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a Dipartimento Scienze Ambientali, Università della Tuscia, I-01100 Viterbo, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unlike the case in other vertebrates, choriogenesis in teleosts is not restricted to the ovary, since the liver also takes part in this process. Egg macromolecular precursors have been found in liver and serum of spawning females by immunoblotting using polyclonal antibodies [4, 13–16, 18, 19]. This suggests that precursors find their way from the production site and are assembled in the ovary.
In a previous work, we described the fine morphology of the mature sea bass egg envelope [20], the polypeptide content of chorion dissolved in 8 M urea, and the partial purification of some chorion macromolecules by gel-filtration chromatography [7]. To further address important questions concerning teleost choriogenesis, we have studied the Mediterranean sea bass Dicentrarchus labrax, an important fish species that is efficiently reproduced and reared in aquaculture. In the present paper, we describe the preparation of a monoclonal antibody (mAb) specific for chorion antigens. The mAb we obtained was used as a probe to study the participation of recognized antigen(s) in choriogenesis and to develop an immunoenzymatic assay for their detection in blood.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Collection of sea bass eggs and the eggshell isolation protocol have already been described [7]. Briefly, mature eggs were collected by gently squeezing the abdomen of sea bass females in their reproductive period (December to March). Chorions were prepared under a stereomicroscope by rupturing the eggs with forceps in 50 mM Tris-HCl pH 7.2, 0.12 M NaCl, 10 mM EDTA, and 1 mM PMSF (TNE). The chorions were then incubated in the same solution with 0.1% Triton X-100 for 15 min at 4°C to remove cytoplasmic material, and centrifuged with cold TNE at 3500 x g for 10 min. Eggshell proteins were partially dissolved in TNE containing 0.6 M NaCl, 1 mM 2-mercaptoethanol, and 8 M urea (S-TNE) by repeated strokes in a 1-ml glass Dounce homogenizer at 4°C. The soluble fraction (homogenate) was separated by centrifuging at 20 000 x g for 20 min at 4°C. The protein concentration in the supernatant was determined by the method of Bradford [21], using BSA (Boehringer, Mannheim, Germany) as standard.
Organ Lysates
Ovarian tissue and liver were dissected from fish (weight 512 ± 73 g) and immediately placed in 0.15 M NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8 mM Na2HPO4 (pH 7.4) (PBS) at 4°C. The tissue was homogenized in a glass-teflon Potter blender (Wheaton Scientific, Milville, NJ) in PBS containing 10 µg/ml each of PMSF, leupeptin, and aprotinin. The homogenates were centrifuged at 2000 x g for 10 min at 4°C, and at 20 000 x g for 20 min at 4°C. Lipids were removed from the resulting supernatants and sera by adding an equal volume of chloroform, shaking vigorously for 5 min, and collecting the aqueous phase. Protein content was determined as above.
Antigen Preparation and Monoclonal Antibody Production
Partially purified sea bass eggshell proteins were obtained by Sephadex G-100 (Pharmacia Biotech Italia, Milan, Italy) gel-filtration chromatography as previously described [7]. Fractions containing 44- to 50-kDa polypeptides were pooled, thoroughly dialyzed at 4°C in 50 mM NH4HCO3, 5 mM NaCl, and 1 mM 2-mercaptoethanol, and lyophilized. One hundred micrograms of lyophilized material resuspended in 100 µl of PBS and emulsified with 100 µl complete Freund's adjuvant (Serva, Heidelberg, Germany) was injected i.p. into three 8- to 10-wk-old BALB/C mice. A similar injection was given 10 days later, followed by two additional boosts at 20 and 30 days with incomplete Freund's adjuvant, and a final boost without adjuvant at 40 days. Three days later, the mice were anesthetized and killed. Spleens were aseptically removed, and splenocytes were obtained by rupturing the organ with a glass pestle over a stainless steel grid in serum-free Dulbecco's Modified Eagle's medium (Gibco Europe, Paisley, Scotland, UK) at 4°C. Three spleens yielded 2.7 x 108 cells; 9 x 107 cells were fused according to standard protocols [22] using polyethylene glycol (PEG 4000, Gibco), with 3 x 107 P3X.ag8.653 mouse myeloma cells. The cells were 97% viable, as determined by trypan blue dye exclusion. After fusion, the cells were plated at 3 x 105 myeloma cells/ml in microculture plates. Positive hybridoma clones were detected by a dot-blot assay using 2 µl (0.2 µg) immunization antigen manually blotted onto nitrocellulose (BA85; Schleicher & Schuell, Dassel, Germany) and 100 µl culture supernatant incubated for 18 h at room temperature. Before addition of the first antibody, the nitrocellulose was saturated for 30 min in PBS containing 3% BSA and 0.1% Triton X-100 (BPT). Antibody binding was detected with horseradish-conjugated goat antimouse IgG (Cappel Europe, Turnhout, Belgium) diluted 1:1500 in BPT for 90 min at room temperature as previously described [23]. For immunoblotting determination, 500 µg of whole eggshell lysate was run on a 10% SDS-PAGE minigel without comb, and electroblotted onto nitrocellulose at 100 mA per gel (1.0 mm thick) for 90 min. Then, 1.5-mm strips of nitrocellulose were cut out, saturated with BPT, and incubated with 1.0 ml of hybridoma culture supernatants as described above. Controls were performed by incubating strips in culture supernatants of the parent myeloma. The hybridoma giving the best staining in immunoblotting and indirect immunofluorescence (IIF) of mature egg cryosections (see below) was selected, cloned by limiting dilution, and screened again by IIF. The hybridoma cell line DLE7 was then established. For the IgG isotype determination, a kit was employed (Sigma, St. Louis, MO) with 2 ml of the hybridoma culture supernatant.
Electrophoresis and Western Blotting
SDS-PAGE was performed according to Laemmli [24] on 10% polyacrylamide slab minigels using proteins ranging from Mr 14 400 to 94 000 (Pharmacia Biotech) as molecular weight reference and colored molecular weight reference standards from Mr 14 300 to 200 000 (Amersham, Little Chalfont, UK) for blotting experiments. The gels were stained with Coomassie blue R-250 dye. For Western blots, the gels were electroblotted onto nitrocellulose membrane for 90 min at 100 mA. After electrophoresis, the nitrocellulose was then saturated for 30 min in BPT and washed twice with PBS before incubation for 3 h with DLE7 as undiluted culture supernatant or with myeloma culture supernatant as control. The nitrocellulose was rinsed twice with PBS and then incubated for 90 min with horseradish peroxidase-labeled goat anti-mouse serum (Cappel) diluted 1:1500 in BPT at room temperature. After extensive rinses with PBS, bound antibody was detected by chemiluminescence (Amersham).
Enzymatic Deglycosylation
The protocol of enzymatic deglycosylation was based on a previous work [25]. Briefly, 5 µg (5 µl) of chorion homogenate in S-TNE from sea bass and trout was incubated with 0.2 units (5 µl) of endoglycosidase F (endo-F; E 872, Sigma) at 37 °C for 18 h in 40 µl 0.2 M acetate buffer at pH 5.4 containing 0.2 % Triton X-100, 0.1 mM 2-mercaptoethanol, and 20 mM EDTA. In control samples, the enzyme was omitted. At the end of incubation, the samples were spiked with 15 µl Laemmli buffer and processed for immunoblotting with DLE7.
Immunofluorescence
For IIF, whole eggs, ovary, and liver pieces from different fish were collected and immediately frozen in liquid nitrogen. They were then embedded in freezing medium (Cambridge Instruments, Heidelberg, Germany), after which 7-µm cryosections were cut at -26°C and placed on glass coverslips previously coated with 0.1 mg/ml poly-L-lysine. The coverslips were air-dried, fixed with 4% paraformaldehyde in PBS pH 7.4 for 15 min at 4°C, washed three times with PBS, and incubated onto Parafilm (American National Can Co., Greenwich, CT) with 100 µl of hybridoma culture supernatant for 45 min at room temperature, or as control, with 100 µl of myeloma culture supernatant for the same time. After three washes with PBS, 100 µl of rhodamine-labeled goat antimouse antibody (Cappel) was added at a 1:300 dilution in PBS for 30 min at room temperature. The coverslips were then washed three times with PBS and mounted with a nonfluorescent mounting medium (BDH Chemicals Ltd., Poole, England). Observations were made with a Zeiss Axiophot microscope equipped with epifluorescence (Carl Zeiss, Inc., Thornwood, NJ). Pictures were taken using Kodak T-max film (Eastman Kodak, Rochester, NY) at 800 ISO.
ELISA Assay
Blood (500 µl/fish, n = 25) was sampled from the caudal vein of animals anesthetized with 1 mg/ml of tricaine methanesulfonate (Sigma) using a syringe, and serum was obtained from clotted blood by centrifuging at 2000 x g for 10 min. Sera were collected during the spawning season (December to February) at different times at three different fish farms. Serum from each animal was aliquoted in small volumes (50 µl) and stored frozen at -70°C. Polystyrene microwells (Nunc, Roskilde, Denmark) were used to adsorb sea bass serum at various dilutions (1:50 to 1:50 000), in 100 µl of carbonate-bicarbonate buffer (50 mM pH 9.4) overnight at 4°C. The wells were washed twice with Tris-HCl (50 mM, pH 7.4) containing 0.05% Tween-20 and 0.15 M NaCl (TTN). The binding sites were saturated with 3% BSA in TTN for 30 min; then 100 µl of DLE7 as undiluted culture supernatant was added for 90 min. The wells were thoroughly washed with TTN and incubated for 90 min with horseradish peroxidase-labeled goat anti-mouse serum (Cappel) diluted 1:3000 in TTN-BSA. After extensive washing with TTN, antibody binding was detected by adding 100 µl/well 0.04% o-phenylenediamine in 50 mM phosphate-citrate buffer (pH 5.0) containing 0.001% H2O2. The reaction was allowed to proceed for 15 min and then was stopped with 50 µl of 3 N sulfuric acid. The absorbance was measured at 492 nm with an automated ELISA reader. Each point was read in triplicate, and controls were performed either by uncoating wells with the antigen or by substituting myeloma culture supernatant for DLE7. Data are given as the mean ± SD of at least two different experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After spleen-myeloma fusion, 148 hybridomas were screened, 27 of which were positive in the dot blot assay using the material purified by gel-filtration chromatography as antigen [7]. Among these, 14 hybridomas recognized polypeptides in Western blot analysis of 8 M urea lysates of chorions. The clone DLE7 was isolated by limiting dilution and gave the most intense staining of eggshell cryosections in IIF. The isotypes for DLE7 showed that this cell line secreted IgG of the IgG1 subclass.
Electrophoresis and Western Blotting
The 10% SDS-PAGE pattern of solubilized eggshell lysate is shown in Figure 1. In a previous study [7], we described the protocol and the yield of the macromolecules released by treatment of isolated sea bass eggshells with S-TNE. Coomassie staining revealed a main triplet at 43, 48, and 50 kDa, and major polypeptides at 100, 120, and 170 kDa (lane a). After electroblotting onto nitrocellulose, the mAb DLE7 immunostained major antigens in the eggshell lysate, including 44-, 48-, and 110-kDa polypeptides (lane b). To assess the sensitivity of solubilized chorion antigens containing glucose and mannose to enzymatic deglycosylation, endoglycosidase-F was used before immunoblotting with DLE7. The results are shown in lane c. A clear decrease in immunostaining was observed for the 48-kDa antigen, whereas the 110-kDa antigen showed a slight decrease in molecular size (about 3 kDa). Results of a control experiment in which the enzyme was omitted is shown in lane d.
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Figure 2 (left panel) shows the 10% SDS-PAGE electrophoretic profile of 50-µg homogenates of ovary (lane a), liver from a nonspawning female (lane b), liver from a spawning female (lane c), serum from the same spawning female (lane d), and serum from a nonspawning female (lane e). Lipids were removed from liver homogenates and sera to avoid interference during electrophoresis. Many polypeptides with different molecular sizes were revealed by Coomassie blue staining. Immunoblotting with DLE7 of the same electroblotted samples revealed a single polypeptide at 90 kDa in the homogenate from liver of a spawning female, and two polypeptides of 180 and 50 kDa in serum from the same female. No positivity was observed in homogenates of ovary and liver or in serum from a nonspawning female (Fig. 2, central panel), or in the control, in which the electroblotted samples were incubated with myeloma culture supernatant (Fig. 2, right panel).
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Indirect Immunofluorescence
Of the hybridomas obtained, the clone DLE7 was the most effective for staining cryosections of ovary; the results can be seen in Figure 3. The cryosections (9 µm thick) showed marked immunofluorescence of the chorion and a negative ooplasm (Fig. 3c) with respect to the control IIF obtained by incubating sections with myeloma culture medium (Fig. 3a). The interference contrast pictures of the same fields (Fig. 3, b and d) show chorions of different diameters (bar = 100 µm).
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The immunoreactivity of DLE7 in liver cryosections is shown in Figure 4. The staining is very intense in sections obtained from a spawning female (Fig. 4d), and the positivity was distributed homogeneously throughout the section. Control staining was obtained by incubating liver cryosections from nonspawning females with DLE7 (Fig. 4b), which produced low background positivity. The interference contrast pictures of same fields are shown in Figure 4, a and c. The fluorescent clumps in Figure 4, b and d, are clusters of self-fluorescent melanomacrophages [8]. A further IIF control was done by incubating cryosections from the thymus of spawning females with DLE7, and these sections showed negative results (data not shown).
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ELISA Assay
The results obtained by using the mAb DLE7 in an indirect ELISA assay of sea bass sera are shown in Figure 5. With this method, the antigen gave a clear and sensitive image. In Figure 5a, sera from females in the spawning season (n = 16, upper line), and from males (n = 9, lower line), were analyzed by adding DLE7 as an undiluted culture supernatant to increasing dilutions of the antigen adsorbed onto plastic wells. In these experiments, sex was ascertained by dissection of the fish. The sex of the fish can be clearly deduced from the sera, since females had higher positivity than males, for which absorbance levels were basal. Data are presented as the mean ± SD of three experiments.
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Figure 5b shows the variation in blood titers of the antigen recognized by DLE7 in sera of adult females during different periods of the year: in the reproductive period (December to March, upper line), and in the nonspawning season (June to September, lower line). Controls were performed in wells that were not coated with DLE7, or by replacing DLE7 with myeloma culture supernatants; in these cases the mean absorbance was 0.05 ± 0.01. The antigen present in serum appeared quite sensitive, since freeze-thawing of the same sample resulted in rapid loss of positivity. To avoid this problem, sera were aliquoted after collection into minimal amounts, stored frozen at -70°C, and used once in ELISA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The aim of the present study was to extend our previous results on sea bass oogenesis [7, 20] by investigating the contribution of extra-ovarian tissues to this vital process. To perform this work, we produced an mAb specific for chorion macromolecular antigens to detect their presence in tissues and body fluids. The mAb obtained came from a panel of hybridomas secreting anti-sea bass chorion IgG, and the clone DLE7 was the most useful for recognizing the immunization antigen by immunoblotting, IIF, and immunoenzymatic analysis. An mAb previously described as specific for pipefish vitelline envelope components [5] failed to recognize the antigen in liver and serum. In immunoblotting analysis, DLE7 stained the immunization antigens (44–50 kDa) and a 110-kDa polypeptide intensely. The high intensity of immunostaining of the 110-kDa polypeptide suggests that it could be derived from covalent modification of antigenic components lower in molecular mass, since an efficient method of chorion hardening based on transglutaminase activity was recently described in rainbow trout [28]. This hypothesis is reinforced by the observation that the sea bass chorion has a high content of glutamic acid [14]. Other researchers recently used an antiserum against vitelline envelope macromolecules to study the endocrine control of oogenesis in Mediterranean sea bass [14]. A remarkable similarity between our results and those of this group can be observed in the immunoblot analysis of chorion proteins with their polyclonal antibody and our mAb, suggesting that certain eggshell components are immunodominant when used as antigen.
Many sea bass eggshell proteins are glycosylated [7], and to test whether antigens recognized by DLE7 were affected by enzymatic deglycosylation, endo-F was incubated with chorion polypeptides. The 48-kDa polypeptide was greatly affected by the enzyme, suggesting that it has a high degree of N-linked glycosylation, whereas the 110-kDa antigen was relatively unaffected by endo-F treatment. The 44-kDa antigen was previously shown to be nonglycosylated with mannose and glucose residues [7]. When DLE7 was used in IIF analysis, a clear positivity for the vitelline envelope was observed in isolated eggshells (data not shown) and in ovary cryosections from an egg-producing female. Liver cryosections also showed high positivity. In the latter case, the positivity was specific, since liver cryosections from nonspawning females (juveniles) were negative. The IIF pattern showed that most, if not all, hepatocytes were positive for DLE7, suggesting that the whole organ is involved in the physiological process of choriogenesis. This observation confirmed that chorion macromolecules are also synthesized in the liver of the sea bass. The next experimental step was to study how they reach the ovary, by immunoblot analysis of organ lysates and serum. DLE7 mapped a single polypeptide cross-reacting at 90 kDa in the liver, and two polypeptides at 180 and 50 kDa in serum from the same spawning female. These antigens were not found in nonspawning females or in males. The 90-kDa polypeptide may be a precursor molecule produced in the liver and released into the blood as a component of lower molecular size like those present in the chorion at 44 to 50 kDa, or approximately half the molecular size of the component present in the liver. The 50-kDa polypeptide present in serum is in the same range as that found in medaka [4, 19], trout [18, 29], sea bass, and sea bream [14]. In this last paper, polypeptides of 90, 48, and 52 kDa were detected by immunoblot in plasma of hormone-treated sea bass. The absence of positivity in ovary lysates is explained by the unsolubility of chorion polypeptides under the conditions employed to obtain organ homogenates. Vitellogenin is the precursor of egg yolk proteins in nonmammalian vertebrates. In teleosts it is produced by the liver and transported by the blood to the ovary [30]. The kinetics of its production may be similar to that of chorion constituents [18]. The molecular mass of the vitellogenin subunit of sea bass according to SDS-PAGE is about 170 kDa [14], and therefore resembles the 180-kDa polypeptide stained by DLE7 in the Western blot of serum. However, a cross-reaction between vitellogenin and the 180-kDa polypeptide can be ruled out by the complete negativity of liver extracts for a 180-kDa polypeptide. This latter finding is supported by the fact that whenever anti-chorion antibodies were tested by Western blot for the presence of antigen in serum, no cross-reaction with vitellogenin was ever obtained [4, 12–14, 18]. We took advantage of the presence of egg antigens in a soluble form in the blood to test whether they can be detected by immunoenzyme analysis. We found that DLE7 enabled rapid and sensitive antigen detection in tiny amounts of blood (minimum 100 µl). The assay was obviously specific for spawning females, but it could also be used for quantitative monitoring of egg antigen levels in males treated with hormones (e.g., estradiol-17ß) or in response to environmental xenobiotics and pollutants [31]. ELISA assays have been recently employed to detect and quantitate vitellogenin and yolk protein in winter flounder [32], thus assessing the use of immunoenzymatic analysis in fish reproduction. The Mediterranean sea bass has only very slight morphological sex differences so that the ELISA assay could be used for sex determination and identification of reproducers. Future studies will be concerned with monitoring the level of oogenesis in single females by measuring antigen levels in blood as an additional tool for studying the reproductive biology of this important fish species.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted: October 29, 1998.
Received: June 1, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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