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Vitellogenin, steroid plasma levels and spawning performanceof cultured female Senegalese sole (Solea senegalensis)

Jose M. Guzman a, Birgitta Norberg b, Jesus Ramos a, Constantinos C. Mylonas c,Evaristo L. Mananos a,*

a Institute of Aquaculture of Torre la Sal, Spanish Council for Scientific Research (CSIC), Torre la Sal s/n, 12595 Cabanes, Castellon, Spainb Institute of Marine Research, Austevoll Research Station, N-5392 Storebø, Norway

c Institute of Aquaculture, Hellenic Center for Marine Research, 71003-Iraklion, Crete, Greece

Received 11 October 2007; revised 4 February 2008; accepted 5 February 2008Available online 10 February 2008

Abstract

The Senegalese sole (Solea senegalensis) is a high value market flatfish, which aquaculture is compromised by severe reproductiveproblems; these are mostly found in soles hatched and raised in captivity (F1 generation). To gain knowledge on the reproductive dys-functions observed in cultured (F1) Senegalese sole, this work aimed at developing a specific vitellogenin (VTG) ELISA, for the mea-surement of plasma VTG levels in this species. Profiles of VTG were correlated with those of sexual steroids and spawningperformance of an F1 broodstock, during three consecutive years. The Senegalese sole VTG (ssVTG) was purified by precipitation withMgCl2–EDTA and anion-exchange chromatography and showed a molecular mass of 172 kDa, by SDS–PAGE. Specific antibodies wereobtained and used to develop a competitive ELISA, which had a sensitivity of 3.6 ng ml�1, and inter- and intra-assay coefficients of var-iation of 9.5% (n = 29) and 6.7% (n = 12), respectively. Annual profiles of plasma VTG showed a major peak at pre-spawning, and asecond minor rise around autumn, which mirrored plasma profiles of both estradiol (E2) and testosterone (T) levels. Spontaneous spawn-ing occurred every year in the spring season, but no fertilized eggs were obtained. In conclusion, this study described, for the first time,the purification and development of a sensitive and specific ELISA for Senegalese sole VTG. The endocrine and spawning data suggestedthat F1 female broodstock showed normal VTG and steroid releasing profiles in captivity with occurrence of spontaneous spawning, butno fertilization of the eggs was recorded.� 2008 Elsevier Inc. All rights reserved.

Keywords: Vitellogenin; Purification; Immunoassay; ELISA; Steroids; Spawning; Reproductive cycle; Aquaculture; Flatfish; Senegalese sole; Solea sen-

egalensis

1. Introduction

Vitellogenin (VTG) is the major yolk protein precursorin oviparous vertebrates, including fish. It is synthesizedin the liver in response to circulating estrogens, and incor-porated into the growing oocytes via specific receptors.Inside the oocyte, VTG is cleaved into smaller yolk pro-teins, phosvitin, lipovitellin, and b-component, which areaccumulated in yolk globules or granules, providing themain nutrient reserves for the developing embryos and

prefeeding larvae (Mommsen and Walsh, 1988; Hiramatsuet al., 2002).

Due to the high levels of synthesis and secretion of VTGduring vitellogenesis, the analysis of plasma VTG is usedas an indicator of ovarian development in female fish brood-stock. Immunoassays for VTG analysis have been developedfor several fish species, including radioimmunoassays(RIAs) (Norberg and Haux, 1988; Tyler and Sumpter,1990) and enzyme-linked immunosorbent assays (ELISAs)(Mananos et al., 1994a,b; Susca et al., 2001; Roy et al.,2004). The ELISAs have the advantage of high sensitivity,similar to RIAs, while avoiding the use of radioactiveisotopes.

0016-6480/$ - see front matter � 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygcen.2008.02.002

* Corresponding author. Fax: +34 964 319509.E-mail address: evaristo@iats.csic.es (E.L. Mananos).

www.elsevier.com/locate/ygcen

Available online at www.sciencedirect.com

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The development of specific immunoassays first requiresthe purification of VTG from a given species. VTGs havebeen purified from several fish species, including flatfishessuch as, common sole (Solea vulgaris) (Nunez Rodrıguezet al., 1989), turbot (Scophthalmus maximus) (Silversandet al., 1993), Atlantic halibut (Hippoglossus hippoglossus)(Norberg, 1995), English sole (Pleuronectes vetulus) (Rou-bal et al., 1997), Barfin flounder (Verasper moseri) (Matsu-bara et al., 1999), Greenback flounder (Rhombosolea

tapirina) (Watts et al., 2003), Smooth flounder (Pleuronec-

tes putnami) (Roy et al., 2004) and California halibut(Paralichthys californicus) (Palumbo et al., 2007).

In addition to their use in studies on reproductivephysiology of fish, VTG ELISAs are used for otherapplications in aquaculture and fish research. Consider-ing the female-specificity of the VTG molecule, ELISAsare used in aquaculture for sex determination, by detect-ing VTG in fish plasma samples; this is especially usefulin fish that do not display secondary sex characters. Inaddition, since VTG is inducible in male and juvenile fishby estradiol-17b, VTG is a commonly used biomarker inecotoxicology, for the detection of estrogen mimickingpollutants in aquatic systems (Sumpter and Jobling,1995). Because of the laborious work to develop specificVTG ELISAs for each species, the use of heterologousimmunoassays is extended in these applications. Never-theless, a homologous ELISA always ensures maximumspecificity, accuracy and sensitivity, which are parametersnecessary for studies in reproductive physiology, wherethe level of VTG normally requires an accuratequantification.

The Senegalese sole is a flatfish that has become a pri-ority species for the diversification of aquaculture in Eur-ope and Mediterranean countries (Dinis et al., 1999;Imsland et al., 2003). However, the culture of this soleis highly limited by important reproductive problems.Although reproduction of wild-caught Senegalese sole isfeasible in captivity, the descendants (F1 generation)which are hatched and raised in captivity, rarely spawnspontaneously, and when eggs are released, they are usu-ally not fertilized. This fact compromises obtaining F2larvae and thus, the creation of captive reared brood-stocks for further aquaculture development and breedingprogrammes.

Studies on the reproductive biology of Senegalese solehave been performed for more than two decades, butthey have been mostly focused on wild-caught brood-stock. Spawning characteristics under natural conditions(Dinis et al., 1999; Anguis and Canavate, 2005), hor-monal induction of spawning (Rodrıguez and Pascual,1982), gonadal development and sex steroid profiles(Garcıa-Lopez et al., 2006a), evaluation of egg quality(Vazquez et al., 1994; Mourente and Vazquez, 1996),and other aspects of broodstock management have beenreported in wild Senegalese sole broodstock maintainedin captivity (reviewed by Dinis et al., 1999). However,research on the reproductive performance of F1 Senegal-

ese sole broodstock in captivity is scarce. Gonadal devel-opment and sex steroid plasma profiles have beenrecently described for male (Garcıa-Lopez et al., 2006b)and female (Garcıa-Lopez et al., 2007) F1 broodstock.Other studies have evaluated the influence of photo-ther-mal (Garcıa-Lopez et al., 2006c) and hormonal manipu-lations on gonadal development, steroid secretion andspawning (Guzman et al., 2005; Agulleiro et al., 2006,2007). However, to date, there is no information onVTG plasma levels in Senegalese sole, either wild-caughtor F1 broodstocks, due to the unavailability of a homol-ogous VTG immunoassay.

The purpose of this study was first, the purificationof Senegalese sole VTG and development of a specificand homologous ELISA and second, the use of thisELISA to describe, for the first time, the seasonalchanges of plasma VTG in F1 Senegalese sole brood-stock. In addition, the VTG plasma profiles were corre-lated with plasma sex steroid profiles, development ofexternal signs of ovarian maturation and spawningperformance.

2. Materials and methods

2.1. Fish broodstock

Senegalese soles (F1 generation) were obtained from spawns of wild-caught Senegalese sole broodstock at the facilities of CIFPA ‘‘El Tor-uno” (Cadiz, Spain), and transported to our facilities at the ‘‘Institutode Acuicultura de Torre la Sal” (Castellon, Spain, 40�N and 0�E) at 2years of age. Soles were tagged with passive integrated transponder tags(Pit-tags, AVID) and sexed by using a heterologous ELISA (Mananoset al., 1994b). Sex was further confirmed by abdominal swelling infemales and by feeling the shape of testes in males during the reproduc-tive season.

Soles were reared in circular fiberglass tanks (4000 L, 1 m depth,4.9 m�2), at a density of 4.3 ± 0.1 kg m�2, without sand substrate andexposed to the natural photoperiod. Tanks were covered with a thin shadymesh, to reduce stress related to an excessive incidence of the sun light;maximum light intensity recorded on the water surface was around 600lux in the summer. Air diffusers were installed in the centre and perimeterof the tanks to ensure aeration; dissolved oxygen was checked regularly(6.9 ± 0.2 ppm). Tanks were maintained in an open flow-through seawater system (salinity �37‰), with a flow rate corresponding to fourrenewals per day. Water supply was obtained from the seashore throughpumping and thus, water temperature of the tanks followed the naturalfluctuations of the region. However, during the summer months of 2005and 2006 and in order to avoid extreme elevations of water temperatures(as detected in the summer of 2004), water supply was obtained from amixture of both the seashore collector (natural temperature) and a littoralwell (constant temperature �17 �C), which provided an open flow-throughsystem with water temperatures of 21.5 ± 0.1 �C. Previous informationhas shown that maintenance of adult Senegalese sole at temperaturesnot higher than 21–22 �C diminishes the incidence of bacterial infectionsin the broodstock (personal communication). Fish were fed ad libitum inthe morning, 5 days a week, using dry pellet (ProAqua) and natural feed,consisting on chopped fresh mussel and frozen squid. Food ration wasadjusted daily depending on remaining food observed in the bottom ofthe tank.

Handling of the fish (routine management and experimentation) wasalways done according to the European Union Directive (EEC, 1986)for the protection of animals used for experimental and other scientificpurposes.

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2.2. Reproductive cycle of cultured F1 senegalese sole

On January 2004, 90 male and female F1 Senegalese sole(849.8 ± 27.0 g) were distributed in four circular fiberglass tanks(4000 L). The study was run in duplicated tanks, using two tanks for endo-crine analysis, with repetitive manipulations and two tanks for spawninganalysis, with no manipulations.

Two tanks (# 43 and 44, 25 fish per tank) were sampled monthly anddevoted to the study of VTG and steroid (E2 and T) plasma levels andevolution of ovarian maturation. Every month (first week), from May2004 to November 2006, fish were lightly anesthetized by immersion in2-phenoxyethanol (0.3 ml l�1), body weight and length recorded, appar-ent gonad development evaluated and a blood sample of 0.8 ml collected.Blood was collected from the caudal vasculature, using heparinizedsyringes and placed on ice-cold heparinized tubes, containing PMSF(1 mM). Plasma was obtained by centrifugation (3000g, 15 min, 4 �C)and stored at �20 �C for further analysis of VTG, E2 and T. Samplingwas avoided some months, because of the incidence of bacterial infectionin the broodstock. Spawning quantity and quality was determined dailyin both tanks.

The other duplicate (tanks # 41 and 42, 20 fish each) was kept underundisturbed conditions (no blood sampling) and devoted exclusively tothe evaluation of spawning performance. These tanks were sampled everyfour months for biometrical measurements (body weight and length),avoiding the spawning period.

During the 3 years of the study, the number of fish decreased due tomortalities associated to the incidence of bacterial infections (i.e., Tenaci-

baculum maritimus and Vibrio spp.). The appearance of infectionsoccurred regularly every year, at early summer (July) and early fall (Octo-ber) and caused mortality of some fish of the affected broodstock tank.Infections were always detected after the spawning period and affectedall broodstock tanks similarly.

2.3. Analysis of spawning performance

Occurrence of spawning was checked daily in all broodstock tanksduring the spawning season. Eggs were collected by using a fine gaugenet placed across the outflow of the tanks. As a measure of egg quality,buoyant eggs from each spawning were separated from non-buoyanteggs in graduated cylinders filled with sea water (salinity �37‰). Buoy-ant eggs from each spawn were incubated for 48 h to determine hatchingrates. For each spawn, a sample of 50 buoyant eggs was examined at thebinocular microscope to determine egg diameter, morphology and fertil-ization. Total number of eggs produced daily was related to the femalebiomass of the tank at that time, to calculate the relative daily fecundity(# of eggs kg�1). Total fecundity for each tank was obtained at the endof the spawning season and related to the female biomass of the tank, tocalculate the relative total fecundity (total # of eggs kg�1). For calcula-tions, the female biomass of tanks # 43 and 44 was obtained everymonth in the corresponding monthly samplings; for tanks # 41 and 42(undisturbed tanks) the female biomass for the spawning season wasobtained from the previous biometrical sampling (early March of eachyear).

2.4. Estimation of the apparent maturational stage of females

A classification code was developed to determine externally the appar-ent stage of gonadal maturation of the females. The fish were classifiedafter visual examination of both sides of the body, as specified in Table1. First, examination of the ocular side, classified the females as stage I,II or III, corresponding to a flat, swollen or highly swollen abdomen,respectively. Second, examination of the blind side, classified the femalesas stage A or B, depending on the absence (A) or not (B) of a clear orangecoloration along the ventral side, which reflected the growth of the ventralovarian lobule.

2.5. Induction of VTG synthesis

VTG synthesis was induced in five male Senegalese sole(733.0 ± 96.8 g) by injection of 17b-estradiol (Sigma). 17b-Estradiol wasdissolved in ethanol: 0.9% NaCl (1:7) at a concentration of 4 mg ml�1

and stored aliquoted at �20 �C. Fish received six intramuscular injections(2 mg 17b-estradiol kg�1 body weight) every other day. Two days after thelast injection, fish were anesthetized and 5 ml of blood from each fish col-lected. Plasma was obtained as previously described and stored at �20 �Cfor further use in VTG purification.

2.6. Purification of Senegalese sole VTG

The Senegalese sole VTG (ssVTG) was purified from plasma of 17b-estradiol treated males following a protocol adapted from the purificationof Atlantic halibut VTG (Norberg, 1995). Before purification, aprotininwas added to the plasma (2.5% v/v) to prevent proteolysis. Plasma wascentrifuged (12,000g, 5 min, 4 �C) to eliminate debris and 1 ml selectivelyprecipitated by serial addition of 20 mM EDTA, pH 7.7 (2 ml), 500 mMMgCl2 (0.1 ml) and distilled water (5 ml), shaking the mixture gently aftereach addition. The resulting mixture was centrifuged (5000g, 30 min, 4 �C)and the precipitate redissolved in 1 ml of 1 M NaCl.

The resuspended precipitate was subjected to anion exchange chroma-tography on a Mono-Q 5/50 GL column (Amersham Bioscience), using aFPLC system. The column was equilibrated with three volumes of 20 mMTris–HCl, pH 8.0 (buffer A) before sample application. The sample waseluted with a linear gradient of NaCl from 0.00 to 0.50 M, followed by1 M NaCl for total removal of adsorbed proteins. The column was washedafter each chromatographic run, with three volumes of 1 M NaCl andthree volumes of distilled water. Fractions of 0.5 ml were collected at aflow rate of 1 ml min�1 and optic density measured at 280 nm. All solu-tions and buffers used in the chromatographic procedure were autoclaved,degassed and filtered through a 0.22 lm filter. Fractions expected to con-tain VTG were dialyzed against buffer A:glycerol (1:1), overnight at 4 �Cand stored at �80 �C for further analysis. Protein content of selected frac-tions was determined by Bio-Rad’s DC protein assay, using bovine serumalbumin (Sigma) as standard.

2.7. Senegalese sole VTG antiserum

Specific antibodies against the purified ssVTG were produced by acommercial company (Agrisera, Sweden). Two rabbits were immunizedwith a first intradermal injection of 100 lg ssVTG, emulsified with FreudComplete Adjuvant and four subsequent intradermal injections of 70 lgssVTG, emulsified with Freud Incomplete Adjuvant, given at 4 weeksintervals. Rabbits were bled 2 weeks before immunization (pre-immuneserum) and 2 weeks after immunizations III, IV and V, in order to performthe corresponding titration tests. Final bleeding was done at 2 weeks afterimmunization V.

2.8. Electrophoresis and Western blot

Samples were analyzed by SDS–polyacrylamide gel electrophoresis(SDS–PAGE) under denaturing conditions, using a Bio-Rad Mini Proteanelectrophoresis system. Samples were diluted in SDS sample buffer

Table 1Classification code of the reproductive stage of F1 female Senegalese soleby external visual examination

Classification Side Observation

I Ocular Totally flat abdomenII Ocular Swollen abdomenIII Ocular Highly swollen abdomen

A Blind No sign of orange colorationB Blind Orange coloration

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(125 mM Tris–HCl, 4% SDS, 20% v/v glycerol, 5% v/v b-mercap-toethanol, 0.02% bromophenol blue) and boiled for 2 min. SDS–PAGEwas performed on 7.5% acrylamide gels for 45 min at room temperature.Gels were stained with Coomassie Brilliant Blue R-250.

For Western blot, proteins were transferred to PVDF membranes(Immobilon-P, Millipore) for 2 h (350 mA), on ice cold 25 mM Tris–HCl buffer containing 192 mM glycine, pH 8.3, using a Bio-Rad TransBlot Cell. The membrane was saturated in 10 mM Tris–saline buffer, pH7.6 (TBS) containing 2% non-fat dry milk, under constant gentle agitation,overnight at 4 �C. After washing with TBS (3 � 10 min), the membranewas incubated with the specific ssVTG antiserum (AbVTG), diluted1:40,000 in TBS, under agitation for 2 h at room temperature. After wash-ing, membrane was incubated with HRP-labelled goat anti-rabbit antibod-ies (GAR-HRP, Bio-Rad), diluted 1:2000, under agitation for 90 min atroom temperature. The antigen–antibody complex was detected by chemi-luminescence (Luminol Reagent, Santa Cruz Biotechnology, Inc) and ana-lyzed in a densitometer (VersaDoc, model 5000, Bio-Rad).

2.9. Senegalese sole VTG ELISA

The purified ssVTG and the specific ssVTG antiserum (AbVTG) wereused to develop a competitive ELISA. The protocol of the ssVTG ELISAwas based on that previously described for the European sea bass VTGELISA (Mananos et al., 1994b). The protocol included the followingsteps:

(1) Coating the wells (Maxisorp, 96-well microplates, Nunc) with0.1 ml well�1 of ssVTG solution (50 ng ml�1, diluted in sodium car-bonate buffer 0.05 M, pH 9.6), overnight at 4 �C. The non-specificbinding (NSB) was determined in wells (triplicate) coated withsodium carbonate buffer.After coating, the wells were washed(3 � 1 min) with PBST (sodium phosphate buffer 0.01 M, pH 7.4,containing 0.9% NaCl and 0.05% Tween-20). This washing stepwas repeated after every step of the assay.

(2) Blocking the wells with 0.2 ml well�1 of PBST-2% (PBST contain-ing 2% of non-fat dry milk), for 30 min at 37 �C, to reducebackground.

(3) Incubation of standard and samples with the specific antibodies(AbVTG, diluted 1:200,000), into the coated wells, overnight at4 �C. Before distributed into the wells (0.1 ml well�1, duplicate),the reaction mixture was prepared in tubes, in PBST-2%, and pre-incubated overnight at 4 �C.

(4) Incubation with 0.1 ml well�1 of GAR-HRP, diluted in PBST-2%at 1:1000, for 45 min at 37 �C.

(5) Color development with 0.1 ml well�1 of TMB reagent (Bio-Rad),in complete darkness at room temperature for 25 min and stoppingthe reaction by addition of 0.1 ml well�1 of 1 N sulphuric acid.Absorbance was measured after 5 min at 450 nm, using a micro-plate reader (Thermomax, Molecular devices, Menlo Park, CA).

2.10. Steroid ELISAs

For steroid analysis, plasma samples were first extracted with alcohol.Ice-cold methanol was added to the plasma (6:1 v/v), shaken and centri-fuged (3000g, 15 min, 4 �C). The pellet was re-extracted twice with200 ll of methanol. Supernatants were pooled, dried and reconstitutedin 120 ll of potassium phosphate buffer (0.1 M, pH 7.4), then stored at�20 �C for analysis.

Levels of estradiol (E2) and testosterone (T) were quantified by ELISA,using a protocol previously validated for Senegalese sole plasma samples(Guzman et al., 2005). In the steroid ELISA, mouse anti-rabbit IgGmonoclonal antibody (Cayman Chemical) coated microtiter plates (Max-isorp, 96-well microplates, Nunc) were incubated (overnight at 4 �C) withthe following mixture (0.2 ml per well): standard (E2 and T) (Sigma–Aldrich) or sample, specific steroid antibody and the acetylcholinesterasetracer (Cayman Chemical). Color development was then achieved by addi-

tion of Ellman’s reagent (34 mM acetylthiocoline, 27 mM dithio-bid-2-nitrobenzoic acid, in 0.1 M potassium phosphate, pH 7.4), in completedarkness at room temperature for 4 h and the absorbance measured at405 nm, using a microplate reader.

The sensitivities of the ELISAs, calculated as Bo � 2 SD (maximumbinding minus twice the standard deviation), were 5.19 pg ml�1 and8.78 pg ml�1 for the E2 and T ELISA, respectively. The intra- (n = 4)and inter-assay (n = 8) coefficients of variation (CV %), at 50% binding,were 5.8% and 6.3% for E2 and 6.1% and 11.3% for T ELISA.

2.11. Statistics

Data are expressed as means ± standard error of the mean (S.E.M.).Differences were examined using One-way ANOVA followed by Stu-dent–Newman–Keuls (SNK) multiple comparison procedure, with signif-icance levels of p < 0.05. When necessary, data were log or Ln-transformed in order to comply with normality and homogeneity of vari-ance, which were tested by Kolmogorov–Smirnov and Bartlett methods,respectively.

The expression of the ELISA results was performed after linearizationof the sigmoid standard curve using the logit transformation (logit (Bi/Bo) = ln(Bi � NSB/Bo � NSB)), where Bi represents the binding of eachpoint, Bo the maximum binding and NSB the non-specific binding. Theparallelism between regression curves was tested by a Student’s t test.Dependence and association between variables was analyzed by simple lin-ear regression and correlation index, respectively. Pearson product-moment was used to examine the significance of regression.

3. Results

3.1. Purification and characterization of senegalese sole VTG

Purification of ssVTG from plasma of E2-treated malesole was performed by selective precipitation withMgCl2–EDTA followed by anion-exchange chromatogra-phy (Fig. 1). The chromatographic elution profile of E2-treated male plasma, after selective precipitation, showeda major homogeneous peak in fraction 18, eluted at a NaClconcentration of approximately 420 mM (Fig. 1A). Thispeak was absent in the chromatographic profile of plasmafrom untreated males (Fig. 1B). Chromatography ofplasma from E2-treated males without a previous precipita-tion with MgCl2–EDTA (Fig. 1C), showed this major peakat fraction 18, together with other minor peaks.

Electrophoresis analysis of fraction 18 showed a majorband of 172 kDa, together with other bands of lowermolecular mass (Fig. 2A, lane 4). The 172 kDa proteinband was present in plasma from vitellogenic femalesand E2-treated males (Fig 2A, lanes 2 and 3, respectively),but not in plasma from untreated males (Fig. 2A, lane1).Based on its inducibility by E2, selective precipitation withMgCl2–EDTA, female-specificity and molecular mass, thisprotein was tentatively identified as Senegalese sole VTG(ssVTG). The ssVTG preparation was used to raise spe-cific antibodies (AbVTG) in rabbits. In Western blot(Fig. 2B), the specific AbVTG immunoreacted withssVTG (Fig. 2B, lane 4) and with plasma from vitellogenicfemales and E2-treated males (Fig. 2B, lanes 2 and 3), butdid not recognize any band from plasma of untreatedmales (Fig. 2B, lane 1), indicating the VTG specificity ofthe AbVTG.

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3.2. Development and validation of Senegalese sole VTG

ELISA

A homologous ELISA was developed for Senegalesesole samples, using the purified ssVTG as standard andthe obtained ssVTG specific antibodies (AbVTG). A ser-ies of tests were performed to optimize the ELISA proto-col, by studying the behaviour of the standard curveunder different temperature and incubation times. Underoptimized assay conditions, the maximum binding (Bo)gave an OD around 1.5 and a non-specific binding(NSB) below 10%.

After optimization, the characteristics of sensitivity, pre-cision, specificity and accuracy of the ssVTG ELISA werecarefully checked. The sensitivity, checked by means oflowest detection limit (Bo � 2 SD) was 3.6 ng ml�1, corre-sponding to 92.3% of binding. The amount of VTG thatcaused 50% of binding (ED50) was 58.8 ng ml�1.

The precision of the assay was tested by means of theintra- and inter-assay coefficients of variation (CV %).Intra-assay variability (IA), tested by measuring replicatesof the same sample in a single ELISA plate, was 6.7%(n = 12). The inter-assay variability (IE), tested by analyz-ing the same sample in different ELISAs analyzed by differ-ent operators and at different days, was 9.8% (n = 29).

The specificity of the assay was tested by cross-reactionof the AbVTG with plasma from untreated males, whichshowed absence of immunoreaction at dilutions as low as1/4 (see Fig. 3).

The accuracy of the ELISA was tested by spike-recoverytest, checking the parallelism between the standard curveand plasma from untreated males spiked with increasingdoses of ssVTG. The calculated recovery rate was96.11 ± 1.12%.

Finally, the assay was validated for Senegalese soleplasma samples by testing the parallelism between the stan-dard curve and serial dilutions of plasma from vitellogenicfemales and E2-treated males (Fig. 3).

3.3. Reproductive cycle of Senegalese sole

The reproductive cycle of cultured Senegalese solebroodstock was studied through three consecutive years,by analyzing spawning performance and VTG and sex ste-roid profiles. The spawning periods of the broodstock areshown in Fig. 4, together with the fluctuations of photope-riod and temperature. The annual spawning characteristicsof the broodstocks are given in Table 2. All 3 years, the F1breeders showed spontaneous spawning, which occurred

Fig. 1. Purification of ssVTG by a one step ion-exchange chromatogra-phy, from MgCl2 precipitated E2-treated male plasma (A). Chromatog-raphy profiles of plasma from untreated male (B) and non-precipitated E2-treated male (C) are shown as reference. An E2-induced protein peak waseluted at fraction 18 (A and C), which was absent in untreated males (B)and selectively precipitated with MgCl2–EDTA (A). Samples were elutedwith 20 mM Tris–HCl, pH 8.0, with a linear gradient 0.00–0.50 M NaCl(dotted line).

Fig. 2. Electrophoresis (A) and Western blot (B) analysis of purifiedssVTG (lanes 4) and plasma samples from: untreated male (lanes 1),vitellogenic female (lanes 2) and E2-treated male (lanes 3). Position ofmolecular mass (MM) standards (kDa) is indicated on the left. Westernblot shows immunoreaction of the AbVTG with vitellogenic femaleplasma (lane 2B), E2-treated male plasma (lane 3B) and purified ssVTG(lane 4B), but not with plasma from untreated males (lane 1B).

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always and exclusively in the spring season. The spring of2004 was the first spawning event of this broodstock, whensoles were 3 years old. Variations in fecundity rates wereobserved between years, being highest in 2006, correspond-ing to the third reproductive season of the broodstock.There was a total absence of egg fertilization in all spawns.The four experimental tanks (undisturbed versus monthlysampled duplicate tanks) did not show differences(p > 0.05) on daily mean fecundity, egg buoyancy (%) oregg diameter (data not shown). The number of females ineach tank, as well as that of males, decreased along the 3

years of the study, due to mortalities associated to the inci-dence of bacterial infections. This affected similarly to allfour tanks.

Seasonal changes of plasma VTG, E2 and T levels,together with the development of the maturation stage offemales are represented in Fig. 5. Along the 3 years ofthe study, the number of analyzed samples per samplingpoint (n) decreased from 26 to 8, due to diminished numberof females per tank, because of mortalities. Maximum lev-els of VTG were detected at the pre-spawning season(around 4 mg ml�1) in April 2005 and February 2006

Fig. 3. Validation of the ssVTG ELISA. The ssVTG standard curve (black circles) shows parallelism with ssVTG spiked male plasma (white circles) andserially diluted plasma samples from vitellogenic females (white triangles) and E2-treated males (black triangles). Plasma from untreated males (blackrhombuses) did not exhibit any cross-reaction in the assay.

Fig. 4. Spontaneous spawning (bars) of F1 Senegalese sole broodstock during three consecutive years (right axis). The amount of spawned eggs waschecked daily and referred to the female biomass (relative daily fecundity, # of eggs kg�1). The fluctuations of photoperiod (dotted line) and thermoperiod(solid line) are shown (left axis).

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(Fig. 5A), decreasing gradually during the spawning perioduntil basal levels (1 mg ml�1) at post-spawning (July 2004–2006). A second minor rise of VTG plasma levels wasdetected in late summer, with a 2-fold increment from pre-vious post-spawning levels.

Plasma levels of sex steroids paralleled those of VTG(Fig. 5B). Both, E2 and T profiles showed maximum con-centrations at the pre-spawning period, decreasing thereaf-ter. Although both steroids increased concomitantly duringthe pre-spawning period, T peaked one month earlier thanE2. Also, a second minor rise was observed in both steroidsat post-spawning (late summer), especially for E2.

First external signs of apparent gonad maturation(stages IIA and B) were observed one month before the ini-tiation of spawning, becoming greatest (stage IIIB) at fullspawning period (Fig. 5C). Just after spawning, all externalsigns of ovarian maturation disappeared (stage IA).Although no spawning was ever recorded in autumn, somefemales showed light externals signs of ovarian maturation(stage IIA) in November 2006.

Plasma levels of both E2 and T were significantly(p < 0.001) and positively correlated with VTG levels(Fig. 6). Both steroids were also significantly (p < 0.001)and positively correlated between them.

4. Discussion

The present study describes, for the first time, the puri-fication of VTG from Senegalese sole and development of aspecific ELISA. The identification of the purified protein asssVTG was based on two main characteristics, the female-specificity of the protein and E2-inducibility in males.

Moreover, the E2 inducible protein was selectively precipi-tated by MgCl2–EDTA and showed chromatographic andelectrophoretic properties similar to VTGs purified fromother teleosts. On the other hand, the natural plasma pro-files detected by using the specific ssVTG ELISA, corre-lated well with the reproductive cycle of femaleSenegalese sole breeders, being related to their spawningseason, profiles of plasma sex steroids and evolution ofexternal signs of ovarian maturation.

The purification method, adapted from that used in thepurification of Atlantic halibut (Norberg, 1995), proved tobe optimal for the purification of ssVTG. During thessVTG purification process, there were no signs of proteindegradation, in spite of performing the chromatographyprocedure at room temperature (�20 �C). It has been pre-viously suggested that susceptibility of VTG to degradationis associated with the water temperature at which the fishspecies naturally lives during its reproductive cycle, beinghigher for cold-temperature spawners than for warm-tem-perature spawners (Norberg, 1995). It is possible that, sincethe Senegalese sole is a warm-water species, its VTG maybe less susceptible to degradation throughout the purifica-tion procedure than other cold-spawner fish species, suchas cod (Silversand et al., 1993), Atlantic halibut (Norberg,1995) or English sole (Lomax et al., 1998), which haveshown higher VTG instability.

When the purified ssVTG was examined by SDS–PAGE, under reducing conditions, a major band of172 kDa and other minor molecular mass bands wererevealed. The 172 kDa band is probably the monomericform of ssVTG, eluted together with some breakdownproducts. The molecular mass of the ssVTG is within the

Table 2Spawning characteristics of F1 Senegalese sole broodstocks, during three consecutive years

Undisturbed 41 42

2004 2005 2006 2004 2005 2006

# of females 7 6 5 8 6 4Sex ratio (f:m) 1:1.8 1:1 1:1 1:1.5 1:1 1:1.5# of spawns 5 1 6 5 3 61st spawning day 28 May 6 April 4 April 27 May 23 April 3 AprilLast spawning day 2 June 6 April 26 April 2 June 25 April 9 MayDaily fecundity 1.37 ± 1.00 3.00 3.51 ± 1.14 2.36 ± 1.51 2.58 ± 1.37 13.94 ± 5.29Total fecundity 6.86 3.00 21.09 11.81 7.76 83.69Egg buoyancy (%) 40.7 ± 5.7 80 25.3 ± 9.2 7.2 ± 6.5 33.4 ± 16.6 12.8 ± 3.51Hatching rate (%) 0 0 0 0 0 0

Sampled 43 44

# of females 14 10 4 12 10 4Sex ratio (f:m) 1:0.7 1:1 1:1 1:0.9 1:1 1:0.7# of spawns 11 7 12 12 1 41st spawning day 15 May 11 April 3 April 28 April 20 May 3 AprilLast spawning day 21 June 25 May 1 June 21 June 20 May 17 AprilDaily fecundity 2.15 ± 0.88 1.65 ± 0.52 10.97 ± 1.85 1.31 ± 0.48 1.69 12.80 ± 4.91Total fecundity 23.71 11.54 131.67 15.81 1.69 51.21Egg buoyancy (%) 19.0 ± 4.8 23.3 ± 14.6 14.1 ± 2.9 30.3 ± 8.8 0 7.1 ± 5.3Hatching rate (%) 0 0 0 0 0 0

Tanks # 41 and 42 were undisturbed, whereas tanks # 43 and 44 were disturbed by monthly sampling.Daily and total fecundities are referred to the female biomass of the tank (relative fecundities) and expressed as # of eggs kg�1 (�103).

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range of that found in other flatfishes, i.e., 185 kDa in tur-bot (Silversand and Haux, 1989), 160 kDa in Atlantic hal-ibut (Norberg, 1995), 175 kDa in winter flounder (Hartlinget al., 1997), 155 kDa in greenback flounder (Sun et al.,2003) and 188 kDa in California halibut (Palumbo et al.,2007).

The developed ssVTG ELISA showed optimal charac-teristics of precision, accuracy, specificity and sensitivity.The precision was ensured by low intra- and inter-assaycoefficients of variation, which were both below 10%. Theaccuracy was demonstrated by high recovery rates inspike-recovery tests, above 95% for all doses, demonstrat-ing the capability to measure real values of the analyte in

the samples. The specificity was demonstrated by theabsence of immunoreaction of the AbVTG with VTG-freecontrol male plasma, both in ELISA and Western blot,even when plasmas were analyzed at high concentrations.Finally, the sensitivity of the ssVTG ELISA (3.6 ng ml�1)was good for the purposes of the study and comparableto other fish VTG ELISA’s, which usually ranged from 1to 15 ng ml�1 (Nunez Rodrıguez et al., 1989; Mananoset al., 1994b; Susca et al., 2001; Roy et al., 2004).

The developed ssVTG ELISA was used to evaluate, forthe first time, plasma VTG levels in Senegalese sole. In thisstudy, VTG analysis was performed over females hatchedand rose in captivity (F1 generation). Female breeders

Fig. 5. Annual profiles of plasma VTG (A), E2 (B, black triangles) and T (B, white triangles) in female Senegalese sole during three consecutive years;horizontal bars at the lower side represent the spawning period of each year. (C) Annual evolution of the maturational stage of the females, by externalexamination (see classification in Table 1); color code, from white to black, shows increasing degree of apparent gonad maturation. * indicates significanthigher levels (p < 0.05) than absolute minimum observed (January 2005 for T; July 2005 for E2 and August 2006 for VTG). Data are expressed asmeans ± SEM (n = 8–26, depending on the sampling).

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showed high circulating VTG levels for three monthsbefore the initiation of the spawning period, suggestingthat vitellogenesis in Senegalese sole is prolonged for atleast 3 months, much before first signs of gonad maturation

become evident externally. Similarly, vitellogenesis in com-mon sole is extended for 3–4 months before initiation ofspawning (Nunez Rodrıguez et al., 1989). Other multiple-spawning fish species, such as red porgy (Pagrus pagrus)(Kokokiris et al., 2001) or common dentex (Dentex dentex)(Pavlidis et al., 2000) also show significant high plasma lev-els of VTG around 3 months before the spawning period.On the contrary, single spawning species, such as the rain-bow trout (Oncorhynchus mykiss) (Bon et al., 1997) orArtic charr (Salvelinus alpinus) (Johnsen et al., 1999), usu-ally exhibit longer vitellogenic periods, showing high VTGlevels up to 7 months before spawning.

The annual VTG profiles in Senegalese sole were some-how different between years (2005 versus 2006), mainlyduring the period of vitellogenesis. In 2005 (second spawn-ing season of the broodstock), plasma VTG increasedslowly and progressively through 4 months, while in2006, plasma VTG sharply increased in 1 month, maintain-ing maximum levels throughout 4 months. In 2005, thiswas associated with reduced external signs of ovarian mat-uration and a very poor spawning performance, as com-pared to 2006. It is difficult to determine precisely theseannual differences, but taken into account that the studywas run in duplicated tanks and culture and photoperiodconditions were constant throughout the years, a potentialinfluence of the natural variations in water temperaturescould be considered. In fact, the winter of 2005 was abnor-mally cold in our region, registering water temperaturesbelow 11 �C in February for several weeks. This was notthe case in 2006, when winter temperatures were milderand more normal to the region. In Senegalese sole, infor-mation concerning the effects of temperature on reproduc-tive processes is scarce, and mainly related to the spawningprocess. Several studies have described the possible effect oftemperature changes on the spawning performance in wildbroodstock (Dinis et al., 1999; Anguis and Canavate,2005). Recently it has been shown that female Senegalesesole kept under constant high temperature (18 �C) hadreduced plasma E2 and T levels as well as a reduced pro-portion of late vitellogenic oocytes (Garcıa-Lopez et al.,2006c). Although in our study no differences in E2 and Tlevels were observed due to lower temperatures in 2005, itmight be possible that these low winter temperatures couldaffect the process of VTG synthesis and further reproduc-tive events. Previous studies in other fish species havedescribed negative effects of low temperatures on VTG syn-thesis, both in vivo and in vitro. In salmonids, reduced andslower VTG synthesis was observed in fish kept at 9 �Ccompared to those kept at 15 �C (MacKay and Lazier,1993). Exposure of rainbow trout hepatocytes to E2 at18 �C induced higher VTG mRNA expression than at14 �C, suggesting increased VTG synthesis due to a temper-ature-dependent increase in metabolism (Pawlowski et al.,2000). An effect of temperature variations during the per-iod of gametogenesis, on the secretion of VTG and sexualsteroids, has been evidenced in European sea bass brood-stock (Mananos et al., 1997). Thus, low water temperature

Fig. 6. Correlation between plasma levels of VTG and E2 (A), VTG and T(B) or E2 and T (C). Linear regression equations and correlationcoefficients are shown in each case. p < 0.001 indicates significantrelationship between the two variables.

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at the onset of vitellogenesis could affect the normaldynamic of VTG synthesis in Senegalese sole, with a possi-ble deleterious effect on further reproductive processes,including oocyte maturation and spawning. Every fish spe-cies might have an optimal range of water temperature forVTG synthesis, and both higher and lower temperaturesaffect the normal development of vitellogenesis (Kim andTakemura, 2003). Further studies will be necessary toclearly establish the appropriate range of temperature dur-ing the period of gametogenesis in Senegalese solebroodstock.

In all 3 years, the maximum VTG concentrationdetected in plasma was around 4 mg ml�1, which is similarto maximum levels described in common sole (3 mg ml�1)(Nunez Rodrıguez et al., 1989) and other teleosts, such asthe European sea bass (3 mg ml�1) (Mananos et al.,1994b), gilthead seabream (1.5 mg ml�1) (Mosconi et al.,1998) or Mediterranean amberjack (Seriola dumerilii)(5 mg ml�1) (Mandich et al., 2004), but lower to thosefound in other flatfishes, such as English sole (15 mg ml�1)(Lomax et al., 1998), Atlantic halibut (32 mg ml�1) (Meth-ven et al., 1992) or greenback flounder (26 mg ml�1) (Sunet al., 2003). In a comparative study over several speciesof Pacific salmonids, maximum VTG concentration wasfound to range from 0.1 mg ml�1 in sockeye salmon(Oncorhynchus kisutch) to 131.6 mg ml�1 in rainbow trout,during the period of ovulation (Benfley et al., 1989). Thevariability of maximum VTG plasma concentration infishes is probably more related to different reproductivestrategies than to phylogenetic relationships.

High circulating levels of VTG were maintained duringthe whole spawning period. The Senegalese sole is a serialspawner, spawning several times during the breeding sea-son, with an ovary that contains clutches of both previ-tellogenic and vitellogenic oocytes after ovulation(Anguis and Canavate, 2005; Guzman et al., unpublishedresults). High levels of VTG during the spawning seasonare probably necessary for continuous oocyte recruitmentto produce multiple batches of spawns. The balancebetween VTG incorporation into the oocytes and liverproduction is probably reflected by gradually decreasingcirculating VTG levels throughout the spawning season(Polzonetti-Magni et al., 2003). A similar VTG profileduring spawning has been reported for other multiplespawning fish, such us gilthead seabream (Mosconiet al., 1998), red porgy (Kokokiris et al., 2001), commondentex (Pavlidis et al., 2000), Mediterranean amberjack(Mandich et al., 2004) or mrigal (Cirrhinus mrigala)(Maitra et al., 2007).

Levels of VTG showed fluctuations in plasma almost allyear-round, except for 2–3 months where they reachedwhat was considered a basal concentration. But even, thesebasal levels were still quite high, as they were never below1 mg ml�1. This would suggest that vitellogenesis in Sene-galese sole is almost a continuous process. Most fish specieshave periods when circulating VTG is undetectable, whichreflect periods of sexual resting normally observed after the

spawning season (Mananos et al., 1994b, 1997; Lomaxet al., 1998; Mandich et al., 2004). Some other fish species,such as the multiple spawner blue fin tuna (Thunnus thyn-

nus) (min VTG �0.5 mg ml�1) (Susca et al., 2001), andthe Florida gar (Lepisosteus platyrhyncus) (min VTG1.54 mg ml�1) (Orlando et al., 2007) show active vitellogen-esis throughout the year, with detectable basal VTG levelsduring the resting period. Species differences in VTG pro-files are probably due to different reproductive strategies.In Senegalese sole, the continuous process of synthesisand release of VTG agrees with the extensive spawning sea-son observed in wild-caught captive reared broodstock;spawning has been observed from March to October, witha main spawning period in spring, and a secondary event inautumn which would coincide with the plasma VTGincreases observed in our F1 broodstock.

Plasma profiles of both sex steroids clearly mirroredthose of VTG. This is consistent with the role of E2 in reg-ulating VTG synthesis (Wallace, 1985) and the role of T asbiosynthetic precursor of E2 (Kagawa et al., 1982). Plasmalevels of both E2 and T have been previously reported inwild-caught and F1 female Senegalese sole (Garcıa-Lopezet al., 2006a, 2007). These authors showed lower plasmasteroid levels in F1 than in wild females, and suggested thatlow steroid concentrations would compromise the acquire-ment of oocyte maturational competence. In our study, theF1 females showed sex steroid levels similar to thosereported for wild-caught populations. This could be relatedto the fact that our F1 broodstock was able to producespontaneous spawns all 3 years, whereas in the studies ofGarcıa Lopez et al. the wild-caught broodstock spawnedbut the F1 did not (Garcıa-Lopez et al., 2006a, 2007). Thisfact could indicate that a certain steroid concentration inplasma would have to be attained for a correct develop-ment of the process of final oocyte maturation andovulation.

The observation of spontaneous spawning in our F1broodstock is not a common feature in captive F1 Senegal-ese sole broodstocks. In most cases, females undergo a sig-nificant ovarian development during spring, but as summerapproaches, ovarian volume decreases without spawning(Garcıa-Lopez et al., 2007). In spite of the success on eggrelease, a complete lack of egg fertilization was observedin our study. Non fertilized spawning is not a rare phenom-enon in cultured fish. In captive flounder (Paralichthys

orbignyanus), absence of fertilized eggs has been associatedto alterations in courtship and spawning behaviour (Bam-bill et al., 2006). In other flatfishes, such as southern floun-der (Paralichthys lethostigma) (Smith et al., 1999) orsummer flounder (Paralichthys dentatus) (Watanabe andCarroll, 2001), high variability in egg fertilization rates iscommon and has been attributed to a low participationof males in spawning events. Alternatively, low fertilizationrates could be associated to inhibited or delayed egg releaseafter ovulation, as suggested in greenback flounder (Pank-hurst and Fitzgibbon, 2006). Overripening of eggs mayimpair the capacity of the eggs to be fertilized, a process

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that can be especially rapid in warm-water species (Bro-mage et al., 1994; Zohar and Mylonas, 2001) such us theSenegalese sole. The partial reabsorption of overripe eggsis a common process and may lead a drastic decrease inbroodstock fecundity calculations, as shown in several fishspecies (Zohar and Mylonas, 2001).

Broodstock fecundities were highly variable betweentanks and between years. The overall spawning perfor-mance of all broodstock tanks was similar, including boththe undisturbed duplicated tanks and the monthly sampledduplicated tanks. This result was somehow unexpected,because it was initially hypothesized that stress associatedto monthly sampling of the fish (i.e., manipulation, anaes-thesia and blood collection) would affect adversely thespawning performance of the sampled fish. It seemed obvi-ous that in Senegalese sole, monthly sampling did not havean important negative effect over the spawning perfor-mance of the broodstock. Nevertheless, it should be consid-ered that, in many cases, the number of spawns per yearwas very low and little variations in daily fecunditiesbetween tanks may influence the annual overall fecundity.The reason for this variability in broodstock fecundity isunknown, but it is something commonly observed in wildSenegalese sole broodstock. Anguis and Canavate (2005)have reported a high dispersion in relative daily fecundityand number of spawns between tanks under similar rearingconditions. Dinis et al. (1999) observed high variations(above 6-fold) in egg production in two consecutive spawnsfrom a wild broodstok. Culture conditions and water tem-perature fluctuations have been identified as potential fac-tors affecting spawning performance, but detailed studiesover these processes are lacking. It seems that in Senegalesesole broodstock, both wild and F1, there is a high variabil-ity in the spawning performance of individual females andthus between broodstock tanks; the reason of this remainstill unknown.

In general, the seasonally changing pattern in daylengthis the most important factor for the cueing and timing ofreproduction in the majority of temperate zone fishes (Bro-mage et al., 2001), as the only variable capable of deliveringan unambiguous ‘‘date signal” (Pankhurst and Porter,2003). In the present study, both plasma VTG and sex ste-roids levels increased just after the increment of daylength(winter solstice) culminating in the apparition of spontane-ous spawns 6 months later. Also, but to a lesser extent, aseasonal rise in VTG and steroids was detected when thedaylight started to decrease (from summer solstice), sup-porting the role of photoperiod on the onset of gonadaldevelopment in Senegalese sole (Dinis et al., 1999; Anguisand Canavate, 2005; Garcıa-Lopez et al., 2006c).

In conclusion, this study described for the first time thepurification and development of a specific ELISA for Sene-galese sole VTG and its use to describe annual variations ofplasma VTG levels in this species. The endocrine resultssuggested that F1 Senegalese sole female breeders shownormal releasing profiles of VTG and sexual steroidsthroughout its reproductive cycle, being able to spawn

naturally, although the total absence of egg fertilizationunderline a severe reproductive alteration in the F1 gener-ation of Senegalese sole.

Acknowledgments

This research was funded by the Spanish Ministry ofEducation and Science (MEC) (Project AGL2003-07670),the Ministry of Agriculture, Fisheries and Food (MAPA)(Project JACUMAR 2006, II National plan for the cultiva-tion of sole) and the Regional Government of Valencia(ACOMP06/211), awarded to E. Mananos. J.M. Guzmanreceived a FPI fellowship from the MEC. The authorsacknowledge the staff of Piscimar s.l. for assistance in fishhusbandry, especially to Vicente Olmos for assistance andconstructive discussion. Authors thank Agrisera AB, espe-cially Joanna Porankiewicz-Asplund, for the collaborativework on the production of the Senegalese sole VTGantiserum.

References

Agulleiro, M.J., Anguis, V., Canavate, J.P., Martınez-Rodrıgues, G.,Mylonas, C.C., Cerda, J., 2006. Induction of spawning of captive-reared Senegalese sole (Solea senegalensis) using different deliverysystems for gonadotropin-releasing hormone agonist. Aquaculture257, 511–524.

Agulleiro, M.J., Scott, A.P., Duncan, N., Mylonas, C.C., Cerda, J., 2007.Treatment of GnRHa-implanted Senegalese sole (Solea senegalensis)with 11-ketoandrostenedione stimulates spermatogenesis and increasessperm motility. Comp. Biochem. Physiol. 147A, 885–892.

Anguis, V., Canavate, J.P., 2005. Spawning of captive Senegal sole (Solea

senegalensis) under a naturally fluctuating temperature regime. Aqua-culture 243, 133–145.

Bambill, G.A., Masakazu, O., Radonic, M., Lopez, A.V., Muler, M.I.,Boccanfuso, J.J., Bianca, F.A., 2006. Broodstock management andinduced spawning of flounder (Paralichthys orbignyanus) (Valenci-ennes, 1893) under a closed recirculated system. Revista de BiologıaMarina y Oceanografica 41, 45–55.

Benfley, T.J., Donaldson, E.M., Owen, T.G., 1989. An homologousradioimmunoassay for coho salmon (Oncorhynchus kisutch) vitello-genin, with general applicability for other Pacific salmonids. Gen.Comp. Endocrinol. 75, 72–78.

Bon, E., Barbe, U., Nunez-Rodrıguez, J., Cuisset, B., Pelissero, C.,Sumpter, J.P., Le Menn, F., 1997. Plasma vitellogenin levels during theannual reproductive cycle of the female rainbow trout (Oncorhynchus

mykiss): establishment and validation of an ELISA. Comp. Biochem.Physiol. 117 B, 75–84.

Bromage, N.R., Bruce, M., Basavaraja, N., Rawa, K., Shield, R., Young,C., Dye, J., Smith, P., Gillespie, M., Gamble, J., 1994. Egg qualitydeterminants in finfish the role of overripening with special reference tothe timing of stripping in the Atlantic halibut (Hippoglossus hippog-

lossus). J. World Aquacult. Soc. 25, 13–21.Bromage, N.R., Porter, M.J.R., Randall, C.F., 2001. The environmental

regulation of maturation in farmed finfish with special reference to therole of photoperiod and melatonin. Aquaculture 197, 63–98.

Dinis, M.T., Ribeiro, L., Soares, F., Sarasquete, C., 1999. A review on thecultivation potential of Solea senegalensis in Spain and in Portugal.Aquaculture 176, 27–38.

EEC, 1986. Council Directive 86/609 EEC for the protection of animalsused for experimental and other scientific purposes. Official J. L358(18/12/1986), 1–28.

Garcıa-Lopez, A., Anguis, V., Couto, E., Canario, A.V.M., Canavate,J.P., Sarasquete, C., Martınez-Rodrıguez, G., 2006a. Non-invasive

J.M. Guzman et al. / General and Comparative Endocrinology 156 (2008) 285–297 295

Author's personal copy

assessment of reproductive status and cycle of sex steroid levels in acaptive wild broodstock of Senegalese sole Solea senegalensis (Kaup).Aquaculture 254, 583–593.

Garcıa-Lopez, A., Fernandez-Pasquier, V., Couto, E., Canario,A.V.M., Sarasquete, C., Martınez-Rodrıguez, G., 2006b. Testiculardevelopment and plasma sex steroid levels in cultured maleSenegalese sole Solea senegalensis Kaup. Gen. Comp. Endocrinol.147, 343–351.

Garcıa-Lopez, A., Pascual, E., Sarasquete, C., Martınez-Rodrıguez, G.,2006c. Disruption of gonadal maturation in cultured Senegalese soleSolea senegalensis Kaup by continuous light and/or constant temper-ature regimes. Aquaculture 261, 789–798.

Garcıa-Lopez, A., Couto, E., Canario, A.V.M., Sarasquete, C., Martınez-Rodrıguez, G., 2007. Ovarian development and plasma sex steroidlevels in cultured female Senegalese sole Solea senegalensis. Comp.Biochem. Physiol. 146A, 342–354.

Guzman, J.M., Marull, E., Ramos, J., Mylonas, C.C., Olmos, V.,Mananos, E., 2005. Induccion hormonal a la puesta del lenguadosenegales (Solea senegalensis) mediante la administracion de sistemasGnRHa de liberacion sostenida. Actas X Congreso Nacional deAcuicultura. Universidad Politecnica de Valencia, Valencia, Spain, pp.490–491.

Hartling, R.C., Pereira, J.J., Kunkel, J.G., 1997. Characterization of aheat-stable fraction of lipovitellin and development of an immunoas-say for vitellogenin and yolk protein in winter flounder (Pleuronectes

americanus). J. Exp. Zool. 278, 156–166.Hiramatsu, N., Matsubara, T., Weber, G., Sullivan, C., Hara, A., 2002.

Vitellogenesis in aquatic animals. Fish Sci. 68, 694–699.Imsland, A.K., Foss, A., Conceicao, L.E.C., Dinis, M.T., Delbare, D.,

Schram, E., Kamstra, A., Rema, P., White, P., 2003. A review of theculture potential of Solea solea and S. senegalensis. Rev. Fish Biol.Fish 13, 379–407.

Johnsen, H.K., Tveiten, H., Willassen, N.P., Arnesen, A.M., 1999. Arcticcharr (Salvelinus alpinus) vitellogenin: development and validation ofan enzyme-linked immunosorbent assay. Comp. Biochem. Physiol.124B, 355–362.

Kagawa, H., Young, G., Nagahama, Y., 1982. Estradiol 17-b productionin isolated amago salmon (Onchorhynchus rhodurus) ovarian folliclesand its stimulation by gonadotropins. Gen. Comp. Endocrinol. 47,361–365.

Kim, B.H., Takemura, A., 2003. Culture conditions affect induction ofvitellogenin synthesis by estradiol-17b in primary cultures of tilapiahepatocytes. Gen. Comp. Endocrinol. 135, 231–239.

Kokokiris, L., Le Menn, F., Kentouri, M., Kagara, M., Fostier, A., 2001.Seasonal cycle of gonadal development and serum levels of vitellogeninof the red porgy, Pagrus pagrus (Teleostei Sparidae). Mar. Biol. 139,549–559.

Lomax, D.P., Roubal, W.T., Moore, J.D., Johnson, L.L., 1998. Anenzyme linked immunosorbent assay (ELISA) for measuring vitello-genin in English sole (Pleuronectes vetulus): development, validationand crossreactivity with other pleuronectids. Comp. Biochem. Physiol.121B, 425–436.

MacKay, M.E., Lazier, C.B., 1993. Estrogen responsiveness of vitello-genin gene expression in rainbow trout (Oncorhynchus mykiss) kept atdifferent temperatures. Gen. Comp. Endocrinol. 89, 255–266.

Maitra, S., Sahu, R., Trehan, N., Garg, S.K., Nath, P., 2007. Circannualvariation in plasma vitellogenin and gonadotropin II levels in relationto annual ovarian cycle in female mrigal, Cirrhinus mrigala. Aquacul-ture 265, 370–384.

Mananos, E., Zanuy, S., Le Menn, F., Carrillo, M., Nunez, J., 1994a. Seabass (Dicentrarchus labrax L.) vitellogenin. I: Induction, purificationand partial characterization. Comp. Biochem. Physiol. 107B, 205–216.

Mananos, E., Nunez, J., Zanuy, S., Carillo, M., Le Menn, F., 1994b. Seabass (Dicentrarchus labrax L.) vitellogenin. II: Validation of anenzyme-linked immunosorbent assay (ELISA). Comp. Biochem.Physiol. 107B, 217–223.

Mananos, E.L., Zanuy, S., Carrillo, M., 1997. Photoperiodic manipula-tions of the reproductive cycle of sea bass (Dicentrarchus labrax) and

their effects on gonadal development, and plasma 17b-estradiol andvitellogenin levels. Fish Physiol. Biochem. 16, 211–222.

Mandich, A., Massari, A., Bottero, S., Pizzicori, P., Goos, H., Marino, G.,2004. Plasma sex steroid and vitellogenin profiles during gonadaldevelopment in wild Mediterranean amberjack (Seriola dumerilii).Mar. Biol. 144, 127–138.

Matsubara, T., Ohkubo, N., Andoh, T., Sullivan, C.V., Hara, A., 1999.Two forms of vitellogenin, yielding two distinct lipovitellins, playdifferent roles during oocyte maturation and early development ofbarfin flounder, Verasper moseri, a marine teleost that spawn pelagiceggs. Dev. Biol. 213, 18–32.

Methven, D.A., Crim, L.W., Norberg, B., Brown, J.A., Goff, G.P., Huse,I., 1992. Seasonal reproduction and plasma levels of sex steroids andvitellogenin in Atlantic halibut (Hippoglossus hippoglossus). Can. J.Aquat. Sci. 49, 754–759.

Mommsen, T., Walsh, P., 1988. Vitellogenesis and ooctye assembly. FishPhysiol. 11A, 347–406.

Mosconi, G., Carnevali, O., Carletta, R., Nabissi, M., Polzonetti-Magni,A.M., 1998. Gilthead seabream (Sparus aurata) vitellogenin: purifica-tion, partial characterization, and validation of an enzyme, andvalidation of an enzymelinked immunosorbent assay (ELISA). Gen.Comp. Endocrinol. 110, 252–261.

Mourente, G., Vazquez, R., 1996. Changes in the content of total lipid,lipid classes and their fatty acids of developing eggs and unfed larvaeof the Senegal sole, Solea senegalensis Kaup. Fish Physiol. Biochem.15, 221–235.

Norberg, B., Haux, C., 1988. An homologous radioimmunoassay forbrown trout (Salmo trutta) vitellogenin. Fish Physiol. Biochem. 5, 59–68.

Norberg, B., 1995. Atlantic halibut (Hippoglossus hippoglossus) vitello-genin: induction, isolation and partial characterization. Fish Physiol.Biochem. 14, 1–13.

Nunez Rodrıguez, J., Kah, O., Geffard, M., Le Menn, F., 1989.Enzymelinked immunosorbent assay (ELISA) for sole (Solea vulgaris)vitellogenin. Comp. Biochem. Physiol. 92B, 741–746.

Orlando, E.F., Binckzik, G.A., Denslow, N.D., Guillete, L.J., 2007.Reproductive seasonality of the female Florida gar, Lepisosteus

platyrhincus. Gen. Comp. Endocrinol. 151, 318–324.Palumbo, A.J., Linares-Casenave, J., Jewell, W., Doroshov, S.I., Tjeer-

dema, R.S., 2007. Induction and partial characterization of Californiahalibut (Paralichthys californicus) vitellogenin. Comp. Biochem. Phys-iol. 146A, 200–207.

Pankhurst, N.W., Fitzgibbon, Q.P., 2006. Characteristics of spawningbehaviour in cultured greenback flounder Rhombosolea tapirina.Aquaculture 253, 279–289.

Pankhurst, N.W., Porter, M.J.R., 2003. Cold and dark or warm and light:variations on the theme of environmental control of reproduction.Fish Physiol. Biochem. 28, 385–389.

Pavlidis, M., Greenwood, L., Mourot, B., Kokkari, C., Le Menn, F.,Divanach, P., Scott, A.P., 2000. Seasonal variations and maturitystages in relation to differences in serum levels of gonad steroids,vitellogenin, and thyroid hormones in the common dentex (Dentex

dentex). Gen. Comp. Endocrinol. 118, 14–25.Pawlowski, S., Islinger, M., Volkl, A., Braunbeck, T., 2000. Temperature-

dependent vitellogenin-mRNA expression in primary cultures ofrainbow trout (Oncorhynchus mykiss) hepatocytes at 14 and 18 8C.Toxicol. In Vitro 14, 531–540.

Polzonetti-Magni, A.M., Mosconi, G., Soverchia, L., Sakae, K., Carne-vali, O., 2003. Multihormonal control of vitellogenesis in lowervertebrates. Int. Rev. Cytol., 239.

Rodrıguez, A., Pascual, E., 1982. Primeros ensayos sobre la utilizacion dela hipofisis del atun Thunnus thynnus en la maduracion y puesta deSolea senegalensis y Sparus aurata. Invest. Pesq. 97, 1–11.

Roubal, W.T., Lomax, D.P., Willis, M.L., Johnson, L.L., 1997. Purifica-tion and partial characterization of English sole (Pleuronectes vetulus)vitellogenin. Comp. Biochem. Physiol. 118B, 613–622.

Roy, R.L., Morin, Y., Coutenay, S.C., Robichaud, P., 2004. Purificationof vitellogenin from smooth flounder (Pleuronectes putnami) and

296 J.M. Guzman et al. / General and Comparative Endocrinology 156 (2008) 285–297

Author's personal copy

measurement in plasma by homologous ELISA. Comp. Biochem.Physiol. 139B, 235–244.

Silversand, C., Haux, C., 1989. Isolation of turbot (Scophthalmus

maximus) vitellogenin by high-performance anion-exchange chroma-tography. J. Chromatogr. 478, 387–397.

Silversand, C., Hyllner, S.J., Haux, C., 1993. Isolation, immunochemicaldetection, and observations of the instability of vitellogenin from fourteleosts. J. Exp. Zool. 267, 587–597.

Smith, T.I.J., McVey, D.C., Jenkins, W.E., Denson, M.R., Heyward,L.D., Sullivan, C.V., Berlinsky, D.L., 1999. Broodstock managementand spawning of southern flounder, Paralichthys lethostigma. Aqua-culture 176, 87–99.

Sumpter, J.P., Jobling, S., 1995. Environ. Health Perspect. 103 (Suppl. 7),173–178.

Sun, B., Pankhurst, N.W., Watts, M., 2003. Development of anenzyme-linked immunoassay for vitellogenin measurement in green-back flounder Rhombosolea tapirina. Fish Physiol. Biochem. 29, 13–21.

Susca, V., Corriero, A., Bridges, C.R., De Metrio, G., 2001. Study of thesexual maturity of female bluefin tuna: purification and partial

characterization of vitellogenin and its use in an enzyme-linkedimmunosorbent assay. J. Fish Biol. 58, 815–831.

Tyler, C.R., Sumpter, J.P., 1990. The development of a radioimmunoassayfor carp, Cyprinus carpio, vitellogenin. Fish Physiol. Biochem. 8, 129–140.

Vazquez, R., Gonzalez, S., Rodrıguez, A., Mourente, G., 1994. Biochem-ical composition and fatty acid content of fertilized eggs, yolk sac stagelarvae and first-feeding larvae of the Senegal sole (Solea senegalensis,Kaup). Aquaculture 119, 273–286.

Wallace, R.A., 1985. Vitellogenesis and oocyte growth in nonmammalianvertebrates. In: Browder, L.W. (Ed.), Developmental Biology. PlenumPublishing Corporation, pp. 127–177.

Watanabe, W.O., Carroll, P.M., 2001. Progress in controlled breeding ofsummer flounder, Paralichthys dentatus, and southern flounder, P.

lethostigma. J. Appl. Aquacult. 11, 89–111.Watts, N., Pankhurst, N.W., Pryce, A., Sun, B., 2003. Vitellogenin

isolation, purification and cross-reactivity in three teleosts species.Comp. Biochem. Physiol. 134B, 467–476.

Zohar, Y., Mylonas, C.C., 2001. Endocrine manipulations of spawning incultured fish: from hormones to genes. Aquaculture 197, 99–136.

J.M. Guzman et al. / General and Comparative Endocrinology 156 (2008) 285–297 297