Estrogen binding sites in the sea scallop: Characterization and possibleinvolvement in reproductive regulation
Chunde Wang a,b,⁎, Roger P. Croll a
a Department of Physiology and Biophysics Dalhousie University Halifax, Nova Scotia, Canada B3H 4H7b Department of Aquatic Biology and Aquaculture, Faculty of Fisheries, Qingdao Agricultural University, Qingdao, Shandong, 266109, China
Received 21 February 2007; received in revised form 25 June 2007; accepted 26 June 2007
Vertebrate type sex steroids have been widely identified inbivalves such as the scallops Pecten hericius (Boticelli et al.,1961), Pecten maximus (Saliot and Barbier, 1971), and Patino-pecten yessoensis (Matsumoto et al., 1997), the mussel Mytilusedulis (Reis-Henriques et al., 1990), and the oyster Crassostreagigas (Matsumoto et al., 1997). The presence of enzymes,precursors and metabolic products indicated that these steroidsare synthesized endogenously (Lehoux and Sandor, 1970; DeLongcamp et al., 1974; Matsumoto et al., 1997). Furthermore,these sex steroids have been found to have physiological effects inbivalves, specifically playing a hormonal role in the regulation ofreproductive processes, as found in vertebrates (for reviews, seeMcEwen, 1978; Gorski, 1979; Dixson, 2001; Croll and Wang, inpress). One of the primary functions seems to be involved ingonadal development and sexual maturation during the reproduc-
⁎ Corresponding author. Department of Aquatic Biology and Aquaculture,Faculty of Fisheries, Qingdao Agricultural University, Qingdao, Shandong,266109, China.
Please cite this article as: Wang, C., Croll, R.P. Estrogen binding sites in the sea sComp. Biochem. Physiol. B (2007), doi:10.1016/j.cbpb.2007.06.008
tive cycles. Injections of estradiol, testosterone and progesteronehave been shown to stimulate oogenesis and spermatogenesis inthe scallop Mizuhopecten yessoensis (Varaksina and Varaksin,1991; Varaksina et al. 1992). Additionally, Wang and Croll (2004)have shown that sex steroids accelerate gonadal development andshift sex ratios as well as cause morphological changes in juvenilescallops. Estradiol has also been reported to induce synthesis ofvitellin, which is one of the major events during gonadaldevelopment, in P. yessoensis and C. gigas (Li et al., 1998;Osada et al., 2003, 2004). The profiles of sex steroids, such asestradiol in C. gigas, P. yessoensis andMya arenaria (Matsumotoet al., 1997; Gauthier-Clerc et al., 2006), progesterone inM. edulisandM. arenaria (Reis-Henriques and Coimbra, 1990; Siah et al.,2002), and testosterone in M. arenaria (Gauthier-Clerc et al.,2006), correlate well with gonadal development and maturation,suggesting possible modulatory roles in these processes. Otherlines of evidence indicate that sex steroids also have stimulatoryeffects on gamete release and spawning. Estradiol potentiated invitro gamete release induced by serotonin (5-HT) in the JapanesescallopP. yessoensis and the sea scallopPlacopectenmagellanicus(Osada et al., 1992; Wang and Croll, 2003). Subsequent in vivostudies have demonstrated that sex steroids have both acute effects
callop: Characterization and possible involvement in reproductive regulation.
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on direct spawning induction and long-term effects on subsequent5-HT-induced spawning (Wang and Croll, 2006).
In vertebrates, most of the actions of steroids are mediatedthrough binding to intracellular steroid receptors, which in turninitiates transcription and synthesis of target genes (for review,see Björnström and Sjöberg, 2005). In invertebrates, however,there have been conflicting lines of evidence concerning theexistence of steroid receptors. Some existing evidence supportsthe presence of steroid receptors in invertebrates, includingmolluscs. For example, in exploring the underlying mechanismsresponsible for the actions of estradiol on 5-HT-induced gameterelease, Wang and Croll (2003) have found that the stimulatingeffects of estradiol can be blocked by tamoxifen, an estrogenreceptor antagonist, as well as by actinomycin D, which is anRNA synthesis inhibitor, and cycloheximide, a proteinsynthesis blocker (Nicol and Goldberg, 1976). Similar resultshave also been reported by Osada et al. (1992) whereactinomycin D blocked the potentiating effects of estradiol on5-HT-induced egg release in P. yessoensis. In fact, estrogenbinding sites have been detected in female soft-shell clams,M. arenaria and Octopus vulgaris (D'Aniello et al., 1996; DiCosmo et al., 2002; Gagne et al., 2003). Despite the aboveevidence, recent molecular cloning of estrogen receptororthologs in gastropods (Thornton et al., 2003, Kajiwaraet al., 2006) and a cephalopod (Keay et al., 2006) raised ques-tions about the nature of estrogen receptors in molluscs. In eachcase, these estrogen receptor orthologs surprisingly do not bindestrogen but instead appear to be constitutively active.
To help clarify these issues, the present study aims tocharacterize these binding sites which might represent estrogenreceptors, and to examine their possible involvement in theregulation of reproduction in sea scallops.
2. Materials and methods
2.1. Animals and chemicals
Sea scallops, P. magellanicus, were obtained from the GreatMaritime Scallop Trading Co. (Chester, NS, Canada). Thescallops were kept in tanks with running natural seawatermaintained at 14–16 °C in a wet lab of the Aquatron Facility ofDalhousie University.
All the steroids used in these studies, including 17β-estradiol, testosterone, and progesterone, were purchased fromSigma Chemical Co. (Mississauga, ON, Canada). Isotope [3H]estradiol (143 Ci/mmol, 250 mCi) was purchased fromAmersham Pharmacia Biotech Inc (Piscataway, NJ, USA). Allother chemicals were also purchased from Sigma Chemical Co.
2.2. Preparation of cell extracts
The protocol for the preparation of cell extracts was adaptedwith modifications from d'Istria et al. (1991) and Cuevas andCallard (1992). Briefly, mature scallops were dissected in coldsea water (4 °C) under a dissecting microscope. The gonadswere removed and minced with razor blades in Petri dishescontaining cold homogenization buffer (40 mM Tris-HCl, pH
Please cite this article as: Wang, C., Croll, R.P. Estrogen binding sites in the sea sComp. Biochem. Physiol. B (2007), doi:10.1016/j.cbpb.2007.06.008
7.4, 1 mM EDTA, 1 mM 2-mercaptoethanol, 30% glycerol).They were then washed twice by mixing with cold homoge-nization buffer followed by centrifugation to remove haemo-lymph and suspended in the same buffer. The minced tissueswere homogenized using 3 strokes of an Ultra-Turrax-AntriebT25 homogenizer (Staufen, Germany) at high speed withcooling on ice between pulses. The homogenate was thencentrifuged at 1000 g at 4 °C for 30 min and the supernatant wasset aside. In another tube, an equal volume of 0.5% dextran-coated charcoal solution (DCC, 0.5% Norit-A Charcoal, 0.05%dextran T-70) to that of the homogenate was centrifuged for10 min at 4 °C. The supernatant of the homogenate was thenmixed with the pellet of the DCC solution and incubated for30 min at 4 °C. The mixture was again spun at 1000 g for 10 minto collect the supernatant. The supernatant was then ultracen-trifuged at 100,000 g at 4 °C for 60 min. This derivedsupernatant is classically referred to as the “cytosolic” fraction(d'Istria et al., 1991; Cuevas and Callard, 1992).
The pellet from the first centrifugation of the homogenateabove was rinsed with washing buffer (10 mM Tris-HCl, 3 mMMgCl2, 0.25 mM sucrose) for three times. After the last wash,the pellet was resuspended and incubated in extraction buffer(0.6 M KCl in homogenization buffer) for 60 min at 4 °C withrotation. The supernatant was then treated with DCC asdescribed above and ultracentrifuged at 100,000 g for 60 minat 4 °C. This second derived supernatant is classically referredto as the “nuclear” fraction (d'Istria et al., 1991; Cuevas andCallard, 1992).
The protein contents of the cell extracts were measuredaccording to the procedures of Bradford (1976). All the extractswere diluted to a protein content of 1 mg/mL using homogeni-zation buffer for cytosolic fractions or extraction buffer fornuclear fractions.
2.3. Saturation binding assays
Varying amounts of radiolabeled ligand, alone or togetherwith a 500-fold excess concentration of radio-inert DES inethanol, were added to glass tubes so that after addition of cellextracts, the concentrations of radiolabeled ligand were 1, 2, 5,7.5, 10, 15 and 20 nM, respectively. Three repeats from thesame cell extract were set for each concentration. After steroidswere air dried, 200 μL of cell extract were added to the tubes,resuspended with steroids, and incubated at 4 °C for 24 h withagitation. After incubation, 200 μL of cold 0.5% DCC solutionwere added into each tube and incubated for 30 min on ice,followed by centrifugation at 1000 g for 15 min to remove freesteroids. A 200 μL aliquot of supernatant was taken to a MiniPoly-Q vial (Beckman, Somer et, NJ, USA) containing 5 mL ofEcolite (+) liquid scintillation cocktail (ICN Biomedicals, Inc.,Costa Mesa, CA, USA) and counted on a 1215 RackBeta IIliquid scintillation counter (LKB Wallac, Turku, Finland) for2 min. Cytosolic and nuclear fractions from both males andfemales were assayed.
The counts of the sample tubes containing only radiolabeledsteroids were measured as “total binding”, whereas thosecontaining both radiolabeled and an excess concentration of
callop: Characterization and possible involvement in reproductive regulation.
Fig. 1. Saturation analysis of [3H] estradiol binding sites in the cytosolic fractionof female scallop gonads (A) and Scatchard plot of the data (B). 200 μl ofcytosolic fraction were incubated with 0.5–20 nM [3H] estradiol in the presenceor absence of a 500 fold excess concentration of DES at 4 °C for 24 h. Boundsteroids were separated from free by DCC treatment. Graphic analysis ofmultiple binding sites was performed according to Rosenthal (1967). Datarepresent mean±SE (n=3).
Fig. 2. Saturation analysis of [3H] estradiol binding sites in the nuclear fractionof female scallop gonads (A) and Scatchard plot of the data (B). 200 μl ofcytosolic fraction were incubated with 0.5–20 nM [3H] estradiol in the presenceor absence of a 500 fold excess concentration of DES at 4 °C for 24 h. Boundsteroids were separated from free by DCC treatment. Graphic analysis ofmultiple binding sites was performed according to Rosenthal (1967). Datarepresent mean±SE (n=3).
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unlabeled steroids were referred to as “nonspecific binding”.The specific binding was the difference between the twomeasures.
Analyses of the binding results were performed according toScatchard (1949). The Scatchard plots were performed byplotting the ratios of bound to free radiolabeled ligand againstthe amounts of bound radioligand. Non-linear regression of thedata was made using SigmaPlot 5.0 (Jandel Scientific Inc.,Chicago, IL, USA). Multiple binding sites were resolvedmanually according to the methods described by Rosenthal(1967). Briefly, two straight lines were found so that the vectorialsum of the points on the lines equaled to the vectorial length ofthe corresponding point on the Scatchard plot. The dissociationconstants (Kd) were the slopes of the lines and the maximumbinding capacity (Bmax) normalized with the protein concentra-tion of cell extract equaled the x-intercepts of the lines.
2.4. Competitive binding assays
Aliquots of 200 μL of cell extracts were incubated with10 nM [3H] estradiol in the absence or presence of competitors
Please cite this article as: Wang, C., Croll, R.P. Estrogen binding sites in the sea sComp. Biochem. Physiol. B (2007), doi:10.1016/j.cbpb.2007.06.008
at increasing concentrations at 4 °C for 24 h with agitation.Competitors included 10–105 nM of unlabeled estradiol,diethylstilbestrol (DES), progesterone and testosterone. Threerepeats were set for each concentration of each competitor. Afterincubation, the samples were treated with DCC solution andtheir radioactivities were counted as described above.
Binding of [3H] estradiol in the absence of the competitorswas referred to as total binding while that in the presence ofDES at the highest concentration (105 nM) was considered asnon-specific baseline binding. Thus, specific binding in thepresence of the competitor was expressed as percentage of totalbinding after the non-specific baseline was subtracted. That is:
Specific bindingk
¼Binding with competitorð Þ�Non‐specific bindingTotal binding� Non‐specific binding
�100k
2.5. Variations of binding capacity with sexual maturation
Scallops at different stages of sexual maturation weredissected separately and their gonadosomatic indices (GSI)
callop: Characterization and possible involvement in reproductive regulation.
Fig. 3. Saturation analysis of [3H] estradiol binding sites in the cytosolic fractionof male scallop gonads (A) and Scatchard plot of the data (B). 200 μl ofcytosolic fraction were incubated with 0.5–20 nM [3H] estradiol in the presenceor absence of a 500 fold excess concentration of DES at 4 °C for 24 h. Boundsteroids were separated from free by DCC treatment. Graphic analysis ofmultiple binding sites was performed according to Rosenthal (1967). Datarepresent mean±SE (n=3).
Fig. 4. Saturation analysis of [3H] estradiol binding sites in the nuclear fractionof male scallop gonads (A) and Scatchard plot of the data (B). 200 μl ofcytosolic fraction were incubated with 0.5–20 nM [3H] estradiol in the presenceor absence of a 500 fold excess concentration of DES at 4 °C for 24 h. Boundsteroids were separated from free by DCC treatment. Graphic analysis ofmultiple binding sites was performed according to Rosenthal (1967). Datarepresent mean±SE (n=3).
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were measured according to Barber and Blake (1991). Thecytosolic and nuclear fractions of the gonads were preparedaccording to the procedures described above. These cell extractswere diluted to a protein concentration of 1 mg protein/mLextract and stored at −74 °C until they were assayed.
Aliquots of 200 μL of cell extracts were incubated with10 nM [3H] estradiol in the absence or presence of a 500×excess concentration of radio-inert DES at 4 °C for 24 h. Afterincubation, the samples were treated with DCC solution andcounted as described above. The total specific binding capacityis the difference between the number of counts per minute(CPM) per microgram of protein in the absence of excess DESand that in the presence of excess DES.
In the first set of experiments, comparisons of total specificbinding capacity in both cytosolic and nuclear fractions weremade between mature (gonads with mature oocytes, GSI=16.9±1.0, n=6) and spent (transparent gonads, with few visibleoocytes, GSI=5.2±0.9, n=6) female scallops.
In another set of experiments, total specific binding in bothcytosolic and nuclear extracts of gonads from scallops atdifferent stages of sexual maturation in both sexes (females withGSI ranging from 3.0 to 22.2, n=15; males with GSI ranging
Please cite this article as: Wang, C., Croll, R.P. Estrogen binding sites in the sea sComp. Biochem. Physiol. B (2007), doi:10.1016/j.cbpb.2007.06.008
from 3.4 to 21.8, n=15) were measured. The specific bindingsof cytosolic and nuclear fractions were compared to obtain therelative binding capacity according to the following formula:
Relative Binding Capacity
¼ Specific binding of cytosolic fractionSpecific binding of nuclear fraction
The relative binding capacity was then plotted against GSI toexamine the variations in the distribution of estrogen bindingsites between the cytosolic and nuclear fraction with sexualmaturation.
3. Results
3.1. Saturation analyses
Females: The existence of estrogen binding sites wasdemonstrated in both cytosolic and nuclear fractions of femalegonad by saturation analysis. As seen from Fig. 1A, the bindingcurve for the cytosolic fraction appeared to consist of twocomponents — one saturated between 5.0–7.5 nM and another
callop: Characterization and possible involvement in reproductive regulation.
Fig. 5. Competitive analysis of binding of [3H] estradiol to the cytosolic (A) andnuclear fractions (B) of female scallop gonads. 200 μl of cell extracts wereincubated with increasing concentrations (10–105 nM) of competitors and 10nM [3H] estradiol at 4 °C for 24 h. Bound steroids were separated from free byDCC treatment. Data were expressed as percentages of specific binding in theabsence of competitor (denoted as Total). Data represent mean±SE (n=3). DES,diethylstilbestrol; E2, estradiol; P, progesterone; T, testosterone.
Fig. 6. Competitive analysis of binding of [3H] estradiol to the cytosolic (A) andnuclear fractions (B) of male scallop gonads. 200 μl of cell extracts wereincubated with increasing concentrations (10–105 nM) of competitors and 10nM [3H] estradiol at 4 °C for 24 h. Bound steroids were separated from free byDCC treatment. Data were expressed as percentages of specific binding in theabsence of competitor (denoted as Total). Data represent mean±SE (n=3). DES,diethylstilbestrol; E2, estradiol; P, progesterone; T, testosterone.
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not saturated below 20 nM. Scatchard analysis revealed twopossible sites: one high-affinity site with limited capacity and onelow-affinity site with high capacity. The dissociation constant(Kd) for the high affinity site was 0.52 nM and its maximumbinding capacity (Bmax) was 62.57 fmol/mg protein (Fig. 1B). Thelow affinity site was not characterized because higher concentra-tions of radiolabeled estradiol were needed in order to do so.
Fig. 2 represents the data for saturation analysis of bindingsites in the nuclear fraction of female gonad. The binding curveindicated the existence of two sites: one was saturated at around2 nM and another was not saturated below 20 nM, as shown inFig. 2A. Scatchard analysis of the data revealed a high affinitybinding site with a Kd of 1.71 nM and a Bmax of 174.3 fmol/mgprotein, and a low affinity binding site with a Kd of 31.84 nMand a Bmax of 1.12 pmol/mg protein (Fig. 2B).
Males: Saturation analysis of the binding sites in thecytosolic fraction of male gonad showed a similar pattern tothat of female gonad (Fig. 3A).
Like in the female gonad cytosol, the binding curve for themale gonadal cytosolic fraction appeared to include two
Please cite this article as: Wang, C., Croll, R.P. Estrogen binding sites in the sea sComp. Biochem. Physiol. B (2007), doi:10.1016/j.cbpb.2007.06.008
components: the first was saturated at around 5 nM and thesecond between 15 nM and 20 nM. Scatchard analysis indicatedtwo binding sites: a high affinity one with a Kd of 0.57 nM and aBmax of 38.3 fmol/mg protein, and a low affinity high capacitysite, the accurate characterization of which required higherconcentrations of radiolabeled estradiol (Fig. 3B).
A similar binding curve to that for female gonad nuclearfraction was also obtained in the nuclear fraction of the malegonad. As shown in Fig. 4A, the first site was saturated ataround 7.5 nM and the second not saturated up to 20 nM.Scatchard analysis also demonstrated the presence of twobinding sites. The dissociation constant for the high affinity sitewas 1.82 nM and its Bmax was 63.97 fmol/mg protein. The lowaffinity site had a Kd of 25.51 nM and a Bmax of 0.293 pmol/mgprotein (Fig. 4B).
3.2. Competitive analyses
Females: Incubation of the female gonad cytosol fractionswith 10 nM of [3H] estradiol alone or in the presence of
callop: Characterization and possible involvement in reproductive regulation.
Fig. 8. Variation of relative specific binding capacity (cytosol/nuclear) withsexual maturation in female (A) and male (B) scallops. Cytosolic and nuclearfractions made from female and male scallop gonads of varying stages of sexualmaturation were incubated with 10 nM of [3H] estradiol in the absence orpresence of 500-fold excess concentration of DES for 24 h at 4 °C. Boundsteroids were separated from free by DCC treatment.
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increasing concentrations of competitors showed that at lowconcentrations (10–103 nM), DES and estradiol, but notprogesterone and testosterone, effectively diminished thebinding of [3H] estradiol (Fig. 5A). Based on their ability toinhibit 50% of total binding, DES had about a 3-fold higheraffinity while progesterone and testosterone had 15- and 30-foldlower affinities than estradiol in competing for the binding sites.
Competitive analysis in female gonad nuclear fractionsrevealed a similar tendency as in the female gonad cytosolicfraction (Fig. 5B). DES and estradiol were good competitors forthe binding sites while progesterone and testosterone were weakin competition for the binding sites at the concentration range of10–103 nM. DES was 10-fold more competent than estradiol tocompete for the binding sites. Progesterone and testosteronewere 80- and 110-fold less competitive than estradiol. At thehighest concentration (105 nM), all four steroids reduced the[3H] estradiol binding drastically.
Males: The competitive curves of the male gonad cytosolicfraction were quite similar to those of the female preparations(Fig. 6A). With increasing concentrations, DES and estradioleffectively decreased [3H] estradiol binding. Progesterone andtestosterone, in contrast, did not compete well for the bindingsites at low concentrations (10–104 nM). At the highestconcentration (105 nM), progesterone and testosterone alsodrastically blocked the total binding. Based on the concentrationof the competitors at which 50% total binding was blocked,DES is 5-fold more competitive than estradiol, and progesteroneand testosterone were 30- and 44-fold less competent thanestradiol.
Similarly, in the nuclear fraction of the male gonad, DES andestradiol competed well with [3H] estradiol for the binding sites(Fig. 6B). Progesterone and testosterone were less effective inblocking the total binding. DES had a 2-fold higher affinity forthe estradiol binding sites than estradiol itself. Progesterone and
Fig. 7. Comparison of binding capacities of cytosolic and nuclear fractions inspent and ripe females. Cytosolic and nuclear fractions made from mature orspent female scallops were incubated with 10 nM of [3H] estradiol in the absenceor presence of 500-fold excess concentration of DES for 24 h at 4 °C. Boundsteroids were separated from free by DCC treatment. Values represent mean±SE(n=6). ⁎⁎Pb0.01, n=6, Student's t-test.
Please cite this article as: Wang, C., Croll, R.P. Estrogen binding sites in the sea sComp. Biochem. Physiol. B (2007), doi:10.1016/j.cbpb.2007.06.008
testosterone were 18- and 45-fold less competent than estradiolin competing for the estradiol binding sites.
In general, DES was about 2–10 times more competitivethan estradiol in binding to estrogen binding sites, progesteroneshowed 20–80-fold less affinity to estrogen binding sites, andtestosterone was 50–130-fold less competent than estradiol.Estrogen binding sites in cytosol and nuclear fraction in bothsexes exhibited same ligand preferences.
3.3. Distribution of estrogen binding sites during sexualmaturation
In the initial experiments, total specific binding capacities ofcytosolic and nuclear fractions of the gonads were comparedbetween mature and spent scallops. As shown in Fig. 7, thebinding capacity of the cytosolic fraction in ripe scallops wasnot significantly different from that of spent ones, but thebinding capacity of nuclear fraction in the ripe scallops was53.8% higher than in the spent ones (Pb0.01, n=12, t-test).
In the second set of experiments, the specific bindings ofcytosolic and nuclear gonadal preparations of scallops atdifferent stages during the reproductive cycle were measured.
callop: Characterization and possible involvement in reproductive regulation.
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Relative binding capacity ratios between cytosolic and nuclearfraction (C/N ratios) were calculated by dividing the specificbinding in the cytosolic fraction by that in the nuclear fraction.The variations of the C/N ratios with GSI for females and malesare shown in Fig. 8A and B, respectively.
In females, when GSI was low, the binding capacity in thecytosolic fraction was lower than that in the nuclear fraction,resulting in a low C/N ratio. At this stage, the gonads of thescallops were almost transparent with very few mature eggs.With the initiation of gametogenesis, the C/N ratio increased toa peak of 1.63 when GSI was about 5. This was the stage whenthe gonads of the scallops began to gain rapid growth. Then theratio decreased and became close to 1.0 when the GSI was about12. At this stage, the scallop gonads were half full, but theintestines were still visible. Immediately before spawning, whenthe scallop gonads were fully distended and the intestine wastotally obscured, the ratio of binding capacity was the lowest,0.56, in the entire cycle (Fig. 8A).
Similar results were obtained in males, as shown in Fig. 8B.At the spent stage when the gonads were generally transparent,the binding capacity of the cytosol was much lower than that ofthe nuclear fraction with a C/N ratio of 0.59. With the gonadaldevelopment, the C/N ratio reached a peak of 2.35 when GSIwas 6. This peak may be correlated with the initiation of therapid growth of gonads. The ratio decreased dramatically after-wards with the C/N ratio reaching 1 when GSI was about 14. Inmale scallops, the gonads at this stage were about 3/4 full andthe intestines were barely visible. The ratio continued to de-crease and the lowest value (0.62) was observed when thegonads were full and no intestines were visible.
4. Discussion
4.1. Characterization and identification of estrogen bindingsites in the sea scallop
We have characterized estrogen binding sites in sea scallopgonads of both sexes. In all preparations including what areclassically referred to as both “cytosolic” and “nuclear” fractions,saturation analyses revealed two populations of estrogen bindingsites, one with low and another with high affinities. The lowaffinity binding sites may be counterparts of type II estrogenreceptors in vertebrates because they have comparable affinities toestrogen, i.e., Kd within the range of 30–50 nM, and a highbinding capacity. Existence of Type II estrogen receptors has beenreported in many vertebrates and their primary functionmay be toconcentrate estrogen and maintain its high availability in the cells(Lopes et al., 1987; Kornyei et al., 1993; Yamamoto et al., 1996).Whether these sites have similar functions in molluscs remains tobe examined in the future.
The dissociation constants (Kd) of the high affinity sites inthe cytosolic fractions in females and males were very close,while those in the nuclear fraction were also close in femalesand males. Furthermore, all these estrogen binding sites havelimited binding capacity (Bmax) for estrogen. It is generallybelieved that the estrogen binding sites in both the cytosolic andnuclear fractions originated from the nuclear fraction but they
Please cite this article as: Wang, C., Croll, R.P. Estrogen binding sites in the sea sComp. Biochem. Physiol. B (2007), doi:10.1016/j.cbpb.2007.06.008
represent the unbound and bound receptors, respectively(Yamashita, 1998). During the preparation of the cell fractions,the unbound binding proteins are extracted with the low ionicstrength homogenization buffer while the bound bindingproteins can only be extracted with the high ionic strengthextraction buffer (homogenization buffer plus 0.6 M KCl).
Competitive binding studies showed that the relative affini-ties for sex steroids in all the fractions are in the same order,that is DESNestradiolNprogesteroneN testosterone. These re-sults indicated that natural and synthetic estrogens are goodcompetitors for these sites, although progesterone and testoster-one, at very high concentrations, can also compete for these sites.Competition for the estrogen binding sites by progesterone andtestosterone has also been reported in O. vulgaris (Di Cosmoet al., 2002). This characteristic seems to be distinct from that ofestrogen receptors in vertebrates (Notides, 1970; Colburn andBuonassisi, 1978).
Regarding the nature of these high affinity binding sites, it ispossible that they are the orthologs of vertebrate estrogenreceptors. In fact, the binding affinity and maximum bindingcapacity values in this study are generally within the range ofthe parameters for vertebrate estrogen receptors (Table 1).Identifications of estrogen receptor orthologs in bivalves lendfurther support for this possibility. Utilizing a polyclonal anti-human ER antibodies, estrogen receptor-like proteins similar tovertebrate ER-α and/or ER-β have been detected in thecytosolic fractions of marine mussels, M. edulis and Mytilusgalloprovincialis and a freshwater mussel, Elliptio complanata(Stefano et al., 2003; Won et al., 2005). Thus it is also possiblethat the two populations of binding sites are similar to ER-α andER-β, as indicated in Canesi et al. (2004). With degenerateprimers designed from known vertebrate estrogen receptorgenes, Croll and Wang (in press) amplified a 710 bp fragmentby PCR from a cDNA library constructed from the gonad of asea scallop. This fragment showed very high similarity withvertebrate estrogen receptor genes. Northern blotting using thecloned fragment as a probe also revealed the expression of a3.1 Kb mRNA in the gonad and liver, but not in the muscle ofthe sea scallop (Wang, 2000; Croll and Wang, in press).However, recent cloning of vertebrate estrogen receptororthologs in gastropods and cephalopod raised the questionon the ligand binding function of these bivalve estrogenreceptor orthologs. The ER orthologs cloned in the sea hareAplysia californica, a marine snail Thais clavigera, and thecephalopod O. vulgaris do not specifically bind estrogens butthey can be constitutively activated in absence of ligand(Thornton et al., 2003; Kajiwara et al., 2006; Keay et al., 2006).Although it is believed that the ligand binding of estrogenreceptors in molluscs may have been lost during the evolutionfrom an ancestral steroid receptor (Thornton et al., 2003; Keayet al., 2006), whether the ligand binding capacity was also lostin bivalve estrogen receptor orthologs is not known. Furtherstudies are thus necessary to determine if these orthologs canbind estrogens.
In addition to binding sites corresponding to classical ver-tebrate intracellular estrogen receptors, at least a portion ofthe binding sites in our study could represent membrane
callop: Characterization and possible involvement in reproductive regulation.
Sea lamprey Testis Cytosol 0.52 56.0 Ho et al., 1987Nuclei 0.39 68.0
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components, either estrogen receptor-related or unrelated. Theexistence of membrane receptors for estrogen is in agreementwith the rapid effects of estrogen reported in bivalves (Canesiet al., 2006; Wang and Croll, 2006). In our studies, we did notattempt to separate the membrane fraction from the cytosolicfraction, thus the binding sites in the cytosolic fraction maycontain membrane components. In fact, estrogen receptor-likeproteins have been identified in the membrane fraction in thepedal ganglion and haematocytes of M. edulis (Stefano et al.,2003; Canesi et al., 2006). Besides, other membrane com-ponents unrelated to classical estrogen receptor have alsobeen suggested. For instance, a G-protein coupled receptor,GPR30, which has high affinity and low binding capacityspecific for estrogens, mediates multiple rapid actions ofestradiol in human cells (Filardo et al., 2000, 2002; Maggioliniet al., 2004; Thomas et al., 2005). The estrogen binding sitesare not likely to represent steroid hormone binding globulin(SHBG) from the haematocytes, if they are present in mol-luscs, because SHBGs usually bind equally to estradiol andtestosterone (Rosener et al., 1991). In fact, the rinsing of theminced tissue with the homogenizing buffer before homoge-nization should remove most of the haematocytes in thepreparation.
Regardless of the identity of these estrogen binding sites, wehave definitely shown here that these sites exist in the sea
Please cite this article as: Wang, C., Croll, R.P. Estrogen binding sites in the sea sComp. Biochem. Physiol. B (2007), doi:10.1016/j.cbpb.2007.06.008
scallops and have many characteristics expected of receptors.These sites could act via the ‘classical’ or ‘genomic’ mechan-isms in which they serve as transcription factors upon activationby estrogen binding. They could also act by cross-talking withother signaling pathways such as protein kinase A-, proteinkinase C-, or Ca2+-mediated signaling (for a review on action ofestrogen through alternative pathways, see Porte et al., 2006).Thus their identification and mechanisms of action should be apriority in future studies.
4.2. Possible involvement of estrogen binding sites in sexualmaturation
Estradiol has previously been implicated in the regulation ofsexual maturation in bivalves (Li et al., 1998; Matsumoto et al.,1997; Osada et al., 2003, 2004; Reis-Henriques and Coimbra,1990; Varaksina and Varaksin, 1991; Varaksina et al. 1992;Wangand Croll, 2004). Thus, as stated above, the concentrations of theestrogen binding sites may also vary with sexual maturation. Toinvestigate this hypothesis, we first compared the bindingcapacity of the bound and unbound sites between spent and ripefemale scallops. No significant difference in the binding capacityof the cytosol was found, but the binding capacity of the nuclearfraction in ripe scallop gonads was much higher than in spentones. These results indicated that there were more bound sites inripe scallops than spent ones, while the number of unbound siteswas not different in ripe and spent animals.
We then examined the distribution of estrogen bindings inthe cytosolic and nuclear fractions in scallops at different stagesof sexual maturation cycle. The results showed that, in bothsexes, the ratio of binding capacity of the cytosol to that of thenuclei (C/N ratio) was low when the GSI was low. A rapidincrease in the C/N ratio was observed with the increase of GSIwhen gametogenesis was initiated. This was followed by acontinuous decrease in the C/N ratio with sexual maturation.The ratio reached its lowest value when the scallops were ripe.
Such changes in the C/N ratio presumably reflect thedynamics of synthesis and binding of these sites. Therefore,these results may indicate that, at the beginning of gametogen-esis, synthesis of estrogen binding sites was very active. Sincethe estrogen level at this stage was relatively low (Reis-Henriques and Coimbra, 1990; Matsumoto et al., 1997; Osadaet al., 2004), most of the synthesized estrogen binding sitesremained unbound in the animal, thus leading to a high C/Nratio. Due to the increases in the estrogen concentration with theprogress of sexual maturation, more and more sites were boundby estradiol, resulting in a high estrogen binding in the nuclearfraction. Thus, although estrogen binding sites may have beencontinuously synthesized, the C/N ratio decreased. The C/Nratio reached its lowest value before spawning when theestrogen level was the highest in the cycle. These results suggestthat the synthesis and binding of estrogen to these sites couldhave been involved in the process of sexual maturation,especially in the initiation of gametogenesis, in both sexes.
In general, it appears that the profiles of the synthesis andestrogen binding to these sites, as well as the levels of estrogen,correlate well with the process of sexual maturation in scallops.
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4.3. Estrogen binding sites might function in male scallops?
Our results demonstrate that a considerable quantity ofestrogen binding sites exist in the male scallop gonad, and thussuggest that they may play roles in the reproduction of males.Although the concentrations of both estrogen binding sites andestrogen are low in males (Reis-Henriques et al., 1990;Matsumoto et al., 1997), it may be still premature to state thatestrogen and estrogen binding sites play lesser important rolesin males than in females of molluscs. In mammals, activation ofestrogen receptor is necessary for the production of sperm inmales. It has been reported that the interaction of estrogenreceptors and estrogen is essential for the initiation ofspermatogenesis and absorption of epididymis fluid in mam-mals (Hess et al., 1997, 2000). A similar effect has also beenreported in molluscs. Varaksina et al. (1992) showed estradiolstimulated spermatogenesis in the scallop M. yessoensis.Administration of estradiol also accelerated spermatogenesisin other molluscan species such as Helix pomatia (Csaba andBierbauer, 1979, 1981).
In our previous studies (Wang and Croll, 2003, 2004, 2006),we have demonstrated that estradiol has effects in male scallops.We have shown that injection of estradiol into undifferentiatedjuvenile scallops resulted in more males than females (Wang andCroll, 2004). In vitro experiments demonstrated that estradiolpromoted sperm release induced by 5-HT treatment and thiseffect can be inhibited by tamoxifen, an antiestrogen, suggestingthe involvement of estrogen binding sites (Wang and Croll,2003). We further showed that estradiol directly stimulatedspawning or indirectly potentiated 5-HT-induced spawning inmale scallops (Wang and Croll, 2006). These results areconsistent with the presence of estrogen binding sites in malegonad preparations. Moreover, in studies of the distribution ofestrogen bindings between the cytosolic and nuclear fractions,our results suggested that synthesis and estrogen binding to thesesites might be involved in sexual maturation of males. Together,these results indicate that estrogen may regulate male reproduc-tion through the activation of estrogen binding sites.
5. Conclusions
The characterization of estrogen binding sites in the seascallops helps to provide a new perspective for the understand-ing of the evolution of estrogen receptors. It is believed that allestrogen receptors evolved from an ancestral steroid receptorwhich binds estrogen, but this binding capability has been lostin the molluscs studied to date (Thornton et al., 2003; Kajiwaraet al., 2006; Keay et al., 2006). If the estrogen binding sites inthe sea scallops are orthologs of classical estrogen receptors, ourresults would suggest that bivalve estrogen receptors are morerepresentative of the ancestral form which still possess estrogenbinding capacity, and that the loss of estrogen binding onlyoccurred elsewhere during the evolution of molluscs. Alterna-tively, if these binding sites are not estrogen receptor orthologs,estrogen must bind to other molecules in bivalves to exert theirfunctions. A novel mechanism can thus be expected for theactions of estrogen in invertebrates.
Please cite this article as: Wang, C., Croll, R.P. Estrogen binding sites in the sea sComp. Biochem. Physiol. B (2007), doi:10.1016/j.cbpb.2007.06.008
The finding that estrogen binding sites exist in bivalves alsohas implications for predicting possible environmental effects ofestrogenic endocrine disruption chemicals (EDCs) on molluscs.Furthermore, these EDCs often exist at low concentrations andidentification of potential EDCs with unknown chemical naturemay be difficult in environmental studies (García-Reyero et al.,2001; López de Alda and Barceló, 2001; Nelson et al., 2007).Utilization of a scallop estrogen binding assay system mayprovide a useful alternative to these problems.
Finally, while the evidence that estrogen binding sites may beinvolved in reproductive regulation in the sea scallops providesnew insight into the endocrinal control of sexual maturation inbivalves, our findings may also have practical application inbivalve aquaculture. With the advances of technologies in thefuture, it may be possible to artificially control the gonadalmaturation using sex steroids during the conditioning of broodstocks.
With implications for understanding the evolution of estrogenreceptors and with possible application in both environmentalstudies and aquaculture, further work on the roles of both sexsteroids and their binding sites is clearly warranted.
Acknowledgment
The authors thank Drs. Catherine Lazier, Jeffrey Ram,Douglas Rasmusson, Ellen Kenchington, Paul Liu and the lateWilliam Moger for their advice and support at many stagesduring this project. This research was funded through researchgrants from the Natural Science and Research Council(NSERC) of Canada.
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