Effects of waterborne exposure of octylphenol and oestrogen on pregnant viviparous eelpout (Zoarces viviparus) and her embryos in ovario
1 Institute of Biology, University of Southern Denmark, Odense,
Denmark
2 Lab. of Pathology and Immunobiology, National Institute of Public Health
and the Environment (RIVM), Bilthoven, The Netherlands
* Author for correspondence (e-mail: tinac{at}biology.sdu.dk)
Accepted 16 September 2002
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Summary |
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Key words: development, embryo, endocrine disrupter, oestrogen, fish, gonad, histology, maternalfoetal relationship, alkylphenol, oestrogen receptor, sex differentiation, vitellogenin mRNA, immunohistochemistry, xeno-oestrogen, viviparous, Zoarces viviparus, eelpout
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Introduction |
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In fish, as in all other vertebrates, oestrogens play an important role in
many reproductive and developmental processes, including sexual maturation and
sexual differentiation (Nakamura et al.,
1998). These processes are therefore likely to be susceptible to
xeno-oestrogenic exposure. In particular, exposure to environmental oestrogens
during the sensitive early life stages of fish, such as the period of
embryonic development and gonadal differentiation, may adversely affect the
later reproductive performance of adult fish.
Various groups of chemicals have oestrogen-like effects, including
alkylphenol poly-ethoxylates and their degradation products, alkylphenols
(APs), such as nonylphenol (NP) and octylphenol (OP) (for a review, see
Servos, 1999). The alkylphenol
poly-ethoxylates belong to one of the largest groups of non-ionic surfactants
and are used as detergents and in many formulated products such as herbicides,
pesticides and paints. The alkylphenols, mainly 4-nonylphenol (4-NP) and
4-tert-octylphenol (4-tOP), have been found in surface waters and
sediments of both freshwater and marine habitats (for a review, see
Bennie, 1999
). In the UK, APs
have been detected at concentrations of up to 180 µg l-1 in
river water and up to 13 µg l-1 in an estuary
(Blackburn and Waldock, 1995
);
however, concentrations of approximately 1-10 µg l-1 seem to be
more common.
Several of the APs, including OP, have oestrogenic activity both in
vitro and in vivo (White et
al., 1994; Jobling et al.,
1996
; Routledge and Sumpter,
1997
; Servos,
1999
). In fish, including the eelpout
(Andreassen and Korsgaard,
2000
), OP has been shown to bind to the oestrogen receptor (ER;
White et al., 1994
), induce
Vtg synthesis in males (White et al.,
1994
; Jobling et al.,
1996
; Routledge et al.,
1998
), induce intersex (Gray
et al., 1999a
) and reduce reproductive success
(Gray et al., 1999b
;
Gronen et al., 1999
).
Sexual differentiation is the process whereby the gonadal cells begin to
specialise in structure and function and, hence, the gonadal phenotype is
expressed. Sexual differentiation includes the formation of the somatic
components of the gonad and the formation and development of the gametes.
Usually, the somatic cells of the teleost gonad differentiate before the germ
cells. The mechanisms controlling and regulating the processes of sexual
determination and sexual/gonadal differentiation in fish have not yet been
fully elucidated and show large differences among species. However, sex
differentiation is mainly thought to be under genetic control
(Nakamura et al., 1998), and
(sex) steroids and steroidogenic enzymes seem to play a crucial role in the
regulation of the process of gonadal differentiation
(Baroiller et al., 1999
).
Endogenous oestrogens have been suggested to act as the natural inducer of
ovarian differentiation, and the enzyme aromatase, which converts androgen to
oestrogen, is probably one of the key enzymes involved in this process in
gonochoristic fish (i.e. sex differentiation is characterised by the early and
direct establishment of one gonadal sex)
(Baroiller et al., 1999
), at
least in some fish species (Nakamura et
al., 1998
; Nagahama,
2000
). Recently, Baroiller and co-workers
(1999
) suggested that the
11-oxygenated-androgen:oestrogen ratio in fish would direct either male
(excess of 11-oxygenated androgens) or female (excess of oestrogens)
differentiation.
It is known that exogenous oestrogens and androgens can manipulate sex
differentiation by overriding endogenous sex-determination mechanisms in the
developing embryo and induce phenotypic sex reversal in some fish species
(Hunter and Donaldson, 1983).
Recently, it has also been demonstrated that exposure to various environmental
oestrogens may alter the normal gonadal development in fish
(Gimeno et al., 1997
;
Gray and Metcalfe, 1997
).
In fish, most studies concerning effects of xeno-oestrogens on the early
life stages of development, such as gonadal differentiation, have focused on
oviparous species. The viviparous eelpout (Zoarces viviparus) is a
recognised model species in aquatic toxicology
(Schladot et al., 1997;
Taylor et al., 1999
), and
there is growing interest in its application in endocrine-disruption research
(Christiansen et al., 1998
;
Andreassen and Korsgaard, 2000
;
Larsson et al., 2000
).
Being viviparous, the eelpout is a suitable species for studying the effect
of endocrine disrupters on maternalfoetal trophic relationships as well
as direct effects on embryos in ovario. It carries a complete brood
and gives birth to well-developed young fish that have completed sexual
differentiation. This makes the eelpout an outstanding model in studies of sex
ratios after exposure to endocrine disruptors, especially in field studies
(Larsson et al., 2000). In
contrast to oviparous fish, any contaminants must be taken up by the mother
fish before the eggs and embryos can be exposed. The eelpout is a
non-migratory fish common in coastal (and brackish) waters in much of northern
Europe. It carries its progeny inside the ovary for approximately five months.
The eggs are released into an ovarian cavity, and fertilisation takes place
immediately after ovulation (late August/early September). Approximately three
weeks after fertilisation, the embryos hatch. The embryos lie freely in the
ovary without any physical connection to the mother fish. During the yolksac
phase (approximately one month), the embryonic growth is partly dependent on
the nutritive external yolk sac. Hereafter, until parturition, the development
of the embryos depends on maternal nutrients in the surrounding ovarian fluid
(Korsgaard and Andersen, 1985
;
Korsgaard, 1986
,
1992
). The eelpout is a
differentiated gonochorist. Gonadal differentiation appears to take place
during the yolk-sac phase (T. H. Rasmussen et al., unpublished observations;
Larsson et al., 2000
).
In the present study, we investigated the effects of waterborne exposure to 4-tOP and the natural oestrogen 17ß-oestradiol (E2) on the mother fish and her embryos during early pregnancy. The objective of the study was to investigate whether 4-tOP and E2 accumulate in the mother fish and are transferred to the embryos in ovario and subsequently disturb the maternalfoetal trophic relationship and affect embryonic development, including gonadal differentiation.
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Materials and methods |
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Experimental design
Fish were treated with 4-tOP (nominal concentrations of 25 µm
l-1 or 100 µg l-1), E2 (nominal
concentration of 0.5 µg l-1) or isopropanol (control) in a
continuous flow-through system. The system consisted of 12 aquaria (501), each
connected to a multi-channel water pump and stock-solution pump. Fresh
seawater was pumped into the aquaria at a flow rate of 2001 day-1.
The compounds were dissolved in 100% isopropanol and applied directly to the
aquaria at a rate of 72 ml day-1. To assure uniform mixing, each
aquarium was fitted with a circulation pump. During the experimental period,
water temperature fluctuated between 10.5°C and 13.5°C. Water samples
were analysed every third day for actual 4-tOP concentrations in the control
and the treatment groups. Each group (control, OP25, OP100 and E2)
consisted of 21 fish with a maximum of 7 fish per aquarium. At day 0, 10
randomly selected untreated fish were sampled. After 17 days of exposure (28
Oct), 7-8 fish from each group were selected randomly from different aquaria
and sampled. At the end of the experiment, at day 35 of exposure (15 Nov), 7-8
fish from each group were chosen, as described above, and sampled.
Sampling procedure
Fish were washed in clean water and anaesthetised (0.2
phenoxyethanol) before the start of sampling. Fish were weighed, measured
(total length) and blood was collected from the caudal vein into heparinized
eppendorf tubes. Ovarian fluid was collected by gently inserting a syringe
directly into the ovarian cavity of the intact ovary. Blood and ovarian fluid
were centrifuged (4 min at 10 000 g at 4°C) and plasma was
divided into aliquots and stored at -80°C until use. Fish were killed by
decapitation, and liver and ovary were carefully removed and weighed. A small
piece of the liver was quick-frozen in liquid N2 and stored at
-80°C. The embryos were carefully dissected out of the ovary,
anaesthetised (0.1
phenoxyethanol), counted and scored for survival.
Ten embryos from each mother fish were randomly chosen and weighed and
measured (total length). Half of the remaining embryos were fixed in
Lillies-fixative (4% neutral-buffered formalin) and the other half were
snap-frozen in liquid N2. A few representative embryos were kept
alive in Ringer solution (190 mmol l-1 NaCl, 4 mmol l-1
KCl, 4 mmol l-1 CaCl2, 10 mmol l-1
NaHCO3, pH 7.5) and used for photographs. Finally, the ovarian sac
was weighed.
Quantification of 4-tOP in water samples and body fluids
Water samples were taken from the out-flow of the aquaria and filtered. To
each sample was added 50 µl internal standard (10 ng µl-1
tert-butylphenol), SDS (sodium dodecyl sulfate) to a concentration of
0.1 mol l-1, and formaldehyde to a concentration of 0.4%. Samples
were left at 4°C until analysis. The extraction and quantification of
4-tOP by liquid chromatographymass spectrometry (LC-MS) followed the
description in Pedersen and Lindholst
(1999).
4-tOP was extracted from plasma as follows: 100 µl of plasma was added
to 50 µl internal standard, diluted 10x in MilliQ-water (reverse
osmosis deionised water) and applied to Sep-Pak C18 extraction
columns previously conditioned as described by Pedersen and Lindholst
(1999). 400 µl of ovarian
fluid was added to 50 µl of internal standard and measured directly on the
LC-MS. Tests were made to assure that this method resulted in similar results
to the column extraction method described for plasma. The limit of
quantification is 100 ng ml-1.
Quantification of 17ß-oestradiol in body fluids
E2 was measured in plasma and ovarian fluid using a commercially
available competitive enzyme-linked immunosorbent assay (ELISA) kit (DRG
Instruments GmbH, Marburg, Germany). The minimal detection limit is 16 pg
ml-1.
Quantification of vitellogenin in plasma
Vtg in plasma was analysed using a direct ELISA as described by Korsgaard
and Pedersen (1998).
Determination of specific E2-binding in liver cytosol
homogenates
Liver of mother fish was homogenised, and cytosolic liver homogenates were
prepared as described by Andreassen and Korsgaard
(2000). Specific binding of
3H-E2 was measured in one-point assays in triplicate as
described previously (Andreassen and
Korsgaard, 2000
).
RT-PCR analysis on liver tissue of mother fish and embryos
RNA was extracted from mother fish liver using TRIzol® (Gibco BRL Life
Technologies, MA, USA), as described by the manufacturers. 1 µg of total
RNA was reverse-transcribed in 20 µl reactions using SuperscriptTM
RNase H- and oligo(dT)12-18 Primer (Gibco BRL) as
described in their optimized protocol. For amplification of
reverse-transcribed Vtg mRNA, the following primers were used: forward primer
5'-CTG TGA AGC TGG AGA AGC AGG - 3' and reverse primer
5'-CTT CGG CTT CAT CCC TCA GG - 3'. These primers were selected
based on a partial Vtg sequence from eelpout (data not shown). As reference
gene, ß-actin was amplified. Primers were selected by aligning
ß-actin sequences from Sparus aurata (X89920), Salmo
salar (AF012125) and Cyprinus carpio (M24113) (GenBank data).
Actin fw3: 5'-GAC GGA CAG GTC ATC ACC AT - 3'; actin revC:
5'-CAC ATC TGC TGG AAG GTG GA - 3'. Verification of the amplified
products as Vtg and ß-actin was obtained by sequencing the PCR product
and aligning with known fish Vtgs and actins (data not shown). 1 µl of the
cDNA was used for the following PCR reaction in 20 µl volumes using 1 unit
of Taq DNA polymerase and 1xPCR-buffer (Sigma, St Louis, MO, USA), 25
pmol of each primer and 200 µmol l-1 of each dNTP. PCR was run
for 4 min at 94°C followed by 15 (Vtg) or 20 (ß-actin) cycles of 30 s
at 94°C, 30 s at 56°C and 1 min at 72°C. A final extension step at
72°C for 7 min was added. The resulting cDNA fragments were resolved on 2%
agarose gels containing 1 µg ml-1 ethidium bromide, and their
molecular size was determined by comparison with size markers (100 bp ladder;
Gibco BRL). Finally, the gel was photographed for documentation.
For determination of Vtg mRNA in embryos, the liver region of whole embryos (a pool of three per mother fish) was homogenised in TRIzol® (Gibco BRL), and total RNA was isolated as described by the manufacturers. The RT-PCR conditions and primers for Vtg and ß-actin were as described above for the mother fish except that the PCR was run as follows: 4 min at 94°C followed by 28 (Vtg) cycles of 30 s at 94°C, 30 s at 56°C and 1 min at 72°C.
Quantification of calcium in body fluids
The concentration of total calcium (free and bound) in plasma and ovarian
fluid was measured by atomic absorption spectrophotometry (Perkin Elmer 2380,
Mountain View, CA, USA; Perkin Elmer, analytical methods for atomic absorption
spectrometry, 1982). The samples were diluted 200x in 0.1%
La2O3.
Determination of free amino acid levels in body fluids
The concentration of ninhydrine-positive substances (NPS), which indicates
the presence of free amino acids, was determined according to the method
described by Moore and Stein
(1948) and measured at 570 nm
with leucine as the standard.
Immunohistochemical analysis of Vtg in embryos
The localisation of Vtg was studied in control-, E2- and
OP-treated embryos by immunohistochemical staining of Vtg using rabbit
polyclonal antibody against eelpout Vtg
(Korsgaard and Pedersen,
1998). The Vtg-antibody was validated for specificity by
incubation on liver and ovary sections from E2-treated female
eelpouts (positive control) and liver sections from untreated male eelpouts
(negative control). Optimal target/background ratio was determined by serial
dilution.
Whole embryos fixed in 4% neutral-buffered formalin were processed by paraffin embedding according to standard procedures. 4 µm sections were cut and arranged on 3-aminopropyltriaethoxysilane-treated slides, dried at 37°C over night, deparaffinized in xylene, and rehydrated in alcohol. Before staining, endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide in methanol for 30 min. The slides were then rinsed (4x4 min) in 0.05% Tween in phosphate-buffered saline (PBS) at room temperature. Next, the slides were incubated with the primary Vtg antibody diluted 1:100 in 1% bovine serum albumin (BSA) in PBS for 60 min in a moist chamber at room temperature. The slides were rinsed (4x4 min) in 0.05% Tween in PBS, incubated with horseradish peroxidase conjugated swine anti-rabbit (DAKO) in 0.05% blocking serum (milk powder) in PBS for 30 min at room temperature and rinsed again for 4x4 min in PBS. Colour visualisation in sections was performed by incubation for 5 min with liquid 3,3-diaminobenzidenitetrahydrochloride (DAB) substrate (0.04% DAB in 0.05mol l-1 Tris/HCl, pH 7.6) (Sigma), with Mayer's haematoxylin as counterstain. The slides were evaluated qualitatively for the presence of Vtg in liver, gut and blood of the embryos using light microscopy. Negative control stainings were performed by incubating with normal rabbit sera instead of the Vtg-antibody. A minimum of four embryos from at least four different mother fish of each treatment group were analysed.
Histological examination by light microscopy
Whole embryos were fixed in Lillies-fixative and processed for histological
examination by light microscopy. After 24 h, embryos were dehydrated through a
series of graded ethanol (50-99%), cleared in xylene, and embedded in
paraffin. Sections (5 µm) were cut so that transverse sections of the
gonads were achieved. The sections were stained in Mayer's haematoxylin and
eosin-Y and analysed using a light microscope.
Transverse sections of the developing gonad were investigated through the whole length of the organ. The number of sections prepared from each embryo varied between 100 and 300 depending on the size of the gonad. Gonads were staged as follows: female gonad in early differentiation, female gonad containing oocytes, presumptive male gonad and abnormal gonad (male or female) as defined in Results. Light micrographs from the anterior, central and posterior regions of the gonad were taken with a digital camera. Four to six embryos from each mother fish were examined. Only embryos from day 0 and day 35 were analysed.
In situ hybridisation of oestrogen receptor mRNA
Fixed, dehydrated and paraffin-embedded embryos from day 0 and after 35
days of exposure were cut on a microtome (6 µm). Transverse sections from
the gonadal region were processed for in situ hybridisation as
described elsewhere (T. K. Andreassen et al., manuscript submitted). A 515 bp
[35S]UTP-labelled sense and anti-sense RNA probe encoding a part of
the E-domain of the cloned eelpout ER (T. K. Andreassen et al.,
manuscript submitted) was prepared and used in the hybridisation
experiments.
Chemicals
4-tOP and E2 [1,3,5(10)-estratriene-3,17ß-diol] were
obtained from Sigma-Aldrich, Steinheim, Germany.
Statistical analysis
Values are expressed as means ± S.E.M. Data were tested for
normality and homogeneity of variance and, if necessary, were
log10-transformed prior to analysis. One-way analysis of variance
(ANOVA; SYSTAT 7.0 for Windows, SPSS Inc., Chicago, IL, USA) followed by the
Tukey test of multiple comparison was used to test differences between groups.
Linear correlation analysis was carried out with Graph Pad Prism (ver. 1.03)
software (San Diego, USA).
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Results |
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Bioconcentration of 4-tOP and E2 in body fluids
High levels of 4-tOP were observed in plasma of pregnant eelpout after 35
days of exposure (Table 1). In
the OP100 group, a mean concentration of 54.9 mg l-1 4-tOP was
measured in plasma. The bioconcentration factors (BCFs) of 4-tOP in plasma
were determined to be approximately 200 and approximately 550 for the OP25 and
OP100 group, respectively (Table
1). Oestradiol-17ß was taken up by the E2-exposed
fish, and, in plasma, a >200-fold increase was detected in comparison with
control plasma after 35 days of exposure
(Table 1). The BCF of
E2 in plasma was approximately 20.
In ovarian fluid, 4-tOP was also found in high concentrations. The mean concentration of 4-tOP in the ovarian fluid increased both with time (results not shown) and dose (Table 1); however, time was only a significant factor for the OP100 group. After 35 days of exposure, concentrations of up to 3.4 mg l-1, with a mean concentration of 1.4 mg l-1, were measured in the OP100 group in ovarian fluid, resulting in a mean BCF of 15 (Table 1). In the OP25 group, levels of 4-tOP were all lower than in the surrounding water; consequently, the BCF was <1. In the ovarian fluid, the concentration of E2 in the E2 group was only approximately twofold the concentration in the control group.
In both plasma and ovarian fluid, 4-tOP bioconcentrated more efficiently than E2, but this was most apparent in the high-dose group.
Induction of oestrogenic biomarkers in pregnant
eelpout:E2-binding activity, Vtg mRNA in liver and Vtg in
plasma
Waterborne exposure of 4-tOP and E2 for 17 days (or 35 days)
up-regulated the Vtg-synthesising apparatus in the pregnant eelpout, as shown
by an up-regulated E2-binding capacity in the liver
(Fig. 2A), induced
sequestration of the Vtg protein into the plasma
(Fig. 2B) and transcription of
the Vtg-encoding gene in the liver (Fig.
2C).
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Oestrogen-binding activity in hepatic liver cytosol preparations was highly induced by the treatment (Fig. 2A). In the 4-tOP-treated fish, a 6-8-fold increase was observed after 17 days of continuous exposure to the compound. A comparable induction of binding activity (sixfold) was found in E2-treated fish.
Vtg in plasma was highly induced after 17 days of exposure to the test compounds (Fig. 2B), and a clear concentration-dependent response was observed. The concentration of Vtg was threefold higher in the OP100 group than in the OP25 group but was comparable with the concentration in the E2 group. A significant positive correlation between the actual concentration of 4-tOP in plasma and the concentration of Vtg in plasma was found (Fig. 3; r2=0.89, P<0.0001, N=23). After 35 days of exposure, a comparable relationship was observed (results not shown).
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Vtg mRNA was induced in all liver samples from E2- as well as 4-tOP-exposed fish (Fig. 2C). A single DNA product of the expected size (215 bp) was produced in the RT-PCR reactions. ß-actin (PCR product at 340 bp) was co-amplified to ascertain that RNA isolation and RT-reaction efficiency was comparable among samples. No induction of Vtg mRNA was seen in any control fish.
Changes in ovarian indices of pregnant eelpout
The gonadosomatic index (GSI) of the control fish increased throughout the
experiment and more than doubled 35 days after the onset of the experiment
(results not shown). In all treated groups, there was a tendency for a reduced
GSI in comparison with control fish, but this was only significant for the
OP25 group (Fig. 4E;
P<0.05, N=8). To get a more elaborate picture of the
development and condition of the ovary, four indices in relation to the ovary
were calculated. These indices describe the mass of the ovarian sac, the
ovarian fluid and the embryos in relation to the total body mass (excluding
ovary) of the mother fish, named the ovarian sac somatic index (OSSI), the
ovarian fluid index (OFI) and the embryo somatic index (ESI), respectively
(Fig. 4). Finally, we
calculated an index describing the proportion of the ovary made up by the
ovarian sac, which we named the ovarian sac mass percentage (OSM). During the
course of pregnancy (11 October to 15 November), OFI, ESI and OSM changed
dramatically and significantly in the control fish (2% to 17%, 12% to 19%, and
14% to 7%, respectively; P<0.05, N=8); by contrast, OSSI
was held at a remarkably constant level (2.3-2.5%). However, all treated fish
showed a tendency towards lower OFI and ESI levels
(Fig. 4A,B) compared with the
control. Furthermore, OSSI was significantly elevated in the OP100 and
E2 groups (Fig. 4C).
OSM values were significantly elevated in all treated groups
(Fig. 4D) after 35 days of
exposure, again showing a concentration-dependent relationship. In a similar
experiment conducted in SeptemberOctober, exactly the same pattern was
observed (results not shown), except that OFI did not seem to change, possibly
because ovarian fluid was very sparse at that time of pregnancy. The amount of
ovarian fluid per embryo (OFI/embryo) for the OP100 group on day 35 was
significantly lower than in controls (result not shown). The same tendency was
observed in the OP25 and E2 groups. There was a significant
positive linear correlation between OFI/embryo and embryonic mass
(r2=0.67, P<0.0001, N=35).
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During the course of the experiment, the hepatic somatic index (HSI) of control fish declined significantly (results not shown). However, a significantly elevated HSI was observed in the OP100 (P<0.001, N=7) and E2 (P<0.01, N=8) groups compared with the control group after 35 days of exposure (Fig. 4F).
No changes in the condition index (Mb/L3x100, where Mb is body mass in g and L is total length in cm) among experimental groups were observed (results not shown).
Exposure to either compound did not induce any significant mortality. Only approximately 10% of the fish in the high dose 4-tOP group died. However, after approximately 14 days of exposure, we started to observe incidences of premature parturition (abortion), a factor first observed in the OP100 and E2 groups and later in the OP25 group.
Changes in components important for the maternalfoetal trophic
relationship: calcium and free amino acids
In plasma, the level of total calcium increased to high levels in the
4-tOP-treated (3-6-fold increase) and E2-treated (fivefold
increase) groups after 35 days of exposure. By contrast, in the ovarian fluid,
a concentration-dependent decrease was observed during the same period in the
4-tOP-exposed fish. Fig. 5
illustrates the relationship between the calcium levels in ovarian fluid and
in plasma and shows a significant negative linear correlation
(P<0.008, N=31). There was, however, no significant
correlation between total calcium concentration and embryonic mass (data not
shown).
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NPS levels (an indicator of the amount of free amino acids) in plasma of all treated groups decreased to approximately 50% of that in the control group (data not shown). In the ovarian fluid, the concentration of NPS was reduced in control fish at day 35 compared with the control fish at day 0, but comparable and very low levels, much lower than in plasma, were seen in all groups, including the control, after the 35 days (data not shown).
Effects on embryonic mortality and growth of embryos
The mortality of the embryos after 35 days of exposure was between 1.8% and
17%. Only mortality of embryos in the OP100 group was significantly higher
compared with the control group. There was a positive correlation between the
concentration of 4-tOP in the ovarian fluid and the mortality ratio of embryos
in the ovary (Fig. 6;
r2=0.684, P<0.0001, N=23).
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At day 0 of the experiment, the embryos were at the late yolk-sac stage. During the experimental period of 35 days, the control embryos had grown, as indicated by an increase in both mass and length. The 35-day control embryos had absorbed the yolk sac and appeared more pigmented compared with the control embryos at day 0. Exposure to 4-tOP or E2 caused a decrease in embryonic growth. Compared with the control embryos at day 35, the mass of embryos from the OP25, OP100 and E2 groups was significantly decreased (Fig. 7A; P<0.01, N=80; P<0.01, N=70; P<0.05, N=80). The length was only significantly reduced in the two 4-tOP groups (Fig. 7B; P<0.05, N=80; P<0.01, N=70). Compared with the control embryos at day 0, the embryos from the three exposed groups had not gained much in mass or length after 35 days of exposure, although they appeared similar to the 35-day control embryos in terms of their degree of pigmentation and the absorbed yolk sac.
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Detection of Vtg induction and oestrogen sensitivity of the
embryos
To investigate if the treatments had induced any measurable oestrogenic
effect on the embryos, the induction of Vtg was examined by RT-PCR
(Fig. 8) and
immunohistochemistry (Fig. 9).
Vtg cDNA was only amplified in the OP100 group, and no detectable Vtg
induction was observed in the other groups
(Fig. 8). In all fish, the
internal standard ß-actin was readily amplified, indicating that RNA
isolation and RT-PCR conditions were identical for all samples.
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In the liver of the OP100-treated embryos, positive immunohistochemical staining of Vtg was found in the cytoplasm of hepatocytes and the blood capillaries (Fig. 9B). By contrast, livers in the control (Fig. 9A), OP25- and E2- treated embryos (not shown) all showed no staining.
In the hindgut, positive staining was seen in the OP100- (Fig. 9D), OP25- and E2-treated embryos, although with a lesser intensity in the OP25 and E2 groups. The staining was restricted to cellular debris in the lumen and to the periphery of the epithelia cells in the hindgut. There was no Vtg staining in the hindgut of the control embryos (Fig. 9C).
Fig. 10 shows the localisation of ER mRNA in early differentiating gonads (day 0), including presumptive male gonads (Fig. 10A,B), and in differentiated female gonads (control day 35; Fig. 10D,E). As a negative control, the sense probe was applied, showing no labelling in the gonads (Fig. 10C,F).
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Effects on gonadal differentiation in the embryos
The gonads of newly hatched embryos appear as an undifferentiated two-lobed
organ situated in a mesentery between the dorsal peritoneal wall (just beneath
the kidneys) and the intestines. This undifferentiated gonad contains
proliferating primordial germ cells (Fig.
11A).
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At day 0 of the experiment, at the late yolk-sac phase of embryonic development, two different types of gonads were observed. Of the analysed embryos, 48% of the gonads were female gonads in an early stage of differentiation (Table 2). The undifferentiated two-lobed gonad had grown and begun to form the ovarian cavity. The ovarian cavity is formed by longitudinal growth, gradual bending towards the mesentery and final fusion of the external ends of the genital ridges in the ventral side of the gonad resulting in a single ovary with an endo-ovarian cavity (Fig. 11B-D). In the central and largest part of the gonad, the ovarian cavity was generally still separated in two parts by a central string (Fig. 11C). The anterior and the posterior ends of the gonad were smaller and hollow (Fig. 11B,D). In some of the embryos, a two-lobed structure was visible in the very posterior end but not in the anterior end of the gonad. These early differentiating female gonads contained proliferating germinal cells surrounded by somatic tissue, but there was no indication of oogenesis and no oocytes were observed.
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The other 52% of the embryos had presumptive male gonads (Table 2). The gonads consisted of a two-lobed organ throughout the complete gonad containing germinal cells and somatic cells (Fig. 11E-G). This two-lobed structure resembles that of the testis of the adult male eelpout. The early male gonad appears very similar to the undifferentiated gonad except for an incipient enlargement of the stromal/somatic tissue connecting the two lobes of the gonad in the posterior end. This enlargement becomes more prominent as the differentiation of the male gonad proceeds. At this early time of differentiation, it was not possible to distinguish the germinal cells in the early female gonads from those of the presumptive male gonad by light microscopy.
In the control embryos at the end of the experiment (day 35), 52% of the gonads were female gonads that had grown in size and had differentiated into single hollow ovaries containing both oogonia and primary oocytes in the perinucleolar stage throughout the ovary (Table 2; Fig. 12A-C). Only the very anterior and posterior ends of the female gonad did not contain oocytes.
|
The other 48% of the embryos had presumptive male gonads, which more or less appeared the same as the presumptive male gonads found at the beginning of the experiment (Table 2; Fig. 12D-F). However, in some of the gonads, the tissue connecting the two lobes at the posterior end had enlarged and the lobes had become more triangular in shape compared with the controls at the beginning of the experiment.
In the embryos from the OP25 and E2 groups, the gonads resembled those of the control embryos, with gonads differentiated into ovaries with oocytes (58% and 49%, respectively) or with overall presumptive male two-lobed gonads (42% and 51%, respectively) (Table 2). However, in the OP100 group, different gonadal structures were found compared with the control embryos at the end of the experiment (day 35). Of the analysed embryos, 46% had normal ovaries with primary oocytes but only 22% of the embryos had normal presumptive male gonads resembling those found in the control embryos (Table 2). The remaining 32% of the embryos had abnormal gonads with atypical structures that had not been observed in the control embryos either at the beginning (day 0) or at the end (day 35) of the experiment. In most of these abnormal gonads, the anterior end of the gonad resembled that of a male gonad having two lobes (Fig. 13A,E). At the central part, the gonad was enlarged, the two-lobed structure had disappeared and the beginning of two hollow cavities had formed in each side of the gonad (Fig. 13B,C,F,G). In some gonads, these cavities had fused into one big cavity more or less resembling the endo-ovarian cavity of the female gonad. In two of the analysed embryos, the gonads looked mostly like male gonads but had either atypical structures at the external ends of the lobes or the lobes had enlarged and started to bend down towards the central string at the posterior end (results not shown). These structurally abnormal gonads contained germinal cells and somatic cells, but no oocytes were observed in any case.
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Discussion |
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The continuous flow-through system developed in the present study was
capable of keeping the actual water concentration of 4-tOP at a constant level
throughout the experiment. The concentrations were, on average, 56-65% of the
nominal concentration. These results are comparable with those obtained by
other groups working with alkylphenols in similar systems
(Nimrod and Benson, 1998;
Harries et al., 2000
). The
difference from nominal concentrations can be attributed to uptake in fish,
volatilisation, microbial breakdown and/or adhesion to the aquarium.
During the experiment, 4-tOP accumulated in the body fluids of the pregnant
mother fish. Very high concentrations of 4-tOP were measured in the blood, and
BCFs of 200-500 were measured after exposure to the actual dose of 65 µg
l-1 (OP100). In trout, a BCF of 91 was measured in the blood after
10 days of exposure to 4 µgl-1 4-tOP, a BCF that included OP
metabolites (Ferreira-Leach and Hill,
2001). How such high BCFs are achieved in the plasma of the
pregnant eelpout is unknown. Earlier studies have shown that during the
experimental period (OctoberNovember) lipids are mobilised to the
plasma from fat stores, making the plasma more lipid
(Korsgaard and Petersen,
1979
). This could increase the solubility of 4-tOP in the plasma.
Furthermore, it is possible that 4-tOP that is accumulated in fat is mobilised
concomitantly with the lipids, adding another source of 4-tOP. The binding of
4-tOP to different plasma proteins might contribute to an elevated solubility
of the compound. But these aspects need further investigation.
We detected the presence of a conjugated metabolite in both blood and
ovarian fluid but quantified only the level of the parent compound 4-tOP and
not the metabolites. It is very likely that 4-tOP is conjugated in the liver
after uptake in the gills. The conjugated metabolites of 4-tOP are probably
not active xeno-oestrogens, as oestrogen metabolites show almost no ER agonism
(Zhu and Conney, 1998).
In the ovarian fluid, a pronounced uptake of 4-tOP was observed. 4-tOP may
be transferred to the ovary because alkylphenols are highly lipophilic
compounds that might pass through ovarian membranes. The ovary appears to be
highly vascularized during pregnancy in eelpout
(Kristoffersen et al., 1973;
Korsgaard, 1983
), thereby
offering the possibility of nutrients, oxygen and lipophilic chemicals such as
4-tOP to enter the ovarian fluid/embryos from the maternal blood. The
accumulation of 4-tOP in the ovarian fluid was higher in the OP100 group than
in the OP25 group, indicating that the maximal capacity of the mother fish
and/or the embryos to metabolise and excrete the compound had been exceeded at
the higher concentration. Fish embryos in general bioaccumulate xenobiotics to
a higher degree than do adult fish, probably because of a less-efficient
metabolising machinery during the earlier life stages
(Monod et al., 1996
). To our
knowledge, no data exist on the bioaccumulation of xeno-oestrogens in other
viviparous species. However, a study using guppy (Poecilia
reticulata), a viviparous fish species with follicular gestation, showed
accumulation of the xenobiotic 3,4-dichloroaniline in ovarian embryos at
levels above that seen in muscle, gill, brain and skin
(Hertl and Nagel, 1993
).
E2 also bioaccumulated in the body fluids, but to a lesser
extent than 4-tOP. This seems reasonable as elaborate endogenous pathways for
the metabolism and excretion of E2 exist
(Zhu and Conney, 1998). As
4-tOP accumulated more efficiently than E2, its oestrogenic potency
in vivo increases as compared with E2.
Induction of the Vtg-synthesising apparatus in pregnant eelpout (ER,
Vtg mRNA and Vtg protein)
As an indicator of the oestrogenic potency of 4-tOP, the induction of the
Vtg-synthesising apparatus was examined. Vtg is widely accepted as a biomarker
of oestrogen exposure in male and juvenile fish
(Sumpter and Jobling, 1995),
and Vtg mRNA induction is regarded as a very sensitive oestrogenic biomarker
(Bowman and Denslow, 1999
). The
induction of Vtg mRNA in liver and also Vtg protein in plasma in all pregnant
mother fish exposed to 4-tOP or E2 was expected and has been
observed in numerous experiments with fish exposed to oestrogens and
xeno-oestrogens, including alkylphenols (e.g.
Flouriot et al., 1995
;
Jobling et al., 1996
;
Lech et al., 1996
;
Christiansen et al., 1998
;
Andreassen and Korsgaard,
2000
). The observed clear linear relationship between the actual
plasma concentration of 4-tOP and Vtg indicates that the bioavailable amount
of 4-tOP determines the vitellogenic response. This observation corresponds
well with the assumption that 4-tOP interacts directly with the hepatic ER in
eelpout (Andreassen and Korsgaard,
2000
) and induces vitellogenesis. This is further supported by the
fact that ER protein levels are induced in accordance with the well-known
auto-regulatory effect of oestrogens at the ER level
(Pakdel et al., 1991
;
MacKay et al., 1996
). In the
present study, a pronounced induction of E2-binding capacity in the
liver cytosol by both 4-tOP and E2 indicated an up-regulation of
the bio-available level of oestrogen receptors. The ability of 4-tOP to induce
the Vtg-synthesising apparatus at all levels (ER, Vtg mRNA and Vtg protein)
signifies the oestrogenic capacity of the compound.
Disturbance of the maternalfoetal trophic relationship: the
ovary, ovarian calcium and amino acids
The ovary of the pregnant eelpout undergoes drastic changes during
gestation (Korsgaard, 1986).
Thus, before and during a short period after hatching, the ovary is
characterised by large fluid-filled follicles and very little fluid present in
the ovarian cavity. However, a sudden shift takes place in the production
and/or distribution of fluid in the ovary after hatching. The amount of fluid
in the ovarian cavity increases significantly, coinciding with less fluid in
the follicles. The condition of the ovary and the resulting ovarian
environment is likely to be an important factor for the normal functioning of
the maternalfoetal trophic relationship and, therefore, the wellbeing
of the embryos.
It is obvious from the present study that oestrogenic exposure has an important impact on ovarian factors, but it is difficult to interpret the consequences of these results. Usually when describing effects on fish gonads and their contents, GSI is used as a general index. However, in this context, this is not adequate as the embryo-filled ovary consists of several components, each reacting differently to oestrogenic exposure. The overall effect of E2 and 4-tOP on the ovary is evident when looking at the OSM index, which summarises the impact on embryos (ESI), ovarian fluid (OFI) and the ovarian sac (OSSI). The changes in ESI and OFI were not statistically significant, presumably due to the large inter-individual variation of pregnant eelpout.
Oestrogen levels in plasma of eelpout peak prior to and decline throughout
pregnancy (Korsgaard, 1994).
Similarly, E2 levels abruptly decline after fertilisation in the
guppy Poecilia reticulata
(Venkatesh et al., 1990
), and
oestrone implants have been shown to suppress ovarian and embryonic
development in the viviparous fish Neoditrema ransonneti
(Ishii, 1960
). These
observations indicate that oestrogen does not have an important role during
pregnancy of viviparous fish or that the absence of oestrogen is important for
the progression of pregnancy. Moreover, Korsgaard
(1983
,
1994
) showed that the
post-ovulatory follicles of Z. viviparus were able to sequester Vtg
in oestrogen-treated individuals, and, in N. ransonneti, oestrone
inhibited the normal development of the ovarian lining during gestation
(Ishii, 1960
). Thus, it is
intriguing to speculate that exposure of pregnant eelpout to oestrogenic
compounds may change the function of the ovary from an organ occupied with the
nutrition and support of embryos to a Vtg-sequestering organ. The incidences
of premature parturition were only observed in the exposed fish but not in the
controls. Oestradiol benzoate has been reported to induce premature birth in
top minnows (Gambusia affinis;
Ishii, 1963
). Furthermore, it
has been proposed that oestrogen is involved in the initiation of parturition
in some viviparous fish species, when birth of the embryos and vitellogenesis
for the next batch of oocytes coincide
(Venkatesh et al., 1990
).
The effects observed on the ovary after E2 and 4-tOP treatment might be attributed to a direct effect mediated by the ER. ER mRNA has been shown to be highly expressed in the ovary of eelpout (T. K. Andreassen et al., manuscript submitted) and to be located in several different ovarian cell types.
The tendency towards lower levels of ovarian fluid in the ovary of the
treated groups could also be of significance, as ovarian fluid is important in
the effective transport of nutrients and oxygen to the embryos. Low oxygen
levels are known to be able to retard the development of fish larvae
(Carlson and Seifert, 1974). It
is believed that nutrient (and oxygen) uptake in the ovary of Z.
viviparus takes place via the highly vascularised follicles
(Kristoffersen et al., 1973
;
Korsgaard, 1983
), the
so-called calyces nutriciae. This transfer of nutrients to the embryos from
the mother fish might be disturbed by the oestrogenic treatment.
Previous investigations have provided evidence that free amino acids are
one of the important nutrients taken up and metabolised by the embryos of
Z. viviparus during their intraovarian development
(Korsgaard and Andersen, 1985;
Korsgaard, 1992
). The
significant decrease in the maternal plasma concentration of free amino acids
(NPS) observed in both E2- and 4-tOP-treated groups (probably due
to an increased demand for amino acids for the hepatic synthesis of Vtg) may
be one of the factors responsible for the observed decrease in embryonic
growth, even if this was not reflected immediately in the concentration of
amino acids (NPS) in the ovarian fluid. We hypothesise that the transfer of
amino acids to the ovary/embryos has been negatively affected as the transfer
is directly related to the embryonic amino acid availability, because, when
first taken up into the ovarian fluid from the maternal circulation, amino
acids are rapidly taken up by the embryos
(Kristoffersen et al., 1973
;
Korsgaard, 1992
).
A marked decline in the concentration of calcium was observed in the
ovarian fluid after 4-tOP and E2 treatment, which significantly
correlated with the observed increase in the concentration of calcium in the
maternal plasma. This indicates that calcium is needed by the maternal
organism for the enhanced hepatic synthesis of Vtg, which is known to
incorporate calcium (Nagler et al.,
1987). Consequently, a different distribution of calcium is
evident in the exposed fish, resulting in low ovarian calcium but high plasma
concentrations. The Vtg-bound calcium in plasma is, however, not available to
ovarian/embryonic uptake. Earlier studies have shown that eelpout embryos take
up calcium in ovario, an uptake that is severely inhibited by
E2 treatment (Korsgaard,
1994
). It is, however, not known if and how such a decline in
calcium availability may affect embryonic development. However, as calcium is
an important component necessary for the development of the skeleton and
various other parts of the body, calcium may play a pivotal role during
embryonic development.
Effects on embryonic growth
Exposure to 4-tOP or E2 inhibited embryonic growth. In a similar
experiment performed during very early pregnancy (SeptemberOctober),
growth was also significantly inhibited by 4-tOP and E2, and
deformities after E2 treatment were observed (results not shown).
Previous studies have reported negative impacts of natural oestrogens
(Johnstone et al., 1978;
Krisfalusi and Cloud, 1996
;
Gimeno et al., 1998
) or
xeno-oestrogens (Ashfield et al.,
1998
; Drèze et al.,
2000
) on growth in different species of fish. Such effects were
suggested to be caused by disruption of the somatotropic axis
(Drèze et al., 2000
).
Thus, the negative effects on embryonic growth observed in the present study
may, in part, be a direct result of the oestrogenicity of the compounds. Being
a viviparous fish in which the embryos are dependent on the ovarian
environment, indirect effects involving the maternalfoetal trophic
relationship of Z. viviparous should also be taken into account.
Thus, the changed physiology of the ovary and the oestrogen-induced metabolic
changes may, as explained above, have a marked effect on this relationship and
hence the growth and development of the eelpout embryos. The positive
correlation between the ovarian fluid index (OFI) and embryonic mass shows the
importance of the ovarian fluid for the normal development of the embryos. A
decrease in ovarian fluid induced by extended exposure to xeno-oestrogens may
thus be critical because the ovarian fluid has a multitude of functions, such
as providing oxygen and nutrients and removing waste products
(Korsgaard and Weber,
1989
).
Increased mortality of embryos was observed in the OP-treated groups,
correlating with the concentration of 4-tOP in the ovarian fluid. This
observation indicates a toxic effect of the compound on the embryos. However,
with a concentration higher than 1000 µg l-1 4-tOP in the
ovarian fluid, lethality among the embryos of this group is to be expected
(Servos, 1999).
Effects on embryonic Vtg synthesis
To investigate whether the 4-tOP in the ovarian fluid had directly affected
the embryos, the induction of Vtg synthesis in the embryos was analysed. As
expected, no Vtg mRNA expression was found in the control embryos. Similarly,
no Vtg mRNA expression was found in the OP25- and E2- treated
groups. Similar results were obtained by immunohistochemistry. However, the
embryos from the OP100 group showed marked induction of Vtg mRNA and protein,
thus confirming that the embryos had indeed been exposed in ovario to
concentrations of 4-tOP high enough to elicit an oestrogenic response. The
fact that no Vtg mRNA or Vtg protein induction was observed in the embryos of
the E2 and OP25 groups could be explained by the relatively low
concentrations of the compounds in ovario and by a lower oestrogen
sensitivity of eelpout embryos compared with adult and juvenile fish.
Earlier studies have shown that the threshold concentration of
E2 for the induction of Vtg is between 27 ng l-1 and 272
ng l-1 in fathead minnow (Pimephales promelas;
Parks et al., 1999) and
between 33 ng l-1 and 212 ng l-1 in sheepshead minnow
(Cyprinodon variegatus; Folmar et
al., 2000
). However, in one study, very low concentrations of
E2 (9 ng l-1) were able to induce Vtg synthesis in
juvenile rainbow trout (Oncorhynchus mykiss;
Thorpe et al., 2000
). In the
E2 group in the present study, an average concentration of 58 ng
l-1 E2 was obtained, while 25 ng l-1
E2 was present in the control ovarian fluid. It is very plausible
that this relatively small increase would not induce a physiological response
in eelpout embryos. In the OP25 group, 5 µg l-1 4-tOP was
detected in the ovarian fluid, which is a fairly low concentration. A
doseresponse study on juvenile rainbow trout using the alkylphenol 4-NP
did not induce Vtg induction at a concentration of 7 µg l-1
(Thorpe et al., 2000
);
however, Jobling et al. (1996
)
observed Vtg induction by exposure of male rainbow trout to 5 µg
l-1 4-tOP for 3 weeks.
Interestingly, strong Vtg-staining was found in the hindgut of the embryos
in all the treated groups, in contrast to the liver where Vtg was induced only
in the OP100 group. This indicates that the Vtg in the hindgut is of maternal
origin. The Vtg probably originates from the ovary (e.g. follicular tissue
containing Vtg), as embryos are known to ingest ovarian material
(Kristoffersen et al., 1973).
This Vtg is taken up by the epithelial cells; it is, however, unknown whether
the hindgut is capable of metabolising the Vtg. Consequently,
oestrogen-treated eelpout embryos might contain Vtg of both embryonic and
maternal origin because of the maternalfoetal trophic relationship
characteristic of the eelpout. A technical consequence is that the ELISA
method widely used for the determination of Vtg in adult fish and oviparous
juvenile fish is not useful for eelpout embryos (whole body homogenates).
Effects on gonadal differentiation
It has been suggested that 17ß-oestradiol may act as the natural
inducer of ovarian differentiation in teleost fish
(Nakamura et al., 1998). The
present study demonstrates that 4-tOP affects the gonadal differentiation of
eelpout embryos exposed in ovario following exposure of the mother
fish to the actual mean concentration of 65 µg l-1 4-tOP (OP100
group). This result correlates well with the observation that only in this
treatment group do embryos show a clear oestrogenic response (induction of Vtg
mRNA and protein).
In the control group at day 0 and at day 35, the ratio of male to female
embryos of the investigated embryos in the mother fish was approximately 1:1,
which agrees with the reported sex ratio from field studies
(Larsson et al., 2000). In the
OP100 group, 46% of the analysed embryos were females; however, only 22% of
the embryos had normal (presumptive) male gonads. The remaining 32% of the
embryos had abnormal gonads in which the anterior end of the gonad generally
appeared as a presumptive male gonad while the central parts and posterior
ends appeared as an early differentiating female gonad with an endo-ovarian
cavity. These observations of female-like reproductive ducts (ovarian
cavities) in `male' embryos after exposure to oestrogenic alkylphenols are
supported by other studies on oviparous fish species. Gimeno et al.
(1997
) reported that exposure
of young genetically male carps (Cyprinus carpio) to
4-tert-pentylphenol during sexual differentiation induced the
formation of an oviduct in almost all fish. Gray and Metcalfe
(1997
) investigated Japanese
medaka (Oryzias latipes) exposed to a range of concentrations of
nonylphenol (NP) from hatching to 3 months of age. They showed that 50% of the
male fish treated with 50 µg l-1 NP developed testis-ova.
Drèze et al. (2000
)
reported that exposure of the viviparous mosquito fish (Gambusia
holbrooki) to 50 µg l-1 4-NP from 3 days post-parturition
to 75 days post-parturition resulted in fish exhibiting female or
undeveloped/atrophied gonads and no fish with normal male gonads. In
accordance with our observations in the OP25 group, exposure to low
concentrations of alkylphenols (0.5-1.9 µg l-1 4-NP) in Japanese
medaka exposed during the first month after hatching had no effect on gonadal
development or sex ratio (Nimrod and
Benson, 1998
). However, no effects on the gonadal development and
the sex ratio were found in offspring of the viviparous guppy (Poecilia
reticulata) after exposure to octylphenol
(Kinnberg et al., in press
).
Recently, Rodgers-Gray et al.
(2001
) found that exposure of
roach (Rutilus rutilus) to graded concentrations of treated sewage
effluents for 150 days during their gonadal differentiation resulted in the
formation of female-like reproductive ducts (ovarian cavities) in male roach
in a dose-dependent manner. The effluents contained low concentrations of
alkylphenols (90-2000 ng l-1), natural oestrogens (5.9-37 ng
l-1) and, presumably, other unidentified endocrine disrupters.
Similar to the present study, Rodgers-Gray and co-workers
(2001
) did not find oocytes in
these feminised male fish. Female-like reproductive ducts have also been
reported in wild roach and gudgeon (Gobio gobio) in UK rivers
(Jobling et al., 1998
;
van Aerle et al., 2001
).
The oestrogenic effects on embryos observed in the present study, such as
induction of Vtg mRNA and protein and the development of female-like ovarian
cavities in `males', might represent direct effects via the oestrogen
receptors in the liver and gonadal tissues, respectively. 4-tOP is known to
bind oestrogen receptors in liver tissue, causing induction of vitellogenesis
in numerous fish species including the eelpout
(Andreassen and Korsgaard,
2000). Recently, oestrogen receptors have also been identified in
testes of adult fish, making direct effects of xeno-oestrogens such as
octylphenol on testes very likely (Loomis
and Thomas, 1999
; Legler et
al., 2000
; T. K. Andreassen et al., manuscript submitted). In
addition, oestrogen receptors have recently been identified during early
development in both male and female transgenic zebrafish Danio rerio
(Legler et al., 2000
). In the
present study, we have shown that ER mRNA expression is present in early
differentiating gonads, including presumptive male gonads, indicating that
functional ER may be present at this stage. This observation makes it possible
for xeno-oestrogens to act directly on the gonads during early sex
differentiation in Z. viviparus. Thus, the critical period of gonadal
differentiation may be very sensitive to the disruption of hormonal
homeostasis by oestrogenic compounds. Administration of oestrogens before
irreversible commitment of the gonads to sex may lead sex ratios towards the
female direction in gonochoristic fish; hence, the genetic determination of
gonadal sex in teleosts may be affected by exogenous sex hormones
(Yamamoto, 1969
;
Nakamura et al., 1998
). As
oestrogens are known to affect the brainpituitarygonadal axis
during development (Kah et al.,
1997
), it is likely that there are alternative ways in which
xeno-oestrogens may affect the gonadal development in the early life stages of
fish.
The timing and duration of exposure directly relates to the severity of the effects by oestrogenic compounds. Therefore, a possible explanation as to why we also observed some male embryos containing apparently normal male gonads, even in the high dose 4-tOP group, could be that the exposure was not conducted sufficiently early during the gonadal differentiation but during the late yolk-sac stage. At the late yolk-sac stage, gonadal differentiation may have progressed to a point where the xeno-oestrogens have reduced effects and hence did not induce the female-like ovarian cavity in all embryos.
Alkylphenols (including 4-tOP) have been detected at concentrations of up
to 13 µg l-1 in an estuary in the UK
(Blackburn and Waldock, 1995);
however, concentrations of approximately 1-10 µg l-1 are more
common (Bennie, 1999
). The
actual concentration of 14 µg l-1 (OP25) is therefore an
environmentally relevant concentration, while the 65 µg l-1
concentration (OP100) is rare in the aquatic environment. In the present
study, severe direct effects on embryos were only observed in the OP100 group;
however, lower and environmentally relevant concentrations (e.g. OP25 group)
have an effect on the viviparous mother fish, resulting in the allocation of
energy from the support of embryonic growth to physiological non-essential
processes (Vtg synthesis) and formation of smaller (and probably less fit)
embryos or, even worse, abortions. In the wild, these kinds of effects may
have consequences at the population level.
Conclusions
In conclusion, this study is the first to demonstrate that an oestrogenic
endocrine disrupter, 4-tOP, can be transferred from the water via the
mother fish to the ovarian fluid of the ovary and can subsequently affect the
embryonic development in ovario in a viviparous teleost species. In
the mother fish, the compound induced vitellogenesis, caused impairment of
normal ovarian development and changed the nutritive status of maternal blood
and ovarian fluid. Embryonic growth was negatively affected, which might, in
part, be attributed to disturbances of the maternalfoetal trophic
relationship. In the embryos, 4-tOP acted as an oestrogenic compound by
inducing Vtg synthesis and causing abnormal male gonads with female-like
ovarian cavities. This study contributes to the increasing evidence that
xeno-oestrogens can impose severe effects on the gonadal differentiation of
fish.
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Acknowledgments |
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