Environmental pollution caused elevated concentrations of oestradiol and vitellogenin in the female flounder, Platichthys flesus (L.)

AOUATIC TllXlCMll6Y ELSEVIER

Aquatic

Toxicology

39 (1997) 195-214

Environmental pollution caused elevated concentrations oestradiol and vitellogenin in the female flounder, Platichthys jlesus (L.) P.A.H.

Jansse9,

J.G.D. Lambert”.“, H.J.Th. GOOS”

A.D.

of

Vethaakb,

‘~Deprrrtment of Experimental Zoology, Research Group Comparative Endocrinology, Univrr.rit)’ of Utrecht. Padualaan 8, 3584 CH Utrrcht, The Netherland. hMini.ytry of Transport, Public Works and Water Management, National Institute for Coastal and Marine ManagementlRIKZ, Section Ecotoxicology, P.O. Bo.u 803Y> 4330 EA Middelhurg, The Netherlands

Received

22 November

1996; revised

18 February

1997; accepted

24 April

1997

Abstract Female and male flounder, Platichthys jest.+ were exposed to various concentrations of polluted harbour dredged spoil in large mesocosms for up to 3 years. The dredged spoil contained a mixture of contaminants representative of pollution concentrations found in the natural environment. Ovarian development, vitellogenesis and steroid hormones in the fish were studied and compared with results from feral fish sampled at a relatively clean field site in the Dutch Wadden Sea. Plasma concentrations of vitellogenin (VTG), established by densitometry in sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, fluctuated during the annual reproductive cycle of the Wadden Sea flounder and reached a maximum during autumn and winter (advanced vitellogenesis). Fish held in the polluted mesocosm for 3 years exhibited premature vitellogenesis, resulting in a high number of oocytes in the yolk granule stage in spring, normally the previtellogenic period of the year. Moreover, VTG was significantly elevated in the plasma of these females relative to the concentrations in fish from the reference mesocosm. The high concentration of plasma VTG in females from the polluted mesocosm coincided with significantly elevated concentrations of testosterone and 17@-oestradiol. The in vitro ovarian production capacity of these steroids, however, was not altered. In feral and pollution-exposed male flounder, no VTG was detected. On the basis of these findings, it was concluded that premature vitellogenesis in the female flounder was a result of elevated 17P-oestradiol concentrations rather than a direct endocrine effect of xeno-oestrogenic contaminants. It is suggested that the elevated 17p-

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oestradiol concentrations are caused blood. 0 1997 Elsevier Science B.V. Kqwwt/s: Pollution:

by decreased

Flounder: Oestradiol: Oestrogen synthesis; Reproductive cycle; Teleost : Vitellogenesis

clearance

rates

Ovary;

Plasma;

of

steroids

Platichthys

in the

~jm~r;

1. Introduction In this study, the flounder, Plutichthys Jesus (L.), has been selected as a model species to determine the effects of aquatic pollution on reproduction. This flatfish is a common species in coastal and estuarine waters as well as in fresh water. Spawning and the associated migration to offshore marine waters take place in winter (Rijnsdorp and Vethaak, 1989). The nurseries and feeding areas of the adults are concentrated in or near estuaries. As the flounder lives in close contact with sediments, it is intensively exposed to environmental pollution (Vethaak and Jol, 1996). Organic contaminants have been shown to accumulate in fat cells and in tissues such as liver and gonads (Von Westernhagen et al., 1981; Von Westernhagen et al., 1987). In several flatfish species, this has been related to reduced reproductive success, e.g. effects on fertilization in the starry flounder, P. stellutus (Spies et al., 1988). and effects on hatching and the number of viable larvae in the flounder, P. fi~sus (Von Westernhagen et al.. 1981 ; Von Westernhagen et al., 1987). Pollutants may have direct efrects on the gonads, resulting in a disturbed development of germ cells. Indirect effects on reproduction, via interference with the regulating hormonal system, have also been suggested (Freeman et al., 1980; Thomas, 1990). In a previous study, it was reported that the long-term exposure of the flounder in a mesocosm to polluted sediment resulted in premature yolk accumulation in the oocytes in the previtellogenic period of the annual reproductive cycle (Janssen et al., 1995). During vitellogenesis, the yolk precursor vitellogenin (VTG) is produced by the liver, transported via the blood and selectively sequestered by the developing oocytes (Tata and Smith, 1979: Wallace, 1985). VTG synthesis and secretion are induced by oestrogens. especially l7b-oestradiol (Ez) (Emmersen and Petersen, 1976; Peute et al., 1985). The VTG plasma concentrations fluctuate throughout the year, with the highest values occurring during the phase of vitellogenesis (Campbell and Idler, 1980). Males are also equipped to synthesize VTG, but normally no production is apparent because of the lack of circulating endogenous oestrogens. The aim of this study was to obtain more data on the premature vitellogenesis of VTG in the reported previously (Janssen et al., 1995). After the characterization flounder, the concentrations of VTG and steroids in plasma were determined and in vitro ovarian oestrogen biosynthesis was studied in fish after long-term exposure to varying degrees of pollution in mesocosms. The results were compared with data on VTG concentrations, steroid concentrations and steroid synthesis during the different phases of ovarian development in feral fish from the Dutch Wadden Sea.

P.A. H. Janssen et allAquatic

2. Materials

Toxicology 39 (1997)

107

195-214

and methods

2. I. Animals

2.1.1. Mesocosm study The effects of long-term exposure to pollution were studied in fish kept in selfsupporting mesocosms of 40 mx40 m x 3 m. A reference mesocosm (mesocosm A) contained relatively clean Wadden Sea sediment and water. An indirectly polluted mesocosm (mesocosm B) also contained Wadden Sea sediment, but the water was polluted as a result of its connection with a directly polluted mesocosm (mesocosm C). The third mesocosm (mesocosm C) contained dredged spoil from Rotterdam harbour. Mesocosms A and B were each stocked with 1200 lyear-old flounder, and mesocosm C was stocked with 400 flounder of the same age. These juvenile flounder were captured at a relatively clean site in the Wadden Sea (Balgzand) in spring 1990. All three basins contained the naturally occurring macro- and meiofauna and, since the fish grew and developed in a comparable manner with the fish from the Wadden Sea, it was decided that additional feeding during the course of the experiment was unnecessary. Physical parameters, such as temperature and salinity, were measured regularly throughout the experiment and no clear differences with the field situation were observed. Of the contaminants in the dredged spoil, polychlorinated biphenyls (PCBs) and polynuclear aromatic hydrocarbons (PAHs) were predominant, with a clear pollution gradient, with the highest values in mesocosm C and the lowest values in mesocosm A. As an example, the ratio of the concentrations of the PCB congener CB-153 in the sediments of mesocosms A, B and C was 1 : 4 : 18 and the corresponding ratio of selected PAHs was 1 : 2 : 5 (Table 1) (Vethaak et al., 1996). Table I Concentrations (mean+SEM) of selected organic pollutants in sediment, mussels and flounder tissue from the three mesocosms during May 1990 to May 1993. C6 PAHs, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, fluoranthene, benzo[ghi]perylene and indenopyrene. C7 PCBs, CB28, CB52, CBIOl, CBI 18, CB153 and CB180. Number of samples analysed is shown in parentheses (after Vethaak et al., 1996) Unit of measurement

Indirectly mesocosm

Reference mesocosm -

86 PAHs Sediment < 63 mm (OGIO cm) Mussels Biliary l-OH pyrene” C7 PCBs Sediment < 63 mm (O-10 cm) Mussels Flounder liver “l-OH

pyrene,

ng (g organic carbon))’ ng (g lipid))’ ng (g lipid))’ ng (g organic carbon)-’ ng (g lipid))’ ng (g lipid))’

1-hydroxypyrene;

indicating

15792?

234 & 12 (7) 6742 100 (7) 1144+210 (4) the exposure

Directly polluted mesocosm

_

1489 (7)

1116& 151 (6) 12827 (52)

polluted

30386 + 1530 (7)

61666?3701

(4)

2543 f 329 (6) 734 f 32 (50)

5688?1698 (3) 2400 2 39 (36)

1019f

4589+390

127 (7)

3278+315 (7) 6207 + 1483 (4) to PAHs

(5)

6380 L 994 (3) 11912+ 1742 (3)

198

P.A. H. Jamsen

et ul.lAquurr~ Tos~olog_~ 39 (1997)

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Fish were sampled in November 1992 and May 1993. In total, 38 adult females (length, 25-37 cm; weight, 1755620 g) and 46 adult males (length, 25532 cm; weight, 155425 g) were examined. 2.1.2. Field stud]? For reference, adult female and male flounder were caught throughout the year in the Blauwe Slenk, an area in the Dutch Wadden Sea known to have a relatively low degree of pollution compared with other Dutch estuaries (Akkerman et al., 1990). Sampling of feral fish was carried out every 2 weeks throughout 1991 and 1992 and, at each sampling, five adult fish of 25-32 cm were selected, as described previously (Janssen et al., 1995). As positive controls for the induction of vitellogenesis, fish captured in March 1994 were placed in 3 m3 basins with recirculating and aerated sea water. After acclimatization for 6 weeks, they received intraperitoneal injections with 500 ng E2 per gram of body weight (100 pg E’ per millilitre of 1% ethanol in 0.8% saline). Control fish were injected with 1% ethanol in 0.8% saline. Subsequent injections took place on days 3, 6 and 9, while sampling followed on day 13. In total, 22 adult females were examined, varying in length and weight from 17 to 37 cm and 50 to 410 g respectively. In addition, five males (27-32 cm; 145-275 g) were examined. 2. I .3. Sumpling procedure Fish were anaesthetized with 0.1% 2-phenoxyethanol, and blood samples were taken from the caudal vein; 6% sodium citrate in 0.7% sodium chloride solution was added as anti-coagulant (50 yl per millilitre of blood). The blood samples were centrifuged (10 min, 8OOg, 4°C) and the plasma was kept in Eppendorf tubes at -20°C until further analysis. Fish were killed by decapitation and the stomach, gut and liver were removed. The gonads were excised, weighed and a small piece of tissue was used for histological examination to identify the phase of ovarian development. The gonadosomatic index (GSI) was calculated as the ratio of the gonadal weight to the body weight without the gonadX 100. 2.2. Histological

method&s

Ovarian tissue was fixed in Smith’s formalin-dichromate fixative, embedded in paraffin and sections, 7 urn thick, were stained with either Mayer’s haematoxylineosin (HE) or Heidenhain’s azokarmin aniline-blue stain (Azan). Ovarian development was classified according to Janssen et al. (1995). 2.3. Characterization

of’.fkmule specljic protein us VTG

To characterize the female specific lipoprotein VTG, density gradient ultracentrifugation was performed. VTG is a very high density lipoprotein (VHDL) which can be separated from other plasma proteins using its specific density.

P. A. H. Janssen et al.lAquatic Toxicology 39 (1997)

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199

Blood samples of female and male flounder, caught in November 1994, were analysed. Plasma (4 ml) was transferred to ultracentrifugation tubes containing 2 g potassium bromide (KBr), resulting in a specific density of 1.300. Another 13 ml KBr solution, with a specific density of 1.300, was added and overlaid to a total volume of 35 ml with KBr solution with a specific density of 1.050. The samples were ultracentrifuged (4 h, 256OOOg, 4°C) in a vertical rotor (Sorvall, TV 850) resulting in a separation of the (1ipo)proteins in the density gradient. The samples were aspirated, starting at the bottom of the tube, at 99 ml h-’ and collected in 0.8 min fractions. The amount of (1ipo)protein was measured by extinction at 280 nm using an extinction meter (Uvicord S, LKB), and the density was simultaneously measured gravimetrically using a conductivity meter. Selected fractions were pooled and concentrated by dialysis against 10 mM 2,4,6_tris(dimethylaminomethyl)phenol (Tris)-1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0. After protein quantification (Lowry et al., 1951) 2040 pg of sample was applied to the gel and the (1ipo)proteins were separated by gel electrophoresis. 2.4. Gel electrophoresis Plasma VTG concentrations were determined by gel electrophoresis followed by densitometric quantification of the specific VTG band. Sodium dodecylsulphatepolyacrylamide gel electrophoresis (SDS-PAGE) was performed by slight modification of the methods reported by Laemmli (1970) and Van Bohemen et al. (1981). Plasma samples were diluted to several concentrations by the addition of Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Subsequently, all samples were diluted 1 : 1 with sample buffer (50 mM Tris, 2 mM EDTA, 1% SDS, 1% (v/v) pmercaptoethanol, 8% (v/v) glycerol, 0.025% bromophenol blue, pH 6.8) and placed into nearly boiling water (10 min, 95°C) to denaturate the proteins. Since B-mercaptoethanol results in the segregation of VTG into its two constituent polypeptide strands (Hara and Hirai, 1978) the molecular weight of the dimer protein was checked by diluting some samples 1 : 1 with sample buffer without B-mercaptoethanol. Running gels (7.5%; pore size, 50 A), 0.75 mm thick, were poured with acrylamide and N’,N’-bis-methylene acrylamide in the ratio 36.5 : 1, running buffer (1.5 M Tris, pH 8.8) and 0.1% SDS. Polymerization was initiated by the addition of 0.05% ammonium persulphate (APS) and 0.05% of the catalyst N,N,N’,N’-tetramethylethylenediamine (TEMED). The stacking gels, 0.75 mm thick, were poured with acrylamide and N’,N’-bis-methylene acrylamide in the ratio 36.5 : 1, stacking buffer (0.5 M Tris, pH 6.8) and 0.01% SDS. Polymerization was initiated by the addition of 0.1% APS and 0.1% TEMED. Two gels were placed together in a Bio-Rad Mini-Protean II electrophoresis cell and 800 ml of electrophoresis buffer (2.5 mM Triss20 mM glycine, pH 8.3, containing 0.01% SDS) was added. Plasma samples of 20 ~1 each were applied to the gel and, in addition, 20 pl of molecular weight marker solution was added to the reference track of each gel as well as 20 ml of a reference plasma sample of an advanced vitellogenic female. Electrophoresis was performed until the front reached

200

P.A. H Janssen et rrl.lAyuatic

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the running gel (40 V, 30 min, room temperature). (120 V) until the tracking dye (bromophenol blue) ( t 1.5 h). Electrophoresis was prolonged for 30 min the high molecular weight proteins. The gels were stained (2 h) with a 0.025% solution 250 in a mixture of methanol, acetic acid and water for 2 X 30 min with methanol, acetic acid and water anol, acetic acid and water (5 : 7 : 88) (overnight). 2.5.

Quant@ution

The voltage was then increased reached the bottom of the gel to obtain a better separation of of Coomassie brilliant blue R(50 : 10 : 40) and destained first (25 : 10 : 65) followed by meth-

of’ VTG

To quantify the bands of interest in the gels. the IBAS image analysis system (KontronJZeiss, Eching, Germany) was used. Gels were scanned with a Panasonic b/w CCD camera (type WC-CDSO), digitized four times and averaged to improve the signal-to-noise ratio (frame size, 640 X 512 pixels: 256 grey concentrations). To delimit the VTG protein band. the dynamic discrimination technique was applied. This method operates with a threshold which depends on the grey concentration of the neighbouring region. In order to verify visually whether the band detection was correct, the delineated bands of interest were displayed in overlay on the monitor over the image and, if necessary. interactively corrected. To determine the intensity of the protein bands, they were measured in both the original image and the background reconstructed image and subtracted from each other. From this integrated optical density (IOD), the relative amount of the protein was calculated against a reference sample from a female in advanced vitellogenesis, applied to each gel. To determine the molecular weight of the identified protein, reference molecular marker proteins were used (Weber and Osborn. 1969: Laemmli, 1970). 2.6. Quun t$icution

of’.stevoidss in the plusmu by rudioimmunous.suy.\

The concentrations of testosterone (T) and Ez in the plasma were quantified using specific radioimmunoassays (RIAs), as described by Schulz (1984, 1985). Extraction of the steroids from 1.5 ml of plasma was performed with 2 X 5 ml ether. After evaporation of the ether. the dry residue was taken up in 500 ~1 assay buffer (phosphate buffer, pH 7.0 with 0.005% sodium azide, 0.9% sodium chloride and O.l’%l gelatin) of which 100. 20 and 5 ~1 were used in each of the two assays. As standards, known amounts of the two steroids were dissolved in steroid-stripped flounder plasma and treated according to the same protocol.

In May 1993, three females were selected from mesocosm A, all being previtellogenie (later perinucleolus phase or cortical alveoli phase), and three from mesocosm C, all being premature vitellogenic. Ovarian oestrogen synthesis was studied as described by Schoonen and Lambert (1986). Briefly, I g of minced ovarian tissue was incubated with 74 kBq [7-“HI-androstenedione in Leibovitz-15 medium, buff-

P.A.H.

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39 (1997)

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201

ered with 15 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid (HEPES) (pH 7.4), for 24 h at room temperature. The synthesized steroids were extracted from the medium with dichloromethane and further isolated and identified by thinlayer chromatography (TLC) using different developing systems. Radioactive areas on TLC plates were located using a Berthold LB 2842 thin-layer radiochromatogram scanner. From the radiochromatogram, the percentage distribution of the tritiated areas, corresponding to the individual steroids, was determined and the percentage yields of T, oestrone (El) and Ez were calculated. 2.8.

Chemicals

Acrylamide, N’,N’-bis-methylene acrylamide, APS, HEPES and Leibovitz-15 medium were obtained from Serva, TEMED from Pharmacia, high and broad range molecular weight electrophoresis calibration kits (HMW and BMW respectively) from Pharmacia and Boehringer, EDTA and Es from Sigma and B-mercaptoethanol, bromophenol blue, Tris and TLC plates (Kieselgel 60 Fz54, 10 cmX 20 cm) from Merck. [7-3H]Androstenedione (specific activity, 685 GBq mmol-‘) was purchased from Amersham and its purity was checked by TLC. Reference steroids were obtained from Sigma. All other chemicals and solvents were of analytical grade and were obtained from Baker or Merck. 2.9. Statistics

Differences between the values of the various fish parameters in the different groups in the injection experiment, in fish from the Wadden Sea in the different phases of ovarian development and in fish from the different mesocosms were tested statistically with the Kruskal-Wallis test. When the significance concentration of 5% was reached, this procedure was followed by the Mann-Whitney U-test to determine which groups were significantly different from each other, again at the 5’%, significance concentration. Correlations between parameters were determined, resulting in a Spearman’s rank order correlation coefficient. All tests were performed using the statistical package Number Cruncher Statistical System (NCSS), version 5.01 (Dr. Jerry L. Hintze, Kaysville, UT, USA). Mean values are given with the standard error of the mean (SEM).

3. Results 3.1. Ovariun development

In the females from November 1992, no severe abnormalities in ovarian structure or oogenesis were observed in fish from the three mesocosms. All ovaries from flounder from the three mesocosms and the Wadden Sea were in the phase of early or advanced vitellogenesis (Figs. 1A and IB).

P. A. H. Jmwn

ct cd.IAquutk

Tosirolo,y~

39 I 1997) I95 -214

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39 (1997)

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Fig. 1. Ovarian development in mesocosm flounder, Platichthysflesus (L.), during November and May (HE, 90X). (A) Ovary from the reference mesocosm (November) in advanced vitellogenesis (vitellogenie oocytes, Vg). Oocytes in late perinucleolus and cortical alveoli stages are present. (B) Ovary from the directly polluted mesocosm (November) in advanced vitellogenesis (vitellogenic oocytes, Vg). Oocytes in late perinucleolus and cortical alveoli stages are present. (C) Ovary from the reference mesocosm (May) in previtellogenesis (cortical alveoli); developing oocytes in early (ePN) and late (IPN) perinucleolus stages and oocytes with cortical alveoli (CA) are situated in the ovarian lamellae. (D) Ovary from the directly polluted mesocosm (May) in premature vitellogenesis; oocytes in yolk granule (UC) stage and all previous stages are present.

In May, all females from the Wadden Sea were in previtellogenesis, as were the females from mesocosms A and B (Fig. l(C)). In contrast, the ovaries of the females from the directly polluted mesocosm contained not only the smaller oocytes, but also oocytes in yolk granule stage. Moreover, compared with ovaries from females from the Wadden Sea and from mesocosms A and B, the oocytes from the directly polluted fish did not develop synchronously (Fig. l(D)). In both November 1992 and May 1993, at least five fish of each sex were sampled; no significant differences were observed between the GSIs of female fish from the reference, indirectly polluted and directly polluted mesocosms (7.09 f 0.69, 7.87kO.51, 10.25k1.95 and 1.77kO.17, 1.98f0.19, 2.28f0.17 respectively), nor between fish from the reference mesocosm and the Wadden Sea (9.40 ? 1.29 and 1.50 + 0.18 for Wadden Sea fish from November and May respectively). 3.2. Characterization of VTG Gradient

ultracentrifugation

of plasma

contr Females

EP-inj.

samples

from

con,,.

Wadden

Sea female

and

EZ-inj Males

Fig. 2. Relative plasma VTG concentrations with respect to a reference sample from a feral female in advanced vitellogenesis (mean + SEM) in female and male flounder, Platichthys Jesus (L.), after 17Boestradiol injection (n given in parentheses); contr., control injection; Ez-inj., 17B-oestradiol injection. **Significant differences of the injected groups with respect to the corresponding control groups (p
204

P. A. H. Juns.wn c’t al. IAquatic~ To.vicology 39 (I 947) 195~-214

male flounder, followed by fractionated sampling of the (lipo)proteins, resulted in a pattern of proteins with different molecular weights, specific for a flounder plasma sample. Selected fractions were analysed on SDS-PAGE gels and showed a female specific protein band in fractions from the density region 1.21-1.25 g ml-‘. Female flounder collected throughout the year in the Wadden Sea were grouped according to their phase of ovarian development: post-spawning, previtellogenesis (late perinucleolus), previtellogenesis (cortical alveoli), early vitellogenesis and advanced vitellogenesis. On the gels, a 190 kDa protein band was detected in females in all phases of ovarian development, fluctuating in intensity throughout the annual cycle. with a maximum during advanced vitellogenesis. In plasma samples from males in all phases of the annual reproductive cycle, this specific band was absent. Omitting P-mercaptoethanol in the procedure resulted in additional bands in the higher molecular weight regions, but the 190 kDa protein was still present in female plasma. After Ez treatment of Wadden Sea flounder captured in March 1994, the female specific protein band increased in intensity (10.82 k4.19) compared with that in saline-treated females (0.49 + 0.19). Moreover, it could not be detected in the plasma of saline-treated males ( < 0.03) but appeared after Ez treatment (3.13 + 0.58) (Fig. 2). These results indicate that the described protein band is the (monomer of) VTG.

PINTG

(IPN,PrevTG

(CA)

early VTG

Phases

of

VE

Post-spawn

ovarian’

development

Fig. 3. Relative VTG concentrations (see Fig. 2) and I7P-oestradiol concentrations (mean ? SEM) of the female Wadden Sea flounder, Platichtlzvs,flr.~u~ (L.). during the different phases of ovarian development. Significant differences of the individual phases with respect to the previous phase: *p
P.A.H.

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Fig 4. Relative plasma VTG concentrations (mean ?z SEM) (see Fig. 2) of mesocosm flounder. Platichthys $esus (L.), in November 1992 and May 1993. Meso A, reference mesocosm; Meso B, indirectly polluted mesocosm; Meso C, directly polluted mesocosm. *Significant differences of the parameters of fish from the indirectly and directly polluted mesocosms from those of the fish from the reference mesocosm: p < 0.05.

3.3. Plasma VTG concentrations In feral fish in the first previtellogenic phase (late perinucleolus), VTG was relatively low (0.20 k 0.05), increased to 0.54 t 0.22 in the second previtellogenic phase (cortical alveoli) and further increased to 1.26 f 0.57 and 2.12 + 0.28 in the early and advanced vitellogenic phases respectively. In the post-spawning phase, VTG had decreased to the relative value of 0.48 k 0.12 (Fig. 3, top). In both November 1992 and May 1993, significant differences were observed in plasma VTG concentrations between fish from the reference mesocosm and those

AB

CD

E

FG

VTG *

Fig. 5. SDS-PAGE gel (7.5%) from flounder, Platichthys j?esus (L.), plasma samples from May 1993 from the mesocosm experiment. Lanes A-C, three females from the reference mesocosm; lane D, standard protein markers; lane E, reference female from the Wadden Sea; lanes F-H, three females from the directly polluted mesocosm. Staining: Coomassie brilliant blue. Ferritine, 220 kDa: myosine. 206 kDa; B-galactase, 116 kDa: phosphorylase B, 106 kDa.

0 ; 9 0

60 55

50 45 40 35 30 25 20 15 10 5 0

MeSO*

Mesoc

Fig. 6. Plasma concentrations and in vitro ovarian biosynthesis of testosterone and the oestrogens of the mesocosm flounder, Platichthys ,jk.w (L.), in May 1993. Meso A, reference mesocosm; Meso B, EZ, 17B-oestradiol; E , , oestrone; indirectly polluted mesocosm: Meso C. directly polluted mesocosm; T. testosterone. Top: plasma concentrations of T and E2 (mean f SEM) determined with specific RIAs. *Significant differences of the steroids of fish from the indirectly and directly polluted mesocosms from those of fish from the reference mesocosm: p
from the directly polluted mesocosm. The directly exposed fish reached VTG values of I .90 f 0.26 in November and an even more pronounced increase to 5.52 k 2.45 in May (Figs. 4 and 5). In plasma samples of fish from the indirectly polluted mesocosm. intermediate values were measured. 3.4. Piu.srna steroid concentrations Plasma concentrations of E2 in feral fish ranged from 58 k9 pg ml-’ in the in the previtellogenic phase Ib, previtellogenic phase Ia, 275 f 107 pg ml-’ 324 + 69 pg ml-i in the vitellogenic phase IIa, 2758 _+659 pg ml-’ in the vitellogenic phase IIb and 65 + 18 pg ml-’ in the post-spawning phase IV (Fig. 3. bottom). In May 1993, premature vitellogenesis was observed in fish from the directly polluted mesocosm C. Therefore Ez and T concentrations were measured in the plasma of the fish from the mesocosm experiment (Fig. 6. top). EZ was significantly

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elevated in fish from mesocosm C (264? 35 pg ml-‘) compared with fish from mesocosm A 116 + 9 pg ml-‘. Fish from mesocosm B showed intermediate concentrations (151? 34 pg ml-l). The concentrations of T varied from 72 ? 9 pg ml-’ in mesocosm A to 138 + 56 pg ml-’ in mesocosm B and 163 + 20 pg ml-’ in mesocosm C, the latter being significantly higher than the value in the reference fish. 3.5. Ovariun oestrogen biosynthesis in vitro In vitro ovarian oestrogen synthesis was studied in flounder collected in May 1993 from mesocosms A and C by incubating ovarian fragments with [7-3H]-androstenedione as precursor. After organic extraction of the incubation medium, about 10% of the radioactivity remained in the water fraction, indicating a minor conversion to the watersoluble steroid conjugates. In all incubations, androstenedione was mainly converted to T ( > 50”/0) and, in minor but reasonable amounts, to the oestrogens El and E2 (5”/~15%). The remaining steroids were all traces of reduced products of androstenedione and T. No clear differences could be demonstrated (Fig. 6, bottom) in oestrogen synthesis between the ovaries of the fish from the reference (A) and directly polluted (C) mesocosms.

4. Discussion In this study, we analysed the possible effects of pollution on the plasma concentrations of VTG, EZ and T and on the in vitro oestrogen biosynthesis in female Aounder after long-term exposure to contaminated dredged spoil under semi-field conditions. As a field reference, female flounder collected in a relatively clean area of the Dutch Wadden Sea were used. Keeping flounder in large-scale mesocosms mimics the natural situation. Under these conditions, female flounder can develop oocytes and release eggs as indicated by the histological analysis of the ovaries collected in November 1992 and May 1993. This is remarkable since normal migration to deeper waters is prevented. However, during the 3 year study, no larvae or successful settlement of juvenile fish were observed in any of the mesocosms. Therefore egg fertilization and hatching under the conditions in the mesocosms seem unlikely. The long-term exposure of flounder in the directly polluted mesocosm to a variety of contaminants resulted in premature VTG incorporation. In contrast with the normal reproductive cycle, with previtellogenesis in May (Janssen et al., 1995) female flounder from the directly polluted mesocosm in May revealed yolk incorporation in approximately one-half of the oocytes. Furthermore, there was a distinct difference in the histological structure of the ovaries from fish sampled in May in the directly polluted mesocosm compared with those from fish sampled in the Wadden Sea. In the ovaries from the directly exposed fish, oogenesis seemed to be more asynchronous, since many of the oocytes were not able to incorporate VTG.

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This suggests that local factors within the ovary may be important for the development of the individual oocytes. Some atretic follicles were observed, but no other structural malformations in relation to the germ cells, as described in other studies (Kime, 1995). VTG is by far the most important yolk protein precursor in all oviparous vertebrates (Wallace, 1985; Selman and Wallace, 1989). In the present study, VTG was identified by a number of parameters. It fluctuated throughout the reproductive cycle of the female flounder, whereas it was absent in males, and, moreover, it was present in female and male flounder plasma after treatment with Ez. Other proteins which originate from the liver and are EZ inducible in both female and male fish are the proteins of the acellular vitelline envelope, outside the plasma membrane of the developing oocyte. However, these proteins have a lower molecular weight, e.g. the vitelline envelope of Atlantic halibut, Hippoglossus hippogfos.su.s, is composed of two major and two minor proteins with molecular weights below 100 kDa (Hyllner et al., 1994). VTG is a glycolipophosphoprotein (Tata and Smith, 1979) with a lower specific density than proteins. This characteristic was used in the density ultracentrifugation study and indicated that the Ep-inducible protein was present in the fraction with a density of 1.21-1.25 g ml-‘. i.e. a VHDL. The last characteristic, which demonstrates that this protein is indeed VTG, is the molecular weight of approximately 190 kDa. This is probably the monomer, since in other fish species monomers with comparable weights have been purified and characterized (Ng and Idler, 1983; Mommsen and Walsh, 1988; Specker and Sullivan, 1994). In the winter flounder, the molecular weight of the monomer was 180 kDa, determined by SDS-PAGE (Nagler and Idler, 1987). and, in the turbot, Scophthulnms ma.\-irnus, the weight was 185 kDa, determined after high-performance anion-exchange chromatography (Silversand and Haux, 1989). In the present study, the 190 kDa protein was also present in the plasma without treatment with P-mercaptoethanol, indicating that other steps in the preparation of the samples were sufficient to disrupt VTG into two monomers. When the flounder from the directly polluted mesocosm were compared with those from the reference mesocosm, the former showed significant elevated plasma VTG concentrations in the vitellogenic period (November), but especially during the previtellogenic period (May). In May. the VTC values in these fish were five to six times higher than those observed in Wadden Sea fish during early vitellogenesis. This extremely high concentration of VTG can be explained by the limited capacity of the young oocytes to incorporate VTG. It is well established that oestrogens are involved in vitellogenesis in teleosts (Aida et al., 1973; Sundararaj and Nath, 1981; Van Bohemen and Lambert, 1981; DeVlaming et al., 1984: Peute et al., 1985), including the flounder (Emmersen and Petersen, 1976; Korsgaard et al., 1983). The premature increase in plasma VTG in flounder after chronic pollution may therefore be due to increased concentrations of endogenous oestrogens. Induced vitellogenesis can, however, also result from the oestrogenic effect of certain pollutants (‘xeno-oestrogens’). Long-term exposure to l%hexachlorocyclohexane (B-HCH), for example, resulted in an increased VTG pro-

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duction in juvenile guppies, Poecilia reticulata (Wester et al., 1985). Food application of a mixture of compounds, including dibenzofurans and dioxins, to female Atlantic salmon, Salmo salar, stimulated ovarian development and induced plasma protein and VTG concentrations (Von der Decken et al., 1992). A variety of compounds were shown to have an affinity for hepatic Ez receptors in rainbow trout (Pelissero et al., 1993). Moreover, alkyl phenols, the final products of the biodegradation of certain detergent components and present in sewage effluents, had oestrogenic effects (induction of vitellogenesis) in the same species, as established in vitro (Jobling and Sumpter, 1993) and in vivo (Purdom et al., 1994). In the present study, no VTG could be detected in feral or pollution-exposed males. This suggests that either xeno-oestrogens are absent in the polluted mesocosms or that male livers are less sensitive to xeno-oestrogens. The latter is supported by the observation that E:! treatment in females is more effective than in males. Moreover, a recent study on the Rotterdam harbour sediment, using the E2 receptor chemical-activated luciferase gene expression assay (ER-CALUX), demonstrated the presence of compounds that can bind to and activate the oestrogen receptor (Legler et al., 1996). To determine the endogenous oestrogen production, E2 and its precursor T were measured in the plasma of female fish from the mesocosms and in feral females. The plasma E2 concentrations in the reference mesocosm in May ( + 125 pg ml-‘) were comparable with those in Wadden Sea flounder during previtellogenesis, indicating a similar hormonal status. In contrast, the E2 concentrations in flounder from the directly polluted mesocosm were doubled, explaining the elevated plasma VTG concentrations and the presence of yolk-containing oocytes. The effects on plasma steroid concentrations after exposure to different sorts of pollution are inconsistent. Increases in plasma Ea, in vitro Ez production and induction of vitellogenesis were observed in Atlantic croaker, Micropogonius undulutus, after cadmium exposure (Thomas, 1989). In stunted landlocked salmon, Sulmo salur se&go, and winter flounder, Pseudopleuronectes americanus, however, short-term acute exposure during the previtellogenic period to high concentrations of crude oil in the sediment had no significant effects on plasma androgens and E2 concentrations (Truscott et al., 1983); English sole, Parophrys vetulus, captured in highly contaminated areas, showed decreased plasma concentrations of E:! (Johnson et al., 1988). The analysis of ovarian steroid synthesis indicated no abnormalities in the production of T, El and EZ between the reference and directly exposed fish. However, there was a difference in steroid synthesis between the ovaries of mesocosm fish and fish from the Wadden Sea. In May, the production of steroid conjugates (mainly glucuronides) was considerably lower in the mesocosm fish (? 10%) than in the Wadden Sea fish (& 30%) (to be published). The reason for the conjugation of steroids in flounder ovary is not yet known, but, in other teleosts, steroid conjugates have been demonstrated to have sexual pheromonal properties (Colombo et al., 1982; Liley and Stacey, 1983; Lambert et al., 1986; Lambert and Resink, 1991). A lower glucuronide production in the mesocosms may indicate a disturbed pheromonal activity related to the inability of the fish to migrate and to display normal spawning. The same phenomenon was observed in male African catfish, Clurias

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guriepinus, where the amount of steroid glucuronides produced by the seminal vesicles of pond fish was reduced in comparison with feral fish (Schoonen et al., 1988). The increased E2 and T concentrations in the fish exposed to pollution cannot be explained by an altered ovarian steroid production capacity. In addition to the regulation of gonadal steroid synthesis, the metabolic clearance of steroids by the liver is another mechanism for the creation of the appropriate hormonal environment for the development of the oocytes (Baroiller et al., 1987). Steroids and xenobiotics, such as PAHs and PCBs, are metabolized in the liver by the cytochrome P450 mixed function oxygenase (MFO) system (Goksoyr and Fiirlin, 1992). Exogenous organic pollutants may interfere with endogenous steroid metabolism and be responsible for a change in plasma steroid concentrations. Interference with the regulating hormonal system has been suggested previously (Freeman et al., 1980; Thomas, 1990) and indications have been found in rats (Waxman, 1988) carp (Cyprinus carpio) (Yano and Matsuyama, 1986) scup (Stenotomus chrysops) (Snowberger and Stegeman, 1987) and Atlantic croaker (Thomas, 1988). The present study confirms these results and suggests that the altered hormonal regulation of flatfish after exposure to environmental contaminants may be a mechanism underlying the reduced reproductive success. as described in other studies. Effects of pollution on fertilization were found in the starry Aounder (P. stehtus) (Spies et al., 1988) and effects on hatching and the number of viable larvae were described for the flounder (P. ,flr.stl.s) (Von Westernhagen et al., 1981; Von Westernhagen et al., 1987). In conclusion, long-term exposure to polluted water and sediment under semifield conditions caused out-of-season elevation of E2 and T blood concentrations in female flounder. Consequently, premature vitellogenesis occurred. Since VTG concentrations were precociously elevated in females only and the ovarian E2 synthesizing capacity was unaltered, it was concluded that the hormonal disruption was caused by a modification of E:! clearance rather than by the oestrogenic action of the pollutants.

Acknowledgements Part of this study was presented at the Fifth International Symposium on the Reproductive Physiology of Fish, July 2-8, 1995, Austin, TX, USA and at the Second Society of Environmental Toxicology and Chemistry (SETAC) World Congress, November 5-9, 1995, Vancouver, Canada. This study was undertaken as part of the project BEON’EFFECTEN, financed by the Ministry of Transport, Public Works and Water Management, National Institute for Coastal and Marine Management/RIKZ, The Hague, The Netherlands. The authors thank J. Granneman, J. Jol, J. Jungman, A. Meijboom and H. Otten for technical assistance. B. Maas, I. Hassing, P. Nicholson, K. Thorpe and J. van Doorn for part of the biochemical analysis, Dr. R. Schulz for providing the steroid

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antisera, Dr. M. Terlou for developing the program for image analysis and F. Kindt and R. Leito for the photographs.

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