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An in vitro vitellogenin bioassay for oestrogenic substances in the medaka (Oryzias latipes)
Aquatic Toxicology 58 (2002) 151– 164
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An in vitro vitellogenin bioassay for oestrogenic substances in the medaka (Oryzias latipes) C. Kordes a,*, E.P. Rieber b, H.O. Gutzeit a a b
Institut fu¨r Zoologie, Technische Uni6ersita¨t Dresden, Mommsenstraße 13, 01062 Dresden, Germany Institut fu¨r Immunologie, Technische Uni6ersita¨t Dresden, Fetscherstraße 74, 01307 Dresden, Germany Received 12 March 2001; received in revised form 19 July 2001; accepted 19 July 2001
Abstract We developed an in vitro bioassay for oestrogenic compounds based on vitellogenin as a biomarker. To quantify the induction of vitellogenin in medaka (Oryzias latipes) we established a sandwich enzyme-linked immunosorbent assay (ELISA). This ELISA was carried out with two different monoclonal antibodies specific for the yolk precursor protein vitellogenin and the high molecular weight yolk proteins (lipovitellin) of medaka. Purified lipovitellin of medaka was used as a standard for the ELISA and could be quantified down to a concentration of 9 ng/ml. The calibration curve was linear up to at least 600 ng/ml and the intra-assay coefficient of variance was 3.2%. The application of the sandwich ELISA was tested using blood samples of males and females as well as primary hepatocyte cultures of medaka. Vitellogenin synthesis in cultured liver cells of males was induced by 1 and 100 nM of the xenoestrogen 17a-ethynylestradiol (EE2). The first production of vitellogenin was detected 6 days after hormone application. In contrast, isolated liver cells of sexually mature females, which were treated in the same manner, showed vitellogenin expression from the beginning of cultivation and its synthesis increased and remained high at 100 nM EE2. However, the induction of vitellogenin synthesis in male hepatocytes in vitro could be maintained for at least one month and this indicated viable and differentiated liver cells. The hepatocyte cultures of male medaka in combination with the sandwich ELISA were shown to be a suitable tool to detect and quantify oestrogenic activity of chemicals. © 2002 Elsevier Science B.V. All rights reserved. Keywords: ELISA; Vitellogenin; Lipovitellin; Medaka; Hepatocytes; Ethynylestradiol
1. Introduction In the last decade much effort has been made to identify anthropogenic substances in the environment which may interfere with the hormonal sys* Corresponding author. Tel.: + 49-351-463-7536; fax: + 49-351-463-7093. E-mail address: [email protected] (C. Kordes).
tem of humans or animals. Numerous xenobiotics have been discovered which act as endocrine disruptors with oestrogenic properties (McLachlan and Arnold, 1996). The chemical structure of these compounds differ widely from natural hormones as illustrated by chemicals with oestrogenic activity like organochlorine pesticides, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, polychlorinated dibenzodioxins, and
0166-445X/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 4 5 X ( 0 1 ) 0 0 2 2 7 - 2
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alkylphenolic compounds (Sumpter et al., 1996). A well-accepted biomarker for oestrogenic compounds in fish is the detection of the yolk precursor protein vitellogenin in male animals (Sumpter and Jobling, 1995; Kime et al., 1999). This phospholipoglycoprotein is produced in the liver of females before the spawning period primarily under control of the natural occurring steroid hormone 17b-estradiol (E2) (Campbell and Idler, 1980; Ng and Idler, 1983). The blood stream transports vitellogenin to the follicles in the ovary, where the protein is taken up by a receptor-mediated process (Tyler and Lancaster, 1993). In the oocytes, vitellogenin is enzymatically cleaved into lipovitellin(s) and phosvitin(s) (Wallace, 1985; Callard and Ho, 1986). These components constitute major parts of the yolk and provide nutrients for the developing offspring. Fish are directly exposed to oestrogenic compounds derived from natural sources or sewage effluents. In several studies, the oestrogenic properties of chemicals were investigated using vitellogenin expression in male fish as a biomarker. Fish species like rainbow trout (Oncorhynchus mykiss) (Jobling and Sumpter, 1993; Pelissero et al., 1993), roach (Rutilus rutilus) (Routledge et al., 1998), fathead minnow (Pimephales promelas) (Giesy et al., 2000), sheepshead minnow (Cyprinodon 6ariegatus) (Folmar et al., 2000), Atlantic salmon (Salmo salar) (Arukwe et al., 2000) and flounder (Platichthys flesus) were used in these studies. The suitability of vitellogenin induction in male fish as a tool to detect the presence of oestrogenic substances in the environment was shown in field studies by Lye et al. (1997) and Allen et al. (1999) with flounder. The small freshwater teleost medaka (Oryzias latipes) was also used to test effects of xenoestrogens on vitellogenesis (Wester and Canton, 1986; Gronen et al., 1999). However, there is no quantitative vitellogenin assay for this species. Medaka is widely used in toxicological and developmental studies and offers many advantages as a model organism. This species exhibit sexual dimorphism, a short generation time, tolerate simple rearing conditions, and the breeding season can be prolonged artificially by altering the light/dark cycle (Yamamoto, 1975). Finally vitellogenin and yolk
proteins of medaka were identified and characterized by Hamazaki et al. (1987) and Murakami et al. (1991). To use the induction of a well-established biomarker for oestrogenic substances in fish like vitellogenin in combination with the advantages offered by the medaka as a model organism, we developed an in vitro bioassay based on primary hepatocyte culture of this species.
2. Materials and methods
2.1. Fish Sexually mature fish of the genus Oryzias latipes (Temminck and Schlegel, 1846) of approximately 3 cm body length were taken for this study from our breeding stock. The fish (strain d-rR with orange red males and colourless females) were reared at 25–27 °C under prolonged daylight (16 h light and 8 h darkness). For further details concerning the rearing conditions see Scholz and Gutzeit (2000).
2.2. Purification of 6itellin Freshly spawned eggs (1 h after spawning) of medaka were collected, counted, and pooled in Eppendorf tubes. Tris/EDTA buffer pH 7.2 (20 mM Tris and 40 mM ethylenediamine tetraacetic acid, Sigma) according to Hamazaki et al. (1987) was added (10 ml/egg). The eggs were homogenized with a pestle and centrifuged at 10 000g for 5 min at 4 °C. The supernatant was collected, sterile filtered through a gauze (pore size 0.2 mm, Schleicher and Schu¨ ll), and stored at − 20 °C. Yolk proteins were purified at 4 °C in two successive steps by medium pressure liquid chromatography (MPLC, Biologic-HR Workstation, Bio-Rad). First the proteins (1 mg per run) were separated at a flow rate of 0.5 ml/min in Tris/ EDTA buffer (see above) using a gel filtration column (Superdex 200 HR 10/30, Amersham Pharmacia Biotech). The eluent was collected in 0.5 ml fractions. The molecular weight of the collected yolk proteins was determined using catalase (232 kDa, Amersham Pharmacia Biotech),
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aldolase (158 kD, Amersham Pharmacia Biotech), and bovine serum albumin (67 kDa, Merck) as reference proteins in separate runs under equal conditions. In a second purification step 1 ml of fractionated vitellin in Tris/EDTA buffer was loaded onto an anion-exchange column (Bio-Scale Q2, Bio-Rad). Bound proteins were eluted using a gradient of ionic strength (0–1 M NaCl in Tris/ EDTA; at a flow rate of 1 ml/min) according to Hamazaki et al. (1987). The eluted proteins were collected in 0.5 ml fractions and the protein concentrations were determined according to Bradford (1976) using bovine serum albumin (Merck) as a standard.
2.3. Gel electrophoresis and Western blot The protein samples were separated by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Native and SDS gels were divided in a stacking gel (2.5% (v/v) 30% acrylamide and 0.8% bisacrylamide, Roth) and a running gel (12% (v/v) acrylamide/bisacrylamide). The preparations of gels, electrode buffer, and sample buffer were prepared essentially as described by Laemmli (1970). For electrophoresis under native conditions, sodium dodecyl sulphate (SDS, Sigma), b-mercaptoethanol (Sigma), and boiling of the samples were omitted. Electrophoresis was carried out at 15 mA per gel over 2–4 h for SDS gels and 6– 7 h for native gels using a power supply (P25) and a gel casting tray of Biometra. Myosin (205 kDa, Sigma) and bgalactosidase (116 kDa, Sigma) were used to determine the molecular weights of the proteins of interest in SDS gels. Following electrophoretic separation the proteins were stained with Coomassie Brilliant Blue R 250 (Sigma) as described by Holtzhauer (1997). For a differential staining of lipophilic proteins, the gels were coloured with Sudan Black B (Sigma) according to Maurer (1971). Unstained gels were placed on nitrocellulose membrane (pore size 0.2 mm, Sartorius) for the preparation of Western blots (Burnette, 1981). The proteins were transferred to the membrane by the wet blot technique at 10 V overnight using a vertical blotting apparatus (BioRad). The immunoblotting was carried out based
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on Beisiegel et al. (1982) and Beisiegel (1986). In our experiments we used 5% (w/v) milk powder (fat free) dissolved in phosphate buffered saline (PBS) and 0.05% (v/v) Tween 20 (Sigma) as a blocking reagent. Culture supernatant with monoclonal antibodies was diluted 1:100 for the antigen detection. A rabbit anti-mouse antibody coupled with horseradish peroxidase (HRP, Dako) was used to label bound immunoglobulins in a final dilution of 1:1000. All of these incubation steps were carried out with gentle shaking for 1 h at room temperature. A substrate solution containing 5 mM 4-chloro-1-naphthol (Sigma), 16.7% (v/ v) methanol, and 0.1% (v/v) hydrogen peroxide in PBS was used for staining protein bands of the antigen.
2.4. Production of monoclonal antibodies Blood samples were collected from two mice of the strain Balb/c x Black 57, diluted 1:10 in PBS, and tested with the indirect ELISA (see below) for reactions with lipovitellin of medaka. These mice showed no reactions and were immunized three times with a total of 220 mg purified lipovitellin during a period of 7 weeks. The antigen was diluted in PBS and mixed with complete Freund’s adjuvants (Gibco) for the priming. Incomplete Freund’s adjuvants (Gibco) was used for the following two boosts with antigen. The increase of antibody concentration in blood serum of mice was controlled prior to each immunization by indirect ELISA (dilution of serum from 1:10 to 1:4000 in PBS). Three days after the last immunization one mouse was killed by injection of 500 ml Trapanal (BYK). Spleen cells were prepared and fused with cells from the myeloma cell line X63 (Ag8.653) essentially as described before (Rieber et al., 1981). Immunoglobulin synthesis in the supernatants of hybridoma cultures was measured with the indirect ELISA. Clones with a high production of antibodies were selected, subcloned, and the cultures expanded. The clones were raised in miniPERM systems (MWCO 12.5 kDa, Vivascience Sartorius) to obtain high antibody concentrations as described by Falkenberg et al. (1995). The antibody subtype of selected clones was de-
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termined by means of a mouse monoclonal antibody isotyping reagent (ISO-2, Sigma) and a rabbit anti-mouse antibody coupled with horseradish peroxidase (HRP, Sigma) in a sandwich ELISA according to manufacturer’s instructions.
2.5. Screening for different epitopes Egg proteins of medaka were digested using the protease V8 (Sigma) of the bacterium Staphylococcus aureus (strain V8; Drapeau, 1976, 1977) to obtain protein fragments for the analysis of antibody binding properties. About 45 mg egg protein were loaded onto an SDS gel and electrophoresis was carried out for a few minutes at an amperage of 15 mA per gel until the egg proteins had just migrated into the stacking gel. The current was switched off and 1 ng of the protease V8 in 1 M Tris buffer (pH 6.8) containing 3 mM SDS, 1 mM EDTA, and 10% (v/v) glycerol was added to the egg protein. The current was then switched on again. Just before the colour front had reached the edge to the SDS running gel (12% (v/v) acrylamide/bisacrylamide), the current was switched off for 30 min to allow protease digestion of the egg proteins. The current was then switched on again for about 2 h. After electrophoresis an immunoblot was carried out using different monoclonal antibodies.
2.6. Purification and biotinylation of monoclonal antibodies Monoclonal antibodies were purified by MPLC at 4 °C based on Ey et al. (1978) using a HighTrap column (1 ml, Amersham Pharmacia Biotech) filled with r-protein A from Staphylococcus aureus. The column was equilibrated with sterile 0.1 M phosphate buffer (pH 7.6) containing 10 mM sodium azide. Culture supernatant (250 ml per run) was loaded onto the column at a rate of 0.2 ml/min. Unbound protein was eluted with 5 ml 1 M phosphate buffer (pH 7.6). Bound immunoglobulins were dissociated from r-protein A by a pH gradient from pH 7.6 to 3.0 (0.1 M citrate buffer with 10 mM sodium azide) at a rate of 0.4 ml/min. The antibodies were collected in
Eppendorf tubes (1 ml fractions) filled with 200 ml 1 M Tris buffer (pH 9). Finally the immunoglobulins were concentrated and the buffer was exchanged to PBS by centrifugation using Centrisart-C4 (MWCO 20 kDa, Sartorius) at 10 000g and 4 °C. Purified monoclonal antibodies were covalently coupled with biotin-7-NHS using a biotin labelling kit (Roche). The biotinylated antibodies were separated from unbound biotin-7-NHS using a Sephadex G-25 column (Roche).
2.7. ELISA procedures 2.7.1. Indirect ELISA Purified lipovitellin of medaka was diluted with coating buffer (50 mM sodium bicarbonate, pH 9.5) to a final concentration of 3 mg/ml. A volume of 100 ml per well of this antigen solution was pipetted into a microtiter plate (96-well culture plate, Costar). The wells were coated with antigen overnight at 4 °C. The plates were washed with PBS containing 0.05% (v/v) Tween 20 (200 ml/ well). Free binding sites in the wells were blocked for 1 h with 5% (w/v) milk powder (fat free) in PBS –Tween (200 ml/well). The microtiter plates were washed again and coated with antibodies against lipovitellin of medaka (100 ml/well) for 30 min. The plates were washed and a rabbit antimouse antibody coupled with HRP (Dako) in a final dilution of 1:1000 in PBS was added (100 ml/well) for 30 min. After a final washing step, o-phenylene diamine (citrate buffer tablets containing OPD from Dako and 0.04% (v/v) hydrogen peroxide) was pipetted into the wells (100 ml/well). The enzyme reaction was stopped after 15 min with 0.5 M sulphuric acid (100 ml/well). The optical density was measured at 490 nm (630 nm as a reference wave length) with a microtiter plate reader (Rainbow, Tecan). 2.7.2. Sandwich ELISA Purified monoclonal antibodies of the clone 1H11 were diluted in PBS to a final concentration of 5 mg/ml. High adsorption microtiter plates (Maxisorp, 96-well, Nunc) were coated with this antibody solution (50 ml/well) overnight at 4 °C. All incubation steps were carried out in a humi-
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dified chamber. The plates were washed with PBS containing 0.05% Tween 20 (300 ml/well) and blocked for 1 h with milk powder solution (300 ml/well). After a second washing step, reference protein (lipovitellin), blood samples or culture supernatants of oestrogen treated hepatocytes (50 ml/well) were incubated for 1 h. The incubation was stopped by washing. A second monoclonal antibody of the clone 2A9 coupled with biotin (1 mg/ml PBS –Tween) was added (50 ml/well) for 1 h. The plates were washed and streptavidin peroxidase (Dianova) was diluted in PBS– Tween to a final concentration of 1 mg/ml. The peroxidase was pipetted into the wells (50 ml/well) and incubated for 1 h. After a final washing step, the subtrate OPD (50 ml/well) was given into the wells for 10 min as described for the indirect ELISA. The enzyme reaction was stopped with 0.5 M sulphuric acid (50 ml/well) and the optical density was measured using the same conditions as mentioned above.
2.8. Preparation of blood probes and organs The fish were anaesthetized and killed with a saturated solution of benzocaine salt (\ 0.04% (w/v) ethyl 4-aminobenzoate, Fluka). An incision was made with a scalpel at the level of the anal fin to sever the Arteria caudalis. Blood was collected with glass capillary tubes (Minicaps, 10 ml, Hirschmann) and added to 20 ml PBS containing 2% (v/v) Liquemin N 25000 (sodium-heparin, AWD) and 67 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF, Sigma). Livers were removed with watchmaker forceps after ventral dissection of the fish, placed into 50 ml PBS, and squashed with a pestle. These samples were centrifuged at 10 000g for 5 min at 4 °C and the supernatants were used for Western blot analysis.
2.9. Primary culture of hepatocytes and application of 17h-ethynylestradiol Livers of several fish were placed in a sterile sieve (pore size 100 mm), washed in sterile PBS, and incubated with 0.05% (w/v) collagenase for 30 min as described by Cao et al. (1996). After enzymatic dissociation with collagenase H
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(Roche) dissolved in DMEM/F12 medium (Dulbecco’s modified Eagle’s medium, nutrient mixture F12, Gibco) the tissues were pressed with the blunt end of a syringe through the sieve. The cell lumps were pipetted several times and filtered through a second sieve (pore size 50 mm). The cells were centrifuged for 3 min at 59g, re-suspended in a defined volume of medium and counted with a hemocytometer (Neubauer). The hepatocytes were cultivated in a 24-well plate (Primaria, Falcon) at a density of approximately 2.5× 105 viable cells per well. Medium (DMEM/ F12 containing L-glutamine, 15 mM HEPES, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mg/ml gentamycin, Gibco; 2% (v/v) Ultroser SF (steroid free), Biosepra) was prepared based on Flouriot et al. (1993) and pipetted to the hepatocytes (1 ml/ well). Half of the volume was changed daily. The cells were cultivated at 25 °C in humidified atmosphere with 5% carbon dioxide. Cell culture media containing 100 and 1 nM 17a-ethynylestradiol (EE2, Merck) were prepared from a stock solution of 1 mM EE2 in dimethyl sulphoxide (DMSO, Sigma). Pure DMSO dissolved in medium (1 ml DMSO per 10 ml medium) was used for control experiments. These experimental media were added to the primary culture after the first day of culture (i.e. after cell attachment).
2.10. Histology and electron microscopy The carcasses (without head and tail) of medaka were fixed with 4% (v/v) formalin in phosphate buffer pH 5.5 (0.28 M sodium dihydrogen phosphate and 0.54 M disodium hydrogen phosphate) for 7 days. Paraffin sections (thickness 3 mm) were made as described by Scholz and Gutzeit (2000). After hydration of the sections, the endogenous peroxidase activity was blocked by treatment with 3% (v/v) hydrogen peroxide in PBS for 10 min. Then the sections were washed in PBS with 0.05% (v/v) Tween 20 and incubated in 5% (w/v) milk powder in PBS–Tween for 1 h. Culture supernatant with monoclonal antibodies was diluted in milk powder solution (1:10) and the sections were incubated for 1 h. They were washed and a rabbit anti-mouse antibody coupled
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with HRP (Dako) in a final dilution of 1:200 in milk powder solution was added. The tissue sections were washed again and incubated in a substrate solution containing 0.8 mM 3,3%-diaminobenzidine tetrahydrochloride dihydrate (DAB, Fluka) and 0.1% (v/v) hydrogen peroxide in PBS. The enzyme reaction was stopped after a few minutes by washing with PBS. The sections were sealed with Aquatex (Merck) and a cover slip. Cells of primary cultures were fixed for scanning and transmission electron microscopy with 2% (v/v) glutaraldehyde (Sigma) and 1% (v/v) osmiumtetroxide (Sigma) in 0.1 M sodium cacodylate buffer (TAAB) for 40 min. For scanning electron microscopy (LEO 420) pieces of the culture dish with attached cells were dehydrated with a graded series of ethanol, critical-point-dried according to Anderson (1951), and coated with gold. Single cell aggregates were orientated in 2% (w/v) agar-agar (Roth), dehydrated with ethanol, embedded in LR-White resin (Sigma), and processed for transmission electron microscopy (EM 208, Philips) as described by Fleig et al. (1991).
2.11. Statistics Student’s distribution was used to identify the lower limit of sensitivity of the sandwich ELISA. The analysis of variance (ANOVA, followed by least significant differences tests, P B0.05) was calculated to define the working range of the assay. The program WinStat (version 1999.2) was used for statistical calculations.
3. Results
3.1. Vitellin preparation Two proteins, which form major components of freshly spawned eggs, were purified by gel filtration and anion-exchange chromatography. Their molecular weights of approximately 220 and 180 kDa were determined by gel filtration chromatography (Fig. 1A; L1 and L2). For comparison, Fig. 1B (lanes 1 and 2) shows unpurified and purified egg proteins in native form. The proteins were
stained with Coomassie Brilliant Blue R 250 (Fig. 1B) or with the lipophilic dye Sudan Black B (not shown). The isolated proteins (Fig. 1B; lane 2) exhibited the same molecular weights and lipophilic quality as lipovitellin(s) of medaka (Murakami et al., 1991). These two yolk proteins were used together for the immunization of mice and served as reference proteins of the indirect and sandwich ELISA.
3.2. Production and specificity of monoclonal antibodies After fusion of lymphocytes with myeloma cells many wells with hybridoma cells showed antibody production against lipovitellin of medaka in the indirect ELISA. We selected five hybridoma clones (1H11, 2A9, 6G6, 4B3, and 5C4) which showed stable and high antibody production. The subtype of the immunoglobulins was determined to be IgG1 in all selected clones. The antibodies of these clones were tested in Western blots of blood and liver samples of sexually mature male and female medaka. The protein samples were separated by SDS and native gel electrophoresis. After native gel electrophoresis, the immunoglobulins recognized a single protein band in blood and liver samples of vitellogenic female medaka, whereas no binding was observed in samples of male medaka (Scholz et al., 1999). Following SDS gel electrophoresis, the antibodies bound to a dominant protein band with a molecular weight of approximately 200 kDa in blood samples of vitellogenic females (Fig. 2; lanes 2 and 5). Hamazaki et al. (1987) reported a molecular weight of 200 kDa for a major protein subunit of medaka vitellogenin under denaturing conditions and these authors determined a molecular weight of 420 kDa for the native form of vitellogenin. In our Western blot experiments, no binding of the monoclonal antibodies was observed when blood samples of male fish were tested (Fig. 2; lane 6). However, there was a clear reaction of the monoclonal antibodies with purified lipovitellin (Fig. 1A; lane 3) and unpurified yolk proteins of medaka eggs (Fig. 2; lane 4), which derived from vitellogenin. The specificity of antibody binding was confirmed using paraffin sections of mature
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female and male medaka (for both sexes n = 3). Vitellogenic oocytes of the ovaries were labelled as well as the capillaries and sinusoids of the female liver (Fig. 3A– C), whereas paraffin sections of male medaka (Fig. 3D) showed no immunohistochemical reaction. Finally, the production of this antigen was induced after oestrogen treatment of hepatocytes in vitro (see below). Taking together, these observations give strong evidence that we raised monoclonal antibodies against vitellogenin of Oryzias latipes.
3.3. Epitope-screening It was necessary to determine the binding properties of the immunoglobulins because a sandwich ELISA required two specific antibodies directed against two different epitopes of the same antigen.
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Therefore, egg proteins were digested by the protease V8 and the resulting protein fragments were separated by SDS-PAGE. In Western blots we found that the clones 2A9, 6G6, and 4B3 show approximately the same binding pattern, while the clones 1H11 and 5C4 had different specificities. Finally, two clones were chosen to produce antibodies against diverse epitopes of medaka lipovitellin and vitellogenin to construct a sandwich ELISA. Purified immunoglobulins of the clone 1H11 were taken to capture antigen and antibodies of the clone 2A9 were biotinylated to label captured protein in the sandwich ELISA.
3.4. Properties of the sandwich ELISA The sandwich ELISA was performed and optimized using purified lipovitellin of medaka (Fig.
Fig. 1. (A) MPLC analysis of medaka yolk proteins (1 mg) produced by gel filtration (Superdex 200 HR 10/30 column; Amersham Pharmacia Biotech). Two lipovitellin(s) with a molecular weight of approximately 220 kDa (L1) and 180 kDa (L2) were major protein components of the medaka eggs. Phosvitin(s) (P) have a low molecular weight (Murakami et al., 1991). (B) Whole yolk (lane 1; 10 mg) and purified lipovitellin(s) (lanes 2 and 3; 1 mg) were separated by native gel electrophoresis (12% acrylamide). The proteins in lanes 1 and 2 were stained with Coomassie Brilliant Blue R 250. Phosvitin(s) (arrowhead) stain weakly with this dye. The two large molecular weight bands above L1 (B, lane 1) are represented in (A) left of and partly overlapping with peak L1 (retention time between 20 and 25 min) as determined by native gel electrophoresis of the collected protein fractions. The dominat proteins of the peaks L1 and L2 are lipovitellin(s). Lane 3 shows a Western blot of these purified lipovitellin(s). Monoclonal antibodies of the clone 1H11 were used to identify the lipovitellin(s). The biotinylated immunoglobulins of the clone 2A9 exhibited the same binding pattern in Western blot analysis of purified lipovitellin (not shown).
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Fig. 2. Yolk proteins (lanes 1 and 4; 24 mg) and blood serum of vitellogenic females (lanes 2 and 5; 3 mg) and sexually mature males (lanes 3 and 6; 3 mg) of the species Oryzias latipes were separated by SDS-PAGE (10% acrylamide). One part of the gel was stained with Coomassie Brilliant Blue R 250 (lanes M, 1, 2 and 3) and with the other part a Western blot was performed (lanes 4, 5 and 6). Lane 2 shows a female-specific protein band in the blood serum with a molecular weight of about 200 kDa, which was detected by the antibodies of the clone 1H11 (lane 5). Myosin (205 kDa, Sigma) and b-galactosidase (116 kDa, Sigma) were used as molecular weight markers in lane M.
1A; lane 2) as reference protein. The lipovitellin concentration of 9 ng/ml was significantly above background (n = 6, P B0.001) and hence defined as the lower detection limit while the working range of the assay started at approximately 20 ng/ml (as determined by ANOVA, n = 6, P B 0.05). The standard curve of the sandwich ELISA was linear up to 600 ng/ml (Fig. 4). The intra-assay coefficient of variation was 3.2% (SD9 0.01; n = 9) and the inter-assay coefficient of variation was 17% (SD90.07; n =9). These parameters were defined by Specker and Anderson (1994).
Fig. 3. Immunohistochemical detection of lipovitellin(s) and vitellogenin with antibodies of the clone 6G6 on paraffin sections of medaka. The antibodies of the clones 2A9 and 4B3 displayed similar binding patterns on paraffin sections. (A) Ovary of mature female with non-vitellogenic (centre) and vitellogenic oocytes (arrows show binding of antibodies at yolk proteins); (B) vitellogenic oocyte (antibody labelling of yolk proteins); (C) liver of mature female (arrows show binding of antibodies in the capillaries and sinusoids); (D) liver of mature male without antibody labelling.
Fig. 3.
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Fig. 4. Calibration curve of the sandwich ELISA based on lipovitellin of medaka (dots show the arithmetic mean; bars show the standard deviation; r 2 = goodness-of-fit; cv = coefficient of variation; n =number of values per concentration).
The application for sandwich ELISA was tested with blood serum of mature females and males of Oryzias latipes. Blood samples of females were diluted in PBS (0.5– 1.5 nl blood serum per well) so that the vitellogenin concentration fell into the linear range of the test system. The concentrations of vitellogenin (expressed in vitellin units) ranged from 125 to 286 mg/ml blood serum (n = 5). Vitellogenin was not detected in blood samples of male medaka (n= 5, 410– 650 nl blood serum per well) by sandwich ELISA.
3.5. Primary culture of hepatocytes The isolated liver cells formed small aggregates (Fig. 5A) which attached to the culture dish during the first 24 h. At least three different cell types could be distinguished when cultivated hepatic cells were analysed by scanning electron microscopy. Spherical hepatocyte-like cells (Fig. 5B), flat fibroblastlike cells (Fig. 5C), and some erythrocytes could be identified. The erythrocytes disappeared within the first week of culture, whereas the number of fibroblast-like cells increased significantly during the culture period. They covered the plate surface between the hepatic cell aggregates. The islets of cell aggregates increased in size during cultivation. After about 20 days of culture, the aggregates
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exhibited flat and in some cases spherical organ-like shapes (Fig. 5D). The cultured hepatocytes remained viable and differentiated for at least one month as determined by their morphology and vitellogenin synthesis (see below). After 27 days of culture organ-like aggregates with diameters of about 500 mm were analysed by transmission electron microscopy. Intact hepatocytes were observed in the centre of these cell aggregates and their ultrastructure displayed typical cell organelles (i.e. endoplasmic reticulum, mitochondria, nucleus, nucleolus, and peroxisomes) and microvilli (Fig. 5E). Furthermore, the hepatocytes reconstructed bile canaliculi with microvilli (Fig. 5F) and formed extensive inter-cellular spaces. Also flat endothelial-like cells were found between the hepatocytes. Fibroblast-like cells were seen in several layers on the surface of the cell aggregates.
3.6. Induction of 6itellogenin in primary culture of hepatocytes The production of vitellogenin was induced in primary cultures of male hepatocytes with 1 and 100 nM 17a-ethynylestradiol (EE2) and there was no induction found in the control group without EE2 (Fig. 6). Vitellogenin synthesis was first detected 6 days after exposure to 100 nM EE2 and the concentration of vitellogenin in the medium rose up to day 17 of culture and then decreased. The concentration of 1 nM EE2 induced consistently smaller amounts of vitellogenin. The increasing standard deviations at 100 nM EE2 in Fig. 6 was mainly due to the drastic decrease of vitellogenin synthesis in one well with hepatocytes at day 21 of culture. The absolute vitellogenin amount in each well was dependent on the frequency of medium exchange (Fig. 6; change of 50% of the medium daily). Higher vitellogenin concentrations were obtained, when 50% of the medium was changed every second day. The observed lag-phase in vitellogenin detection was not affected by this alteration. This experiment was also performed with hepatocytes of males, which were cultured in medium containing 10% foetal calf serum (FCS) instead of 2% Ultroser SF (steroid free). In this case the induction of vitellogenin showed the same pattern
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Fig. 5.
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tions declined to 30 ng/ml (vitellin units) after 14 days. The concentration of 1 nM EE2 was not sufficient to maintain the high vitellogenin synthesis of female hepatocytes in this experiment.
4. Discussion
Fig. 6. Induction of vitellogenin with 1 and 100 nM 17aethynylestradiol in primary cultures of hepatocytes of male medaka. The cells were cultured with DMEM/F12 supplemented with 2% Ultroser SF (steroid free). Vitellogenin was measured by sandwich ELISA (symbols show the arithmetic mean of triplicates and bars represent the standard deviations).
as described above, but the production of vitellogenin was about 3-fold (1 nM EE2) and 5-fold (100 nM EE2) higher than in medium supplemented with Ultroser SF. This indicated that additional factors of the FCS are able to enhance vitellogenin synthesis. Because of the defined composition we preferred Ultroser SF in vitellogenin induction experiments. Hepatic cells of mature females were cultivated in medium containing 2% Ultroser SF and treated with 1 and 100 nM EE2 in order to test, if the observed delay in vitellogenin expression was due to our cell isolation procedure or culture conditions. In this experiment vitellogenin was detected from the beginning of cultivation at both concentrations of EE2 as well as in the control group. When exposed to 100 nM EE2, the vitellogenin concentrations increased from about 12 000 up to 20 000 ng/ml (vitellin units), while cultivation with 1 nM EE2 and in the control group the concentra-
The female-specific protein vitellogenin is a well accepted biomarker for oestrogenic substances in male teleost oviparous fish. To produce antibodies against vitellogenin of medaka (Oryzias latipes), a source of this protein as antigen was required for immunization of mice and as a reference protein in a quantitative assay. Unfortunately medaka is to small to get sufficient amounts of blood (1–7 ml per fish) to purify vitellogenin. Furthermore, we want to avoid the production of vitellogenin in ascites of male fish by 17b-estradiol (E2) treatment as reported by Hamazaki et al. (1987). Therefore, we relied on the structural similarities of vitellogenin and its lipovitellin derivative. This antigen source was taken to generate monoclonal antibodies against vitellogenin of medaka as outlined by Scholz et al. (1999). Purified lipovitellin was also used to establish an ELISA so that a highthroughput method for the analysis of oestrogenic chemicals becomes available. Since the first ELISA was published by Engvall and Perlmann (1971), this sensitive method is widely used in laboratories to quantify antigens. We decided to construct a sandwich ELISA based on two different monoclonal antibodies to improve specificity and sensitivity of the assay. The usage of lipovitellin from medaka as a reference protein in our ELISA was found to be a suitable alternative to vitellogenin. There was no evidence that lipovitellin limits the application of the sandwich ELISA for the identification of oestrogenic substances. Our assay was as sensitive as competitive ELISAs established for vitellogenin of striped
Fig. 5. Primary culture of medaka hepatocytes (pictures A –D were made by scanning electron microscope and pictures E and F by transmission electron microscope). (A) Aggregates of hepatocytes (bright clusters) at day 5 of culture; (B) aggregate of hepatocytes at day 5 of culture in detail; (C) flat fibroblast-like cell at day 5 of culture; (D) part of a large cell aggregate at day 27 of culture; (E) hepatocytes in the centre of an aggregate at day 27 of culture (cm = cell membrane, en =endothelium-like cells, er =endoplasmic reticulum, itc = inter-cellular space, mic = microvilli, mit = mitochondrion, nu = nucleus, lip =lipid vacuoles); (F) bile canaliculus with microvilli (mic) at day 27 of culture.
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bass (Morone saxatilis) (Kishida et al., 1992), English sole (Pleuronectes 6etulus) (Lomax et al., 1998), rainbow trout (Bon et al., 1997) or brown trout (Salmo trutta) (Sherry et al., 1999), which were based on polyclonal antibodies. Monoclonal antibodies, however, have the advantage that large quantities of immunoglobulins with strictly defined binding properties can be produced and hence standardized test systems can be established. To provide evidence that substances of interest are able to induce vitellogenin synthesis in male medaka and therefore show oestrogenic properties, we chose an in vitro test system based on primary culture of male hepatocytes. For critical validation of the sensitivity and reliability of this test system, we used the environmental xenoestrogen 17a-ethynylestradiol (EE2). This potent anthropogenic oestrogen from contraceptive pills was detected in sewage water in concentrations up to 7 ng/l (Desbrow et al., 1998; Larsson et al., 1999). Ju¨ rgens and Johnson (1999) reported that EE2 is about ten times more stable than E2. Therefore, it is important to investigate the effects of this synthetic oestrogen on fish despite its low concentration. In our in vitro test system 1 and 100 nM EE2 induced vitellogenin synthesis in hepatocytes of mature male medaka for at least one month. This fact as well as the intact cell morphology assured us of the validity of this in vitro bioassay. The long-term vitellogenin synthesis was also found in cultured hepatocytes of other species like rainbow trout, which were treated with oestrogens (Flouriot et al., 1993). Also the behaviour of hepatic cells after isolation and during cultivation (i.e. attachment and aggregate formation) in our assay seemed to be very similar to the in vitro system of rainbow trout (Flouriot et al., 1993). However, the delay in induction of vitellogenin synthesis in male hepatocytes of medaka in vitro was remarkable. The earliest vitellogenin production was detected 6 days after EE2 exposure. This lag-phase was presumably due to an oestrogen-dependent induction and up-regulation of oestrogen receptor (ER) in hepatic cells. As
described by Mommsen and Lazier (1986), the treatment of cultured hepatocytes of male salmon (Salmo salar) with E2 resulted in an increase in ER expression. Flouriot et al. (1996) also found in E2-stimulated hepatic cells of rainbow trout cultured in vitro an increase of ER followed by vitellogenin induction. In addition, a delayed proliferation of isolated liver cells, which starts after day 6 of culture according to Cao et al. (1996), might have contributed to the late vitellogenin detection in our experiments. Recovery from the cell isolation procedure seemed to be of minor importance for the observed delay in vitellogenin synthesis, because the production of the yolk precursor protein started immediately, increased, and remained high, when liver cells of mature females were isolated and treated with EE2. Hamazaki et al. (1987) reported that vitellogenin became detectable in blood serum of adult non-spawning female and male medaka after 3–4 days of oestrogen treatment (food dusted with 1 mg E2). Apparently, the delay reflects a biological process that can be observed in vivo and in vitro. However, the induction of vitellogenin synthesis in male hepatocytes was confirmed to be a useful tool to investigate oestrogenic effects of chemicals. This method was able to detect the presence of low concentrations of 17aethynylestradiol. In addition, the long-term production of this yolk precursor protein can also be used as an indicator for differentiated and viable fish hepatocytes in primary culture.
Acknowledgements We wish to thank Heidrun Gebauer and Christine Gra¨ fe for their help in the production of monoclonal antibodies. We are also grateful to Doris Zschernig for preparing paraffin sections and to Dr Richard Fleig for his support with the electron microscope techniques. We also thank Dr Stefan Scholz for helpful discussions and establishing our breeding stock of medaka. Finally, we acknowledge Nadja Bachmann for her support in maintaining our fish stock.
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Kishida, M., Anderson, T.R., Specker, J.L., 1992. Induction by b-estradiol of vitellogenin in striped bass (Morone saxatilis): characterization and quantification in plasma and mucus. Gen. Comp. Endocrinol. 88, 29 –39. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680 – 685. Larsson, D.G.J., Adolfson-Erici, M., Parkkonen, J., Pettersson, M., Berg, A.H., Olsson, P.-E., Fo¨ rlin, L., 1999. Ethynylestradiol—an undesired fish contraceptive? Aquat. Toxicol. 45, 91 –97. Lomax, D.P., Roubal, W.T., Moore, J.D., Johnson, L.L., 1998. An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole (Pleuronectes 6etulus): development, validation and cross-reactivity with other pleuronectids. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 121 (4), 425 –436. Lye, C.M., Frid, C.L.J., Gill, M.E., McCormick, D., 1997. Abnormalities in the reproductive health of flounder Platichthys flesus exposed to effluent from sewage treatment works. Mar. Pollut. Bull. 34, 34 –41. Maurer, H.R., 1971. Disc Electrophoresis. Walter de Gruyter, Berlin (Holtzhauer, M., 1997. Biochemische Labormethoden, 3rd ed. Springer, Berlin, p. 70). McLachlan, J.A., Arnold, S.F., 1996. Environmental estrogens. Am. Sci. 84, 452 –461. Mommsen, T.P., Lazier, C.B., 1986. Stimulation of estrogen receptor accumulation by estradiol in primary cultures of salmon hepatocytes. FEBS Lett. 195 (1/2), 269 –271. Murakami, M., Iuchi, I., Yamagami, K., 1991. Partial characterization and subunit analysis of major phosphoproteins of egg yolk in the fish, Oryzias latipes. Comp. Biochem. Physiol. B 100 (3), 587 –593. Ng, T.B., Idler, D.R., 1983. Yolk formation and differentiation in teleost fishes. In: Hoar, W.S., Randall, D.J., Donaldson, E.M. (Eds.), Fish Physiology, vol. IX. Academic Press, New York, pp. 373 –404. Pelissero, C., Flouriot, G., Foucher, J.L., Bennetau, B., Dunogues, J., Le Gac, F., Sumpter, J.P., 1993. Vitellogenin synthesis in cultured hepatocytes: an in vitro test for estrogenic potency of chemicals. J. Steroid Biochem. Mol. Biol. 44, 263 – 272. Rieber, E.P., Lohmeyer, J., Schendel, D.J., Riethmu¨ ller, G., 1981. Human T cell differentiation antigens characterizing a cytotoxic/suppressor T cell subset. Hybridoma 1, 59 –69.
Routledge, E.J., Sheahan, D., Desbrow, C., Brighty, G.C., Waldock, M., Sumpter, J.P., 1998. Identification of estrogenic chemicals in STW effluent. 2. In vivo responses in trout and roach. Environ. Sci. Technol. 32, 1559 – 1565. Scholz, S., Kordes, C., Rieber, E.P., Gutzeit, H.O., 1999. Molekulare Marker der Geschlechtsdifferenzierung des Medakas (Oryzias latipes): Methoden zur Untersuchung von o¨ strogenen Umweltchemikalien. In: Oehlmann, J., Markert, B. (Eds.), O8 kosystemare Ansa¨ tze in der O8 kotoxikologie. Ecomed, pp. 546 – 549. Scholz, S., Gutzeit, H.O., 2000. 17a-Ethynylestradiol affects reproduction, sexual differentiation and aromatase gene expression of the medaka (Oryzias latipes). Aquat. Toxicol. 50, 363 – 373. Sherry, J., Gamble, A., Fielden, M., Hodson, P., Burnison, B., Solomon, K., 1999. An ELISA for brown trout (Salmo trutta) vitellogenin and its use in bioassays for environmental estrogens. Sci. Total Environ. 225 (1/2), 13 – 31. Specker, J.L., Anderson, T.R., 1994. Developing an ELISA for a model protein — vitellogenin. Biochem. Mol. Biol. Fishes 3, 567 – 578. Sumpter, J.P., Jobling, S., 1995. Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. Environ. Health Perspect. 107, 173 – 178. Sumpter, J.P., Jobling, S., Tyler, C.R., 1996. Oestrogenic substances in the aquatic environment and their potential impact on animals, particularly in fish. In: Taylor, E.W. (Ed.), Toxicology of Aquatic Pollution: Physiological, Cellular and Molecular Approaches. Cambridge University Press, Cambridge, UK, pp. 205 – 224. Temminck, C.J., Schlegel, H., 1846. Siebold’s fauna Japonica. P. 224, Pl. 102, Fig. 5. Tyler, C.R., Lancaster, P., 1993. Isolation and characterization of the receptor for vitellogenin from follicles of the rainbow trout, Oncorhynchus mykiss. J. Comp. Physiol. B 163, 225 – 233. Wallace, R.A., 1985. Vitellogenesis and oocyte growth in non mammalian vertebrates. In: Browder, L.W. (Ed.), Developmental Biology: A Comprehensive Synthesis. 1. Oogenesis. Plenum Press, New York, pp. 127 – 177. Wester, P.W., Canton, J.H., 1986. Histopathological study of Oryzias latipes (medaka) after long-term b-hexachlorcyclohexane exposure. Aquat. Toxicol. 9, 21 – 45. Yamamoto, T.-O., 1975. Medaka (Killifish): Biology and Stains. Kaigaku Publishing Company, Tokyo.
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An in vitro vitellogenin bioassay for oestrogenic substances in the medaka (Oryzias latipes) C. Kordes a,*, E.P. Rieber b, H.O. Gutzeit a a b
Institut fu¨r Zoologie, Technische Uni6ersita¨t Dresden, Mommsenstraße 13, 01062 Dresden, Germany Institut fu¨r Immunologie, Technische Uni6ersita¨t Dresden, Fetscherstraße 74, 01307 Dresden, Germany Received 12 March 2001; received in revised form 19 July 2001; accepted 19 July 2001
Abstract We developed an in vitro bioassay for oestrogenic compounds based on vitellogenin as a biomarker. To quantify the induction of vitellogenin in medaka (Oryzias latipes) we established a sandwich enzyme-linked immunosorbent assay (ELISA). This ELISA was carried out with two different monoclonal antibodies specific for the yolk precursor protein vitellogenin and the high molecular weight yolk proteins (lipovitellin) of medaka. Purified lipovitellin of medaka was used as a standard for the ELISA and could be quantified down to a concentration of 9 ng/ml. The calibration curve was linear up to at least 600 ng/ml and the intra-assay coefficient of variance was 3.2%. The application of the sandwich ELISA was tested using blood samples of males and females as well as primary hepatocyte cultures of medaka. Vitellogenin synthesis in cultured liver cells of males was induced by 1 and 100 nM of the xenoestrogen 17a-ethynylestradiol (EE2). The first production of vitellogenin was detected 6 days after hormone application. In contrast, isolated liver cells of sexually mature females, which were treated in the same manner, showed vitellogenin expression from the beginning of cultivation and its synthesis increased and remained high at 100 nM EE2. However, the induction of vitellogenin synthesis in male hepatocytes in vitro could be maintained for at least one month and this indicated viable and differentiated liver cells. The hepatocyte cultures of male medaka in combination with the sandwich ELISA were shown to be a suitable tool to detect and quantify oestrogenic activity of chemicals. © 2002 Elsevier Science B.V. All rights reserved. Keywords: ELISA; Vitellogenin; Lipovitellin; Medaka; Hepatocytes; Ethynylestradiol
1. Introduction In the last decade much effort has been made to identify anthropogenic substances in the environment which may interfere with the hormonal sys* Corresponding author. Tel.: + 49-351-463-7536; fax: + 49-351-463-7093. E-mail address: [email protected] (C. Kordes).
tem of humans or animals. Numerous xenobiotics have been discovered which act as endocrine disruptors with oestrogenic properties (McLachlan and Arnold, 1996). The chemical structure of these compounds differ widely from natural hormones as illustrated by chemicals with oestrogenic activity like organochlorine pesticides, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, polychlorinated dibenzodioxins, and
0166-445X/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 4 5 X ( 0 1 ) 0 0 2 2 7 - 2
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alkylphenolic compounds (Sumpter et al., 1996). A well-accepted biomarker for oestrogenic compounds in fish is the detection of the yolk precursor protein vitellogenin in male animals (Sumpter and Jobling, 1995; Kime et al., 1999). This phospholipoglycoprotein is produced in the liver of females before the spawning period primarily under control of the natural occurring steroid hormone 17b-estradiol (E2) (Campbell and Idler, 1980; Ng and Idler, 1983). The blood stream transports vitellogenin to the follicles in the ovary, where the protein is taken up by a receptor-mediated process (Tyler and Lancaster, 1993). In the oocytes, vitellogenin is enzymatically cleaved into lipovitellin(s) and phosvitin(s) (Wallace, 1985; Callard and Ho, 1986). These components constitute major parts of the yolk and provide nutrients for the developing offspring. Fish are directly exposed to oestrogenic compounds derived from natural sources or sewage effluents. In several studies, the oestrogenic properties of chemicals were investigated using vitellogenin expression in male fish as a biomarker. Fish species like rainbow trout (Oncorhynchus mykiss) (Jobling and Sumpter, 1993; Pelissero et al., 1993), roach (Rutilus rutilus) (Routledge et al., 1998), fathead minnow (Pimephales promelas) (Giesy et al., 2000), sheepshead minnow (Cyprinodon 6ariegatus) (Folmar et al., 2000), Atlantic salmon (Salmo salar) (Arukwe et al., 2000) and flounder (Platichthys flesus) were used in these studies. The suitability of vitellogenin induction in male fish as a tool to detect the presence of oestrogenic substances in the environment was shown in field studies by Lye et al. (1997) and Allen et al. (1999) with flounder. The small freshwater teleost medaka (Oryzias latipes) was also used to test effects of xenoestrogens on vitellogenesis (Wester and Canton, 1986; Gronen et al., 1999). However, there is no quantitative vitellogenin assay for this species. Medaka is widely used in toxicological and developmental studies and offers many advantages as a model organism. This species exhibit sexual dimorphism, a short generation time, tolerate simple rearing conditions, and the breeding season can be prolonged artificially by altering the light/dark cycle (Yamamoto, 1975). Finally vitellogenin and yolk
proteins of medaka were identified and characterized by Hamazaki et al. (1987) and Murakami et al. (1991). To use the induction of a well-established biomarker for oestrogenic substances in fish like vitellogenin in combination with the advantages offered by the medaka as a model organism, we developed an in vitro bioassay based on primary hepatocyte culture of this species.
2. Materials and methods
2.1. Fish Sexually mature fish of the genus Oryzias latipes (Temminck and Schlegel, 1846) of approximately 3 cm body length were taken for this study from our breeding stock. The fish (strain d-rR with orange red males and colourless females) were reared at 25–27 °C under prolonged daylight (16 h light and 8 h darkness). For further details concerning the rearing conditions see Scholz and Gutzeit (2000).
2.2. Purification of 6itellin Freshly spawned eggs (1 h after spawning) of medaka were collected, counted, and pooled in Eppendorf tubes. Tris/EDTA buffer pH 7.2 (20 mM Tris and 40 mM ethylenediamine tetraacetic acid, Sigma) according to Hamazaki et al. (1987) was added (10 ml/egg). The eggs were homogenized with a pestle and centrifuged at 10 000g for 5 min at 4 °C. The supernatant was collected, sterile filtered through a gauze (pore size 0.2 mm, Schleicher and Schu¨ ll), and stored at − 20 °C. Yolk proteins were purified at 4 °C in two successive steps by medium pressure liquid chromatography (MPLC, Biologic-HR Workstation, Bio-Rad). First the proteins (1 mg per run) were separated at a flow rate of 0.5 ml/min in Tris/ EDTA buffer (see above) using a gel filtration column (Superdex 200 HR 10/30, Amersham Pharmacia Biotech). The eluent was collected in 0.5 ml fractions. The molecular weight of the collected yolk proteins was determined using catalase (232 kDa, Amersham Pharmacia Biotech),
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aldolase (158 kD, Amersham Pharmacia Biotech), and bovine serum albumin (67 kDa, Merck) as reference proteins in separate runs under equal conditions. In a second purification step 1 ml of fractionated vitellin in Tris/EDTA buffer was loaded onto an anion-exchange column (Bio-Scale Q2, Bio-Rad). Bound proteins were eluted using a gradient of ionic strength (0–1 M NaCl in Tris/ EDTA; at a flow rate of 1 ml/min) according to Hamazaki et al. (1987). The eluted proteins were collected in 0.5 ml fractions and the protein concentrations were determined according to Bradford (1976) using bovine serum albumin (Merck) as a standard.
2.3. Gel electrophoresis and Western blot The protein samples were separated by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Native and SDS gels were divided in a stacking gel (2.5% (v/v) 30% acrylamide and 0.8% bisacrylamide, Roth) and a running gel (12% (v/v) acrylamide/bisacrylamide). The preparations of gels, electrode buffer, and sample buffer were prepared essentially as described by Laemmli (1970). For electrophoresis under native conditions, sodium dodecyl sulphate (SDS, Sigma), b-mercaptoethanol (Sigma), and boiling of the samples were omitted. Electrophoresis was carried out at 15 mA per gel over 2–4 h for SDS gels and 6– 7 h for native gels using a power supply (P25) and a gel casting tray of Biometra. Myosin (205 kDa, Sigma) and bgalactosidase (116 kDa, Sigma) were used to determine the molecular weights of the proteins of interest in SDS gels. Following electrophoretic separation the proteins were stained with Coomassie Brilliant Blue R 250 (Sigma) as described by Holtzhauer (1997). For a differential staining of lipophilic proteins, the gels were coloured with Sudan Black B (Sigma) according to Maurer (1971). Unstained gels were placed on nitrocellulose membrane (pore size 0.2 mm, Sartorius) for the preparation of Western blots (Burnette, 1981). The proteins were transferred to the membrane by the wet blot technique at 10 V overnight using a vertical blotting apparatus (BioRad). The immunoblotting was carried out based
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on Beisiegel et al. (1982) and Beisiegel (1986). In our experiments we used 5% (w/v) milk powder (fat free) dissolved in phosphate buffered saline (PBS) and 0.05% (v/v) Tween 20 (Sigma) as a blocking reagent. Culture supernatant with monoclonal antibodies was diluted 1:100 for the antigen detection. A rabbit anti-mouse antibody coupled with horseradish peroxidase (HRP, Dako) was used to label bound immunoglobulins in a final dilution of 1:1000. All of these incubation steps were carried out with gentle shaking for 1 h at room temperature. A substrate solution containing 5 mM 4-chloro-1-naphthol (Sigma), 16.7% (v/ v) methanol, and 0.1% (v/v) hydrogen peroxide in PBS was used for staining protein bands of the antigen.
2.4. Production of monoclonal antibodies Blood samples were collected from two mice of the strain Balb/c x Black 57, diluted 1:10 in PBS, and tested with the indirect ELISA (see below) for reactions with lipovitellin of medaka. These mice showed no reactions and were immunized three times with a total of 220 mg purified lipovitellin during a period of 7 weeks. The antigen was diluted in PBS and mixed with complete Freund’s adjuvants (Gibco) for the priming. Incomplete Freund’s adjuvants (Gibco) was used for the following two boosts with antigen. The increase of antibody concentration in blood serum of mice was controlled prior to each immunization by indirect ELISA (dilution of serum from 1:10 to 1:4000 in PBS). Three days after the last immunization one mouse was killed by injection of 500 ml Trapanal (BYK). Spleen cells were prepared and fused with cells from the myeloma cell line X63 (Ag8.653) essentially as described before (Rieber et al., 1981). Immunoglobulin synthesis in the supernatants of hybridoma cultures was measured with the indirect ELISA. Clones with a high production of antibodies were selected, subcloned, and the cultures expanded. The clones were raised in miniPERM systems (MWCO 12.5 kDa, Vivascience Sartorius) to obtain high antibody concentrations as described by Falkenberg et al. (1995). The antibody subtype of selected clones was de-
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termined by means of a mouse monoclonal antibody isotyping reagent (ISO-2, Sigma) and a rabbit anti-mouse antibody coupled with horseradish peroxidase (HRP, Sigma) in a sandwich ELISA according to manufacturer’s instructions.
2.5. Screening for different epitopes Egg proteins of medaka were digested using the protease V8 (Sigma) of the bacterium Staphylococcus aureus (strain V8; Drapeau, 1976, 1977) to obtain protein fragments for the analysis of antibody binding properties. About 45 mg egg protein were loaded onto an SDS gel and electrophoresis was carried out for a few minutes at an amperage of 15 mA per gel until the egg proteins had just migrated into the stacking gel. The current was switched off and 1 ng of the protease V8 in 1 M Tris buffer (pH 6.8) containing 3 mM SDS, 1 mM EDTA, and 10% (v/v) glycerol was added to the egg protein. The current was then switched on again. Just before the colour front had reached the edge to the SDS running gel (12% (v/v) acrylamide/bisacrylamide), the current was switched off for 30 min to allow protease digestion of the egg proteins. The current was then switched on again for about 2 h. After electrophoresis an immunoblot was carried out using different monoclonal antibodies.
2.6. Purification and biotinylation of monoclonal antibodies Monoclonal antibodies were purified by MPLC at 4 °C based on Ey et al. (1978) using a HighTrap column (1 ml, Amersham Pharmacia Biotech) filled with r-protein A from Staphylococcus aureus. The column was equilibrated with sterile 0.1 M phosphate buffer (pH 7.6) containing 10 mM sodium azide. Culture supernatant (250 ml per run) was loaded onto the column at a rate of 0.2 ml/min. Unbound protein was eluted with 5 ml 1 M phosphate buffer (pH 7.6). Bound immunoglobulins were dissociated from r-protein A by a pH gradient from pH 7.6 to 3.0 (0.1 M citrate buffer with 10 mM sodium azide) at a rate of 0.4 ml/min. The antibodies were collected in
Eppendorf tubes (1 ml fractions) filled with 200 ml 1 M Tris buffer (pH 9). Finally the immunoglobulins were concentrated and the buffer was exchanged to PBS by centrifugation using Centrisart-C4 (MWCO 20 kDa, Sartorius) at 10 000g and 4 °C. Purified monoclonal antibodies were covalently coupled with biotin-7-NHS using a biotin labelling kit (Roche). The biotinylated antibodies were separated from unbound biotin-7-NHS using a Sephadex G-25 column (Roche).
2.7. ELISA procedures 2.7.1. Indirect ELISA Purified lipovitellin of medaka was diluted with coating buffer (50 mM sodium bicarbonate, pH 9.5) to a final concentration of 3 mg/ml. A volume of 100 ml per well of this antigen solution was pipetted into a microtiter plate (96-well culture plate, Costar). The wells were coated with antigen overnight at 4 °C. The plates were washed with PBS containing 0.05% (v/v) Tween 20 (200 ml/ well). Free binding sites in the wells were blocked for 1 h with 5% (w/v) milk powder (fat free) in PBS –Tween (200 ml/well). The microtiter plates were washed again and coated with antibodies against lipovitellin of medaka (100 ml/well) for 30 min. The plates were washed and a rabbit antimouse antibody coupled with HRP (Dako) in a final dilution of 1:1000 in PBS was added (100 ml/well) for 30 min. After a final washing step, o-phenylene diamine (citrate buffer tablets containing OPD from Dako and 0.04% (v/v) hydrogen peroxide) was pipetted into the wells (100 ml/well). The enzyme reaction was stopped after 15 min with 0.5 M sulphuric acid (100 ml/well). The optical density was measured at 490 nm (630 nm as a reference wave length) with a microtiter plate reader (Rainbow, Tecan). 2.7.2. Sandwich ELISA Purified monoclonal antibodies of the clone 1H11 were diluted in PBS to a final concentration of 5 mg/ml. High adsorption microtiter plates (Maxisorp, 96-well, Nunc) were coated with this antibody solution (50 ml/well) overnight at 4 °C. All incubation steps were carried out in a humi-
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dified chamber. The plates were washed with PBS containing 0.05% Tween 20 (300 ml/well) and blocked for 1 h with milk powder solution (300 ml/well). After a second washing step, reference protein (lipovitellin), blood samples or culture supernatants of oestrogen treated hepatocytes (50 ml/well) were incubated for 1 h. The incubation was stopped by washing. A second monoclonal antibody of the clone 2A9 coupled with biotin (1 mg/ml PBS –Tween) was added (50 ml/well) for 1 h. The plates were washed and streptavidin peroxidase (Dianova) was diluted in PBS– Tween to a final concentration of 1 mg/ml. The peroxidase was pipetted into the wells (50 ml/well) and incubated for 1 h. After a final washing step, the subtrate OPD (50 ml/well) was given into the wells for 10 min as described for the indirect ELISA. The enzyme reaction was stopped with 0.5 M sulphuric acid (50 ml/well) and the optical density was measured using the same conditions as mentioned above.
2.8. Preparation of blood probes and organs The fish were anaesthetized and killed with a saturated solution of benzocaine salt (\ 0.04% (w/v) ethyl 4-aminobenzoate, Fluka). An incision was made with a scalpel at the level of the anal fin to sever the Arteria caudalis. Blood was collected with glass capillary tubes (Minicaps, 10 ml, Hirschmann) and added to 20 ml PBS containing 2% (v/v) Liquemin N 25000 (sodium-heparin, AWD) and 67 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF, Sigma). Livers were removed with watchmaker forceps after ventral dissection of the fish, placed into 50 ml PBS, and squashed with a pestle. These samples were centrifuged at 10 000g for 5 min at 4 °C and the supernatants were used for Western blot analysis.
2.9. Primary culture of hepatocytes and application of 17h-ethynylestradiol Livers of several fish were placed in a sterile sieve (pore size 100 mm), washed in sterile PBS, and incubated with 0.05% (w/v) collagenase for 30 min as described by Cao et al. (1996). After enzymatic dissociation with collagenase H
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(Roche) dissolved in DMEM/F12 medium (Dulbecco’s modified Eagle’s medium, nutrient mixture F12, Gibco) the tissues were pressed with the blunt end of a syringe through the sieve. The cell lumps were pipetted several times and filtered through a second sieve (pore size 50 mm). The cells were centrifuged for 3 min at 59g, re-suspended in a defined volume of medium and counted with a hemocytometer (Neubauer). The hepatocytes were cultivated in a 24-well plate (Primaria, Falcon) at a density of approximately 2.5× 105 viable cells per well. Medium (DMEM/ F12 containing L-glutamine, 15 mM HEPES, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mg/ml gentamycin, Gibco; 2% (v/v) Ultroser SF (steroid free), Biosepra) was prepared based on Flouriot et al. (1993) and pipetted to the hepatocytes (1 ml/ well). Half of the volume was changed daily. The cells were cultivated at 25 °C in humidified atmosphere with 5% carbon dioxide. Cell culture media containing 100 and 1 nM 17a-ethynylestradiol (EE2, Merck) were prepared from a stock solution of 1 mM EE2 in dimethyl sulphoxide (DMSO, Sigma). Pure DMSO dissolved in medium (1 ml DMSO per 10 ml medium) was used for control experiments. These experimental media were added to the primary culture after the first day of culture (i.e. after cell attachment).
2.10. Histology and electron microscopy The carcasses (without head and tail) of medaka were fixed with 4% (v/v) formalin in phosphate buffer pH 5.5 (0.28 M sodium dihydrogen phosphate and 0.54 M disodium hydrogen phosphate) for 7 days. Paraffin sections (thickness 3 mm) were made as described by Scholz and Gutzeit (2000). After hydration of the sections, the endogenous peroxidase activity was blocked by treatment with 3% (v/v) hydrogen peroxide in PBS for 10 min. Then the sections were washed in PBS with 0.05% (v/v) Tween 20 and incubated in 5% (w/v) milk powder in PBS–Tween for 1 h. Culture supernatant with monoclonal antibodies was diluted in milk powder solution (1:10) and the sections were incubated for 1 h. They were washed and a rabbit anti-mouse antibody coupled
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with HRP (Dako) in a final dilution of 1:200 in milk powder solution was added. The tissue sections were washed again and incubated in a substrate solution containing 0.8 mM 3,3%-diaminobenzidine tetrahydrochloride dihydrate (DAB, Fluka) and 0.1% (v/v) hydrogen peroxide in PBS. The enzyme reaction was stopped after a few minutes by washing with PBS. The sections were sealed with Aquatex (Merck) and a cover slip. Cells of primary cultures were fixed for scanning and transmission electron microscopy with 2% (v/v) glutaraldehyde (Sigma) and 1% (v/v) osmiumtetroxide (Sigma) in 0.1 M sodium cacodylate buffer (TAAB) for 40 min. For scanning electron microscopy (LEO 420) pieces of the culture dish with attached cells were dehydrated with a graded series of ethanol, critical-point-dried according to Anderson (1951), and coated with gold. Single cell aggregates were orientated in 2% (w/v) agar-agar (Roth), dehydrated with ethanol, embedded in LR-White resin (Sigma), and processed for transmission electron microscopy (EM 208, Philips) as described by Fleig et al. (1991).
2.11. Statistics Student’s distribution was used to identify the lower limit of sensitivity of the sandwich ELISA. The analysis of variance (ANOVA, followed by least significant differences tests, P B0.05) was calculated to define the working range of the assay. The program WinStat (version 1999.2) was used for statistical calculations.
3. Results
3.1. Vitellin preparation Two proteins, which form major components of freshly spawned eggs, were purified by gel filtration and anion-exchange chromatography. Their molecular weights of approximately 220 and 180 kDa were determined by gel filtration chromatography (Fig. 1A; L1 and L2). For comparison, Fig. 1B (lanes 1 and 2) shows unpurified and purified egg proteins in native form. The proteins were
stained with Coomassie Brilliant Blue R 250 (Fig. 1B) or with the lipophilic dye Sudan Black B (not shown). The isolated proteins (Fig. 1B; lane 2) exhibited the same molecular weights and lipophilic quality as lipovitellin(s) of medaka (Murakami et al., 1991). These two yolk proteins were used together for the immunization of mice and served as reference proteins of the indirect and sandwich ELISA.
3.2. Production and specificity of monoclonal antibodies After fusion of lymphocytes with myeloma cells many wells with hybridoma cells showed antibody production against lipovitellin of medaka in the indirect ELISA. We selected five hybridoma clones (1H11, 2A9, 6G6, 4B3, and 5C4) which showed stable and high antibody production. The subtype of the immunoglobulins was determined to be IgG1 in all selected clones. The antibodies of these clones were tested in Western blots of blood and liver samples of sexually mature male and female medaka. The protein samples were separated by SDS and native gel electrophoresis. After native gel electrophoresis, the immunoglobulins recognized a single protein band in blood and liver samples of vitellogenic female medaka, whereas no binding was observed in samples of male medaka (Scholz et al., 1999). Following SDS gel electrophoresis, the antibodies bound to a dominant protein band with a molecular weight of approximately 200 kDa in blood samples of vitellogenic females (Fig. 2; lanes 2 and 5). Hamazaki et al. (1987) reported a molecular weight of 200 kDa for a major protein subunit of medaka vitellogenin under denaturing conditions and these authors determined a molecular weight of 420 kDa for the native form of vitellogenin. In our Western blot experiments, no binding of the monoclonal antibodies was observed when blood samples of male fish were tested (Fig. 2; lane 6). However, there was a clear reaction of the monoclonal antibodies with purified lipovitellin (Fig. 1A; lane 3) and unpurified yolk proteins of medaka eggs (Fig. 2; lane 4), which derived from vitellogenin. The specificity of antibody binding was confirmed using paraffin sections of mature
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female and male medaka (for both sexes n = 3). Vitellogenic oocytes of the ovaries were labelled as well as the capillaries and sinusoids of the female liver (Fig. 3A– C), whereas paraffin sections of male medaka (Fig. 3D) showed no immunohistochemical reaction. Finally, the production of this antigen was induced after oestrogen treatment of hepatocytes in vitro (see below). Taking together, these observations give strong evidence that we raised monoclonal antibodies against vitellogenin of Oryzias latipes.
3.3. Epitope-screening It was necessary to determine the binding properties of the immunoglobulins because a sandwich ELISA required two specific antibodies directed against two different epitopes of the same antigen.
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Therefore, egg proteins were digested by the protease V8 and the resulting protein fragments were separated by SDS-PAGE. In Western blots we found that the clones 2A9, 6G6, and 4B3 show approximately the same binding pattern, while the clones 1H11 and 5C4 had different specificities. Finally, two clones were chosen to produce antibodies against diverse epitopes of medaka lipovitellin and vitellogenin to construct a sandwich ELISA. Purified immunoglobulins of the clone 1H11 were taken to capture antigen and antibodies of the clone 2A9 were biotinylated to label captured protein in the sandwich ELISA.
3.4. Properties of the sandwich ELISA The sandwich ELISA was performed and optimized using purified lipovitellin of medaka (Fig.
Fig. 1. (A) MPLC analysis of medaka yolk proteins (1 mg) produced by gel filtration (Superdex 200 HR 10/30 column; Amersham Pharmacia Biotech). Two lipovitellin(s) with a molecular weight of approximately 220 kDa (L1) and 180 kDa (L2) were major protein components of the medaka eggs. Phosvitin(s) (P) have a low molecular weight (Murakami et al., 1991). (B) Whole yolk (lane 1; 10 mg) and purified lipovitellin(s) (lanes 2 and 3; 1 mg) were separated by native gel electrophoresis (12% acrylamide). The proteins in lanes 1 and 2 were stained with Coomassie Brilliant Blue R 250. Phosvitin(s) (arrowhead) stain weakly with this dye. The two large molecular weight bands above L1 (B, lane 1) are represented in (A) left of and partly overlapping with peak L1 (retention time between 20 and 25 min) as determined by native gel electrophoresis of the collected protein fractions. The dominat proteins of the peaks L1 and L2 are lipovitellin(s). Lane 3 shows a Western blot of these purified lipovitellin(s). Monoclonal antibodies of the clone 1H11 were used to identify the lipovitellin(s). The biotinylated immunoglobulins of the clone 2A9 exhibited the same binding pattern in Western blot analysis of purified lipovitellin (not shown).
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Fig. 2. Yolk proteins (lanes 1 and 4; 24 mg) and blood serum of vitellogenic females (lanes 2 and 5; 3 mg) and sexually mature males (lanes 3 and 6; 3 mg) of the species Oryzias latipes were separated by SDS-PAGE (10% acrylamide). One part of the gel was stained with Coomassie Brilliant Blue R 250 (lanes M, 1, 2 and 3) and with the other part a Western blot was performed (lanes 4, 5 and 6). Lane 2 shows a female-specific protein band in the blood serum with a molecular weight of about 200 kDa, which was detected by the antibodies of the clone 1H11 (lane 5). Myosin (205 kDa, Sigma) and b-galactosidase (116 kDa, Sigma) were used as molecular weight markers in lane M.
1A; lane 2) as reference protein. The lipovitellin concentration of 9 ng/ml was significantly above background (n = 6, P B0.001) and hence defined as the lower detection limit while the working range of the assay started at approximately 20 ng/ml (as determined by ANOVA, n = 6, P B 0.05). The standard curve of the sandwich ELISA was linear up to 600 ng/ml (Fig. 4). The intra-assay coefficient of variation was 3.2% (SD9 0.01; n = 9) and the inter-assay coefficient of variation was 17% (SD90.07; n =9). These parameters were defined by Specker and Anderson (1994).
Fig. 3. Immunohistochemical detection of lipovitellin(s) and vitellogenin with antibodies of the clone 6G6 on paraffin sections of medaka. The antibodies of the clones 2A9 and 4B3 displayed similar binding patterns on paraffin sections. (A) Ovary of mature female with non-vitellogenic (centre) and vitellogenic oocytes (arrows show binding of antibodies at yolk proteins); (B) vitellogenic oocyte (antibody labelling of yolk proteins); (C) liver of mature female (arrows show binding of antibodies in the capillaries and sinusoids); (D) liver of mature male without antibody labelling.
Fig. 3.
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Fig. 4. Calibration curve of the sandwich ELISA based on lipovitellin of medaka (dots show the arithmetic mean; bars show the standard deviation; r 2 = goodness-of-fit; cv = coefficient of variation; n =number of values per concentration).
The application for sandwich ELISA was tested with blood serum of mature females and males of Oryzias latipes. Blood samples of females were diluted in PBS (0.5– 1.5 nl blood serum per well) so that the vitellogenin concentration fell into the linear range of the test system. The concentrations of vitellogenin (expressed in vitellin units) ranged from 125 to 286 mg/ml blood serum (n = 5). Vitellogenin was not detected in blood samples of male medaka (n= 5, 410– 650 nl blood serum per well) by sandwich ELISA.
3.5. Primary culture of hepatocytes The isolated liver cells formed small aggregates (Fig. 5A) which attached to the culture dish during the first 24 h. At least three different cell types could be distinguished when cultivated hepatic cells were analysed by scanning electron microscopy. Spherical hepatocyte-like cells (Fig. 5B), flat fibroblastlike cells (Fig. 5C), and some erythrocytes could be identified. The erythrocytes disappeared within the first week of culture, whereas the number of fibroblast-like cells increased significantly during the culture period. They covered the plate surface between the hepatic cell aggregates. The islets of cell aggregates increased in size during cultivation. After about 20 days of culture, the aggregates
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exhibited flat and in some cases spherical organ-like shapes (Fig. 5D). The cultured hepatocytes remained viable and differentiated for at least one month as determined by their morphology and vitellogenin synthesis (see below). After 27 days of culture organ-like aggregates with diameters of about 500 mm were analysed by transmission electron microscopy. Intact hepatocytes were observed in the centre of these cell aggregates and their ultrastructure displayed typical cell organelles (i.e. endoplasmic reticulum, mitochondria, nucleus, nucleolus, and peroxisomes) and microvilli (Fig. 5E). Furthermore, the hepatocytes reconstructed bile canaliculi with microvilli (Fig. 5F) and formed extensive inter-cellular spaces. Also flat endothelial-like cells were found between the hepatocytes. Fibroblast-like cells were seen in several layers on the surface of the cell aggregates.
3.6. Induction of 6itellogenin in primary culture of hepatocytes The production of vitellogenin was induced in primary cultures of male hepatocytes with 1 and 100 nM 17a-ethynylestradiol (EE2) and there was no induction found in the control group without EE2 (Fig. 6). Vitellogenin synthesis was first detected 6 days after exposure to 100 nM EE2 and the concentration of vitellogenin in the medium rose up to day 17 of culture and then decreased. The concentration of 1 nM EE2 induced consistently smaller amounts of vitellogenin. The increasing standard deviations at 100 nM EE2 in Fig. 6 was mainly due to the drastic decrease of vitellogenin synthesis in one well with hepatocytes at day 21 of culture. The absolute vitellogenin amount in each well was dependent on the frequency of medium exchange (Fig. 6; change of 50% of the medium daily). Higher vitellogenin concentrations were obtained, when 50% of the medium was changed every second day. The observed lag-phase in vitellogenin detection was not affected by this alteration. This experiment was also performed with hepatocytes of males, which were cultured in medium containing 10% foetal calf serum (FCS) instead of 2% Ultroser SF (steroid free). In this case the induction of vitellogenin showed the same pattern
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Fig. 5.
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tions declined to 30 ng/ml (vitellin units) after 14 days. The concentration of 1 nM EE2 was not sufficient to maintain the high vitellogenin synthesis of female hepatocytes in this experiment.
4. Discussion
Fig. 6. Induction of vitellogenin with 1 and 100 nM 17aethynylestradiol in primary cultures of hepatocytes of male medaka. The cells were cultured with DMEM/F12 supplemented with 2% Ultroser SF (steroid free). Vitellogenin was measured by sandwich ELISA (symbols show the arithmetic mean of triplicates and bars represent the standard deviations).
as described above, but the production of vitellogenin was about 3-fold (1 nM EE2) and 5-fold (100 nM EE2) higher than in medium supplemented with Ultroser SF. This indicated that additional factors of the FCS are able to enhance vitellogenin synthesis. Because of the defined composition we preferred Ultroser SF in vitellogenin induction experiments. Hepatic cells of mature females were cultivated in medium containing 2% Ultroser SF and treated with 1 and 100 nM EE2 in order to test, if the observed delay in vitellogenin expression was due to our cell isolation procedure or culture conditions. In this experiment vitellogenin was detected from the beginning of cultivation at both concentrations of EE2 as well as in the control group. When exposed to 100 nM EE2, the vitellogenin concentrations increased from about 12 000 up to 20 000 ng/ml (vitellin units), while cultivation with 1 nM EE2 and in the control group the concentra-
The female-specific protein vitellogenin is a well accepted biomarker for oestrogenic substances in male teleost oviparous fish. To produce antibodies against vitellogenin of medaka (Oryzias latipes), a source of this protein as antigen was required for immunization of mice and as a reference protein in a quantitative assay. Unfortunately medaka is to small to get sufficient amounts of blood (1–7 ml per fish) to purify vitellogenin. Furthermore, we want to avoid the production of vitellogenin in ascites of male fish by 17b-estradiol (E2) treatment as reported by Hamazaki et al. (1987). Therefore, we relied on the structural similarities of vitellogenin and its lipovitellin derivative. This antigen source was taken to generate monoclonal antibodies against vitellogenin of medaka as outlined by Scholz et al. (1999). Purified lipovitellin was also used to establish an ELISA so that a highthroughput method for the analysis of oestrogenic chemicals becomes available. Since the first ELISA was published by Engvall and Perlmann (1971), this sensitive method is widely used in laboratories to quantify antigens. We decided to construct a sandwich ELISA based on two different monoclonal antibodies to improve specificity and sensitivity of the assay. The usage of lipovitellin from medaka as a reference protein in our ELISA was found to be a suitable alternative to vitellogenin. There was no evidence that lipovitellin limits the application of the sandwich ELISA for the identification of oestrogenic substances. Our assay was as sensitive as competitive ELISAs established for vitellogenin of striped
Fig. 5. Primary culture of medaka hepatocytes (pictures A –D were made by scanning electron microscope and pictures E and F by transmission electron microscope). (A) Aggregates of hepatocytes (bright clusters) at day 5 of culture; (B) aggregate of hepatocytes at day 5 of culture in detail; (C) flat fibroblast-like cell at day 5 of culture; (D) part of a large cell aggregate at day 27 of culture; (E) hepatocytes in the centre of an aggregate at day 27 of culture (cm = cell membrane, en =endothelium-like cells, er =endoplasmic reticulum, itc = inter-cellular space, mic = microvilli, mit = mitochondrion, nu = nucleus, lip =lipid vacuoles); (F) bile canaliculus with microvilli (mic) at day 27 of culture.
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bass (Morone saxatilis) (Kishida et al., 1992), English sole (Pleuronectes 6etulus) (Lomax et al., 1998), rainbow trout (Bon et al., 1997) or brown trout (Salmo trutta) (Sherry et al., 1999), which were based on polyclonal antibodies. Monoclonal antibodies, however, have the advantage that large quantities of immunoglobulins with strictly defined binding properties can be produced and hence standardized test systems can be established. To provide evidence that substances of interest are able to induce vitellogenin synthesis in male medaka and therefore show oestrogenic properties, we chose an in vitro test system based on primary culture of male hepatocytes. For critical validation of the sensitivity and reliability of this test system, we used the environmental xenoestrogen 17a-ethynylestradiol (EE2). This potent anthropogenic oestrogen from contraceptive pills was detected in sewage water in concentrations up to 7 ng/l (Desbrow et al., 1998; Larsson et al., 1999). Ju¨ rgens and Johnson (1999) reported that EE2 is about ten times more stable than E2. Therefore, it is important to investigate the effects of this synthetic oestrogen on fish despite its low concentration. In our in vitro test system 1 and 100 nM EE2 induced vitellogenin synthesis in hepatocytes of mature male medaka for at least one month. This fact as well as the intact cell morphology assured us of the validity of this in vitro bioassay. The long-term vitellogenin synthesis was also found in cultured hepatocytes of other species like rainbow trout, which were treated with oestrogens (Flouriot et al., 1993). Also the behaviour of hepatic cells after isolation and during cultivation (i.e. attachment and aggregate formation) in our assay seemed to be very similar to the in vitro system of rainbow trout (Flouriot et al., 1993). However, the delay in induction of vitellogenin synthesis in male hepatocytes of medaka in vitro was remarkable. The earliest vitellogenin production was detected 6 days after EE2 exposure. This lag-phase was presumably due to an oestrogen-dependent induction and up-regulation of oestrogen receptor (ER) in hepatic cells. As
described by Mommsen and Lazier (1986), the treatment of cultured hepatocytes of male salmon (Salmo salar) with E2 resulted in an increase in ER expression. Flouriot et al. (1996) also found in E2-stimulated hepatic cells of rainbow trout cultured in vitro an increase of ER followed by vitellogenin induction. In addition, a delayed proliferation of isolated liver cells, which starts after day 6 of culture according to Cao et al. (1996), might have contributed to the late vitellogenin detection in our experiments. Recovery from the cell isolation procedure seemed to be of minor importance for the observed delay in vitellogenin synthesis, because the production of the yolk precursor protein started immediately, increased, and remained high, when liver cells of mature females were isolated and treated with EE2. Hamazaki et al. (1987) reported that vitellogenin became detectable in blood serum of adult non-spawning female and male medaka after 3–4 days of oestrogen treatment (food dusted with 1 mg E2). Apparently, the delay reflects a biological process that can be observed in vivo and in vitro. However, the induction of vitellogenin synthesis in male hepatocytes was confirmed to be a useful tool to investigate oestrogenic effects of chemicals. This method was able to detect the presence of low concentrations of 17aethynylestradiol. In addition, the long-term production of this yolk precursor protein can also be used as an indicator for differentiated and viable fish hepatocytes in primary culture.
Acknowledgements We wish to thank Heidrun Gebauer and Christine Gra¨ fe for their help in the production of monoclonal antibodies. We are also grateful to Doris Zschernig for preparing paraffin sections and to Dr Richard Fleig for his support with the electron microscope techniques. We also thank Dr Stefan Scholz for helpful discussions and establishing our breeding stock of medaka. Finally, we acknowledge Nadja Bachmann for her support in maintaining our fish stock.
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