Induction, isolation and a characterization of the lipid content of plasma vitellogenin from two Salmo species: Rainbow trout (Salmo gairdneri) and sea trout (Salmo trutta)

Comp. Biochem. Physiol. Vol. 81B, No. 4, pp. 869-876, 1985 Printed in Great Britain

0305-0491/85 $3.00+ 0.00 © 1985 Pergamon Press Ltd

INDUCTION, ISOLATION AND A CHARACTERIZATION OF THE LIPID CONTENT OF PLASMA VITELLOGENIN FROM TWO SALMO SPECIES: RAINBOW TROUT (SALMO GAIRDNERI) AND SEA TROUT (SALMO TRUTTA) BIRGITTA NORBERG and CARL HAUX Department of Zoophysiology, University of G6teborg, P.O. Box 250 59, S-400 31 G6teborg, Sweden (Tel: 031-853000) (Received 2 January 1985) Abstract--1. Vitellogenin synthesis is induced in juvenile rainbow trout (Salmo gairdneri) and juvenile sea

trout (Salmo trutta) by estradiol-17fl. 2. A purification procedure for vitellogenin from trout plasma by precipitation with MgCI2-EDTA and subsequent anion exchange chromatography on DEAE-Sephacel is described. 3. The total lipid contents of purified rainbow trout and sea trout vitellogenins are 18 and 19%, respectively. Approximately 2/3 of the lipids are phospholipids, while the remainder consists of triglycerides and cholesterol. Phosphorus determinations on delipidated vitellogenin yield a phosphorus content of 0.6370 in rainbow trout and 0.58% in sea trout vitellogenin. 4. Native (dimeric) vitellogenins from rainbow trout and sea trout both have an apparent molecular weight of 440,000, when estimated by gel filtration on Sepharose 6B.

INTRODUCTION The development of the non-mammalian oocyte starts with the transformation of the oogonium into a primary oocyte. The cell grows and increases in volume many thousandfold before maturation and ovulation. The enormous growth of the primary oocyte is mainly due to the incorporation of the egg yolk protein precursor, vitellogenin. This large protein is synthesized in the liver, in response to estrogen stimulation, secreted and transported in the blood to the ovary, where it is sequestered and cleaved into the yolk proteins lipovitellin and phosvitin. The yolk proteins are stored and serve as a nutrient reserve for the developing embryo (Follett and Redshaw, 1974; Wallace, 1978). Vitellogenin is a high molecular weight protein, ranging from 250,000 to 600,000 in different species. It contains variable amounts of lipids, carbohydrates and phosphate, and is capable of binding divalent cations, predominantly calcium. In teleosts, vitellogenin has been isolated and partly characterized in several species (Pluck et al., 1971; Nath and Sundararaj, 1981; de Vlaming et aL, 1980). In rainbow trout, Salmo gairdneri, Hara and Hirai (1978) and Campbell and Idler (1980) have isolated plasma vitellogenin from both sexually mature females and estrogen-treated juveniles by precipitation and subsequent gel chromatography. Further characterization by biochemical, immunological and electrophoretic methods demonstrated a strong relationship between plasma vitellogenin and the yolk proteins phosvitin and lipovitellin isolated from mature rainbow trout eggs. The total lipid content of teleost vitellogenins appears to be around 20% (Campbell and Idler, 1980; de Vlaming et al., 1980; this study). However, limited

information is available on the lipid composition of fish vitellogenins (Lrger et al., 1981). In view of the observed increases in different plasma lipids after estradiol administration (Bailey, 1957; Plack and Pritchard, 1968; Takashima et al., 1972; de Vlaming et al., 1977; Emmersen et al., 1979; de Vlaming et al., 1979; Wiegand and Peter, 1980), as well as during vitellogenesis (Ho and Vanstone, 1961; Petersen and Korsgaard-Emmersen, 1977), the relationship between plasma vitellogenin and plasma lipids remains to be investigated. The aim of the present study was to develop a simple method for isolation of an intact and homogenous vitellogenin fraction of high purity from blood plasma of estrogen-treated juvenile trout and to make a chemical characterization of the purified vitellogenin fraction, with special regard to the lipid composition. A comparison was made between vitellogenins from two Salmo species, Salmo gairdneri (rainbow trout) and Salmo trutta (sea trout).

MATERIALS AND METHODS Fish and hormone treatment Juvenile rainbow trout, Salmo gairdneri, with an average weight of 100 g, were obtained from a hatchery (Antens laxodling AB) close to G6teborg. Juvenile sea trout, Salmo trutta, with an average weight of 60 g, were supplied by the Salmon Research Institute, ~lvkarleby, Sweden. The fish were acclimated for at least 1 week in basins with filtered, aerated and recirculated freshwater at a temperature of 10°C. The photoperiod was 12 hr light/12 hr dark. No food was given before or during the experiments. At the start of the experiments, the fish were transferred to glass aquaria filled with 501 of filtered and aerated freshwater. The water was renewed every second day and the water temperature fluctuated between 8 and 12°C.

869

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B1RGITTA NORBERG and CARL HAUX

To induce vitellogenin synthesis, the fish were given weekly, i.p. injections of estradiol-17fl (Sigma Chemicals Co), at a dose level of 10 mg estradiol-17fl/kg body wt/week. The hormone was finely dispersed in peanut oil by using an ultrasonic bath. The injected volume was 0.2 ml. Control fish were not injected.

Sampling of blood and isotope Blood was collected 18-21 days after the first injection. To monitor the recovery of vitellogenin, 185 kBq of carrier-free 32p-orthophosphate (Amersham) was i.p. injected 24hr prior to blood sampling in some of the estrogenized rainbow trout and sea trout, as well as in some control sea trout. No inhibitors of proteolytic enzymes were used initially. To avoid proteolysis in the samples in subsequent experiments, the blood was either directly mixed with a saturated solution of the serine protease inhibitor phenylmethylsulfonylfluoride (PMSF, Sigma Chemicals Co) in 0.9~o NaC1, in a volume ratio of 2: 1, or the fish were injected in the caudal vessels 20 min before sampling with a trypsin inhibitor, aprotinin (Sigma Chemical Co). The aprotinin was dissolved in 0.9~o NaCI at a concentration of 10-20 trypsin inhibitor units (TIU)/ml and the injected volume was 100 #1. Blood was drawn from the caudal vessels using a heparinized, precooled syringe. The blood was centrifuged and the resulting plasma preparation used immediately. All preparative procedures were carried out at 4°C. Chromatography, precipitation and electrophoresis of vitellogenin The plasma preparation obtained as described above was either directly loaded onto a DEAE-Sephacel column or further processed by selective precipitation of vitellogenin, according to the procedures described below. DEAE-Sephacel (Pharmacia Fine Chemicals) was packed into a 28 x 2.6cm column. The column was equilibrated with at least 3 vol of 0.050 M Tris-HC1, pH 8.0 (starting buffer). The temperature was kept at 4°C. Immediately after centrifugation, 2-3ml of plasma was applied to the column. Unbound substances were eluted with 1 vol of starting buffer. To separate the plasma proteins, a linear NaC1 gradient (0.004).30 M NaC1 in starting buffer) was generated with a three-channel peristaltic pump (Pharmacia P3), maintaining a flow of 100 ml/hr through the column. Fractions of 8.0ml were collected and the absorbance at 280 nm measured with a Perkin-Elmer Lambda 3 spectrophotometer. The fractions corresponding to the main peak were pooled, concentrated on an ultrafiltration membrane (Amicon XM 100), dialyzed overnight against 200 vol of distilled water and finally freeze-dried. The freeze-dried protein was stored desiccated at -20°C. After each run, the column was washed with 500ml of 0.20 M Na-citrate containing 0.2~o Triton X-100 and reequilibrated with at least 1000 ml of starting buffer, according to the procedure of Wallace (1965). Vitellogenin was precipitated by mixing 1.0 ml of plasma with 4.0 ml of 0.020 M EDTA, pH 7.7, and subsequently adding 0.3 ml of 0.50 M MgC12. After centrifugation, the precipitate was dissolved in 1.0ml of 1.0M NaC1 and reprecipitation of the vitellogenin fraction performed by dilution with 10.0 ml of distilled water. The resulting precipitate was collected by centrifugation, dissolved in 0.50 M NaC1, diluted with 5.0 ml of distilled water, applied to a DEAE-Sephacel column and chromatographed as described above (Wiley et al., 1979). Occasionally, the dilution of the sample with distilled water prior to chromatography resulted in protein precipitation. This was remedied by dropwise addition of 0.20 M EDTA, pH 7.7, until the solution became dear. Sepharose 6B (Pharmacia Fine Chemicals) was packed into a 90 x 2.6cm column and equilibrated with several volumes of 0.020 M Tris-HCl, 0.35 M NaCI, pH 8.0 (eluant buffer). Gel filtration was performed at 4°C, at a flow rate

of 22ml/hr. Fractions of 7.3ml were collected and the absorbance at 280 nm measured as described previously. The column was calibrated for molecular weight determination with aldolase (158,000), catalase (232,000), ferritin (440,000) and thyroglobulin (669,000) (Pharmacia Fine Chemicals). Polyacrylamide gel electrophoresis was performed according to the procedure of Crambach et al. (1976), system 2860.0.X, with the modifications made by Wiley et al. (1979). The gels were stained for proteins with 0.2570 Coomassie Brilliant Blue R250 in 5070 methanol-10~o acetic acid. Phosphate determinations The analytical procedures for estimation of lipid and protein bound phosphate were modified from previously described methods (Bailey, 1957; Follett and Redshaw, 1968; Wallace and Jared, 1968; Craik, 1978). A plasma aliquot of 25 #1 was added to 5.0 ml of ice-chilled 2070 trichloroacetic acid (TCA) and the mixture centrifuged 5 rain at 2000 g. The precipitate was successively extracted with 2.5ml hot (80°C) ethanol (9970), 2.5ml chloroform:ether:ethanol (1:2:2), 2.5ml acetone and 2.5ml ether. An aliquot of 2.0 ml of the combined extracts was evaporated to complete dryness and lipid bound phosphate (i.e. phospholipids) determined according to Bartlett (1959). The remaining precipitate was dried overnight and digested at a temperature of 180°C for 3 hr in 0.50ml of a mixture of concentrated sulphuric acid: perchloric acid (6070) : water (28:7.5:64.5). Occasionally, an extra hour was necessary to obtain a clear solution after cooling. The whole solution was used for the determination of protein bound phosphate and the assay followed the procedure described by Bartlett (1959). Protein labelled with 32p was measured by a slight modification of the method of Mans and Novelli (1961). Samples of 100 #1 from each of the chromatographic fractions were applied to individual glass microfibre filters (Whatman GF/B, diameter 24 mm), placed in scintillation vials. After the discs were dried, protein was precipitated with 1.0 ml of a cold mixture of 1070 TCA and 1~o phosphotungstic acid. The discs were allowed to stand for 1 hr at 4°C and then washed twice with ethanol:ether (3:1), twice with ether and air-dried overnight. Ten ml of scintillation cocktail (Instagel, Packard) was added to each vial and the samples counted for radioactivity in a fl-scintillation counter (LKB 215 Rackbeta). Other analytical procedures Lipids were extracted twice from a 2070 (w/v) solution of vitellogenin in 0.9700NaCI with methanol :chloroform: NaCI (0.9~o) (2:1:1). Lipid determinations were carried out in the chloroform phase. The total lipid content was estimated in two ways: gravimetrically and by the charring method of Marsh and Weinstein (1966). Phospholipids were determined by a modification of the method of Bartlett (1959). The amount of phospholipids was calculated by multiplying the phosphorus value with 25, using lecithin, which contains 4~o phosphorus as a 'standard phospholipid'. Cholesterol was determined by the method of Webster (1962) and triglycerides by the method of Carlsson (1963). Total plasma protein was determined by the biuret method (Henry et al., 1957). When the protein concentration of a preweighed and dissolved amount of vitellogenin is measured by the Biuret method, the absorbance is only 8070 of that of an equal amount of preweighed and dissolved bovine serum albumin. RESULTS

Induction and isolation o f vitellogenin Following injection with estradiol-17fl, the plasma protein levels increased in r a i n b o w t r o u t from 3.8 + 0.1 g/100 ml (n = 4) in controls to 12.9 -+ 1.0 g/

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Fig. 1. Polyacrylamide gel electrophoresis of sea trout plasma and various vitellogenin preparations. (a) Plasma from control sea trout. (b) Plasma from estradiol-treated sea trout. (c) DEAE-Sephacel-purified vitellogenin from sea trout plasma. (d) Mg2+-EDTA-precipitated protein. (e) Vitellogenin precipitated with Mg2+-EDTA and chromatographed on DEAE-Sephacel. The electrophoresis was performed according to Crambach et al. (1976), system 2860.0.X and the gels were stained for protein with Coomassie Brilliant Blue R250. Arrows indicate vitellogenin dimer.

871

100 ml (n = 8) in estradiol-treated fish. In sea trout, the levels increased from 4.6 + 0.8 g/100 ml (n = 4) in controls to 10.5 + 0.7 g/100 ml (n = 6) in estrogenized fish. No sex differences regarding the increase in plasma protein levels were observed. Polyacrylamide gel electrophoresis on slab gels of plasma from estrogen-treated and control fish confirmed that the increase in plasma protein levels was due to the appearance of a new, high molecular weight protein, absent from plasma of controls (Fig. 1). When plasma of estradiol-treated fish, injected with nP-orthophosphate was chromatographed on DEAE-Sephacel, in the absence of protease inhibitors, a large peak containing protein-bound radioactivity eluted at a chloride ion concentration of 0.20M. Small amounts of protein-bound radioactivity eluted after the main peak, indicating that some degree of proteolysis had occurred in the sample. This was more pronounced in sea trout than in rainbow trout (Fig. 2). Chemical analysis of the fractions revealed a large phosphoprotein and phospholipid containing peak eluting at 0.20M C1 (Fig. 3). In the presence of either the serine protease inhibitor PMSF or the trypsin inhibitor aprotinin, the amount of phosphoprotein eluting after the main peak was reduced to a minimum (Figs 3 and 4).

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873

Isolation of trout vitellogenin Table 1. Chemical composition of vitellogenins from some different vertebrate species Salmo gairdneri a* b'l" Protein-bound phosphorus (%)¶ Total lipids (%) Phospholipids (%) Triglycerides (%) Cholesterol (%) Molecular weight ( × 103)**

0.63 18.0 11.0 4.0 2.0 4.4

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No phosphoprotein containing peak eluting at 0.20 M C1- was detected when plasma from control trout was subjected to DEAE-Sephacel chromatography (Fig. 5). Selective precipitation of vitellogenin with magnesium chloride, in the presence of EDTA, followed by chromatography on DEAE-Sephacel, showed that essentially all of the precipitated protein eluted at 0.20M CI-. A small amount of contaminating material was present, however, which eluted immediately before the main peak (Fig. 6). The elution patterns for the precipitated vitellogenin were similar for both trout species. A further evaluation of the purity of the isolated vitellogenin was made by polyacrylamide gel electrophoresis (Fig. 1). Precipitation with magnesium chloride, or DEAE-Sephacel chromatography alone, yielded contaminated fractions, while a combination of the methods appeared to give a purified vitellogenin fraction. Chemical characterization

Two different methods for the determination of total lipid content were used: the charring method of Marsh and Weinstein (1966) and gravimetric determination after extraction and evaporation of the solvent. Both methods yielded a total lipid content of

18% (w/w) in rainbow trout and of 19% in sea trout. In rainbow trout, 61% of the lipids consisted of phospholipids, 22% of triglycerides and 11% of cholesterol. The values for sea trout were 73% phospholipids, 16% triglycerides and 11% cholesterol (Table 1). The phosphorus content in delipidated rainbow trout vitellogenin was determined to 0.63% (w/w) and in sea trout vitellogenin to 0.58% (Table 1). Gel chromatography on Sepharose 6B of purified vitellogenin gave an apparent molecular weight 440,000 for vitellogenin from both trout species, the elution volume being similar to that of ferritin (440,000 mol. wt) (Table 1, Fig. 7). DISCUSSION Several different procedures have been developed for the isolation and purification of amphibian and avian vitellogenins (Wallace, 1965; Wiley et al., 1979). A precipitation step, followed by gel filtration and/or ion exchange chromatography are often involved to obtain pure and homogeneous vitellogenin. More or less modified, these methods have been used to purify vitellogenins from fish blood plasma (Hickey and Wallace, 1974; Korsgaard-Emmersen and Petersen,

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874

BIRGITTA NORBERG and CARL HAUX

1976; Hara and Hirai, 1978; Idler et al., 1979; Campbell and Idler, 1980; Nath and Sundararaj, 1981). However, for some fish species studied, the vitellogenin appears to be sensitive to proteolysis and easily denaturated during isolation procedures (Hickey and Wallace, 1974; de Vlaming et al., 1980). This could be due to the different chemical composition and structure of fish vitellogenins compared to other vertebrate vitellogenins. The use of the serine protease inhibitor PMSF could prevent the disintegration of vitellogenin (Hickey and Wallace, 1974). However, as recently demonstrated in a thorough characterization of goldfish vitellogenin, even when great precautions were taken, proteolysis could not be completely eliminated (de Vlaming et al., 1980). In the present study, 32p-orthophosphate was used as a specific marker for trout vitellogenin. There was only a slight tendency for the labelled vitellogenin to show any degree of proteolysis, manifested as protein-bound radioactivity eluting after the main peak (Fig. 2). Sea trout vitellogenin appeared to be more susceptible to proteolysis than rainbow trout vitellogenin. Repeated runs utilizing either 32p-orthophosphate or non-labelled phosphoprotein as specific indicators of vitellogenin gave replicable and identical results. To avoid heterogeneous elution profiles, PMSF was added to the blood immediately after sampling. This treatment largely prevented proteolysis, but since the low solubility and high toxicity of PMSF makes it inconvenient to use routinely, the trypsin inhibitor aprotinin was preferred in subsequent experiments. Aprotinin and PMSF were equally effective in preventing proteolysis of trout vitellogenin. It could thus be concluded from the initial isolation and purification work, that rainbow trout vitellogenin is less sensitive to proteolysis than sea trout vitellogenin or goldfish vitellogenin. These findings corroborate previous studies on rainbow trout, where the isolated vitellogenin was reported to be homogeneous, although the obtained plasma or serum containing vitellogenin was deep frozen and stored before subsequent purification and analysis (Campbell and Idler, 1980). To enrichen vitellogenin from plasma before chromatography, an initial precipitation step has often been introduced. Precipitation can be achieved by the addition of dimethylformamide (DMF), CaC12, CaCI2-EGTA or MgC12-EDTA to the plasma. Since DMF is toxic and calcium ions may activate proteolytic enzymes (Wiley et al., 1979), addition of EDTA followed by MgCI 2 was tested. Precipitation occurred, but only when the total plasma protein levels were above 7-8 g/100 ml, as measured by the Biuret method. This indicates that the concentration of vitellogenin is critical for the precipitation process. De Vlaming et al. (1980) found no precipitation of goldfish vitellogenin when EDTA and MgCI2 were added and they suggested that this was due to the low phosphorus content of goldfish vitellogenin, compared to amphibian vitellogenin. However, the present results indicate that this hypothesis may not be valid, as the phosphorus contents of goldfish, rainbow trout and sea trout vitellogenin are very similar. The present study confirms previous work showing that fish vitellogenins have a total lipid content of

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about 20~ (Table 1). This is distinctly more than observed for non-teleost vitellogenins (e.g. Wallace, 1970). The largest proportion of the lipid content of trout vitellogenin consists of phospholipids (approximately 2/3), while the remainder seems to be mainly triglycerides and cholesterol (Table 1). As large amounts of vitellogenin is accumulated in the growing oocytes, it becomes apparent that the lipids included in the viteUogenin molecule contribute significantly to the total fat content of the mature eggs. Furthermore, the increases in total plasma lipids, including phospholipids, triglycerides and cholesterol, that have been demonstrated in fish treated with estradiol (Bailey, 1957; Ho and Vanstone, 1961; de Vlaming e~ al., 1977; Emmersen et al., 1979; de Vlaming et al., 1979; Wiegand and Peter, 1980) could at least partly be due to the presence of vitellogenin. However, it is not known to what extent the lipids contained in vitellogenin contribute to the increases in total plasma lipids, as estradiol may also cause a mobilization of extra-hepatic fat depots (Wiegand and Peter, 1980). The phosphorus contents of vitellogenin from rainbow trout and sea trout are very similar (0.63 vs 0.58~o) and nearly identical to the values previously reported for rainbow trout (Campbell and Idler, 1980) and goldfish vitellogenin (de Vlaming et al., 1980). The few determinations reported of the phosphorus content of isolated fish vitellogenins, together with indirect evidence, suggest that teleosts reproducing in freshwater produce vitellogenins that are low in phosphorus compared with non-teleost vitellogenins (Wallace and Jared, 1968; de Vlaming et al., 1980).

Isolation of trout vitellogenin The functional role of phosphorylated proteins is not clear, but it has been proposed by Rosenstein and Taborsky (1970) that the binding energy of phosphate-protein is high and could be utilized during the embryonic development. However, this may not be the case with the eggs of marine teleosts, where the phosphorus content of the oocytes declines during a late stage of oocyte maturation and is low in fully mature eggs. (Craik, 1982). Attempts to determine the molecular weight of fish vitellogenins have indicated large species differences as well as significant differences within the same species. The large intraspecific variation is, most likely, due to varying isolation procedures as well as different methods for molecular weight determination. This was demonstrated for rainbow trout vitellogenin, where estimations made by ultracentrifugation, gel chromatography and gradient gel electrophoresis gave molecular weights of 342,000, 440,000 and 470,000, respectively (Campbell and Idler, 1980). In addition, Hara and Hirai (1977) reported a molecular weight of 600,000 for rainbow trout vitellogenin subjected to gel filtration on a Sepharose 6B column. In the present study, gel chromatography on Sepharose 6B yielded an apparent molecular weight of 440,000 for both rainbow trout and sea trout vitellogenin. This value is in accordance with that found by Campbell and Idler (1980). However, the determination of molecular weights of lipoproteins tends to give overestimations, due to 'abberant behaviour' on gel columns (Rodbard, 1976). As recent studies have shown that the monomeric form of rainbow trout vitellogenin has a molecular weight of 170,000 (Chen, 1983), it is apparently the dimeric form that is present in plasma. Acknowledgements--We gratefully acknowledge Ms Birgitta Vallander for drawing the figures, Docent Ake Larsson for valuable comments on the manuscript and FK Ulf-Peter Wichardt for kindly supplying the sea trout. The investigation was financed by Lars Hiertas Minne, HiertaRetzius fond frr vetenskaplig forskning, Anna Ahrenbergs fond, Kungl. och Hvitfeldtska Stipendieinr/ittningen and Stiftelsen Wilhelm och Martina Lundgrens minne. REFERENCES

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