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 the

production 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 lM) dissolved in ethanol (0.1%). The mRNA expressions of steroidogenic acute regulatory (StAR) protein, P450-mediated cholesterol side-chain cleavage (P450scc), aromatase isoforms, 3b-hydroxysteroid

dehydrogenase (3b-HSD), Cyp11b-, 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-17b

(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 lM NP exposure at day 3 paralleled P450Arom isoforms mRNA expression at the same time interval. The expression patterns of other steroidogenic protein and enzyme genes, such as StAR, P450scc, 3bHSD and Cyp17 inversely paralleled this pattern, displaying consistent decreased transcript levels. These

findings 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 relevant

biochemical 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 wildlife

species 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 are

believed 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 L1 total NP has

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

180 lg L1 have been detected in rivers in the United Kingdom

(UK; Blackburn and Waldock, 1995), 60 lg L1 in rivers in Switzerland (Ahel et al., 1994) and 17 lg L1 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 L1 in river waters and 1 mg kg1 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/chemosphere
aquatic 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 estrogen

receptor (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 and

proteins 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 brain

functions 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 (P450arom) 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 17a-hydroxylase/17,20 lyase (Cyp17), 3b-hydroxysteroid dehydrogenase (3bHSD), and 21- and 11b-hydroxylases (Cyp21 and Cyp11b) 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 have

not 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 previtellogenic

ovarian tissues to NP would produce differential expression patterns of steroidogenesis- and growth factor-related genes, and

alterations 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 RNA

purification, 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-N0

-[2-ethanesulfonic acid] (Hepes) were purchased from Sigma Chemical (St. Louis, MO, USA). Iscript cDNA Synthesis Kit and

iTaqTMSybr Green supermix with ROX were purchased from BioRad laboratories (Hercules, CA, USA) and GenerulerTM 100 bp DNA

ladder and dNTPs from Fermentas GmbH (Germany). Estradiol17b, testosterone and 11-ketotestosterone enzyme immunoassay

(EIA) kits were purchased from Cayman chemical company (Ann

Arbor, 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 the

department 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 live

animals 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 lM) in a humidified

incubator at 10 C. The control group received ethanol (carrier

vehicle) and the final concentration of ethanol in all exposure

groups 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, embedded

and 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 to

standard 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 random

primers 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 3b-HSD, P450aromA and

B, Cyp11b, 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-ll DNA amplification reaction contained

12.5 ll of iTAQTMSYBR Green Supermix with ROX (Bio-Rad), 1 ll of

cDNA, 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) 989–998
standard plots of Ct versus log copy number. The criterion for using

the standard curve is based on equal amplification efficiency (usually >90%) with unknown samples and this is checked prior to

extrapolating unknown samples to the standard curve. The standard plots were generated for each target sequence using known

amounts 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-17b (E2), testosterone (T) and 11-ketotestosterone (11-KT) were measured by EIA.

Ovarian tissues of equal sizes were thawed on ice, homogenized

in 500 ll of 0.1 M sodium phosphate buffer and centrifuged at

14,000g for 15 min. The supernatants were purified by extraction

using 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. Enzyme

immunoassay 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 mL1

, T; 6 pg mL1 and 11-KT; 1.3 pg mL1. 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 groups

were 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 lM NP, while 1 and 50 lM NP did not produce significant changes, compared to control at this time interval (Fig. 1A).

At day 7 of exposure, an apparent concentration-dependent

decreasing trend of StAR mRNA was observed, albeit not significant

(Fig. 1A). 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 lM NP

(Fig. 1B). In contrast, an apparent concentration-dependent increase (significant at 50 lM) in P450scc mRNA expression was observed after NP treatment at day 7 (Fig. 1B).

The expression of P450AromA (Fig. 1C) and P450AromB (Fig. 1D)

showed similar modulations patterns after NP exposure at day 3,

displaying increased levels after exposure to the two highest NP

concentrations (10 and 50 lM). At day 7, P450AromA mRNA levels

were slightly elevated in all NP exposure groups, compared to control level, albeit not significantly (Fig. 1C). For P450AromB, a significant increase of mRNA levels was observed for samples treated

with 1 and 50 lM NP, while 10 lM NP showed no effect compared

to control (Fig. 1D). The mRNA levels of 3b-HSD were modulated

after NP exposure (Fig. 2A). For example, 1 and 50 lM NP produced

increased 3b-HSD mRNA levels at day 3 of exposure, while 10 lM

NP decreased non-significantly, compared to control at this time

interval (Fig. 2A). At day 7, a non-significant increasing pattern

after NP exposure was observed for 3b-HSD mRNA, compared to

control (Fig. 2A). The expression of Cyp11b showed an increasing

pattern of expression after NP exposure (Fig. 2B). However, this

pattern was more apparent at day 7 of exposure, where a significant 2-fold increase was observed after NP exposure at 1 and

10 lM, compared to control (Fig. 2B). The mRNA expression of

Cyp17 was similar to the effect seen on 3b-HSD levels, and exposure to 10 lM NP produced a significant reduction in Cyp17 mRNA

levels at day 3, compared to control at this time interval (Fig. 2C).

At day 7 of exposure, no differences in Cyp17 mRNA expression

were observed between NP treated and control samples (Fig. 2C).

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. 3A). IGF-1

mRNA increased for samples exposed to 1 and 50 lM 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 sequencea Amplicon size

(nucleotides)

Annealing

temperature (C)

GenBank Accession

number Forward Reverse

3b-HSD CAGTGAGCTTCACAGGGTCA CCTCCTTCTTGGTCTTGCTG 206 60 AF468975

Cyp11b 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

P450AromA GGGCACTGTCTGATGATGTC GGGCTTGAGGAAGAACTCTG 104 60 DQ361037

P450AromB CTGACCCCTCTGGACACG TCTCGTTGAGAGGCACCC 96 55 DQ361038

P450ssc TGGAGTCCTGCTCAAGAATG TTATGTACTCGGGCCACAAA 141 63 DQ361039

StAR AGGATGGATGGACCACTGAG GTCTCCCATCTGCTCCATGT 163 63 DQ415678

a Sequences are given in the 50

–30order.

T.M. Kortner et al. / Chemosphere 77 (2009) 989–998 991
time intervals (significantly at day 7) reaching a 4-fold induction at

day 7 compared to control (Fig. 3A). In contrast, treatment with

10 lM NP did not produce changes in IGF-1 mRNA (Fig. 3A). 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 lM NP, while 10 lM NP significantly

suppressed IGF-2 mRNA expression, compared to control at this

time interval (Fig. 3B). At day 7, a non-significant increase of IGF2 mRNA expression was observed for samples exposed to 1 lM

NP, whereas no alterations were observed for samples treated with

10 and 50 lM NP at the same time interval (Fig. 3B). For the IGF-1

receptor (IGF1-R), while treatment with 1 and 50 lM NP showed a

moderate increase in expression level, 10 lM NP produced decreased mRNA levels, compared to control at day 3 (Fig. 3C). No dif0

100

200

StAR (%of control)

C 1 10 50 C 1 10 50

Day 3 Day 7

0

100

200

300

P450scc (% of control)

C 1 10 50 C 1 10 50

Day 3 Day 7

0

150

300

450

P450aromA (% of control)

C 1 10 50 C 1 10 50

Day 3 Day 7

0

120

240

360

P450aromB (% of control)

C 1 10 50 C 1 10 50

Day 3 Day 7

A B

C D

* *

*

* *

*

*

*

Fig. 1. Expression of StAR (A), P450scc (B), P450AromA (C) and P450AromB (D) mRNA levels in cultured ovary of Atlantic salmon exposed to NP at 0 (control), 1, 10 and 50 lM

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

400

3β-HSD (%of control)

Day 3 Day 7

0

100

200

300

Cyp11β (%of control)

A B

*

* *

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 3b-HSD (A), Cyp11b (B) and Cyp17 (C) mRNA levels in cultured ovary of Atlantic salmon exposed to NP at 0 (solvent control), 1, 10 and 50 lM 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) 989–998
ferences in IGF-R mRNA expression were observed between NP

treated and control samples at day 7 of exposure (Fig. 3C).

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 lM

NP for 3 and 7 d, while 1 and 50 lM 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. 4B).

At day 3, the T level was slightly below control levels at 1 lM NP

exposure, followed by an apparent concentration-dependent increase that was significantly different from control at 50 lM NP

(Fig. 4B). At day 7, significantly higher tissue T levels were observed for all NP concentrations (Fig. 4B). For 11-KT, tissue samples

exposed for 3 d to 1 and 10 lM NP showed significantly decreased

levels of 11-KT, compared to control, while 50 lM NP restored 11-

KT levels back to control levels (Fig. 4C). At day 7, NP produced significant increases of tissue 11-KT levels, compared to control

(Fig. 4C). No significant differences in 11-KT levels were observed

between different NP concentrations at day 7 (Fig. 4C).

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 lm. A representative cellular localization of the StAR and

P450scc proteins in cultured ovary sampled at day 7 in control

and after exposure to 50 lM 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 anthropogenic

chemicals 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 steroidogenesis

may 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 transcript

changes, 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 agents

that 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

C

IGF-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 lM 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) 989–998 993
may 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 lM NP

decreased StAR and P450scc mRNA levels at day 3 (no effect was

observed with 1 and 50 lM NP at the same time interval), whereas

the opposite effect was observed on P450Arom 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

C

E2 (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-17b (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 lM 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 mL1 (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 lM (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 lm. Abbreviations: cyt: cytoplasma, fc: follicular cells, nl: nucleoli. nu: nucleus.

994 T.M. Kortner et al. / Chemosphere 77 (2009) 989–998
The 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 alterations

in 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 17b-estradiol

(E2) play a role in stimulating the delivery of cholesterol to its site

of 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 about

the 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 cholesterol

transfer 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 association

with 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 P450Arom isoforms transcripts, a general increasing pattern after NP exposure was observed, with 10 lM NP producing the

most pronounced effect at day 3 of exposure. The increased levels

of P450Arom 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

P450Arom, it was unexpected that these transcripts were regulated

in an inverse manner relative to P450Arom. In addition, a similar

decrease was observed for other steroidogenesis-related transcripts, such as 3b-HSD and Cyp17. A potential explanation for

the observed decrease in transcript abundance for genes upstream

of P450Arom 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 this

study alone, we can not fully explain this potentially important aspect of NP-impaired steroidogenesis in teleosts. Recently, the effects of (xeno)estrogens on P450Arom 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).

Cyp11b 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, Cyp11b 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, Cyp11b 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 suggest

that 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 Cyp11b mRNA levels in previtellogenic Atlantic salmon

ovarian tissues, indicating that gonads of immature salmon have

the potential to produce 11-KT (see below). Additionally, the significant increases of Cyp11b mRNA levels after NP exposure suggest that xenoestrogens can impair this step in the steroidogenic

pathway, 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

development 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 (lM) 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) 989–998 995
fish 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 lM 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, 3b-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 in

these 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 synthesized

from testosterone via an aromatization step catalyzed by the cytochrome P450 aromatase enzyme. In this study, cellular E2 levels

were elevated after 10 lM NP exposure. These results paralleled

the P450Arom isoforms mRNA expression at day 3 of exposure,

whereas they inversely paralleled the expression of other steroidogenic protein and enzyme genes (StAR, P450scc, 3b-HSD and

Cyp17). 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 that

NP 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 lM 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 also

shown 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 Cyp11b mRNA levels in the salmon previtellogenic ovary with apparently high susceptibility to EDCs. Additionally, we show that NP can modulate these potentially important

molecular 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 regulating

pathways 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 study

and recent studies from our laboratory and elsewhere. An overwhelming number of adverse effects, including decreased fertility

in wildlife species and mammals and decreased hatching success

in 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.

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