# Modulation of salmon ovarian steroidogenesis and growth factor responses by the xenoestrogen, 4-nonylphenol

Trond M. Kortner, Siv-Hege Vang, Augustine Arukwe *

Department of Biology, Norwegian University of Science and Technology (NTNU), Høgskoleringen 5, 7491 Trondheim, Norway



### article info

Article history: Received 27 April 2009 Received in revised form 8 July 2009 Accepted 30 July 2009 Available online 22 August 2009

Keywords: Steroidogenesis Previtellogenic oocytes Nonylphenol Floating agarose Endocrine disruption
Salmon 

### abstract

Endocrine-disrupting chemicals are known to influence organismal reproductive processes, including theproduction and regulation of gonadal steroids. This study evaluated the effects of a xenoestrogen (nonylphenol: NP) on salmon ovarian steroidogenesis and growth factors using an in vitro organ culture system. Ovarian tissues were cultivated for 3 and 7 d with different concentrations of NP (0 (control), 1, 10 and

50 

### l M) dissolved in ethanol (0.1%). The mRNA expressions of steroidogenic acute regulatory (StAR) protein, P450-mediated cholesterol side-chain cleavage (P450scc ), aromatase isoforms, 3 b -hydroxysteroid dehydrogenase (3 b -HSD), Cyp11 b -, Cyp17 and 21-hydroxylase, and insulin-like growth factors (IGF-1 and IGF-2) and IGF1-receptor (IGF1-R) were quantified by real-time PCR. Tissue levels of estradiol-17 b (E2), testosterone (T) and 11-ketotestosterone (11-KT) were quantified using enzyme immunoassays.Our data show that nominal NP levels produced time- and concentration-specific effects on the expression of steroidogenesis- and IGF-related transcripts in salmon ovarian tissues. Tissue levels of ovarian E2, T and 11-KT were significantly modulated after NP exposure. Interestingly, elevated ovarian E2 levels
after 10 

### l M NP exposure at day 3 paralleled P450 Arom isoforms mRNA expression at the same time interval. The expression patterns of other steroidogenic protein and enzyme genes, such as StAR, P450scc, 3 b

HSD and Cyp17 inversely paralleled this pattern, displaying consistent decreased transcript levels. Thesefindings show that NP (an ubiquitous environmental pollutant) can produce variations in gonadal steroidogenesis and growth regulating responses with potential consequences for overt fecundity in teleosts.

2009 Elsevier Ltd. All rights reserved.

1. Introduction

The underlying biochemical pathways that affect receptor mediated responses in fish reproduction have been suggested to represent a potential and strong target for various endocrine-disrupting chemicals (EDCs ( Rodriguez-Mozaz et al., 2004 ). But, the complete mode of actions for xenoandrogens and xenoestrogens on relevantbiochemical and physiological mechanisms have not been described in full detail ( Goksøyr et al., 2003; Rotchell and Ostrander 2003; Bonefeld-Jørgensen et al., 2007; León et al., 2008 ) and are still subject for systematic investigations both in our laboratory ( Lyssimachou et al., 2006; Kortner and Arukwe 2007b; Lyssimachou and Arukwe 2007; Vang et al., 2007 ) and elsewhere ( Rodriguez-Mozaz et al., 2004; Porte et al., 2006 ). Research on wildlifespecies show that the reproductive system, together with the associated endocrine and neural controls, can be very susceptible to alterations by environmental exposures to a variety of chemicals, including pharmaceuticals and physical agents ( Arukwe and Goksøyr 2003; Nash et al., 2004 ). Chemical compounds known to mimic the effects of endogenous estrogens in laboratory and field studies include synthetic steroids such as ethynylestradiol (EE2) used in contraceptive pills ( Nash et al., 2004 ) and alkylphenol polyethoxylates (APEs) such as nonylphenol (NP) (Kwak et al., 2001 ). In sewage treatment work (STW) effluents, steroidal estrogens arebelieved to be responsible for, at least in part, the feminized responses in some wild fish species (Jobling et al., 2002 ). Several
studies have identified NP as the most critical metabolite of APEs because of its enhanced resistance toward biodegradation, toxicity,estrogenic effects, and ability to bioaccumulate in aquatic organisms ( Ahel et al., 1994 ). Concentration of 330 

### lg L

1 total NP hasbeen reported in wastewaters from a sewage treatment works discharging into the river Aire (UK), and concentrations as high as

180 

### lg L

1 have been detected in rivers in the United Kingdom (UK; Blackburn and Waldock, 1995 ), 60 

### lg L

1 in rivers in Switzerland ( Ahel et al., 1994 ) and 17 

### lg L

1 in a river estuary in Croatia ( Kvestak et al., 1994 ). It has been shown that the concentration of NP in the environment is decreasing, although concentrations
of 4.1 

### lg L

1 in river waters and 1 mg kg

1 in sediments are still apparent ( Soares et al., 2008 ). Since high concentrations of alkylphenolic compounds are found in estuaries, it is plausible to assume that these compounds could impose an estrogenic risk to

0045-6535/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi: 10.1016/j.chemosphere.2009.07.075

* Corresponding author. Tel.: +47 73 596265; fax: +47 73 591309.

E-mail address: arukwe@bio.ntnu.no (A. Arukwe). Chemosphere 77 (2009) 989–998

Contents lists available at ScienceDirect


# Chemosphere

journal homepage: www.elsevier .com/locate/chemosphereaquatic organisms, a risk that may not be restricted to areas close to the outlets of municipal and industrial discharges. So far, substantial amount of research on endocrine toxicology has mainly focused on estrogenicity that involves direct estrogenreceptor (ER) mediated effects. In contrast, mechanisms of endocrine disruptions mediated through pathways that involve regulatory proteins and/or enzymes in the steroidogenic pathways are not well explored ( Arukwe 2008 ). Nevertheless, key enzymes andproteins involved in steroidogenesis are increasingly being considered as important targets for EDCs ( Sanderson 2006 ) and have recently become subject for systematic investigations. For instance, we have previously reported that NP induced transcriptional changes of the steroidogenic acute regulatory (StAR) protein and cytochrome P450-mediated side-chain cleavage enzyme (P450scc ) in Atlantic salmon brain ( Arukwe 2005 ) and Atlantic cod ovary ( Kortner and Arukwe 2007b ). In another study, NP modulated Atlantic salmon brain and liver aromatase isoforms ( Meucci
and Arukwe 2006 ). Elsewhere, significant insights have been gained on the potential effects of fungicides on these pathways in the fathead minnow ( Pimephales promelas ) ( Ankley et al., 2007; Villeneuve et al., 2007a,b ). Gonadal steroids are known to modulate both the synthesis and release of gonadotropin by the pituitary and influence several brainfunctions that are apparently responsible for gender-specific differences in the regulation of hypothalamic-pituitary-adrenal (HPA) secretions and hypothalamus-pituitary-gonadal-liver (HPGL) axis. The rate-limiting step in acute steroid production is
the movement of cholesterol across the mitochondrial membrane by the StAR protein, with subsequent conversion to pregnenolone by P450scc ( Stocco 2000 ). Although the StAR, P450scc and aromatase (P450 arom ) are the main proteins involved in the early steroidogenic pathway and have so far received the most attention in studies conducted on teleosts, other proteins such as 17 

### a -hydroxylase/17,20 lyase (Cyp17), 3 b -hydroxysteroid dehydrogenase (3 b

HSD), and 21- and 11 b -hydroxylases (Cyp21 and Cyp11 b ) are also key enzymes in steroidogenesis ( Miller 1988 ). Essentially nothing is known about the potential effects of NP on these enzymes, and their expression patterns during early gametogenesis in fish havenot been investigated in detail. Furthermore, ovarian steroidogenesis is stimulated by insulin-like growth factor 1 (IGF-1) ( Kagawa et al., 2003; Campbell et al., 2006 ), and the high ovarian expression of IGF-1, insulin-like growth factor 2 (IGF-2) and their receptors suggests an important role of the products of these genes during ovarian development ( Campbell et al., 2006 ). The objective of this work was to investigate the effects of the xenoestrogen, NP on Atlantic salmon ( Salmo salar ) gonadosteroidogenic pathways by quantifying the expression patterns of genes involved in steroidogenesis and oocyte growth after in vitro exposure. We hypothesized that exposure of salmon previtellogenicovarian tissues to NP would produce differential expression patterns of steroidogenesis- and growth factor-related genes, andalterations in ovarian steroid levels. This will improve our understanding of the molecular mechanisms and the chemical mode of actions that may cause impaired steroidogenesis with potentially negative impacts on reproduction in fish species.

2. Materials and methods

2.1. Chemicals and reagents

4-nonylphenol (85% of p -isomers) was purchased from Fluka chemika–biochemika (Buchs, Switzerland). Trizol reagent for RNApurification, TA cloning kit and Leibovitz L-15 medium were purchased from Gibco-Invitrogen life technologies (Carlsbad, CA,USA). Bovine serum albumin (BSA) and N-[2-hydroxyethyl]piperazine-N 0 -[2-ethanesulfonic acid] (Hepes) were purchased from Sigma Chemical (St. Louis, MO, USA). Iscript cDNA Synthesis Kit and iTaq TM Sybr  Green supermix with ROX were purchased from BioRad laboratories (Hercules, CA, USA) and Generuler TM 100 bp DNAladder and dNTPs from Fermentas GmbH (Germany). Estradiol17 b , testosterone and 11-ketotestosterone enzyme immunoassay (EIA) kits were purchased from Cayman chemical company (AnnArbor, MI, USA). All other chemicals were of the highest commercially available grade.

2.2. Animals and floating agarose in vitro culture technique

Juvenile, randomly picked Atlantic salmon ( Salmo salar ), 190–310 g body weight were purchased from Lundamo Hatcheries, Norway and kept in aerated 70 L aquaria at 10  C and 12:12 h light:- dark photoperiod at the animal holding facilities at thedepartment of Biology, Norwegian University of Science and Technology (NTNU). The organ culture technique employed was based
on the in vitro agarose floating method that has been tested and validated in our laboratory ( Kortner and Arukwe 2007a ). The experiments/procedures described were conducted in accordance with the laws and regulations controlling experiments with liveanimals in Norway. Briefly, juvenile female salmon were anesthetized, sacrificed and washed in 70% ethanol. Ovarian tissues from several individuals (4–6) were excised, cut into small pieces (1  1  1 mm) and grown in 6-well dishes on a floating agarose
substrate covered with a nitrocellulose membrane in basal culture
media. The basal culture medium consisted of Leibovitz L-15 medium supplemented with 0.1 mM L-glutamic acid, 0.1 mM L-aspartic acid, 1.7 mM L -proline, 0.5% BSA and 10 mM Hepes (pH 7.4). Pieces of tissue were cultured randomly, ensuring that each exposure contained gonadal pieces from at least four different female fish.The tissue were exposed for 3 and 7 d with different nominal concentrations of NP (0 (control), 1, 10 and 50 

### l M) in a humidified
incubator at 10  C. The control group received ethanol (carrier vehicle) and the final concentration of ethanol in all exposuregroups was 0.1% (v/v). The medium was changed at day 3 for tissues cultured for 7 d. After cultivation, tissues pieces were collected in triplicates ( n = 3) and were snap-frozen in liquid nitrogen and stored at

80  C until further processing. Additionally, tissues from all experimental groups were fixed, embeddedand sectioned by regular techniques. Immunohistochemical analysis was performed as described earlier ( Vang et al., 2007 ).

2.3. Quantitative (real-time) PCR

Total RNA was extracted using the Trizol reagent according tostandard procedure as recommended by the manufacturer (Gibco-Invitrogen life technologies, Carlsbad, CA, USA). Total cDNA for the real-time PCR reactions were generated from 1 

### lg total RNA from all samples using a combination of poly-T and randomprimers from a iScript cDNA Synthesis Kit as described by the manufacturer (Bio-Rad). Quantitative (real-time) PCR was used for evaluating gene expression profiles for 3 b -HSD, P450 aromA and

B , Cyp11 b , Cyp17, Cyp21, IGF-1, IGF-2, IGF1-R, P450scc and StAR. For each treatment, the expression of individual gene targets was analyzed using the Mx3000P Real-Time PCR System (Stratagene, La Jolla, CA, USA). Each 25- 

### l l DNA amplification reaction contained

12.5 

### l l of iTAQ TM SYBR Green Supermix with ROX (Bio-Rad), 1 

### l l ofcDNA, and 200 nM of each forward and reverse primer. The threestep real-time PCR program included an enzyme activation step at

95  C (3 min) and 40 cycles of 95  C (15 s), 55–65  C (30 s) (depending on the primers used; see Table 1 ), and 72  C (20 s). Controls lacking a cDNA template were included to determine the specificity of target cDNA amplification. Cycle threshold (Ct) values obtained were converted into mRNA copy number using 990 T.M. Kortner et al. / Chemosphere 77 (2009) 989–998standard plots of Ct versus log copy number. The criterion for usingthe standard curve is based on equal amplification efficiency (usually >90%) with unknown samples and this is checked prior toextrapolating unknown samples to the standard curve. The standard plots were generated for each target sequence using knownamounts of plasmid containing the amplicon of interest, as described previously ( Arukwe 2005 ). Data obtained from triplicate runs for target cDNA amplification were averaged and expressed as percentage of control samples.

2.4. Steroid hormone analysis

Ovarian tissue concentrations of estradiol-17 b (E2), testosterone (T) and 11-ketotestosterone (11-KT) were measured by EIA. Ovarian tissues of equal sizes were thawed on ice, homogenized

in 500 

### l l of 0.1 M sodium phosphate buffer and centrifuged at
14,000 g for 15 min. The supernatants were purified by extractionusing organic solvent to prevent the interference of lipids and proteins in the analysis. Briefly, samples were mixed with ethyl acetate/hexane (50:50, 4  the sample volume) by vortexing. After phase separation, the ethyl acetate/hexane phase was transferred to a clean tube. This step was repeated three times. Finally, the ethyl acetate/hexane was evaporated by heating to 30  C. The dry extract was reconstituted in EIA buffer by vortexing. Enzymeimmunoassay was performed according to the manufacturer’s protocol. Steroid hormone levels were quantified against a standard curve that was linearized using a logit transformation of B/B0 (bound sample/maximum bound). The sensitivity of the EIAs were E2; 19 pg mL

1 , T; 6 pg mL

1 and 11-KT; 1.3 pg mL

1 . The intraand inter-assay variations for all EIAs employed were

 10% and

 8%, respectively. Except for a 2.2% cross-reactivity with 11-KT in the T EIA, cross-reactivities between 11-KT, T and E2 EIAs and their respective steroid were 100%, and other steroids (11-KT, T and E2) were all < 0.01%.

2.5. Statistical analysis

Statistical analyses were conducted with GraphPad Prism, version 5.00 (GraphPad Software Inc. 2007). Statistical differences between carrier solvent control and exposure groups were performed using One-way ANOVA after testing for normality and variance homogeneity. Significant differences between exposure groupswere analyzed using the Tukey’s Multiple Comparison Test. The level of statistical significance was set at p < 0.05. 3. Results

3.1. Modulation of steroidogenic gene expressions

StAR mRNA expression significantly decreased after 3 d exposure to 10 

### l M NP, while 1 and 50 

### l M NP did not produce significant changes, compared to control at this time interval ( Fig. 1 A). At day 7 of exposure, an apparent concentration-dependent decreasing trend of StAR mRNA was observed, albeit not significant ( Fig. 1 A). For P450scc mRNA, the effect of NP at day 3 of exposure was similar to the effect observed for StAR expression, showing significantly decreased levels after exposure to 10 

### l M NP ( Fig. 1 B). In contrast, an apparent concentration-dependent increase (significant at 50 

### l M) in P450scc mRNA expression was observed after NP treatment at day 7 ( Fig. 1 B). The expression of P450 AromA ( Fig. 1 C) and P450 AromB ( Fig. 1 D) showed similar modulations patterns after NP exposure at day 3, displaying increased levels after exposure to the two highest NP concentrations (10 and 50 

### l M). At day 7, P450 AromA mRNA levelswere slightly elevated in all NP exposure groups, compared to control level, albeit not significantly ( Fig. 1 C). For P450 AromB , a significant increase of mRNA levels was observed for samples treated
with 1 and 50 

### l M NP, while 10 

### l M NP showed no effect compared to control ( Fig. 1 D). The mRNA levels of 3 b -HSD were modulated after NP exposure ( Fig. 2 A). For example, 1 and 50 

### l M NP produced
increased 3 b -HSD mRNA levels at day 3 of exposure, while 10 

### l M NP decreased non-significantly, compared to control at this time interval ( Fig. 2 A). At day 7, a non-significant increasing pattern after NP exposure was observed for 3 b -HSD mRNA, compared to control ( Fig. 2 A). The expression of Cyp11 b showed an increasing pattern of expression after NP exposure ( Fig. 2 B). However, thispattern was more apparent at day 7 of exposure, where a significant 2-fold increase was observed after NP exposure at 1 and

10 

### l M, compared to control ( Fig. 2 B). The mRNA expression of Cyp17 was similar to the effect seen on 3 b -HSD levels, and exposure to 10 

### l M NP produced a significant reduction in Cyp17 mRNA levels at day 3, compared to control at this time interval ( Fig. 2 C). At day 7 of exposure, no differences in Cyp17 mRNA expression were observed between NP treated and control samples ( Fig. 2 C). The expression levels of Cyp21 in salmon previtellogenic ovary were barely detectable and too low for quantification (data not shown).

3.2. Modulation of insulin-like growth factors gene expressions

Ovarian mRNA levels of IGF-1 showed a similar expression pattern at day 3 and 7 after exposure with NP, although with higher abundance levels at day 7, compared with day 3 ( Fig. 3 A). IGF-1 mRNA increased for samples exposed to 1 and 50 

### l M NP at both Table 1 Primer pair sequences, accession numbers, amplicon size and annealing temperature conditions for genes quantified with real-time PCR.

Target Gene Primer sequence a Amplicon size (nucleotides) Annealing temperature (  C) GenBank Accession
number Forward Reverse

3 b -HSD CAGTGAGCTTCACAGGGTCA CCTCCTTCTTGGTCTTGCTG 206 60 AF468975 Cyp11 b AGGAGGTGGTAGTGGGGG CCCCAGCCATGAGTTCAG 118 62 DQ352841 Cyp17 GGCTGGGTCTACTGCTCTTG GCTTCTGGAAGAGGACATGC 144 60 NM_001124747 Cyp21 AAGAGGTGGCCAGGAGAGAT GTCAGGACCACTCCCTGTGT 157 60 EU246942

IGF-1 ACTGTGCCCCTGTCAAGTCT TTTGTCTTGTCTGGGTGCTG 150 60 EF432852 IGF1-R AGAGACGTGTGTGGCCATTA CACCACGTGATGACAGTTGA 112 60 DQ358691

IGF-2 ATTGCGCTGGCACTTACTCT CTCCACGATACCACGGTTCT 171 60 M95184

P450 AromA GGGCACTGTCTGATGATGTC GGGCTTGAGGAAGAACTCTG 104 60 DQ361037

P450 AromB CTGACCCCTCTGGACACG TCTCGTTGAGAGGCACCC 96 55 DQ361038

P450ssc TGGAGTCCTGCTCAAGAATG TTATGTACTCGGGCCACAAA 141 63 DQ361039

StAR AGGATGGATGGACCACTGAG GTCTCCCATCTGCTCCATGT 163 63 DQ415678

a Sequences are given in the 5 0 –3 0 order. T.M. Kortner et al. / Chemosphere 77 (2009) 989–998 991time intervals (significantly at day 7) reaching a 4-fold induction at day 7 compared to control ( Fig. 3 A). In contrast, treatment with

10 

### l M NP did not produce changes in IGF-1 mRNA ( Fig. 3 A). An almost identical expression pattern was observed for IGF-2 mRNA levels at day 3 of exposure, showing moderate increased levels after exposure to 1 and 50 

### l M NP, while 10 

### l M NP significantly suppressed IGF-2 mRNA expression, compared to control at this time interval ( Fig. 3 B). At day 7, a non-significant increase of IGF2 mRNA expression was observed for samples exposed to 1 

### l M NP, whereas no alterations were observed for samples treated with
10 and 50 

### l M NP at the same time interval ( Fig. 3 B). For the IGF-1 receptor (IGF1-R), while treatment with 1 and 50 

### l M NP showed a moderate increase in expression level, 10 

### l M NP produced decreased mRNA levels, compared to control at day 3 ( Fig. 3 C). No dif

### 0 100 200StAR (%of control)



### C 1 10 50 C 1 10 50 Day 3 Day 7

0 100 200 300P450scc(% of control)



### C 1 10 50 C 1 10 50 Day 3 Day 7

0 150 300 450P450aromA(% of control)



### C 1 10 50 C 1 10 50 Day 3 Day 7

0 120 240 360P450aromB (% of control)



### C 1 10 50 C 1 10 50 Day 3 Day 7 

# B A

D C * * *

* *

* *

*

Fig. 1. Expression of StAR (A), P450scc (B), P450 AromA (C) and P450 AromB (D) mRNA levels in cultured ovary of Atlantic salmon exposed to NP at 0 (control), 1, 10 and 50 l M and sampled at day 3 and 7 of exposure. Messenger RNA (mRNA) levels were quantified using real-time PCR with specific primer pairs. Data are given as mean values expressed as percentage (%) of control ( n = 3) ± standard error of the mean (SEM). Asterisks denote exposure groups that are significantly different from the solvent control ( p < 0.05).



### 0 100 200 300 4003β-HSD (%of control) 

### Day 3 Day 7

0 100 200 300Cyp11β(%of control) 

# B A

* * *

0 100 200 300 

# CCyp17 (%of control)



### * C 1 10 50 C 1 10 50 C 1 10 50 C 1 10 50

C 1 10 50 C 1 10 50 Day 3 Day 7

Day 3 Day 7

Fig. 2. Expression of 3 b -HSD (A), Cyp11 b (B) and Cyp17 (C) mRNA levels in cultured ovary of Atlantic salmon exposed to NP at 0 (solvent control), 1, 10 and 50 l M and sampled at day 3 and 7 of exposure. Messenger RNA (mRNA) levels were quantified using real-time PCR with specific primer pairs. Data are given as mean values expressed as percentage (%) of control ( n = 3) ± standard error of the mean (SEM). Asterisks denote exposure groups that are significantly different from the solvent control ( p < 0.05). 992 T.M. Kortner et al. / Chemosphere 77 (2009) 989–998ferences in IGF-R mRNA expression were observed between NP treated and control samples at day 7 of exposure ( Fig. 3 C).

3.3. Modulation of steroid hormone levels

Levels of the steroid hormones (E2, T and 11-KT) extracted from cultivated salmon ovarian tissues were differentially modulated after NP exposure. In general, a similar pattern of NP modulation was observed for the androgens (T and 11-KT), whereas a different pattern of effect was observed for the estrogen (E2) ( Fig. 4 ). Tissue levels of E2 were significantly increased after exposure to 10 

### l M NP for 3 and 7 d, while 1 and 50 

### l M NP did not produce changes in tissue E2 levels during these time intervals, compared to the control ( Fig. 4A). NP produced an apparent concentration-dependent increase of T levels at both 3 and 7 d of exposure ( Fig. 4 B). At day 3, the T level was slightly below control levels at 1 

### l M NPexposure, followed by an apparent concentration-dependent increase that was significantly different from control at 50 

### l M NP ( Fig. 4 B). At day 7, significantly higher tissue T levels were observed for all NP concentrations ( Fig. 4 B). For 11-KT, tissue samples exposed for 3 d to 1 and 10 

### l M NP showed significantly decreased levels of 11-KT, compared to control, while 50 

### l M NP restored 11- KT levels back to control levels ( Fig. 4 C). At day 7, NP produced significant increases of tissue 11-KT levels, compared to control ( Fig. 4 C). No significant differences in 11-KT levels were observed between different NP concentrations at day 7 ( Fig. 4 C).

3.4. Ovarian histology and cellular detection of StAR and P450scc proteins

Histological evaluations showed that all gonadal tissues cultivated for 3 and 7 d, either in the presence or absence of NP concentrations displayed normal histology, comprising mainly previtellogenic oocytes with a diameter of approximately

150 

### l m. A representative cellular localization of the StAR and

P450scc proteins in cultured ovary sampled at day 7 in control and after exposure to 50 

### l M NP is shown in Fig. 5 and a subjective evaluation of protein staining intensity pattern for all exposure
conditions is shown in Table 2 . Using rabbit polyclonal antisera prepared against synthetic peptide sequences, the tissue detection
of the StAR and P450scc were mainly observed in follicular cells of the oocyte boundary layer in both control and NP exposed tissues. Qualitative immunohistochemical staining intensities showed that NP exposure produced increased StAR and P450scc protein stainings in follicular cells of oocyte membranes, compared to control ( Fig. 5 ).

4. Discussion

It has been established that a large number of anthropogenicchemicals released into the environment may disrupt the endocrine homeostasis in wildlife species, leading to hormonal imbalance with potential consequences for general health, fecundity and reproduction. Endocrine toxicology research on xenoestrogens has so far focused mainly on responses mediated through the ER. In this study, we present data showing that ovarian steroidogenesismay be susceptible of harmful effects of EDCs such as NP. By comparing NP effects at the transcriptional level with effects observed on steroid hormone production and ovarian histology, we may gain further insight into the complex interactions between transcriptchanges, chemical mode of actions and overall organismal reproduction and health consequences.

4.1. Modulation of steroidogenic gene expressions

In mammals, StAR and P450scc are rapidly synthesized in response to acute tropic hormone stimulation and cAMP, and agentsthat increase steroid biosynthesis also increase StAR mRNA expression ( Stocco 2001 ). Current knowledge about StAR is mostly based on mammalian studies, whereas the relationship between StAR gene expression and steroid production in fish is not well described ( Stocco et al., 2005; Hagen et al., 2006; Hecker et al., 2006; Villeneuve et al., 2007a,b ). Indeed, StAR expression data suggest that the molecular mechanisms of StAR synthesis and regulation in fish 0 100 200 300 400 500

0 100 200 300

0 50 100 150 200 

# A B

CIGF-1 (%of control)IGF-2 (%of control)IGF1-R (% of control) 

### * *

*

* 

### Day 3 Day 7 Day 3 Day 7

Day 3 Day 7 C 1 10 50 C 1 10 50 C 1 10 50 C 1 10 50

C 1 10 50 C 1 10 50

Fig. 3. Expression of IGF-1 (A), IGF-2 (B) and IGF1-R (C) mRNA levels in cultured ovary of Atlantic salmon exposed to NP at 0 (solvent control), 1, 10 and 50 l M and sampled at day 3 and 7 of exposure. Messenger RNA (mRNA) levels were quantified using real-time PCR with specific primer pairs. Data are given as mean values expressed as percentage (%) of control ( n = 3) ± standard error of the mean (SEM). Asterisks denote exposure groups that are significantly different from the solvent control ( p < 0.05). T.M. Kortner et al. / Chemosphere 77 (2009) 989–998 993may differ from what has been previously observed in mammals ( Goetz et al., 2004 ). Herein, we showed that ovarian StAR and

P450scc mRNA expressions were modulated by NP exposure in a time- and concentration-specific manner. Interestingly, 10 

### l M NP
decreased StAR and P450scc mRNA levels at day 3 (no effect was observed with 1 and 50 

### l M NP at the same time interval), whereas the opposite effect was observed on P450 Arom isoforms expression. At day 7 of exposure, NP produced an apparent concentration-dependent decrease and increase in StAR and P450scc mRNA expression, respectively. 0 15 30 45

0 1 2 3 4 5

0.0 1.5 3.0 4.5 

# A B

CE2 (pg/ml)T (pg/ml)11-KT (pg/ml) 

### * * *

* * *

* * * * * 

### Day 3 Day 7 Day 3 Day 7

Day 3 Day 7 C 1 10 50 C 1 10 50 C 1 10 50 C 1 10 50

C 1 10 50 C 1 10 50

Fig. 4. Tissue levels of estradiol-17 b (E2; A), testosterone (T; B) and 11-ketotestosterone (11-KT; C) in Atlantic salmon ovarian tissue exposed to NP at 0 (control), 1, 10 and

50 l M and sampled at day 3 and 7 of exposure. Steroid hormone levels were measured by EIA. Data are given as mean values and expressed as pg mL

1 ( n = 3) ± standard error of the mean (SEM). Asterisks denote exposure groups that are significantly different from the solvent control ( p < 0.05).

Fig. 5. Cellular localization of the StAR and P450scc proteins in Atlantic salmon ovary exposed to NP at 0 (control, panel A and B) and 50 l M (panel C: StAR protein, and D:

P450scc ), respectively, and sampled at day 7 of exposure. The figures are representative histological and immunostained tissue sections for the study period, as there were consistent differences between the control and exposure groups, at all sampling days. Positive protein staining was observed mainly in follicular cells of the oocyte boundary. Bar equals 50 l m. Abbreviations: cyt: cytoplasma, fc: follicular cells, nl: nucleoli. nu: nucleus. 994 T.M. Kortner et al. / Chemosphere 77 (2009) 989–998The unique time (see day 3 and 7) and concentration differences
in NP effects on the StAR and P450scc mRNA patterns observed in the present is interesting and are in accordance with previous observations from our laboratory and elsewhere ( Hecker et al.,2006; Meucci and Arukwe 2006; Lyssimachou et al., 2006; Villeneuve et al., 2007a,b; Vang et al., 2007; Arukwe, 2008 ). The mechanisms underlying the chemical modulations of StAR and P450scc in steroidal tissues are not fully understood. Previous studies have shown that ACTH signal transduction involves cAMP ( Quabius et al., 1997 ), in addition to cAMP-dependent protein kinases A and C ( Leblond and Hontela 1999 ). The effects of endocrine-disrupting chemicals (EDCs) may also be interpreted as alterationsin steroidogenesis and steroidal regulation of the normal development and function of the male and female reproductive tracts, in addition to their effects as direct ER agonists ( Villeneuve et al., 2007a,b ) and these responses are mostly regulated through feedback mechanisms. In this regard, the impairment of steroidogenic enzyme activity and/or protein expressions is one potentially important mechanism or mode-of action (MOA). For example, the possibility that endogenous estrogens such as 17 b -estradiol (E2) play a role in stimulating the delivery of cholesterol to its siteof cleavage on the matrix side of the inner mitochondrial membrane, where the P450scc complex is located, was previously proposed by Holt (1989) . Despite their integral role(s) in developmental and reproductive processes, little is known aboutthe effects and mechanisms of EDCs on gonadal, neural and interrenal steroids. In teleosts, gonadal steroids are known to modulate both the synthesis and release of gonadotropins by the pituitary, and to influence several brain functions that are apparently responsible for gender-specific differences in the regulation of the hypothalamus-pituitary-gonadal (HPG) axis and dependent on the integrity of the reproductive systems. Although the StAR
and P450scc are the main proteins involved in the early steroidogenic pathway, it is possible that several other integral aspects of the entire hormonal signal transduction systems are involved in their regulation, independent of contaminant exposure. The protein expression pattern for the StAR and P450scc did not parallel mRNA levels in the present study. Despite that the protein analysis were less quantitative, compared with the real-time PCR method, there are several aspects of the StAR protein chemistry that may explain these differences. Firstly, the StAR protein and

P450scc are synthesized rapidly in response to acute tropic hormone stimulation, and this happens regardless of steroidogenic organ or tissue. Secondly, it was previously demonstrated in early mammalian studies that the StAR protein is a 30-kDa protein that is first synthesised in the cytosol as a 37-kDa precursor in response to the activation of cAMP protein kinase-A intracellular signalling pathways ( Artemenko et al., 2001 ). Thirdly, newly synthesised StAR was recently shown to be an effective mediator of cholesteroltransfer protein, which is often present in low levels with an estimated very high effectivity in excess of 400 cholesterol molecules for each StAR molecule ( Artemenko et al., 2001 ). Lastly, ovarian StAR was shown by Kusakabe et al. (2002) to peak in associationwith oocyte maturation and ovulation, coinciding with the production peaks for maturational steroid hormones in brook trout ( Salvelinus fontinalis ). As of these biological aspects of the StAR may contribute to the discrepancy between mRNA and protein levels observed in the present study.
For the P450 Arom isoforms transcripts, a general increasing pattern after NP exposure was observed, with 10 

### l M NP producing the most pronounced effect at day 3 of exposure. The increased levels
of P450 Arom mRNA are in compliance with the increased cellular E2 levels (see below) and previous studies assessing NP effects in juvenile salmon ( Meucci and Arukwe 2006 ). However, given that
StAR and P450scc regulate steroidogenic processes upstream of

P450 Arom, it was unexpected that these transcripts were regulated
in an inverse manner relative to P450 Arom . In addition, a similardecrease was observed for other steroidogenesis-related transcripts, such as 3 b -HSD and Cyp17. A potential explanation for the observed decrease in transcript abundance for genes upstream
of P450 Arom would be that teleost fish may have different mechanisms of transcriptional regulations for these genes, that may involve feedback signalling to the pituitary, or even short feedback loops within the organ itself. However, based on the data from thisstudy alone, we can not fully explain this potentially important aspect of NP-impaired steroidogenesis in teleosts. Recently, the effects of (xeno)estrogens on P450 Arom gene expression seem to differ depending on developmental stage, cellular context, species and chemicals, and controlled by different promoters and second messenger pathways that provide various molecular and cellular targets for xenobiotic interactions ( Filby et al., 2006 : Cheshenko et al., 2008 ). Cyp11 b is a multifunctional enzyme in the steroid hormone biosynthetic pathways, converting 11-deoxycortisol to cortisol and deoxycorticosterone to corticosterone in steroidogenic tissues ( Miller 2005 ). Additionally, Cyp11 b is the main enzyme in the biosynthesis of the male potent androgen, 11-ketotestosterone (11- KT), which appears to control many aspects of spermatogenesis ( Kusakabe et al., 2002 ). The non-aromatizable androgen 11-KT has traditionally been regarded as male-specific hormone ( Borg

1994 ). Recent studies have shown that significant amounts of 11- KT circulate in the blood of female teleosts ( Lokman et al., 2002 ). Furthermore, Cyp11 b has been localized in the ovary of the honeycomb grouper ( Epinephelus merra ) ( Alam et al., 2005 ). Very recent studies from our laboratory have shown that 11-KT can induce growth and development of previtellogenic oocytes in Atlantic cod ( Kortner et al., 2008 ). Taken together, these reports suggestthat non-aromatizable androgens such as 11-KT may play significant roles in the regulation of early previtellogenic oocyte growth and development in teleost fish. The present study demonstrates significant Cyp11 b mRNA levels in previtellogenic Atlantic salmon ovarian tissues, indicating that gonads of immature salmon havethe potential to produce 11-KT (see below). Additionally, the significant increases of Cyp11 b mRNA levels after NP exposure suggest that xenoestrogens can impair this step in the steroidogenicpathway, potentially resulting in an abnormal gonadal development which may have severe consequences for the entire reproductive potential.

4.2. Modulation of the insulin-like growth factor pathway

Evidence from a variety of fish species indicate that the IGF system may play important roles in regulating ovarian development ( Kagawa et al., 1994; Negatu et al., 1998; Weber and Sullivan 2000; Weber and Sullivan 2005 ), and ovarian steroidogenesis can be stimulated by IGF-1 ( Maestro et al., 1997; Kagawa et al.,

2003 ). It has been shown that IGF-1 can promote growth and developmen t of previtellogenic oocytes ( Campbell et al., 2006; Lokman et al., 2007 ). In mammals, sex steroids are known to affect the IGF system, but information regarding a similar interaction in Table 2 Subjective summary of immunohistochemical protein expression patterns of the StAR
and P450scc in follicular cells of salmon previtellogenic oocytes exposed in vitro to nonylphenol (NP).

NP ( l M) Day 3 Day 7

Control 1 10 50 Control 1 10 50

StAR + ++ ++ +++ + ++ ++ +++

P450scc ++ ++ +++ +++ + ++ +++ +++

+++ strong positive reaction; ++ moderate positive reaction; + weak reaction; - no

reaction. T.M. Kortner et al. / Chemosphere 77 (2009) 989–998 995fish is limited. It has been suggested that the ovarian IGF system may be regulated by sex hormones related to reproductive phase ( Gioacchini et al., 2005 ), thus rendering the IGF pathways susceptible to chemical alterations. In the present study, we observed a general decrease in mRNA expression for IGF-1, IGF-2 and IGF1-R
after 10 

### l M NP exposure at day 3. This pattern was inversely correlated with E2 levels, but correlated positively with 11-KT levels. Interestingly, this is the opposite of what was typically observed in mammals, as E2 is believed to control IGF-1 production. Generally,when all these differential responses are put into a broader context, the relationship between the StAR protein, P450scc, IGF system, 3 b -HSD and 11-KT at day 3 of exposure to NP represents a potentially novel aspect supporting our earlier hypothesis on the role of androgens on the growth of previtellogenic oocytes that may be susceptible to chemical disruption. These observations are consistent with our recent data showing that aromatizable (T) and non-aromatizable (11-KT) androgens produce differential gene expression patterns, whose functional products (proteins) modulate previtellogenic oocyte growth in cod ( Kortner et al.,

2008 ). The role of androgens in previtellogenic oocyte development is supported by the observation that the early ovarian growth of species-specific critical stage progressed in hypophysectomized freshwater turtle ( Chrysemys picta ), indicating that gonadotropins (GtHs) were not necessary ( Ho et al., 1982 ). It has also been demonstrated that 11-KT induced an increase in the growth of previtellogenic oocytes in eel ( Rohr et al., 2001; Lokman et al., 2007 ) and cod ( Kortner et al., 2008 ). In mammals, androgens also modify the intra-ovarian gene expression as demonstrated by increased mRNA abundance of IGF-1 and IGF1-R in rhesus monkey ovarian follicles up to early antral stage ( Vendola et al., 1999 ). Given that this happens despite the diversity in follicular development inthese species, the findings in the present study suggest a generalized effect of EDCs in vertebrates, at least with the methods applied in our studies. The effects of EDCs on early follicular growth in fish form a fundamental basis for the understanding of the roles/effects of xenoandrogens on early oocytes growth and fecundity through modification of gene expression patterns for growth factors and steroidogenesis. These are still subject of systematic investigations in our laboratory.

4.3. Modulation of ovarian steroid hormone levels

E2 is the main estrogen secreted by the ovary and is synthesizedfrom testosterone via an aromatization step catalyzed by the cytochrome P450 aromatase enzyme. In this study, cellular E2 levels
were elevated after 10 

### l M NP exposure. These results paralleled
the P450 Arom isoforms mRNA expression at day 3 of exposure,whereas they inversely paralleled the expression of other steroidogenic protein and enzyme genes (StAR, P450scc, 3 b -HSD andCyp17). Furthermore, the inversely paralleled levels of IGF-1, IGF2 and IGF1-R are interesting, suggesting that these growth factors may be susceptible to endocrine disruption, affecting ovarian development.The androgen (T and 11-KT) levels showed an apparent concentration-dependent increase when exposed to NP, suggesting thatNP may potentially modulate steroidogenesis by targeting the initial and rate-limiting step that involves the StAR protein and is supported by the parallel effects seen in StAR and P450scc expressions. Recently in our laboratory, it was reported that NP reduced E2 and 11-KT levels in Atlantic cod ( Gadus morhua ) previtellogenic oocyte cultures exposed to 50 and 100 

### l M NP after 14 d of cultivation, and that these effects paralleled StAR and P450scc mRNA levels ( Kortner and Arukwe 2007b ). Furthermore, the presence of significant 11-KT levels in Atlantic salmon previtellogenic ovarian tissue is intriguing (see discussion above). Earlier studies have alsoshown that 11-KT can promote growth and development of previtellogenic oocytes in the Australian eel ( Auguilla australis ) ( Rohr et al., 2001; Lokman et al., 2007 ). This phenomenon was also observed very recently in our laboratory, using an Atlantic cod previtellogenic oocyte culture system ( Kortner et al., 2008 ). Interestingly, we reported that transcription levels of StAR and

P450scc were modulated after androgen exposure, further indicating the importance of steroidogenic genes and protein in early ovarian development in teleosts ( Harris et al., 2001; Kortner et al., 2008 ). The present study adds new information on the effects of androgens in immature female fish, demonstrating the presence of both 11-KT and Cyp11 b mRNA levels in the salmon previtellogenic ovary with apparently high susceptibility to EDCs. Additionally, we show that NP can modulate these potentially importantmolecular actors of early oocyte growth in teleost fish, with potentially narrow window of response.

5. Conclusions

The findings in the present study indicate that NP can produce variations in key gonadal steroidogenic and growth regulatingpathways that may lead to hormonal imbalance, affecting reproduction and/or overt fecundity. Given that steroidogenesis involves more than androgen to estrogen conversion or direct ER effects,studies on other steroidogenic pathways are of integral physiological significance, as has been demonstrated in the present studyand recent studies from our laboratory and elsewhere. An overwhelming number of adverse effects, including decreased fertility in wildlife species and mammals and decreased hatching successin fish, birds, and reptiles have been purportedly attributed to environmental contaminant exposure, particularly those with endocrine-disrupting potentials ( Colborn et al., 1993; Arukwe and Goksøyr 1998; Rotchell and Ostrander 2003; Fairbrother et al.,

2004 ). When the findings of this in vitro study are extrapolated to the whole organismal level, it is possible that NP may alter the entire negative feedback control of steroid hormone synthesis. This involves GtHs and their receptors in the brain ( Bieniarz and Epler

1992 ), making the entire HPGL axis susceptible to chemical impact ( Hutchinson and Pickford 2002; Yadetie and Male 2002 ) with serious health, developmental and reproductive consequences.

Acknowledgement

This study was financed by the Norwegian Research Council (NFR) Contract Number 165073/V40.

References

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