Tilapia (Oreochromis niloticus) vitellogenins: development of homologous and heterologous ELISAs and analysis of vitellogenin pathway through the ovarian follicle

Tilapia (Oreochromis niloticus) Vitellogenins:Development of Homologous and HeterologousELISAs and Analysis of Vitellogenin PathwayThrough the Ovarian Follicle

PAP NDIAYE1, JEAN FORGUE2, VALERIE LAMOTHE3, CHANTAL CAUTY4,PHILIPPE TACON4, PIERRETTE LAFON5, BLANDINE DAVAIL2,ALEXIS FOSTIER4, FRANC-OISE LE MENN2, AND JESUS NUNEZ6�1IFAN, Universite Cheikh Anta Diop, Dakar, Senegal2Laboratoire de Genomique et de Physiologie des Poissons, Universite deBordeaux I, 33405 Talence, France3Unite Micronutriments Reproduction Sante, ENITAB, 1 cours du general deGaulle, 33175 Gradignan cedex, France4INRA SCRIBE, campus de Beaulieu, 35042 Rennes cedex, France5Laboratoire des Regulations Neuroendocriniennes, EA 2972, Universite deBordeaux I, 33405 Talence cedex, France6IRD, Groupe Aquaculture Continentale Mediterraneenne et Tropicale (GAMET),UR 175 CAVIAR, Av. Agropolis, 34033 Montpellier cedex, France

ABSTRACT Vitellogenin (VTG) of Oreochromis niloticus was again purified, due to theconflicting results found in the literature. Three purification processes have been used:electrophoresis and electro-elution, double chromatography (gel filtration and ion-exchangechromatography) and single ion-exchange chromatography. Using SDS-PAGE we confirmed in allcases the presence of two polypeptidic forms of plasma VTG of 130 kDa (VTG1) and 170 kDa (VTG2).We raised polyclonal antibodies against each VTG form and we demonstrated the complete cross-reactivity of each antibody with both forms of VTG by Enzyme Immuno-Assay (EIA) and Westernblots. The homologous ELISAs developed exhibited a detection limit of 6 ng.ml�1, equivalent to60 ng.ml�1 of plasma VTG and allowed us to quantify the total plasma VTG of O. niloticus with highspecificity and sensitivity. Using photonic and electron immunomicroscopy, we followed the pathwayof VTG into the ovarian follicle (OF) demonstrating that VTG enters the oocyte at stage 3 of OFdevelopment, at the same time as cortical alveoli and lipid globules appear. Heterologous ELISAsperformed on other cichlid species allowed us to quantify plasma VTG in Oreochromis aureus andSarotherodon melanotheron and to detect it in Hemichromis fasciatus, Hemichromis bimaculatusand Tilapia zillii, constituting a reliable tool for monitoring the presence of xeno-estrogens in theenvironment of these fish species. J. Exp. Zool. 305A:576–593, 2006. r 2006 Wiley-Liss, Inc.

The tilapia Oreochromis niloticus belongs to thePerciform order and Cichlidae family. This specieswas originally restricted to the Nile Basin and thenorth tropical areas of Africa, from Senegal to thecentral Great Lakes (Philipart and Ruwet, ’82).This species, well adapted to wide variations ofecological factors, has been able to colonizeextremely diverse biotopes (Pullin and Lowe-McConnell, ’82). At present, O. niloticus is widelyspread throughout Africa, South, Central andNorth America as well as Asia, owing to itsaquaculture. Even more, the species is now reared

in temperate countries using power plants hotwater effluents. Its world production was aroundone million tonnes in 2003 (FAO statistics) and iswidely tipped to become an even bigger player inthe international aquaculture area (Young andMuir, 2002).

Published online 13 April 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jez.a.290.

Received 15 August 2005; Accepted 24 January 2006

�Correspondence to: J. Nunez, IRD, Groupe Aquaculture Con-tinentale Mediterraneenne et Tropicale (GAMET), UR 175, CAVIAR,Av. Agropolis, 34033 Montpellier cedex, France. E-mail: [email protected]

r 2006 WILEY-LISS, INC.

JOURNAL OF EXPERIMENTAL ZOOLOGY 305A:576–593 (2006)

Despite numerous publications concerning thereproductive cycle, endocrinology of reproduction,fecundity and sexual behavior or strategy ofreproduction of this species (Babiker and Ibrahim,’79; Fishelson and Yaron, ’83; Baroiller andJalabert, ’89; Kestemont et al., ’89; Tacon et al.,’95, ’96, 2000; Baroiller et al., ’97; Hines et al., ’99;Duponchelle et al., ’99, 2000), few data areavailable about gametogenesis (Dadzie, ’74; Alveset al., ’83; Nakamura et al., ’93; Francolini et al.,2003) and particularly vitellogenesis. In fish, as inother oviparous vertebrates, embryonic and earlylarval development relies on egg yolk nutrients.The main yolk precursor, synthesized by the liverunder estrogenic induction and transported in theplasma, is vitellogenin (VTG), a large phospholi-poglycoprotein, which is incorporated specificallyinto oocytes by receptor-mediated endocytosis(Stifani et al., ’90; Chan et al., ’91; NunezRodriguez et al., ’96; Davail et al., ’98; Hiramatsuet al., 2004).

Fish VTG has been quantified for the first timeby ELISA in Solea vulgaris (Nunez Rodriguezet al., ’89) and since then for various other fishfamilies. Published studies on O. niloticus VTGstructure are conflicting. In previous work (Chanet al., ’91; Lee et al., ’92; Buerano et al., ’95), it hasbeen reported that the two VTGs present in thisspecies were immunochemically different. Thisencouraged us to start the purification of O.niloticus VTGs to be able to detect or to quantifyall forms of VTGs present over again.

The aim of this study was to obtain efficientimmunological tools in order (i) to investigate thedegree of homology between the two forms of VTG(VTG1 and VTG2) possibly present in this species,(ii) to follow the process of VTG internalizationinto the oocyte by immunocytochemistry, (iii) toquantify VTG during a sexual cycle using a specifichomologous ELISA and (iv) to develop hetero-logous ELISAs for other related Cichlid species.

MATERIAL AND METHODS

Fish, hormonal treatment, blood samplingand yolk extraction

Females and males of O. niloticus, weighingfrom about 30 g for immature juveniles to about120 g for the breeders, were obtained from eitherthe Nianga fish farm (Dagana, Senegal), Institutdes Savanes, (Bouake, Ivory Coast) or GAMETexperimental facility (Montpellier, France) andtransported to laboratories under oxygen aeration.

They were reared in 200-l aquaria provided withaerated dechlorinated tap water in natural orexperimental conditions of temperature (241C)and photoperiod, and fed once daily. After accli-matization for 2 weeks, 12 juvenile males wereinjected intra-peritoneally every 2 days during20 days with 4.5mg.g�1 of body weight of17b-estradiol (Sigma) using a stock solution ofestradiol in absolute ethanol (30 mg ml�1) diluted10-fold in cotton oil. Fish blood was collected bycaudal puncture in heparinized plastic centrifugetubes just before the first estrogen injection (t0),every 2 days before each estrogen injection and2 days after the last injection (t14). The blood wascentrifuged immediately at 10,000 g for 5 min, at41C. Plasma samples were pooled, aliquoted andstored at �201C. Vitellogenic follicles were col-lected on vitellogenic females, opened understereo-microscope and yolk samples diluted inNaCl. 12% were centrifuged for 5 min, at 2,000g.The intermediate translucent phase was used asyolk extract. Wild mature males were caught inthe Senegal river, their plasma immediatelyprepared as described above and stored at�201C. Ten females weighing between 50 and150 g were sampled at 3-days postspawning inter-vals, between days 1 and 27 of the normalreproductive cycle. The fish were caught, anesthe-tized, blood-sampled for VTG ELISA and weighed.Following decapitation, the ovaries were removedand weighed for calculation of the gonadosomaticindex (GSI) (ovarian weight�100/body weight).

Other Cichlidae species—Oreochromis aureus,Sarotherodon melanotheron, Tilapia dageti, Tila-pia guineensis, Tilapia zillii, Hemichromis bima-culatus and Hemichromis fasciatus—were caughtin the wild in Senegal and plasma of vitellogenicfemales was sampled.

Microscopy

As soon as the female was caught and anesthe-tized with MS222, the bulb was sectioned, and theheart still beating, the abdominal cavity wasopened for fixation of ovaries, one for photonicmicroscopy, the other for electron microscopy.

For photonic microscopy, one ovary was sec-tioned and immersed in Bouin Holland’s fluid.Ovarian lamellae were isolated under a stereo-microscope. After 72 hr fixation and then dehydra-tion in graded ethanol, pieces were embedded incytoparaffin using butanol as vehicle. For vitello-genic ovarian follicles (OFs), blocks were treatedduring 48 hr with ethanol-60%/glycerine (9:1) in

ISOLATION AND ELISA OF NILE TILAPIA VITELLOGENINS 577

J. Exp. Zool. DOI 10.1002/jez.a

order to facilitate the slicing of yolk (Baker, ’45).Serial sections of 7 mm thickness were stained withGroat eosin–hematoxylin, Masson trichrome orHeidenhain azan.

For electron microscopy, the second ovary wastreated at the same time by fixative solution(glutaraldehyde 3%, para-formaldehyde 2%, CaCl28 mM, sodium cacodylate 0.1 M, pH 7.3) either byimmersion (juvenile) or perfusion of the ovarianartery for 30 min (vitellogenic female) and quicklydissected under a stereo-microscope. A comple-mentary fixation of small pieces of ovarianlamellae of about 1 mm3 was performed overnightin fresh fixative at room temperature underdegassed atmosphere. Osmolarity of the fixativefluid was 320 mOsm, when O. niloticus plasma was300 mOsm. Except for immunocytochemistry, apostfixation was performed for 1 hr in 1% OsO4 incacodylate buffer containing 1.5% potassium fer-ricyanide. The fixed samples were then dehy-drated through graded ethanol and propyleneoxide, and embedded in Epon 812 resin.

The semi-thin sections were stained with 0.1%toluidine blue. Ultra-thin sections were contrastedby uranyl acetate and lead citrate.

Electrophoresis

SDS-PAGE (SDS 0.1% polyacrylamide gel 7.5%)was performed using Miniprotean II device(Biorad) with SDS-Tris-glycine buffer, for 1 hrat 120 V for two gels, according to Laemmli (’70).b-mercapto-ethanol was used as reducing agent.Relative molecular weights of separated proteinswere determined by comparing their mobility withstandard proteins using an electrophoresis cali-bration kit (Biorad HMW). Gels were stained withCoomassie Brilliant blue G 250.

VTG purification

All procedures were performed at 41C. In someexperiments, aprotinin and leupeptin or PMSF(1 mM) were used as anti-protease agents.

Electro-elution procedure

Plasma from an end-course estrogenized male(t14) was run on SDS-PAGE. After Coomassie bluestaining, each of the two bands correspondingto the putative VTGs (VTG2 and VTG1) was cutout, minced in 0.5 ml electro-elution buffer((NH4)HCO3 0.4 M, SDS 2%, pH 8.2) and imme-diately put in an electro-elution chamber. Proteinswere electro-eluted through a 10,000 Da cutoffmembrane by a 50 V, 10 mA current. At the end of

the electro-elution, protein was concentrated upto 500 mg.ml�1 on Amicon cell (10,000 Da cutoffmembrane), aliquoted and stored at �301C.

Double chromatography procedure

Gel filtration. Three milliliters of VTG-inducedplasma (t14) were mixed with 2 ml of chromato-graphy buffer (Tris 100 mM, CaCl2 1 mM, pH 7.8)and centrifuged during 5 min at 10,000g. Thesupernatant was deposited onto a 100� 2.5 cm gelfiltration equilibrated column of Sepharose6B (Pharmacia LKB). A 60 ml.h�1 buffer flowrate was applied and fractions of 10 ml werecollected. Fractions containing VTG were identi-fied by SDS-PAGE, and were pooled and con-centrated to 5 ml on the Amicon cell (10,000 Dacutoff membrane).

Ion-exchange chromatography. Previously sepa-rated proteins were applied to a 50� 1.8 cm2

column of DEAE-trysacryl M (IBF) equilibratedwith the previous buffer (flow rate: 36 ml.h�1).After 2 hr of washing, bound plasma proteins wereeluted by 200 ml of a linear gradient of Tris buffer(Tris 100–300 mM, CaCl2 1 mM, pH 7.8). Fractionscontaining VTG were selected by SDS-PAGE,pooled and concentrated using the Amicon cell(10,000 Da cutoff membrane) up to a final concen-tration of 2 mg.ml�1, aliquoted and stored at�301C.

Single chromatography procedure

Single DEAE chromatography. One milliliter ofinduced plasma (t14) was applied on a columncontaining 1.6 ml of ion-exchange resin DEAEFractogel (Merck) equilibrated with Tris buffer(Tris 50 mM, CaCl2 2 H2O 2 mM, pH 8.2) at a flowrate of 36 ml.h�1. After washout of unboundmolecules, the bound proteins were eluted with160 ml of linear gradient of NaCl (NaCl 0–500 mM,Tris 50 mM, CaCl2 2 mM, pH 8.2) at the same flowrate. Eluted fractions (3 ml) containing VTG wereselected by SDS-PAGE, pooled, aliquoted andstored at �301C.

For each procedure, absorbance at 280 nm ofevery eluted fraction was measured.

Immunization and anti-serum harvesting

Twelve-week-old rabbits received injections ofemulsified VTG solution (50% VTG at 200mg.ml�1

in 9% NaCl, and 50% Sigma Freund’s completeadjuvant). Each 1 ml subcutaneous injection,performed in 8–10 points on the back, was givenevery week during 4 weeks and twice during thenext month. Blood was sampled before immuniza-

P. NDIAYE ET AL.578


tion protocol and 1 week after each injection, andserum was obtained after 6 hr of blood clottingat 201C.

Immunizations were performed using VTGpurified by double chromatography (putativeVTG1) and by electro-elution (putative VTG2).

Before use, anti-sera were harvested with VTG-free lyophilized liver and plasma obtained fromwild males. One milliliter of anti-serum wasincubated with 10 mg of lyophilized liver and10 mg of lyophilized plasma for 3 hr at 201Cfollowed by 15 min centrifugation at 2,000g. Thesupernatant was incubated again overnight at 41Cwith the same amount of lyophilized liver andplasma and centrifuged for 15 min at 2,000g and41C. The final supernatant was recovered as anti-serum and stored at �201C.

Immunochemical techniques

Antigen–antibody dilution test

The binding kinetics and the specific activity ofantibodies were performed using Enzyme Immu-no-Assay (EIA). A range of diluted antigen coatedonto microtitration plates (Nunc maxisorp) wascrossed with a range of diluted anti-sera. Theimmunocomplexes were incubated with peroxi-dase anti-peroxidase (PAP) complexes. The perox-idase activity was revealed in the dark by adding200ml per well of orthophenylene-diamine solution(0.05%) in citric acid buffer (0.1 M, pH 5) in thepresence of 0.05% H2O2, giving an orange color.The reaction was stopped after 20 min by adding50ml per well of H2SO4, 4 M.

Immunocytochemistry

Photonic immunocytochemistry

Dewaxed paraffin slices were treated succes-sively with lithium carbonate (1%), hydrogenperoxide (30%), distilled water and phosphatebuffer (PB) (NaHPO4 0.1 M, pH 7.4). Saturationof non-specific binding sites was achieved byincubation in blocking solution (1% normal pigserum in PB) during 20 min. The slides were thenincubated for 1 hr with the primary antibody(anti-VTG1 1:1,000 in blocking solution), washedin PB and incubated for 1 hr in horse–radishperoxidase (HRP) conjugated with swine anti-rabbit IgG (1:250 in blocking solution). Antigen–antibody complexes were revealed as above.

Electron immunocytochemistry

Ultra-thin sections were deposited on gold gridsand incubated for 15 min in PB. Saturation of non-

specific binding sites was performed during 20 minin PB containing 0.3% of normal goat serum and0.1% of ovalbumin. After washing six times withPB, grids were incubated for 1 hr in PB containinganti-VTG1 (1:1,000). Then, after washing threetimes, the grids were incubated for 40 min in PBcontaining (1:100) goldlabelled goat anti-rabbitIgG (Amersham). Finally, after washing threetimes, grids were dried and observed under anelectron microscope (Phillips 200).

Western blot

After electrophoresis, the proteins were trans-ferred onto PVDF (polyvinyldifluoride) membranes(Amersham). Before use, PVDF membranes wereimmersed quickly in methanol and distilled water,and protein transfer was performed using Biorad’sprocedure for 1 hr at 100 V.

Western blot was processed under shackingin TBS-T (Tris 20 mM, NaCl 137 mM, pH 7.6,Tween-20 0.3%) at room temperature, and eachwashing step was performed four times in TBST-Tfor 10 min. PVDF blots were blocked overnight at41C in 5% defatted milk in TBS-T. Selectedantibody was then added in the blocking solution(1:40,000 or 1:60,000) for 2 hr. After washing,HRP-conjugated goat anti-rabbit IgG (Dako) wasadded at 1:10,000 in blocking solution for 1 hr.Western blots were revealed after washing withchemioluminescence reagents (Chemiglow, TMWEST).

ELISA procedure

The assay was performed in 96-well NuncMaxisorp microtitration plates as follows usingchromatography-purified putative VTG1 or electo-eluted purified putative VTG2.

1. Antigen coating: Purified VTG or lyophilizedVTG-free male plasma (100–400 ng.ml�1 insodium carbonate buffer, 0.05 M, pH 9.6) wascoated overnight at 41C.

2. Pre-incubation: In parallel, samples and serialdilutions of the reference purified VTG (from1000 to 7.81 ng) diluted in PBST-NPS (0.01 MPB, 0.15 M NaCl, 0.05% Tween 20, pH 7.4,containing 4% of normal pig serum) were pre-incubated in separate tubes overnight at 371Cwith specific antibody diluted (1:40,000 to1:160,000) in the same buffer.

3. Saturation: After removing the coating solu-tion, non-specific binding was lowered byadding PBST-NPS for 30 min at 371C.



4. Washing: After every step of the assay, thewells were extensively washed three timeswith PBS-T.

5. Incubation: Each pre-incubation solution wasdispensed in duplicate into the microplatewells, and PBST-NPS added into the fournon-specific binding wells. After incubationfor 2 hr at 371C, the plates were washed.

6. Second antibody incubation: Each well receivedswine anti-rabbit immunoglobulins (Dako) di-luted in PBST-NPS (1:2,500) during 45 min at371C, then washed.

7. PAP incubation: Each well received PAP com-plexes (Dako) diluted in PBST-NPS (1:5,000)for 45 min at 371C, then washed.

8. Revelation: The peroxidase activity wasrevealed as described for antigen–antibodydilution test.

9. Absorbance measurement: The absorbance ofeach well was measured after 10 min at 492 nmusing a Titertech multiskan plate reader.

The results of absorbance measurements wereexpressed using two models. In the non-linearmodel, results were expressed as Bi/B0� 100 as afunction of log (dose), where Bi represented thebinding of each standard or sample point and B0

the maximum binding obtained when no free VTGwas present in the well. The linear model wasperformed by logit–log transformation (Rodbardand Lewald, ’70) expressed as logit (Bi/B0) orlog (B0�Bi/Bi�N) as a function of the log(dose),where N was the non-specific binding. Theparallelism between regression curves was testedby covariance analysis (Snedecor and Cochran,’89; Sokal and Rohlf, ’96) with the Statgraphics 4.0Software package.

RESULTS

Kinetics of new proteins appearance

Total plasma proteins concentration exhibited ahuge increase, from around 45 mg.ml�1 inthe control plasma (t0) to around 130 mg.ml�1 atthe end of hormonal treatment (t14). SDS-PAGE(7.5% gel) of treated fish plasma (Fig. 1) showedthat the dramatic increase of total proteinswas mainly linked to the emergence of two newbands, of 130 and 170 kDa. They appeared fromday 2 of treatment and became particularlyintense from day 6, corresponding to the third17b-estradiol injection, to the end of the treat-ment (day 14), corresponding to the seventh17b-estradiol injection.

Purification of Nile tilapia VTG

Purification by electro-elution

The two major bands induced by estrogenictreatment were assumed to represent two VTGforms. The electro-eluted fractions submitted toSDS-PAGE (Fig. 2) showed that each purifiedprotein appeared as a single band located atthe same position as in the electrophoregramof 17b-estradiol-treated fish plasma. Both VTG

Fig. 1. SDS-PAGE follow-up of 17b-estradiol treatment.tn: Plasma sample control with n corresponding to thenumber of days of treatment (0–14).

Fig. 2. Electro-eluted VTGs SDS-PAGE. kDa: molecularmass markers; contr; Pl: free-VTG male plasma; Pl1E2:plasma from 17b-estradiol-treated fish, diluted (1:100); VTG1,VTG2: electro-eluted VTGs.

P. NDIAYE ET AL.580


fractions exhibited almost no plasma proteincontamination.

Purification by double chromatography

The elution profile of the gel filtration on 6BSepharose (Fig. 3a) of 17b-estradiol-treated juve-nile male plasma showed three successive peaks,peak 1 corresponding to the void volume. Electro-phoretic control of a selection of eluted fractions(Fig. 3b) indicated that the rising part of peak 2contained a major band of 130 kDa as well as othercontaminating plasma proteins.

Fractions containing the highest amounts ofVTG (27–34) were pooled, concentrated on theAmicon cell (membrane cutoff 50,000 Da) anddeposited on top of a DEAE-M trisacryl ion-exchange column. The elution profile is shown in

Figure 3c. The first peak corresponded to theunbound material eluted in the void volume. Thefollowing peaks (2, 3 and 4) corresponded tothe peaks eluted by application of the linear Trisbuffer gradient.

A selection of eluted fractions were subjected toSDS-PAGE (Fig. 3d). The 130 kDa containingfractions were located in the decreasing partof peak 3, with a maximum intensity observedin fraction 52. Eluted fractions from 50 to 56considered as VTG1 were pooled, concentrated onthe Amicon cell and frozen at �201C.

Purification by single Fractogelion-exchange chromatography

The elution buffer volume recommended by themanufacturer was increased by 100 times in

Fig. 3. (a) Sepharose 6B gel filtration chromatogram. Solid black points correspond to eluted fractions tested on SDS-PAGE.(b): SDS-PAGE of Sepharose 6B gel filtration fractions. Numbers indicate the eluted fractions from Sepharose 6B gel filtrationchromatography. MW: molecular mass markers (kDa); PL1E2: plasma from E2-treated fish. (c): Ion-exchange chromatogram.Solid black points (13, 37, 50, 52, 64) correspond to eluted fractions tested on SDS-PAGE. Gray points (51, 53, 54, 55, 56) andgray point 52 (with between brackets scripts) correspond to vitellogenic eluted fractions pooled to obtain VTG1. (d): SDS-PAGEof DEAE Tris-acryl fractions. Numbers indicate eluted fractions from DEAE Tris-acryl chromatography. MW: molecular massmarkers (kDa); Pl1E2: plasma from 17b-estradiol-treated fish; pool chr.1: pool of gel filtration vitellogenic fractions applied onthe ion-exchange column.



proportion of the large amount of VTGs induced inestradiol-treated fish. The elution profile (Fig. 4a)revealed three different peaks. The first onecorresponded to the unbound material eluted inthe void volume. The two other well-separatedpeaks were eluted following the application of alinear gradient (0–1 M) NaCl.

The electrophoretic control is shown in Figure 4b.A 130 kDa band was eluted with peak 2 in frac-tion 35 as putative VTG1. It appeared wellisolated with few plasma proteins contamination.A 170 kDa band was eluted in peak 3 and appearedstrongly contaminated. We selected fraction 62 asputative VTG2.

Charaterization of the VTGs

Electrophoretic characterization

Samples of the different VTG preparations wererun simultaneously in SDS-PAGE. Electrophore-tic properties of the two 130 and 170 kDa proteins

remained identical in the presence (lanes b, d, fand h) or in the absence (lanes a, c, e and g) ofb-mercapto-ethanol as reducing agent (Fig. 5).This demonstrated that the heavier 170 kDa bandwas not a dimer of the lighter 130 kDa one as itseemed the case for Salvelinus alpestris VTG(lanes g and h) tested in parallel.

Immunochemical characterization

Two antibodies have been developed in rab-bits, against the 170 kDa (putative VTG2)electro-eluted protein and against 130 kDa (puta-tive VTG1) protein purified by double chromato-graphy (Sepharose 6B and DEAE-Trisacryl).

Characterization by Western blotting. Both anti-bodies have been tested on Western blots. Bothantibodies recognized yolk proteins contained inO. niloticus yolk extract that definitively provestheir yolk origin. They also recognized the twoestradiol-induced new bands exhibited by treatedmales or vitellogenic females (Fig. 6).

Fig. 4. (a): Fractogel ion-exchange chromatography. Solidblack points correspond to eluted fractions tested on SDS-PAGE. Gray points with between brackets numbers corre-spond to VTG1 (fraction 35) and to VTG2 (fraction 62). (b):SDS-PAGE of Fractogel chromatography fractions. kDa:Molecular mass marquers; Pl C: VTG-free male controlplasma; Pl1E2: plasma from 17b-estradiol-treated fish. Num-bers indicate the fraction number of eluted fractions fromFractogel chromatography.

Fig. 5. SDS-PAGE of Oreochromis niloticus (a–f) andSalvelinus alpestris (g, h) electro-eluted VTGs. (a, c, e, g).Without b-mercapto-ethanol; (b, d, f, h) with b-mercapto-ethanol.

Fig. 6. Blots (a) and corresponding Western blots (b)revealed with (A) anti-VTG1 chr. antibody and (B) anti-VTG2 el. antibody. (1) Oreochromis niloticus vitellus extract;(2) plasma from vitellogenic female; (3) plasma from 17b-estradiol-treated fish; (4) VTG-free male control plasma.

P. NDIAYE ET AL.582


Each of two antibodies recognized both forms ofVTG (Fig. 7), VTG1 purified by double chromato-graphy (a) and VTG2 purified by electro-elution(b). Furthermore, the lack of any signal in wildmale plasma, used as negative control, showedthat antibody-harvesting procedures eliminatedthe rabbit gamma globulins directed towardscontaminating plasma proteins. We demonstratedthat each antibody strongly recognized its specificVTG, and the other form.

Characterization by EIA. The good parallelismof logit–log transformed binding curves (Fig. 8)between serial dilutions of O. niloticus VTG1 and

VTG2 purified by DEAE Fractogel, and VTG1

purified by double chromatography, using electro-eluted VTG2 for coating and anti-VTG2 electro-eluted as antibody, demonstrated the cross-reac-tivity between all kinds of VTGs and anti-VTGs.

Same kind of results were obtained with serialdilutions of electro-eluted VTG2, using VTG1

purified by double chromatography for coatingand anti-VTG1 purified by double chromatographyas antibody (results not shown), indicating thatsimilar cross-reactivity existed between the twoVTGs and anti-VTG1.

Homologous ELISA of O. niloticus

ELISA was established using one or the otherantibody obtained, each of them exhibiting a goodrecognition of the two VTGs as well as the vitellusextract components. The specificity was attestedby the parallel curves obtained using anti-VTG1

(Fig. 9) between O. niloticus VTG standard, serialvitellogenic female plasma dilutions and the lackof displacement with VTG-free wild male plasma.

The detection limit was 6 ng.ml�1 after over-night coating with 400 ng.ml�1 induced maleplasma, specific antibody diluted 1:40,000 andincubation time of 2 hr at 371C. This was equiva-lent to 60 ng.ml�1 in plasma, since it has to bediluted 1:10 to avoid any non-specific competition(results not shown). Inter- and intra-assay varia-tions were similar around 12% in 50% of binding.

This ELISA allowed us to measure plasma VTGduring a sexual cycle of wild females duringgonadal development between two successivespawnings (Fig. 10). VTG plasma profile and GSI

Fig. 8. Logit–log tranformation of binding curves usinganti-VTG2 antibody of three different VTG preparations:VTG Chr (VTG1 prepared by DEAE chromatography seeFig. 3c and d), VTG2 FG and VTG1 FG (VTG prepared byDEAE Fractogel chromatography (see Fig. 4a and b).

Fig. 7. Western blots with (a) anti-VTG1 chr. antibody;(b) anti-VTG2 el. antibody. C pl: VTG-free male controlplasma; pl1E2: plasma from 17b-estradiol-treated fish; chr.VTG1: VTG1 obtained by double chromatography; el. VTG2:VTG2 obtained by electro-elution.

Fig. 9. Homologous ELISA for Oreochromis niloticus.Binding curves of VTG standard (VTG1 FG), mature femaleand VTG-free male serial plasma dilutions using anti-VTG1

antibody.



exhibit similar profiles: they appeared to decreaseslightly during the first 6 days of the mothermouthbrooding (MG), they levelled out from day12 to day 21 during the mother guardian phase (G)and then continuously increased until day 27,at the spawning ending the cycle.

Heterologous ELISAs of various Cichlidae

In order to assess VTG homology betweendifferent tilapia species, serial dilutions of femaleplasma from seven different species within thefour closerelated genera of the Cichlidae Family(Sarotherodon, Oreochromis, Tilapia and Hemi-chromis) have been tested in the O. niloticus VTGELISA system (Fig. 11). Slopes of the serialdilution curves and statistical significance arepresented in Table 1.

Parallelism of regression lines indicated similarinteraction between heterologous VTG andO. niloticus VTG antibody and the different VTGsof the species tested. Only two species (O. aureusand S. melanotheron) gave parallel curves toO. niloticus VTG. From the other species tested,three gave parallel slopes between them(H. fasciatus, H. bimaculatus and Tilapia zilii)but different from O. niloticus VTG. Finally,T. dagetti had a completely unparallel slope withall other species tested. As a negative control,plasma from a wild male of S. melanotheron gaveno displacement (slope 5�0.001).

Histology, electron microscopy andimmunocytochemistry

The data presented here deal with the descrip-tion in O. niloticus of the two Stages 3 and 4, of OFdevelopment reflecting the accumulation of yolk inthe primary oocyte.

The diameters of Stage 3 ranged from 200mm(early) to 600mm (late). At the photonic level(Fig. 12a), the nucleus exhibited numerous nu-cleoli illustrating the amplification of the nuclearorganizer genes, which started during the pre-vious previtellogenic stages. The ooplasm wastypically organized in three concentric rings. Theouter ring was constituted by cortical alveolilocated at the vicinity of numerous Golgi

Fig. 11. Logit–log transformed competition curves ob-tained with serial dilutions of plasma of different vitellogenicfemale tilapia species. O.n.: Oreochromis niloticus; O.a.:Oreochromis aureus; S.m. F: Sarotherodon melanotheronfemale; H.b.: Hemichromis bimaculatus; H.f.: Hemichromisfasciatus; S.m. M: Sarotherodon melanotheron male; T.d.:Tilapia dageti; T.z.: Tilapia zillii.

TABLE 1. Slope comparison of binding curves obtainedwith the serial plasma dilutions of tilapia species

after logit–log transformation

O. n. O. a. S. m. H. b. H. f. T. z. T. d.

O. n. 0.324 0.120 0.000 0.002 0.001 0.000O. a. NS 0.279 0.000 0.000 0.002 0.000S. m. NS NS 0.000 0.000 0.006 0.000H. b. �� 0.722 0.762 0.000H. f. �� NS 0.556 0.000T. z. �� NS NS 0.000O. n. ��

P values of paired comparisons (upper triangle), and significance levels(lower triangle). See Fig. 11 for other details.

Fig. 10. Changes in GSI and plasma VTG concentrationduring an inter-spawning interval in wild females of Oreo-chromis niloticus. S: spawning, MB: mouthbrooding period,fr: fry release, G: guarding period. Bars indicate standarddeviation, n 5 10 for all samples.

P. NDIAYE ET AL.584


complexes. The median ring enclosed numerousgranules. The inner ring was constituted by thefirst appearance of lipid globules. At the outersurface of the oocyte, a thin dark layer appearedlocated under the granulosa epithelium (Fig. 12b),due to the onset of deposition of the zonaradiata externa.

The diameters of Stage 4 OF varied from 600to more than 2,000 mm. At the electron microscopelevel, illustrated in Figure 13a and b with atransient stage, at the limit stage 3–stage 4, thezona radiata appeared as electron-dense materialdeposited on the oocyte surface between the oocytemicrovillosities (omv) of the oolemma protrudingtowards the granulosa cells (gc): the internal zonaradiata (izr), located under the homogenouselectron-dense external zona radiata (ezr), exhib-ited successive inner reticulated layers in a highlyordered and twisted fiber-arched arrangement.Numerous pinocytotic pits appeared pinching offthe oolemma giving rise to clathrin-coated vesicles(horizontal arrows) entering the peripheralooplasm. In the peripheral ooplasm a lot ofmultivesicular bodies (mvb) were intermingledwith small yolk globules (ygl). The crystallinearrangement of yolk platelets was exhibited intothe largest ygl formed (Fig. 13c). Smooth densecored Golgian vesicles were observed in theperipheral ooplasm (arrowheads), dealing withthe elaboration of the zona radiata (Fig. 13a and b).

Immunohistochemistry using anti-VTG1-Chrshowed that the granules in the median ring ofStages 3 and 4 OF contained VTG-like compounds

(Fig. 14a), as revealed by the dark brown color,when previtellogenic Stages 1 and 2 remaineduncolored.

Ultrastructural immunocytochemistry allowedus to identify the traffic of VTGs in full Stage 4OF. Gold labelling shows the presence of vitello-genic compounds appearing in the channels of thezona radiata along the omv (Fig. 14b) and stronglypresent in mvb, ygl and platelets of larger ygl(Fig. 14c). O. niloticus species allowed us toillustrate the succession of evolutive forms oforganelles limited by a membrane and containingVTG and/or its derivative products: coated pits,

Fig. 13. Ultrastructure of young stage IV ovarian follicle:(a, b) peripheral ooplasm and zona radiata, (c) highermagnification of yolk globules. ca: cortical alveolus; ezr:external zona radiata; izr: internal zona radiata; lgl: lipidglobule; m: mitochondria; mvb: multivesicular body; omv:oocyte microvillies; poopl: peripheral ooplasm; pp: pinocytoticpit; ygl: yolk globule; ypl: yolk platelet.

Fig. 12. Cytology of Stage 3 ovarian follicle: (a) generalview, (b) details of the peripheral area. bl: Basement lamina;ca: cortical alveolus; ezr: externa zona radiata; gc: granulosacell; tc: thecal cell; lg: lipid globule; N: nucleus; n: nucleolus;oog: oogonia; ygr: yolk granule.



then in coated vesicles, mvb, yolk granules, ygland finally large ygl with platelets.

DISCUSSION

VTG purification

In the past two decades, purification of VTGfrom estradiol-induced fish plasma using variouschromatographic techniques was published in alarge number (Mananos et al., ’94b; Bon et al., ’97;Korsgaard and Pedersen, ’98; Mosconi et al., ’98;Parks et al., ’99; Brion et al., 2000; Holbech et al.,2001; Takemura and Kim, 2001; Fukada et al.,

2003; Hennies et al., 2003; Malgalhaes et al.,2004). Analysis of this literature showed thatcombination of chromatographic techniques leadsto good results and encouraged us to use thisapproach.

We chose to undertake the purification of VTGfrom plasma without a preliminary concentrationstep. Indeed, previous reports concerningO. niloticus VTG purification mentioned the pre-cipitation of VTG by EDTA prior to purification byanion-exchange chromatography on DEAE Sepha-dex (Chan et al., ’91) or electro-elution from PAGE(Buerano et al., ’95). This procedure, used toconcentrate the VTG before its purification, was

Fig. 14. Immunocytochemistry: (a) photonic immunocytochemistry of VTG labelling in ovary; (b, c) electron immunocy-tochemistry of VTG labelling in OF. ca: cortical alveolus; ezr: external zona radiata; izr: internal zona radiata; mvb:multivesicular body; omv: oocyte microvillies; poopl: peripheral ooplasm; st: stage; yst: young stage; sloped arrow: pinocytotic pit.

P. NDIAYE ET AL.586


largely used for various teleosts (Norberg andHaux, ’85; Copeland and Thomas, ’88; Ding et al.,’89; Silversand et al., ’93). It is now welldocumented that any change in the plasma, suchas addition of distilled water or EDTA-Mg21,modifies the interactions between VTG andCa21, and disrupts the quaternary structure ofthe VTG, which is then generally precipitated withaggregation of its subunits (Ding et al., ’89). Thefollowing process of redissolving the VTG does notlead, in all the species studied, to the initial nativeprotein tri-dimensional structure (Ansari et al.,’71; De Vlaming et al., ’80; Kanungo et al., ’90;Tyler and Sumpter, ’90).

Gel filtration was performed as the first of a two-steps procedure, taking advantage of the largedifference in electrophoretic mobility between thetwo new estrogen-induced bands as seen on SDS-PAGE. Surprisingly, as shown in Figure 3b, theband of higher molecular mass corresponding toVTG2 did not appear during the elution and wasundetectable on PAGE. This phenomenon has notyet been described in the literature. The hypoth-esis which could be advanced might be that VTG2

remained adsorbed on the Sepharose gel, due to itshydrophobic properties. Nevertheless, VTG1, wellpurified after DEAE chromatography, was used toraise the anti-VTG1 antibody.

In other respects, by operating the two otherpurification techniques—electro-elution and sin-gle DEAE chromatography—we isolated twoforms of VTGs, VTG1 and VTG2, exhibiting thesame electrophoretic migration patterns in SDS-PAGE as in the induced plasma after estradioltreatment.

VTG characterization

Electrophoretic identification

In the recent literature, the existence of eitherone or two forms of VTG in O. niloticus has beendiscussed several times.

In a previous work (Chan et al., ’91), thepresence in female plasma of two native and verydistinct protein bands, Vg and H, was shown, theformer only being identified as VTG. Indeed, theauthors claimed that Vg was the only estrogen-inducible phosphoprotein in O. niloticus, theprotein H being unlabelled when E2-induced fishwere injected with 32P-orthophosphate. Theirapparent molecular weights in native conditionswere respectively around 300 and 400 kDa. Sincein native PAGE the electrophoretic mobility is notlinearly related to the molecular mass (Brion

et al., 2000), these two proteins could be equiva-lent to VTG1 and VTG2 identified in our study as130 and 180 kDa proteins.

Lee et al. (’92), studying the degree of homologybetween VTG from various Perciformes claimedthat O. niloticus VTG appeared in a singlemolecular form under native PAGE conditions,but exhibited two polypeptide forms of 220 and130 kDa under denaturing-reducing conditions.This was in part questioned by Buerano et al.(’95) who have identified in the same species twoforms of VTG both in native and in SDS-PAGEconditions. The two prominent polypeptides,EIpp2 (120 kDa) and EIpp1 (185 kDa), detectedby these authors in denaturing conditions couldcorrespond, respectively, to our VTG1 and VTG2.

The presence of two VTGs was demonstrated inclosely related species. Two VTGs of 130 and180 kDa were shown on SDS-PAGE by Ding et al.(’89) in estrogen-treated O. aureus and confirmedby Lim et al. (’91). Other works (Kishida andSpecker, ’93, ’94) identified by SDS-PAGE, inOreochromis mossambicus, two plasmatic vitello-genic components designated as tVTG-130 andtVTG-200 according to their apparent molecularmasses. In O. mossambicus as well, Takemuraand Kim (2001) identified two VTGs with appar-ent molecular masses of 140 and 210 kDa bySDS-PAGE.

In summary, we conclude that in O. niloticus,as well as in closely related species, two formsof vitellogenic polypeptides exist in maturefemales and in estrogen-treated males: VTG1

and VTG2.

Immunological characterizationby Western blot

We raised two antibodies in rabbits, one againstthe putative VTG1 purified by a two-step chroma-tography and the other against putative VTG2

purified by electro-elution. We harvested the anti-VTG1 antibody with VTG-free plasma from wildmale caught in the Senegal river. Results showedthat the harvesting procedure removed the rabbitgamma globulins directed towards the few plasmaproteins contaminating the purified VTG1.

The two antibodies recognized O. niloticusvitellus extract, which means that 130 and180 kDa proteins are plasma precursors of yolk.Each antibody recognized the two VTGs whateverthe purification protocol used.

Western blot demonstrated that the storage ofthe purified VTGs at �801C and a fortiori at



�201C lead to a rapid degradation, numerousbands of lower molecular mass being labelled byanti-VTGs on Western blots. The quicker theVTGs are used after their purification, the lessthey are broken down. If experimentations wereperformed within around 24 hr following VTGsisolation, results would be the same with orwithout the use of anti-proteolytic agents duringthe VTGs purification. Nevertheless, we haveobserved that VTGs purified by electro-elutionappeared less degradable than VTGs purified bychromatography. This observation was cleared uptaking into account the presence of a trypsin-likeprotease in the plasma of O. niloticus (Inaba et al.,’97) whose activity is strongly inhibited byleupeptin and aprotinin: during isolation of pro-teins by electrophoresis which is quicker than bychromatography, the time of contact of VTGs withprotease is shorter than during isolation bychromatography, and the action of the trypsin-like protease is lower.

Immunological characterization by EIA

The cross-reactivity previously demonstratedbetween the two antibodies and their correspond-ing antigens was largely confirmed by crossed EIAs.

In contrast with previous data, our results showthat both antibodies cross-react with each VTGform. It has been shown (Chan et al., ’91) that anantibody directed against the purified Vg (corre-sponding to VTG1) did not recognize the heavierband H (corresponding to VTG2) induced at thesame time by estrogen treatment of males.Similarly, Buerano et al. (’95) found that the twovitellogenic polypeptides (EIpp1 and EIpp2) wereimmunochemically different. We may concludefrom those diverging results that each of ourpolyclonal antibodies recognize a larger diversityof epitopes, including some shared by both formsof VTG.

Use of homologousand heterologous ELISAs

One or the other of the two specific antibodiesrecognizing the two forms of VTG present in thisspecies can be used to quantify by ELISA the totalplasma VTG of O. niloticus. The O. niloticus VTGELISA exhibited high specificity and sensitivityand was comparable to other ELISAs developedfor other fresh water or marine fish species, Soleavulgaris (Nunez Rodriguez et al., ’89), Acipenserbaeri (Cuisset et al., ’89), Dicentrarchus labrax(Mananos et al., ’94a), Onchorhynchus mykiss

(Mourot and Le Bail, ’95; Bon et al., ’97), Zoarcesviviparous (Korsgaard and Pedersen, ’98), Sparusaurata (Mosconi et al., ’98), Pimephales promelas(Parks et al., ’99), Salmo trutta (Sherry et al., ’99),Danio rerio (Holbech et al., 2001), Cyprinus carpio(Hennies et al., 2003), Acanthogobius flavimanus(Ohkubo et al., 2003), Lepomis macrochirus(Cheek et al., 2004), C. carpio, D. rerio and Oryziaslatipes (Nilsen et al., 2004; Nozaka et al., 2004).

We have used this ELISA to quantify plasmaVTG during wild female sexual cycle, i.e., duringthe inter-spawning period. We have shown thatthe plasma VTG profile was in correlation with theGSI, reflecting itself the development of thevitellogenic oocytes batch spawned at the end ofthe cycle (Tacon et al., ’96). All the data obtainedunderlined that the MG and guard of the fry bythe female play an inhibitory role upon ovariandevelopment (Tacon et al., 2000).

This assay might be used to quantify VTG in twoother species (S. melanotheron and O.aureus),since plasma dilution curves of heterologousELISAs were parallel to O. niloticus VTG stan-dard curve, indicating that the VTG structure inthese three species was similar. Nevertheless, thelack of parallelism between O. niloticus and theother species tested is not a damning result, andthe presence of VTG in males or fry can bequalitatively tested in order to denounce thepresence of xeno-estrogens in the environment.

Similar heterologous immunoreactions havebeen shown in other fish families. A carp anti-VTG recognized VTG of common carp (C. carpio),roach (Rutilis rutilis) and bream (Abramis brama)using RIA (Tyler and Sumpter, ’90). Anothercommon carp anti-VTG used in ELISA recognizedVTG of fathead minnow (P. promelas) (Mylchreestet al., 2003). More recently, both anti-rainbowtrout (O. mykiss) VTG and anti-masu salmon(O. masu) VTG also recognized cutthroat trout(O. clarki), white-spotted char (Salvelinus leuco-maensis) and Sakalin taimen (Hucho perryi) VTGs(Fukada et al., 2001).

Endocytosis of VTG into the OF

We have traced the uptake of VTG by the OFduring vitellogenesis at the photonic and electro-nic microscope level using VTG immunodetectionwith anti-VTG1 antibody. An ultrastructuralapproach confirms in this species that Stage 3 ischaracterized by the onset of oocyte vitellogenesis,where VTG starts to enter the OF at the sametime when lipid globules and cortical alveoli

P. NDIAYE ET AL.588


appear (Le Menn et al., 1999). Once again, weobserved in this fish that initiation of yolkaccumulation into the oocyte was concomitantwith deposition of the zona radiata externa. Atthis stage, yolk is present in the oocyte, but lipidaccumulation is largely predominant over VTGendocytosis: type I vitellogenesis. In the followingStage 4, the yolk components are the same as inStage 3, but the proportion of lipid globules andygl is inverted and characterizes type II vitello-genesis. It corresponds to the rapid growth phaseof the OF development, where endocytosis of VTGis considerable and during which the zona radiatainterna is deposited.

These observations concerning O. niloticus arein agreement with those previously described inother teleostean or chondrichtyan species: Fundu-lus heteroclitus (Wallace and Selman, ’81; Selmanand Wallace, ’83, ’86), Salmo trutta (Breton et al.,’83), Solea vulgaris (Nunez Rodriguez, ’85), D.labrax (Zanuy et al., ’89), Acipenser baeri (Cuissetet al., ’89) and Gobius niger (Le Menn et al., ’99).

Our ultrastructural approach coupled with im-munocytochemistry detection of the VTG allowsus to claim that in all species studied the yolkentering the oocyte occurs earlier than photonichistology leads to believe.

What gene(s) code(s) for VTGsin O. niloticus?

In animals, VTGs are members of the large lipidtransfer protein superfamily, which could arisefrom an ancestral gene ensuring a pivotal eventin the intra-cellular and extra-cellular transfer oflipid and liposoluble substances (Babin et al., ’99).Recent results have demonstrated the presence ofmultiple VTG genes (vtg) in some teleost fishspecies (Buisine et al., 2002; Wang et al., 2005).Comparative genomic analyses suggest two wholegenome duplication events occurring one beforeand one after the divergence of ray-finned andlobe-finned fishes, prior to the teleost radiation(Jaillon et al., 2004; Van de Peer, 2004; Dehal andBoore, 2005; Sawaguchi et al., 2005; Woods et al.,2005). In teleost fish, the current model for vtgamplification has been demonstrated in Salmoni-dae, where ancestral vtg duplications gave rise totwo paralogous gene clusters, VtgA and VtgB(Buisine et al., 2002). Salmonids harboring a lowvtg copy number reflect an ancestral state,whereas salmonids harboring a high vtg copynumber reflect the latest state of differentiation.The ultimate state of differentiation appears in all

Oncorhynchus species which have lost, in thecourse of evolution, VtgB-related genes, andexhibit a unique VtgA locus containing a set of20 highly similar genes tandemly arranged (Tri-chet et al., 2000). The zebrafish (D. rerio) genomecontains at least seven vtg encoding heterologousVTGs with three distinct types mapped onto twodifferent chromosomes. Northern blot resultsshow that all seven vtg were expressed predomi-nantly in the liver and adipose tissue of female fishor E2-induced males (Wang et al., 2005). In thisspecies, lipovitellin I, phosvitin and lipovitellin II,parts of the yolk derivative proteins (Wahli, ’88),are encoded by vtg1 (vg1) type, which is mosthighly expressed.Two vtg transcripts have beencloned in F. heteroclitus (LaFleur et al., ’95;GenBank acession number UO7055) in Oryziaslatipes (Tong et al., 2004; GenBank accessionnumbers: AB064320 and AB074891) and inAcanthogobius flavimanus (Ohkubo et al., 2003).Multiple vtg transcripts have been demonstratedin Gambusia affinis (Sawaguchi et al., 2005). A vtgtranscript was characterized from the tilapia,O. aureus (Lim et al., 2001), and the structureof the corresponding gene determined (Teo et al.,’98). However, in a previous study (Lee et al., ’94),the authors found the presence of twomajor groups of OaVg cDNA exhibiting a 7%intra-specific divergence in the vtg family.Takenaltogether, these results support the presence ofat least two vtg-related genes in the genome ofOreochromis genus, in accordance with the char-acterization of two forms of plasma VTG in O.aureus (Ding et al., ’89), in O. mossambicus(Kishida and Specker, ’93; Takemura and Kim,2001) and in O. niloticus (this study).

CONCLUSION

In this paper we have clearly identified twoforms of VTG in O. niloticus by SDS-PAGE. Thesetwo proteins have apparent molecular masses of130 kDa (VTG1) and 170 kDa (VTG2). They arepresent in mature female or in estrogen-treatedfish plasma.

We have raised two antibodies against each formof VTG. Using EIA and Western blotting, wehave demonstrated the complete cross-reactivityof these antibodies with both forms of VTG. Eachof them is able to recognize each of the twoplasma VTGs.

With anti-VTG1, using photonic and electronimmunocytochemistry, we have confirmed on thisspecies that oocyte yolk accumulation is initiated



at OF Stage 3, as soon as the oocyte synthesis ofcortical alveoli occurs and lipid globules appear inthe ooplasm. On this species, as for numerousprevious ones, we have seen that initiation ofoocyte vitellogenesis is concomitant with firstdeposition of the zona radiata.

Using one or the other antibody, we havedeveloped specific ELISAs with high sensitivityallowing in the plasma the detection of the VTGsas a whole. We have used the ELISA to follow thevariation of plasma VTG during wild femalesexual cycle and demonstrated that VTG profilewas perfectly correlated with the evolution ofthe GSI, with a pause during the maternal MGand guard.

We demonstrated that using O. niloticus VTG ascoating and O. niloticus anti-VTG as antibody,this ELISA might be used for several other relatedspecies to quantify (O. aureus and S. melanother-on) or detect (H. fasciatus, H. bimaculatus andT. zillii) plasma VTG. These heterologous ELISAsconstitute a reliable tool to monitor the presenceof xeno-estrogens in the environment of thesefish species. Indeed, recent studies show thatO. niloticus (Leanos-Castaneda et al., 2002; Par-ente et al., 2004) as well as other Cichlidae(Okoumassoun et al., 2002) may be submitted toxenobiotic aggressions in their natural habitat asknown for many other fish species (Sumpter, ’98;Kime et al., ’99; Hutchinson and Pickford, 2002).

ACKNOWLEDGMENTS

We wish to thank Pr. Patrick Babin (Labor-atoire de Genomique et Physiologie des Poissons,Universite de Bordeaux I) for critical readingof the manuscript and for granting access to hislab, Christophe Cochet from the IRD-GAMET forhelp with VTG induction and Pr. Patrick Cottin(ISTAB, Universite de Bordeaux I) for helpfuladvices on chromatrography.

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Tilapia (Oreochromis niloticus) vitellogenins: development of homologous and heterologous ELISAs and analysis of vitellogenin pathway through the ovarian follicle