Vitellogenin in the blood plasma of male cod (Gadus morhua): A sign of oestrogenic endocrine disruption in the open sea?

MARINE ENVIRONMENTAL RESEARCH Marine Environmental Research 61 (2006) 149–170 www.elsevier.com/locate/marenvrev

Vitellogenin in the blood plasma of male cod (Gadus morhua): A sign of oestrogenic endocrine disruption in the open sea? Alexander P. Scott a,*, Ioanna Katsiadaki a, Peter R. Witthames b, Ketil Hylland c, Ian M. Davies d, Alistair D. McIntosh d, John Thain e a

d

Centre for Environment, Fisheries and Aquaculture Science (Cefas), Weymouth Laboratory, Barrack Road, Weymouth, Dorset, DT4 8UB, UK b Cefas, Pakefield Road, Lowestoft, Suffolk, NR33 0HT, UK c NIVA, P.O. Box 173, Kjelsa˚s, N-0411, Oslo, Norway Fisheries Research Services, Marine Laboratory, 375 Victoria Road, Aberdeen, AB11 9DB, UK e Cefas, Remembrance Avenue, Burnham-on-Crouch, Essex, CM0 8HA, UK Received 9 May 2005; received in revised form 25 August 2005; accepted 31 August 2005

Abstract An ELISA for cod vitellogenin (VTG) has been set up using cod lipovitellin for plate coating and standardisation. The assay has been applied to plasma samples collected from male and female cod caught in three distinct areas around the UK, three areas off the Norwegian coast and also to cod reared initially at an aquaculture site and subsequently maintained at a research station. The aim of the study was to determine whether there were any signs of oestrogenic endocrine disruption in a fish species living offshore. VTG induction was found in male cod caught in the North Sea, the Shetland Box area, in Oslofjord and also in cultivated fish. There was a strong relationship between concentrations of VTG and fish size. There was no evidence that the presence of VTG in the plasma of males is a natural part of their life cycle. On the other hand, the size of fish at which these elevated VTG concentrations appear (ca. 5 kg) is about the size that cod change from feeding primarily on benthic invertebrates to mainly other fish, both benthic and pelagic. The possibility is suggested that large cod pick up oestrogenic endocrine disrupters through the food chain. Crown Copyright
2005 Published by Elsevier Ltd. All rights reserved. *

Corresponding author. Tel.: +44 1305 206637; fax: +44 1305 206601. E-mail address: [email protected] (A.P. Scott).

0141-1136/$ - see front matter. Crown Copyright
2005 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2005.08.003

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Keywords: Cod; Endocrine disruption; Vitellogenin; Oestrogens; Estrogens; Marine fish

1. Introduction There are large numbers of examples of xenoestrogenic endocrine disruption in freshwater fish and, until very recently, only a few in estuarine or marine environments (Oberdorster & Cheek, 2001). However, the number of examples in the marine environment is now large and growing. Male flounder (Platichthys flesus) caught in industrialised estuaries of the UK and the Netherlands have been found to have elevated (in some cases grossly elevated) concentrations of vitellogenin (VTG) in their plasma (Allen et al., 1999a, 1999b; Kirby et al., 2004; Kleinkauf, Scott, Stewart, Simpson, & Leah, 2004; Lye, Frid, & Gill, 1998; Lye, Frid, Gill, & McCormick, 1997; Matthiessen et al., 1998; Vethaak et al., 2002). Some male flounder with elevated VTG concentrations have also been caught in the open sea (Allen et al., 1999a), but these were hypothesised to be fish that had recently emigrated from a contaminated estuary. In estuarine and coastal areas of Japan, many male marbled sole (Pleuronectes yokohamae) (Hashimoto et al., 2000), common goby (Acanthogobius flavimanus) (Ohkubo et al., 2003) and grey mullet (Mugil cephalus) (Hara, Matsubara, & Soyano, 2001) have also been found with unexpectedly high concentrations of VTG in their plasma. Moving away from the coast and into the open sea, the presence of VTG and zona radiata protein (Zrp) in blood plasma (and/or immunohistochemical evidence for enhanced liver production of VTG and Zrp) has been confirmed in swordfish (Xiphias gladius) caught in the Mediterranean (Desantis et al., 2005; Fossi et al., 2001; Fossi et al., 2004; Fossi et al., 2002) and off the coast of South Africa (Desantis et al., 2005) but not fish in the Pacific Ocean (Desantis et al., 2005). Similarly, many male tuna (Thunnus thynnus) caught in the Mediterranean have VTG in their plasma (Fossi et al., 2002) while tuna (Thunnus obesus) caught in the Pacific Ocean (Hashimoto et al., 2003) do not. In addition to direct evidence of oestrogenic effects provided by the presence of VTG and Zrp in plasma, there is also indirect (circumstantial) evidence provided by the presence of intersex gonads (Allen et al., 1999a; Cho et al., 2003; De Metrio et al., 2003; Lye et al., 1997) and feminised secondary sexual characteristics (Kirby et al., 2003) in males of some of these marine species. Despite the many claims for endocrine disruption in fish, in many (but not all) cases, interpretation of the evidence is hampered by the fact that, because it is not known what is ÔnormalÕ, it is difficult to know whether reported observations are ÔabnormalÕ (Sumpter & Johnson, 2005). In the marine environment particularly, it is difficult to identify a totally unpolluted ÔcontrolÕ site. In order to measure VTG in fish plasma, it is necessary to develop sensitive and specific immunoassays. Surprisingly, although antisera for cod (Gadus morhua) VTG were produced over ten years ago (Silversand, Hyllner, & Haux, 1993; Yao & Crim, 1996) and the antiserum from the former study is commercially available, there is only one published report of the development and application of an enzyme-linked immunosorbent assay (ELISA) for cod VTG (Hylland & Haux, 1997). Part of the problem can be ascribed to the fact that cod VTG is reputedly unstable during lyophilisation or long-term storage (Silversand et al., 1993). In order to carry out measurements of cod VTG, a fresh batch of

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VTG (for plate coating) needed to be prepared each time that an assay was carried out (Hylland & Haux, 1997). A pool of cod plasma was used for standardisation. Clearly, it would be more convenient (and there would be no risk of degradation of the protein during frozen storage of the plasma) if one had a powder that could be stored for many years in a dry (and hence stable) form. The same problem was encountered by Hartling, Pereira, and Kunkel (1997) when trying to set up an ELISA for vitellogenin of the winter flounder (Pleuronectes americanus). These authors overcame the problem by using lipovitellin (LV), instead of VTG, as a standard and a coating for ELISA plates. In their study, they found that LV could be freeze-dried without damage and could conveniently be heat-treated (to destroy protease activity). LV is the most abundant protein in the ovaries and can thus be prepared in large amounts and in a highly purified form. The present paper reports on the application of an ELISA for cod LV to determine whether signs of oestrogenic endocrine disruption (exemplified by elevated VTG concentrations in males and immature females) are detectable in wild cod in the North Sea. Other possible causes of elevated VTG concentrations are discussed. 2. Materials and methods 2.1. Preparation of cod VTG Cod VTG was purified and lyophilised in an identical fashion to that described for flounder VTG (Scott & Hylland, 2002). Blood plasma rich in VTG was generated by adding powdered E2 to melted cocoa butter (ca. 40 C) at a concentration of 50 mg/ml. The powder was then evenly dispersed in the butter by ultrasonication and injected intramuscularly into a male cod (ca. 1 kg) at the rate of 400 ll (20 mg) per kg of fish. The cod, which had been adapted to laboratory conditions following its capture in the North Sea, was first anaesthetised with 2-phenoxyethanol in seawater (0.1% v/v). Three weeks later, it was anaesthetised again, killed by a blow to the head and blood collected from the caudal vein. In order to minimise any possible degradation of VTG (Silversand et al., 1993), the 2 ml syringes that were used for the blood collection were rinsed with a saline solution containing 8 Trypsin Inhibitor Units (TIU)/ml of aprotinin and 500 IU/ml heparin (sodium salt). Blood was transferred to collection tubes, on ice, which contained 50 ll of the same solution. A maximum of 2.5 ml blood was added to each tube, centrifuged at 2000 rpm and 4 C for 15 min and the plasma removed, frozen and stored in liquid nitrogen. 2.2. Preparation of antiserum The antiserum (R283) used in this study was generated as described previously (Scott & Hylland, 2002) by injection into a rabbit of 3 mg lyophilised VTG suspended in 2.5 ml phosphate buffer and added to 2.5 ml of Complete FreundÕs Adjuvant (CFA). Boost immunisations followed at four weekly intervals using incomplete FreundÕs Adjuvant. 3. Preparation of cod LV The procedure was based on that described for the purification of LV from winter flounder (Hartling et al., 1997). A portion of frozen ovary (160 g) from a fully sexually

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mature female cod was homogenised in 800 ml 0.5 M NaCl (1/5;w/v) with a Turbex grinder. The extract was left at room temperature for 30 min and then centrifuged for 30 min at ca. 1000g (to remove membrane proteins). The supernatant was placed in a flask in a heated water bath until the temperature reached 60 C. It was then cooled under a running tap and centrifuged again at ca. 1000g for 30 min. The supernatant was removed, mixed with 10 volumes of ice-cold distilled water and left overnight at 4 C. The next morning, most of the liquid was decanted and discarded. The slurry from the bottom of the container was transferred to 50 ml plastic tubes and centrifuged at 1000g for 20 min. The remaining liquid was discarded and the precipitates (between 5 and 10 ml per tube) frozen and stored at
20 C. The crude LV was purified by size exclusion chromatography on a HR 10/30 Superdex column (Amersham Biosciences). Before any sample was loaded onto the column, it was equilibrated with 50 mM Tris–HCl, pH 7.6 containing 1% sodium chloride (w/v) and 0.01% sodium azide. Approximately 500 ll of the LV precipitate was thawed out, redissolved in 500 ll of the column buffer and then passed through a 22 lm filter. Just over 150 ll of this filtrate was then used to load a 100 ll loop and injected on to the column at a flow rate of 0.5 ml/min. The procedures were carried out at room temperature. There were several minor peaks and one very dominant LV peak. The fraction collector was set to collect only the fractions from this peak. The procedure was repeated five times and the eluted protein solution combined, desalted on a PD-10 column with deionised water and lyophilised. 3.1. Confirmation that the ELISA detects VTG in plasma Two sexually quiescent cod, one male and one female, weighing approximately 1 kg, were implanted with E2 as described above. Just prior to injection and at weekly intervals afterwards, finishing at four weeks, the fish were anaesthetised and a 5 ml blood sample was collected and added to a tube containing heparin (50 ll; 1000 U/ml) and 25 TIU aprotinin. The blood was mixed, centrifuged and the plasma snap frozen in liquid nitrogen. It was then stored at
70 C . At a later date, the plasma samples were thawed, 150 ll mixed with 150 ll of column buffer, passed through a 22 lm filter and 100 ll injected onto the HR 200 column as described above for the purification of LV. Fractions were collected in Ôdeep wellÕ polystyrene multiwell plates every minute between 13 and 36 min. The fractions were then stored at
20 C until they could be assayed for VTG by ELISA (see below). 3.2. SDS–PAGE Lyophilised VTG and LV were dissolved at a concentration of 1 mg/ml in 50 mM carbonate–bicarbonate buffer; mixed (1:5 v/v) with 100 mM Tris–HCl buffer (pH 8.5) containing 10 mM EDTA, 8 M urea, 2% sodium dodecyl sulphate (SDS; w/v) and 200 mM b-mercaptoethanol; and heated at 70 C for 30 min. Aliquots of the above solutions were further diluted with Laemmli sample buffer (62.5 mM Tris–HCl, 25% glycerol (v/v), 2% SDS (w/v), 0.01% bromophenol blue (w/v), pH 6.8) at 1:4 (v/v) and heated at 105 C for 5 min prior to electrophoresis. Electrophoresis (SDS–PAGE) was performed using precast Tris–HCl polyacrylamide gels (4% stacking gel, 7.5% resolving gel, 2.6% crosslinker, 0.375 mM Tris–HCl, pH 8.8). A high molecular weight (212–53 kDa) SDS calibration kit was also employed. Electrophoresis was carried out at 180 V (constant voltage) for 30 min

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with a Bio-Rad Power Pack (200) power supply and a mini-Protean (Bio-Rad Laboratories Ltd.) dual slab electrophoresis cell. After separation, the gels were stained with Coomassie brilliant blue for 30 min. 3.3. ELISA procedure Disposables, equipment and basic procedures for ELISA were identical to those used for the assay of stickleback (Gasterosteus aculeatus) spiggin (Katsiadaki, Scott, Hurst, Matthiessen, & Mayer, 2002) with the exception that, for coating plates, 1 mg heat-treated LV was dissolved in 500 ll distilled water followed by 500 ll of coating buffer. A 400 ll aliquot was added to 100 ml of coating buffer and 100 ll of this dilute solution was then added to all wells. The only other important exception was that the first well in each set of four 10-fold dilutions received 100 ll buffer and 50 ll of plasma or standard (not 135 and 15 ll). Also, after all dilutions had been made, 65 ll of antiserum R283 at a dilution of 1:15,000 (v/v) was added to all wells. 3.4. Assay of 17b-estradiol For steroid assay, plasma aliquots (100 ll) were shaken vigorously with 100 ll distilled water and 4 ml diethyl ether for two min. The aqueous layer was allowed to settle and then frozen in liquid nitrogen. The solvent phase was poured into a glass tube (12 mm · 75 mm) and allowed to evaporate in a water bath at 45 C. The residue was redissolved in 400 ll radioimmunoassay (RIA) buffer and measured as described previously for E2 (Scott, Bye, Baynes, & Springate, 1980; Scott, Mackenzie, & Stacey, 1984). Two aliquots of 100 ll were used for assay of E2. The coefficient of interassay variation was <10%. 3.5. Collection of cod plasma samples Plasma was collected from male and female wild cod (weighing between 130 and 10,000 g; n = 140) caught by trawling during two research vessel cruises in the North Sea and English Channel (in the first week of July in both 2001 and 2003). The most northerly site was at Farne Deep (5529 0 N0107 0 E) and the most southerly at Rye Bay (5039 0 N0141 0 E). A further collection of male and female cod (108–9400 g; n = 183) plasma was made from cod collected in the Shetland Box area of the North Sea (centred around 610 0 N010 0 E) in July, 2002. Collections of male cod only (152–7190 g; n = 100) were made from the Irish Sea (within an area bounded by 5219 0 N337 0 W and 5443 0 N66 0 W) in March, 2003. All fish were killed by a blow to the head, weighed and measured and a blood sample taken with a heparinised syringe from the caudal vein. One year old Arcto-Norwegian cod reared at Parisvatnet, Norway from captive broodstock were brought to the Institute of Marine Research, Matre in the autumn of 2002. From this time until February 8 2003, these fish were held in a 40 m3 tank supplied continuously with fresh sea water and fed dry food (Felleskjøpet: ÔCod pelletsÕ; 10% fat, 53% protein, 18.5% carbohydrates) in a natural photoperiod. After February 8, the fish were divided between two tanks and exposed to either a natural or constant photoperiod as part of another experiment not reported here. The fish were not fed again until the end of the experiment. A number of fish were taken from these tanks between February 19 and March 21 to provide plasma for assay of VTG and E2.

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Plasma samples were taken from cod caught by trawling at three sites off the Norwegian coast: two reference sites (Lofoten and Varanger; 6812 0 N 1448 0 E and 6955 0 N 2930 0 E, respectively) and one area known to be moderately influenced by contaminants (Inner Oslofjord; 5946 0 N 1035 0 E). These cod were sampled as part of the Norwegian Joint Assessment and Monitoring Programme (JAMP). All fish were collected during September or October. As for the above fish, all cod were killed by a blow to the head and a blood sample taken with a syringe pretreated with heparin and aprotinin. Plasma and blood cells were separated within 5 min and the plasma frozen in liquid nitrogen. The approximate weight to length relationship of all wild male cod in the present paper was: 1 kg = 37 cm; 2 kg = 59 cm; 4 kg = 74 cm; 5 kg = 80 cm; 7.5 kg = 93 cm; 10 kg = 104 cm. 3.6. Statistics Variables were checked for homogeneity of variances (LeveneÕs test) prior to statistical analyses. Appropriate transforms were used where required. If homogeneity of variances could not be achieved, non-parametric Mann–Whitney or Kruskal–Wallis analyses were used to compare groups (Sokal and Rohlf, 1981). In the cases where variances were homogenous, ANOVAs or ANCOVAs were used to test differences between groups with vitellogenin or estradiol as dependent variables (Sokal and Rohlf, 1981). Sampling area was always an explanatory variable, weight was included as covariate for some of the analyses for VTG. The level of significance for rejection of H0: no difference between groups was set to 0.05. 4. Results 4.1. Physical properties of lyophilised VTG and LV The lyophilised VTG split into three bands on SDS–PAGE (Fig. 1). The band with the highest mass was approximately 164 kDa and this was assumed to be unfragmented VTG. It should be noted that the complete lyophilised preparation (a mixture of all three bands) was used for the induction of antiserum R283. Another antiserum was also raised to the 164 kDa band. However, this antiserum, and also the commercially available anti-cod VTG serum, had titres that were ca. eight times lower (and yielded less sensitive standard curves) than R283. The LV ran as a single band on SDS–PAGE (Fig. 1). It ran slightly ahead (116 kDa) of the middle band from the VTG preparation (135 kDa). 4.2. Confirmation that the ELISA detects VTG in plasma Plasma samples from either a wild female cod or from an E2-injected male cod gave dilution curves that were parallel to LV over the steepest most accurate part of the curve (Fig. 2). The coefficient of intra-assay (essentially Ôbetween plateÕ) variability was examined at least three times (n = 6, 10 and 11) and was 13.4%, 9.6% and 11.9%. The coefficient of inter-assay variability was established using seven different male plasma samples (range 0.3–87.2 lg/ml) that were measured on separate occasions (between three and five times each), yielding values between 11.5% and 37.2%.

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Fig. 1. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis separation of lyophilised preparations of cod vitellogenin and lipovitellin. Molecular weight markers (kilodaltons): a, 212; b, 170; c, 116; d, 76, e, 53.

Absorbtion at 405 nm

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Effluent volume (ml) Fig. 3. Size exclusion chromatography on Superdex 200 HR of plasma taken from a male and female cod sampled 0, 14 and 28 days after being implanted with 17b-oestradiol; dashed line = UV absorbtion at 280 nm of the male fish (arbitrary units); thin solid line = VTG concentration in fractions of male cod plasma; thick solid line = VTG concentration in fractions of female cod plasma. The UV peak between 19 and 20 min is due to aprotinin.

E2 treatment dramatically increased the amounts of immunoreactive material in plasma of both a male and female cod (from <1 to >20,000 lg/ml over three weeks). When the plasma samples were subjected to gel filtration and the fractions were then assayed with the LV-based ELISA, it could be seen (Fig. 3) that, prior to treatment, there was little to no activity in any of the fractions. After two and four weeks, however, there was a large build up of immunoreactive material in the expected elution position of VTG. This was matched by the appearance of a peak of UV adsorption. 4.3. Measurements of VTG in cod plasma There were marked differences in mean VTG concentrations in male cod between the three major sampling sites. However, when these were plotted against the weight of the fish (Fig. 4(a)) it could be seen that these differences were governed more by the size of

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Fig. 4. Concentrations of vitellogenin in plasma of male cod caught in three different areas around the UK plotted against the body weight of the fish with (a) vitellogenin concentrations shown on a linear scale and (b) vitellogenin concentrations shown on a logarithmic scale (with regression line). h, North Sea (n = 82); n, Shetland Box (n = 85); s, Irish Sea (n = 99).

the fish than the region of capture. Up to a mass of about 5 kg (=80 cm), VTG concentrations were low and in some cases below the limits of detection of the assay (0.01 lg/ml). However, above 5 kg, there were many fish with noticeably elevated VTG concentrations. When VTG data were plotted logarithmically, it could be discerned that the increase in VTG concentrations did in fact start below 5 kg and that the relationship between VTG and fish weight was more of an exponential form (Fig. 4(b)). The coefficient of correlation (R2) of log VTG concentration to body weight was 0.65. An attempt was made to determine whether there was a statistical difference in VTG concentrations between all regions (after correction for fish weight). Unfortunately, it was not possible to obtain homogeneous variances for fish between all sites. This was because many of the smaller fish from the Shetland Box area had values that were at or below the detection limit. Estimations of age were made on several of the large male cod caught in the North Sea in 2003 (by counting rings in one of the otoliths). The largest fish (10 kg) was 5 years old and the second largest (7.5 kg) was 3 years old. Most of the other males that were examined were 2 years old. Female cod were only collected from two of the regions. Although there was a significant correlation of log VTG to body weight in females from the Shetland Box (R2 = 0.334; n = 101; p > 0.05) there was none in the females from the North Sea (R2 = 0.041; n = 94). There were many small North Sea females (<5 kg) that had higher VTG concentrations than those found in males of the same size range (Fig. 5). Unfortunately, due to too many values from the Shetland Box area being close to the detection limit, it was not possible to determine whether or not there was a statistical difference between the two regions. All of the males from Norwegian coastal sites weighed <2 kg. For the two most northern sites, male cod had VTG concentrations that were as expected for fish of that size (Fig. 6). Many of the male cod from the inner Oslofjord, however, had VTG concentrations (1–10 lg/ml) that were significantly higher than those from the other two regions, and from Irish and North Sea fish. Two out of the twelve males caught at Lofoten were 6 years old, three were 5 years old and four were 4 years old.

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Fig. 5. Concentrations of vitellogenin in plasma of female cod caught in two different areas of the North Sea plotted against the body weight of the fish. The small black symbols are the values for wild male cod already shown in Fig. 4 and included here for comparative purposes. h, North Sea (n = 94); n, Shetland Box (n = 101).

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Body weight (g) Fig. 6. Concentrations of vitellogenin in plasma of male cod caught at three different sites off the coast of Norway. The small black symbols are the values for wild male cod (already shown in Fig. 4 and included here for comparative purposes). n, Varanger (n = 10); s, Lofoten (n = 12); h, Oslofjord (n = 13).

Reproductively mature farmed female cod had, as expected, very high (>5 mg/ml) VTG concentrations (Fig. 7). Some of the males, however, had VTG concentrations that were significantly elevated in comparison to those found in wild males of an equivalent weight (<2 kg), excluding those caught in the Oslofjord.

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Body weight (g) Fig. 7. Concentrations of vitellogenin in plasma of farmed female (s; n = 51) and male (h; n = 23) cod plotted against the body weight of the fish. The small black symbols are the values for wild male cod (already shown in Fig. 4 and included here for comparative purposes).

4.4. Measurement of E2 in cod plasma There was no obvious size-related trend in E2 concentrations in either the males (Fig. 8) or immature females (Fig. 9). There was also no correlation (R2 < 0.02; n > 200) between E2 and VTG concentrations in either group. There were no statistical differences in E2 0.40 0.35

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Fig. 8. Concentrations of 17b-oestradiol in plasma of male cod caught in three different areas around the UK plotted against the body weight of the fish. These are the same fish as shown in Fig. 4. h, North Sea; n, Shetland Box; s, Irish Sea.

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Body weight (g) Fig. 9. Concentrations of 17b-oestradiol in plasma of female cod caught in two different areas of the North Sea plotted against the body weight of the fish. These are the same fish as shown in Fig. 5. h, North Sea; n, Shetland Box.

concentrations between males and females in either the North Sea or Shetland Box areas. There was also no significant difference between males in the North and Irish Sea. There was a significant difference, however, for both males and females, between the Shetland Box area and the two southern regions. When compared with the concentrations found in mature farmed females (Fig. 10), these differences were relatively small. These 100.00

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Body weight (g) Fig. 10. Concentrations of 17b-oestradiol in plasma of female (s) and male (h) farmed cod plotted against the body weight of the fish. These are the same fish as shown in Fig. 7. The small black symbols are the values for wild male cod (already shown in Fig. 8 and included for comparative purposes).

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differences were not due to inter-assay variability (ca. 10%), but could possibly have been due to Ôinter-extractionÕ variability (as the samples from the different sites were collected in different years and were extracted at separate times). 5. Discussion 5.1. Characteristics of the ELISA The ELISA that is described in the present paper is not the first to be described for cod VTG. Hylland and Haux (1997) devised and applied a method in which the coating material was freshly prepared, prior to each assay, by Superdex 200 fractionation of plasma from an E2-treated fish. The same plasma sample (without fractionation) was used as a standard. It was decided at the start of the present study, that a stable lyophilised standard would be more convenient for routine immunoassay of VTG. In addition to being easier to handle and quicker to prepare, a lyophilised standard would have the advantage of longterm stability and inter-laboratory portability. We have confirmed (Fig. 1) that, unlike LV, cod VTG breaks up into three bands when it is lyophilised. We do not know the reason. The damage does not appear to occur during the purification stage nor even during longterm frozen (
20 C) storage in distilled water (own unpublished results). It has been speculated that fish VTGs in general are prone to damage because VTG is a natural precursor of smaller fragments (such as LV) and thus designed for proteolytic cleavage (Arukwe & Goksoyr, 2003). Protease inhibitors are often included during the initial stages of VTG purification (though there is no clear proof that they are effective). It appears that, for some fish species like rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio), VTG is robust during lyophilisation and, for others like cod and perch (Perca fluviatilis) it is not (Hennies, Wiesmann, Allner, & Sauerwein, 2003; Silversand et al., 1993). Success in obtaining intact VTG appears to owe little to measures taken to prevent proteolytic cleavage and more to choice of species. However, one laboratory has recently claimed that they have had Ôsuccess in finding conditions for stabilising VTG by lyophilisation, although this has not been a straightforward task, and different species behave differently in this processÕ (Arukwe & Goksoyr, 2003). It is interesting to compare the present results for the purification of cod VTG with those of a previous study (Silversand et al., 1993). These authors estimated 167 ± 5 kDa for the mass of cod VTG (cf. estimate of 164 in this study). They did not lyophilise their preparation. However, they did note that, when stored frozen with SDS in the buffer, it broke completely into two bands with masses of ca. 130 and 60 kDa. These look to be the same size as the two faster-moving bands that were present in our lyophilised preparation of VTG. 5.2. A natural endogenous cause for elevated VTG concentrations in large male cod? VTG is produced in the liver of fish. The liver cells possess oestrogen receptors and will not synthesis VTG unless these receptors are activated. In the normal female reproductive cycle, the main oestrogen that binds to these receptors is E2. The source of the E2 is the follicular cells that surround the oocytes. The synthesis of E2 is, in its turn, dependent upon stimulation by gonadotropin (also called Follicle Stimulating Hormone; FSH) from the pituitary gland (Antonopoulou, Bornestaf, Swanson, & Borg, 1999). FSH is normally

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only secreted during the stage known as secondary vitellogenesis (when the oocytes build up their reserves of egg yolk protein). In a temperate fish species such as the cod, this stage lasts from about October to March (Dahle, Taranger, Karlsen, Kjesbu, & Norberg, 2003; Norberg, Brown, Halldorsson, Stensland, & Bjornsson, 2004). In the intervening months, when the fish are mainly feeding, there should, in theory, be little or no secretion of FSH by the pituitary gland with the consequence that there should be little or no secretion of E2 by the ovarian follicles and hence little or no production of VTG by the liver. All of the females shown in Fig. 4 were caught in June/July. Histological examination of their ovaries (results not shown) confirmed that all oocytes were in a primary growth stage (i.e., there was no evidence that any of the oocytes in any of the fish had advanced as far as the Ôcortical alveoliÕ stage that heralds the start of the reproductive cycle). Although E2 was detectable in all the plasma samples, concentrations were at levels previously found in reproductively quiescent cod (Dahle et al., 2003) and carp (Matsumoto et al., 2002). There was also no correlation at all between E2 and VTG concentrations. The reason why fish can have a little bit of E2 in their blood and not trigger VTG synthesis is possibly because fish blood contains a specific sex steroid binding protein (Pasmanik & Callard, 1986). In most fish, this protein has an affinity and capacity for E2 that ensures that, at the concentrations found in the present study, only 1% would be free (i.e., unbound) and able to interact with the receptor (Scott, Pinillos, & Huertas, 2005). This small amount of E2 is probably insufficient to trigger VTG synthesis. One other reason for suspecting an exogenous cause is that small (<5 kg) female cod from the North Sea had a far higher frequency of elevated VTG concentrations than those from the Shetland Box area. If VTG induction were part of a normal physiological phenomenon, this difference would perhaps not be expected. The only hint of caution is that, in the first batch of female cod that were examined in this study, caught in the southern North Sea in July 2001, it was found (Scott, Katsiadaki, & Thain, 2002) that the three females with the highest VTG concentrations in their plasma had slightly heavier ovaries than the rest of the females. This would perhaps suggest that, despite the absence of histological signs or of elevations in E2 concentrations, the reproductive cycle had indeed been initiated in these fish. Unfortunately, ovaries were not weighed during subsequent sampling trips. In males, although both the testes and the brain are probably capable of synthesising E2, the concentrations of E2 that were found in the plasma of males, especially those that were essentially immature, were the same as those found in the females caught at the same time. The same finding was made in carp (Matsumoto et al., 2002). As with females, there was also no relationship of E2 concentrations to either fish size or VTG concentrations in the males. Also, if endogenous E2 was the cause of elevated VTG concentrations in male cod, then it might be expected that males caught in March at the height of the spawning season (Irish Sea) would have much higher E2 and VTG concentrations than those caught in June/July (e.g., North Sea and Shetland Box). However, this was very obviously not the case. It is still possible that the elevated VTG concentrations in large male cod are caused by an endogenous natural mechanism that remains to be discovered. One possibility could be that, as the males grow older, the liver receptors become hypersensitive to the small amount of E2 that are present in the blood. Another could be that the liver cells gain the ability to make VTG without the intervention of E2. Yet another could be that male cod start secreting a yet-to-be-identified oestrogen as they grow older. However, this is

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pure speculation and we are unaware that any of these possibilities has yet been demonstrated in any other species. Furthermore, a large size in cod does not necessarily imply old age. There are extreme differences in the size–age relationship in cod from different Atlantic stocks (Garrod, 1988). Consistent with what is known about the size–age relationship for the cod that were sampled in the present study, the largest male cod that was caught in the North Sea (that had a mass of 10 kg and a VTG concentration of 154 lg/ml) was only 5 years old. Just under half of the males that were caught at Lofoten off the coast of Norway (mean body weight of only 0.75 kg) were 5 and 6 years old and yet had low to undetectable concentrations of VTG. In other words, the appearance of VTG in the blood plasma of large male cod does not seem, on the basis of these data, to be an aging phenomenon – but a size-related phenomenon. More data are needed to confirm this, however. There have been several recorded observations in the seventeenth and nineteenth centuries (Howes, 1893; Masterman, 1895) of hermaphroditic cod (i.e., simultaneous presence of testis and ovaries; also termed ÔintersexÕ). There are many papers that imply, through their titles and content, a link between this phenomenon and that of VTG induction (Jobling, Nolan, Tyler, Brighty, & Sumpter, 1998; Kirby et al., 2004). Oestrogens admittedly have an important role to play in ÔfeminisationÕ of gonadal tissue (Krisfalusi & Nagler, 2000). However, sexual differentiation in fish is very plastic. Other types of compounds (e.g., aromatase inhibitors, androgens and anti-androgens), plus even a change in temperature, are able to readily alter fish gender (Al-Ablani & Phelps, 2002; Baroiller, Guiguen, & Fostier, 1999) whereas only oestrogens (or more specifically compounds that in one way or another activate the oestrogen receptor) are so far known to be involved in the induction of VTG (Sumpter & Jobling, 1995). Field studies of flounder in the UK have indicated a poor association between those sites that have a particularly high degree of VTG induction and those where there are hermaphroditic fish (Allen et al., 1999b). For example, at a site with a prolonged history of high VTG induction (the Tees estuary), hermaphrodites have yet to be recorded (Kirby et al., 2004). Although it is possible in the present study that the larger males (>5 kg) were in fact undetected hermaphrodites (as only a small portion of each gonad was examined histologically), it has already been explained above why the possession of ovarian tissue is not necessarily an explanation for the presence of VTG. Yet one other possible ÔnaturalÕ explanation for elevated VTG concentrations is that E2 is transferred via the water from sexually mature females to males. This could have been a reason for elevated VTG concentrations in the cultivated males in the present study (because they were held in the same tanks as the mature females). Fish-to-fish transfer of steroids has been demonstrated in closed tank systems (Budworth & Senger, 1993; Vermeirssen & Scott, 1996). However, it is unlikely to occur to any great extent in fish that are free-swimming in the ocean (and also, as for most of the fish in the present study, reproductively immature). 5.3. Perhaps the material that cross-reacts in the assay is not VTG? There is a possibility that the material that is being detected in the large male cod is not VTG but some other protein that cross-reacts with the antiserum. In order to disprove this, the cross-reacting protein would need to be isolated and subjected to partial sequence analysis. Until such time, however, it is necessary to rely on indirect evidence of the probable specificity of the assay used in the present study. Firstly, the present ELISA uses an

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antiserum raised against VTG extracted from plasma and a coating material (LV) extracted from the ovary. It is unlikely that a protein entirely unrelated to VTG would be present in both preparations. Secondly, VTG concentrations measured by the present ELISA were found in a previous study (Scott et al., in press) to be highly correlated (R2 = 0.88; n = 92) with those measured by a totally different type of ELISA for cod VTG. 5.4. Are elevated VTG concentrations in male cod perhaps generated by a procedural error in the ELISA? Although the coefficient of interassay variation in the present study is relatively high, this variation is nowhere high enough to explain the differences between VTG concentrations between small and large male cod. Also, all measurements on samples from each site were made within a single assay (i.e., all samples from, for example, the Shetland Box, had the same batch of standard and reagents and done at the same time, and thus were subject only to inter-plate variation). All samples were also assayed in the order that they were collected (which did not relate to fish size) and in several cases the results were confirmed by repeat assay. 5.5. Are elevated VTG concentrations in large male cod generated by something in the diet – for example, ingestion of other fish that are reproductively mature? It has long been speculated that fish might be able to pick up physiologically active amounts of oestrogens by eating other reproductively mature fish (Pelissero & Sumpter, 1992). However, there has been no direct proof that oestrogens ingested in this way are capable of inducing VTG synthesis. Most North Sea fish species (whether prey or predator) spawn in winter and spring in order to take advantage of the plankton blooms in the spring and summer (Daan, Bromley, Hislop, & Nielsen, 1990). It thus seems unlikely that the large cod that were caught in June/July in the present study would have had many reproductively mature fish in their diet. There have only been a few attempts to directly assess whether fish flesh is a rich source of oestrogens. One such study (Pelissero, Cuisset, & Le Menn, 1989) reported astonishingly high amounts (1 lg/100 g) of E2 in commercial fish meals. However, a more recent study (Matsumoto, Kobayashi, Moriwaki, Kawai, & Watabe, 2004) reported zero oestrogenic activity in commercial batches of brown and white fish meal. Most, if not all, of the oestrogenic activity that has been reported in commercial fish feeds appears to derive from phytoestrogens (see the following section) and not from the fish component of the diet. 5.6. Are elevated VTG concentrations in large male cod generated by something in the diet – for example, phytoestrogens? It is now well-established that Ôsteroid-likeÕ substances (phytoestrogens) derived from plants can, when ingested by fish, trigger the synthesis of VTG (Latonnelle, Le Menn, Kaushik, & Bennetau-Pelissero, 2002; Pelissero & Sumpter, 1992). It is also wellestablished that most commercial fish diets are oestrogenic and that this is mainly due to the fact that they are very rarely if ever made up solely of animal protein and oil but contain varying amounts of soybean oil cake, wheat flour or corn-gluten meal (Matsumoto et al., 2004). Although it is possible that large wild cod have a diet that is richer in

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phytoestrogens than small cod, there is no evidence to support this hypothesis. The prevalence of phytoestrogens in commercial fish diets, however, suggests that this is a possible cause of elevated VTG concentrations in males from the fish farm site. This needs to be examined, however. 5.7. Are elevated VTG concentrations in large male cod generated by xenoestrogens in the diet and/or water? In most published cases (Pickering & Sumpter, 2003; Tyler, Jobling, & Sumpter, 1998) where elevated VTG concentrations have so far been found in the blood plasma of male fish in the wild, an environmental cause has been pinpointed (e.g., a sewage treatment works or factory effluent). Most, if not all of these examples have come from freshwater and estuarine situations. In the present study, the fish have been caught far out at sea (i.e., well away from obvious point sources of pollution) and this at first sight seems perplexing. How could endocrine disrupters in the open sea be available at concentrations that are high enough to have a noticeable effect on VTG concentrations in male cod? One possible way is for the fish to pick up the endocrine disrupters via their food rather than via the water. One study of the feeding ecology of cod (Mattson, 1990) show that young cod start off feeding on free-swimming organisms in the main water column. However, as they increase in size, they root more often and deeper into the bottom for larger prey, eventually preying almost exclusively on other fish and decapods. Another two studies (Daan, 1973; Kikkert, 1993) carried out on fish from the North Sea also show that, as cod grow, they progressively include more and more other (mainly bottom-dwelling) fish in their diet. It is plausible that these bottom-dwelling fish could be the source of the endocrine disrupters that have been picked up from feeding on the worms and other bottomdwelling organisms that live in the sediment. It is known that oestrogenic endocrine disrupters in estuaries occur at higher concentrations in the sediment than in the water column (Thomas, Balaam, Hurst, Nedyalkova, & Mekenyan, 2004). It is also known that bottom-living organisms are able to recycle and bioaccumulate xenobiotics that are bound in the sediments (Farrington, 1991). There is direct evidence that diet is, in some cases, a more important source of xenobiotics than direct uptake from the water. In a study on the spot (Leiostomus xanthurus), fish exposed to PCB-contaminated sediments and fed a daily diet of polychaetes (Nereis virens) from the same sediment accumulated more than twice the PCB whole-body residues than fish exposed to similar conditions but fed uncontaminated polychaetes (Rubenstein, Gilliam, & Gregory, 1984). In the same study, it was shown that fish isolated from direct contact with PCB-contaminated sediment did not significantly accumulate PCB residues compared with fish allowed contact with sediment. In another study, flounder that were placed for three weeks in a cage in an oestrogen-contaminated estuary, showed no induction of VTG synthesis. However, when flounder in the laboratory were fed for three weeks on mussels (Mytilus edulis) that had been harvested from the same estuary, there was an induction of VTG synthesis (Allen et al., 2002). As discussed above, the link between VTG concentrations in female cod and fish size is not nearly as clear as that for males. This is because many, but not all, of the small female cod (especially those caught in the North Sea) had elevated VTG concentrations. Although these fish could have been producing VTG as part of their natural reproductive cycle, another reason could be that the females are more sensitive to exogenous endocrine

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disrupters than the males and thus respond at a smaller size. This possibility is supported by the finding that immature female rainbow trout fed E2 in their diet produced three- to fourfold more VTG than immature males (Carlson & Williams, 1999). In the study that reported VTG induction in male swordfish in Mediterranean and South Africa waters but not in the Pacific Ocean (Desantis et al., 2005), it was mentioned that persistent organic pollutants (POPs) such as PCB and DDT were present at higher concentrations in the Mediterranean and South African waters than in the Pacific Ocean. The fact that fish from these regions were actually being exposed to organic contaminants was backed up by the finding that cytochrome P4501A (CYP1A) was also induced in their livers. This enzyme is required for the deactivation of planar xenobiotics. A build up of persistent oestrogenic POPs in the lipid of the cod – that would then be released periodically – could presumably be an explanation for the results in the present paper. A study was carried out in 2002 to determine whether the oil industry might be a source of oestrogenic endocrine disrupters in the open sea (Scott et al., in press). Cod were deployed at four stations at varying distances from an oil field in the North Sea. Six weeks later, the cod were retrieved and blood samples taken for measurement of VTG. Mean VTG concentrations in males caged closest (500 m distance) to the rig were significantly higher than at the other three stations. However, the elevation was marginal (mean of 0.26 lg/ml in comparison to the mean of 42 lg/ml for all fish >5 kg/ml in the present study). Although these results seem to suggest that, in terms of exposure to putative waterborne endocrine disrupters, the oil industry poses a low risk to cod, it is dangerous to generalise from a single experiment. A more comprehensive study (Thomas, Balaam, Hurst, & Thain, 2004a), carried out on 81 separate offshore platforms, showed large differences between sites in the amounts of oestrogen receptor agonists that were present in produced water effluents (ranging from <0.03 to 91 ng/L E2 equivalent). Isomeric mixtures of C1 to C5 and C9 alkylphenols contributed to the majority of the ER agonist potency in these produced water effluents (Thomas, Balaam, Hurst, & Thain, 2004b). A study has also been carried out on cod exposed to a 50% dilution of domestic sewage effluent showing that, after 21 days, most of the cod had significantly elevated VTG concentrations (Pickering & Sumpter, 2003). This finding, that mirrors the results of numerous studies of freshwater fish exposed to sewage effluents (Pickering & Sumpter, 2003), could be a reason for the elevated VTG concentrations that were found in males captured in Oslofjord (because it is an area of high human density). 5.8. Future studies As will be apparent from the above discussions, firm proof is still needed that elevated VTG concentrations in male cod are due to endocrine disruption. One obstacle to obtaining this proof will be that, when sampling in the wild, one cannot be sure that any site is ÔnormalÕ (Sumpter & Johnson, 2005). In other words, it may prove impossible to find a control population of cod that has not in some way been affected by human activity. However, this obstacle can probably be overcome gathering a wide geographical and seasonal range of data on VTG concentrations in cod – at the same time gathering correlative data on age and gut content analysis. Another approach would be to look for signs of VTG induction in other species that feed on the benthic food chain. A third approach would be to screen extracts of bile, liver and fat tissue of large and small cod for oestrogenicity. If greater activity were to be found in the extracts from large cod, it might be possible to

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directly identify the causative agent(s). Even if this approach failed, then it might still be possible to screen extracts of the food organisms upon which the large cod feed. 5.9. If elevated VTG concentrations are due to endocrine disrupters, what are the possible adverse implications? The degree of VTG induction noted in cod in the present study is moderate in comparison to the degree of VTG induction previously noted in flounders in some UK estuaries (Allen et al., 1999b). Also, it is restricted mainly to large fish that will already have gone through at least one cycle of reproduction. This would suggest that population effects, if any, are likely to be low. However, if what is being found in large cod is due to contaminants picked up via the diet, there then could possibly be implications for the other species that form part of the codÕs food chain. Yet another possible adverse effect is on the health of predators that feed on the large cod. In our opinion, these last two reasons are the most important drivers for further research to determine the cause of the elevated VTG concentrations noted in the present study. Acknowledgements We acknowledge financial support from Marine and Water Division, DEFRA, UK and the Cefas seedcorn research programme. We gratefully acknowledge: Dr. Olav Kjesbu for provision of plasma samples from farmed cod and the numerous technical staff in laboratories and ships for assistance with collection of samples. Aquaculture facilities to keep farmed cod was supported by European Community Project Q5RS-2002-01825. The authors declare that they have no competing financial interests. References Al-Ablani, S. A., & Phelps, R. P. (2002). Paradoxes in exogenous androgen treatments of bluegill. Journal of Applied Ichthyology, 18, 61–64. Allen, Y., Balaam, J., Bamber, S., Bates, H., Best, G., Bignell, J., et al. (2002). Endocrine disruption in the marine environment (EDMAR). London, UK: EDMAR Secretariat, Department for Environment, Food and Rural Affairs, 67pp. Allen, Y., Matthiessen, P., Scott, A. P., Haworth, S., Feist, S., & Thain, J. E. (1999a). The extent of oestrogenic contamination in the UK estuarine and marine environments – further surveys of flounder. Science of the Total Environment, 233, 5–20. Allen, Y., Scott, A. P., Matthiessen, P., Haworth, S., Thain, J. E., & Feist, S. (1999b). Survey of estrogenic activity in United Kingdom estuarine and coastal waters and its effects on gonadal development of the flounder Platichthys flesus. Environmental Toxicology and Chemistry, 18, 1791–1800. Antonopoulou, E., Bornestaf, C., Swanson, P., & Borg, B. (1999). Feedback control of gonadotropins in Atlantic salmon, Salmo salar, male parr. I. Castration effects in rematuring and nonrematuring fish. General and Comparative Endocrinology, 114, 132–141. Arukwe, A., & Goksoyr, A. (2003). Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption. Comparative Hepatology [online] 2003, 2:4, http://www.comparative-hepatology.com/content/2/1/4. Baroiller, J.-F., Guiguen, Y., & Fostier, A. (1999). Endocrine and environmental aspects of sex differentiation in fish. Cellular and Molecular Life Sciences, 55, 910–931. Budworth, P. R., & Senger, P. L. (1993). Fish-to-fish testosterone transfer in a recirculating-water system. The Progressive Fish-Culturist, 55, 250–254.

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