICW" L b

P M % 0.52 f 0.11 100 0.05 f 0.01 67 t 1 0.42 f 0.16 55 -t 8 0.45 f 0.14 57 f 5 0.06 f 0.01 63 f 8 3.05 f 1.45 57 f 5 1.32 f 0.49

>loo 47 f 4 12 f 5

0.13 f 0.05 61 f 11 4.2 f 0.7 49 1- 9

a Calculated by linear regression transformation of the data plotted in Fig. 6. *Represents the difference 100 (%) - X (%), where X equals percentage of [3H]5HT specific binding at maximal analog concentration

e ND, not determined. tested (100 pM).

originality of their pharmacological profiles, significant dif- ferences in their homology with known mammalian 5HT receptors. Indeed, it has recently been reported that all three 5HT-dro receptors appear much closer to the mammalian 5HTlA than to the 5HT2 receptors (14). Based on this obser- vation, the authors postulated that the "5HTIA-like" and "5HTz-like" families of receptors diverged early in evolution (ie. before the separation betweeen vertebrates and inverte- brates). In contrast, the 5HT binding sites that we described in the plasma membrane fraction share some pharmacological properties of both 5HT1~ (8-OH DPAT is at least 10 times more potent than 5HT as required according to 5 H T 1 ~ phar- macology) and 5HT2 (ritanserin and mianserin are equipotent

as required for a 5HT2 profile) mammalian receptors. There- fore, based on our pharmacological data, it seems reasonable to assume that the Spisuln oocyte plasma membrane 5HT binding sites appeared very early in evolution and are prob- ably a common "ancestor" of the 5HTIA and 5HT2-like fami- lies of 5HT receptors. However, this hypothesis needs further confirmation from molecular cloning of the Spisula membrane binding sites. In contrast, although they possess some char- acteristics required for a 5HTz profile, the vitelline envelope 5HT binding sites have no common properties with the mam- malian 5 H T 1 ~ receptor. Interestingly, according to their high sensitivity to MDL 12222, the binding sites of both VE and Mb fractions appear to be similar to the 5HT3 mammalian

Serotonin Receptors and Meiosis Reinitiation in Clam Oocytes 7989

receptors. It thus remains to be elucidated whether both the plasma membrane and the vitelline envelope 5HT binding sites originate from one common ancestor binding site which was equally sensitive to the compounds, respectively selective for either 5HT2 or 5HT3 types of mammalian serotonin recep- tors. According to such a hypothesis, 5HT binding sites of the plasma membrane should have evolved from this ancestor by acquisition of the 5HTIA-like characteristics.

Our biochemical data raised the question about the respec- tive physiological roles of the 5HT binding sites described in vitelline envelope and plasma membrane of Spisula oocytes. The answer appears clear for the membrane binding sites. Indeed, the pharmacological profile of these binding sites, as described herein, was almost identical with what we previ- ously reported for the 5HT-induced meiosis reinitiation proc- ess (15); the efficiencies of most compounds tested upon GVBD (taken from our previous study, see Ref. 15) correlated well with their efficiencies to inhibit the [3H]5HT-specific binding. Moreover, the K d value of 5HT binding sites in Spisula oocyte plasma membrane estimated as 0.23 f 0.08 FM is a value consistent with 5HT concentration that is effective in inducing oocyte maturation in this species (15; present study). In addition, the half-association time of 7.4 f 0.2 min that we determined for these binding sites is consistent with the time necessary to produce 50% of 5HT-dependent GVBD (15). Taken altogether, these striking similarities betweeen the [3H]5HT binding kinetics and the biological response to 5HT argue for the physiological involvement of the 5HT plasma membrane binding sites in transduction of 5HT ac- tions on Spisula oocytes. Therefore, these plasma membrane binding sites fulfill the criteria of Bradley's classification of 5HT receptors (25) and can be called 5HT6 type.

In contrast, the physiological role of the vitelline envelope 5HT binding sites is less obvious. The fact that oocytes devoid of vitelline envelope can still respond to 5HT and the absence of correlation between the efficiencies of all the active analogs to inhibit [3H]5HT-specific binding in the VE fractions and their efficiencies to elicit GVBD (see Ref. 15) would suggest that the 5HT binding sites of the vitelline envelope do not participate in transduction of the 5HT signal. Such an as- sumption is further supported by the inconsistency between the half-time of association of these binding sites (t lIz = 11.9 +. 0.9 min) and the time of GVBD occurrence after 5HT application (8-12 min) as well as the discrepancy existing between the nanomolar affinity of the VE binding sites for 5HT and the micromolar concentration of 5HT necessary to induce GVBD. However, oocytes deprived of their vitelline envelope could respond to the same extent as intact oocytes only in the presence of, at least, a 10-fold higher concentration of the neurohormone. This latter result would suggest that, although the vitelline envelope 5HT binding sites are not necessarily and not directly involved in the transduction of 5HT effects on meiosis reinitiation, they may play some accessory role susceptible to facilitate the physiological re- sponse of the Spisula oocyte to 5HT. It is reasonable to assume that the VE binding sites could be able to "capture" 5HT from the surrounding medium, due to their higher affinity for 5HT, and thus to concentrate this neurohormone around its plasma membrane receptors. The existence of analogous bind- ing protein-dependent systems, which are localized in the

outer membrane of bacteria and play a role in the "concentra- tion" of sugars from the medium, has already been proposed (26). Dealing with such a hypothetical role for the vitelline envelope 5HT binding sites, it might be relevant that imip- ramine, a nonselective blocker of the mammalian 5HT mem- brane binding-transporter systems, is at least 10 times more efficient on the VE than on the Mb fraction binding sites. Similarly, the cooperation between low and high affinity binding sites specific for nerve growth factor are believed to facilitate the expression of full biological response to nerve growth factor (27).

In conclusion, in the present study, we reported the exist- ence of two distinct 5HT binding sites in the oocytes of s. solidissima. These two binding sites display different localiza- tion, different kinetic characteristics, and different pharma- cological profiles. A functional correlate could be established only for binding sites of plasma membrane. We propose therefore that this new class of 5HT-specific binding sites be called 5HTs receptors. It remains to be confirmed whether vitelline envelope binding sites play a physiological role in 5HT-induced meiosis reinitiation by facilitating 5HT5 recep- tor activation.

Acknowledgments-We thank Drs. RLmi Quirion and Emmanuel Moyse for stimulating discussions and Daniel Lajeunesse for useful suggestions. We acknowledge the assistance of Pierre Rivailler and Marie Leclerc in Na/K-ATPase activity assays and of Jean DBsilets, Karin Julliard, and Emmanuel Moyse in transmission electron mi- croscopy analysis. We are also indebted to Marie Antoinette Maillard and Renee Merdy for editorial assistance, to Johanne Noel for pre- paring the figures, and to Glynis Thoiron for English revision.

REFERENCES 1. Aprison, M. H., and Ferster, C. B. (1960) Experientio 16,159-160 2. Lucki, I., Nobler, M. S., and Frazer, A. (1984) J. Phurmacol. Exp. Ther.

3. Quirion, R., Martel, J. C., Robitaille, Y., Etienne, P., Wood, P., Nair, N. P.

4. Eskin, A., and Takahashi, J. S. (1983) Science 220.82-84

6. Bicker, G., and Menzel, R. R (1989) Nature 337,33-39 5. Kravitz, E. A. (1988) Science 2 4 1 , 1775-1781

8. Matsutani, T., and Nomura, T. (1987) Gen. Comp. Endocrinol. 67, 111- 7. Gibbons, M. C., and Castagna, M. (1984) Aquaculture 40, 189-191

2 2 8 , 133-139

V., and Gauthier, S. (1986) Can. J. Neurol. Sci. 13 , 503-510

l l Q LA"

9. Hirai, S., Kishimoto, T., Koide, S., and Kanatani, H. (1988) J. Exp. Zool.

10. Osanai, K., and Kuraishi, R. (1988) Bull. Mar. Biol. Stn. Asamushi 18,45-

11. Peroutka, S. J. (1988) Trends Neurosci. 11, 496-500 12. Bockaert, J., Sebben, M., and Dumnius, A. (1990) Mol. Phurmacol. 37 ,

246,318-329

56

408-411 13. Witzi P., Amlaiky, N., Plassat, J. L., Maroteaux, L., Borelli, E., and Hen,

14. Saudou, F., Boschert, U., Amlaiky, N., Plassat, J. L., and Hen, R. (1992)

15. Krantic, S., Dube, F., Quirion, R., and Guerrier, P. (1991) Deu. Biol. 146 ,

R. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,8940-8944

EMBO J. 11 , 7-17

AOl-AQQ 16. 17. 18. 19.

20.

21. 22.

24. 23.

25.

26.

27.

Maul, G. G. (1980) Ex Cell Res. 129,431-438 Post, R. L., and Sen, k? K. (1967) Methods Enzymol. 10 , 762-768

Fillion, G., Fillion, M. P., Spirakis, C., Bahers, J. M., and Jacob, J. (1976) Rebhun, L. I., and Sharpless, T. K. (1964) J. Cell Biol. 22,488-491

Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J.

Mc Pherson, G. A. (1983) Comput. Prog. Biomed. 17,107-114 Sanders-Bush, E. (1988) The Serotonin Receptors, Humana Press Inc.,

-"A ."

Life Sci. 18, 65-74

Biol. Chem. 193,265-275

Cliftnn N.1 Amlaiky, N., and Caron, M. G. (1985) J. Biol. Chem. 260,1983-1986 Bandivdekar, A. H., Segal, S. J., and Koide, S. S. (1991) Inuertebr. Reprod.

Deu. 19 , 147-150 Bradley, P. B., Engel, G., Feniuk, W., Fozard, J. R., Hum hrey, P. P. A,,

Middlemeiss, P. N., Mylecharne, E. J., Richardson, B. g., and Saxena,

Dudler, R. Schmidhauser C., Parish, R. W., Wettenhall, R. E. H., and P. (1986) Neorophnrmolagy 25,563-576

Bothwell, M. (1991) Cell 65,915-918 Schmidt: T. (1988) EMEO J. 7,3963-3970

"_ _"", - .-