Cloning and developmental expression of the …web.macam.ac.il/~gad100/AAA CV Gad 2010...

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General and Comparative Endocrinology 138 (2004) 271–280

GENERAL AND COMPARATIVE

ENDOCRINOLOGY

Communication in Genomics and Proteomics

Cloning and developmental expression of the cytochrome P450aromatase gene (CYP19) in the European eel (Anguilla anguilla)

Itai Tzchoria,b,*, Gad Deganib,c, Avshalom Hurvitzb, Boaz Moava

a Department of Zoology, Tel Aviv University, Tel Aviv 69978, Israelb MIGAL, Galilee Technological Center, Kiryat Shmona 10200, Israelc School of Science and Technology, Academic Tel-Hai College, Israel

Received 23 February 2004; revised 7 June 2004; accepted 21 June 2004

Available online 13 August 2004

Abstract

To characterize the involvement of the aromatase gene during the process of sex determination in the European eel (Anguilla

anguilla), the expression of its gonadal form was determined during various developmental stages. The cloned cDNA from the Euro-

pean eel gonad (EeCYP19) contains an open reading frame of 1539bp, encoding a deduced protein of 513 residues. The predicted

amino acid sequence shows 97% identity with that of the Japanese eel, and 59–69% of identity with those of the CYP19 gonadal and

brain forms of other teleost fish. Two potential initiation sites (ATG) were found downstream of the first ATG codon. A fluorescent-

based method of real-time PCR was developed to quantify EeCYP19 expression. The expression levels of EeCYP19 in the gonads of

adult males were approximately 12- and 30-fold lower than the levels in adult females and juvenile eels previously treated with E2,

respectively. Expression of aromatase was found only in a single specimen in the control group. In contrast, no difference was found

among sexes in the aromatase expression in the brain. Treatment with aromatase inhibitor (AI) of juvenile eel resulted in the total

loss of aromatase expression in the gonads and brains. The results of this work revealed that AI treatment not only reduces the

synthesis of estradiol, but reduces the expression levels of EeCYP19 as well. No evidence for the presence of a distinct extra-gonadal

(brain) form of aromatase in the European eel could be provided.

� 2004 Elsevier Inc. All rights reserved.

Keywords: European eel; Sex determination; Estradiol-17b; CYP19; Expression; Aromatase inhibitor; Real-time PCR

1. Introduction

Steroids play an important role in gonadal differenti-

ation in lower vertebrates (Bogart, 1987; Devlin and

Nagahama, 2002; Yamamoto, 1969). Treating fish with

exogenous sex steroid hormones during early develop-

mental stages may cause sex-inversion in spite of

the genotypic sex. It has been demonstrated in manyfish species that exogenous androgens generally have a

masculinizing effect, while exogenous estrogens have

0016-6480/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygcen.2004.06.007

* Corresponding author. Present address: Bldg 6B, LMGD/

NICHD, National Institutes of Health, Bethesda, MD, USA. Fax:

+301-402-0543.

E-mail address: [email protected] (I. Tzchori).

feminizing effects (Yamazaki, 1983). However, the

mechanism of action of these steroids on sex differentia-

tion is not well understood.

Estrogens are C-18 steroids indispensable for normal

sexual development of the female, but have been associ-

ated with sexual function in male too (Devlin and Naga-

hama, 2002). The synthesis of estrogens is dependent on

the activity of the steroidogenic enzyme P450 aromatase(CYP19). CYP19 catalyses aromatization of the A ring

of C19 steroids to form the phenolic ring characteristic

of estrogens, with concomitant loss of the C19 angular

methyl group as formic acid. CYP19 activity is an

important modulator of E2 concentration and is critical

to the regulation processes controlled by E2 (Brodie,

1991).

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272 I. Tzchori et al. / General and Comparative Endocrinology 138 (2004) 271–280

In the mammalian brain, the local formation of estro-

gens has been shown to influence the sexual differentia-

tion of neural structure and to modulate neuroendocrine

function and sexual behavior (Lephart, 1996). In lower

vertebrates, such as fish, CYP19 is predominantly ex-

pressed in the gonad and in the brain. It has becomepossible to reduce estrogen biosynthesis by inhibiting

the activity of aromatase, by using nonsteroidal aroma-

tase inhibitors such as fadrozole (CGS 16949A; Steele

et al., 1987). Fadrozole is a reversible competitive inhib-

itor, whereby both substrate and inhibitor compete for

the same site on the enzyme (Steele et al., 1987). In adult

mammalian and non-mammalian vertebrates, disrup-

tion of CYP19 was also shown to impair gonadal devel-opment and alter hormone synthesis (Afonso et al.,

1999). Treatments with aromatase inhibitors during sex-

ual differentiation resulted in skewed sex ratio in mam-

mals (Schieweck et al., 1988; Steele et al., 1987),

reptiles (Wibbles and Crews, 1994), and fish (Afonso

et al., 1999; Guiguen et al., 1999; Ijiri et al., 2003; Kwon

et al., 2000; Piferrer et al., 1994).

The cDNAs encoding CYP19 have been character-ized in a range of species, including mammals (Chen

et al., 1988; Corbin et al., 1988) and birds (McPhaul

et al., 1988). Among teleost fish, the CYP19 cDNA

has been cloned from the ovary of rainbow trout (Tana-

ka et al., 1992), catfish (Trant, 1994), medaka (Fukada

et al., 1996), Nile tilapia (Chang et al., 1997), goldfish

(Gelinas et al., 1998; Tchoudakova and Callard, 1998),

zebrafish (Kishida and Callard, 2001), European seabass (Dalla Valle et al., 2002), and Japanese eel (Ijiri

et al., 2003). Interestingly, teleost fish express two sub-

types of CYP19 cDNA. The isozyme, CYP19B, has

been isolated from the brain of goldfish (Gelinas et al.,

1998), tilapia (Cruz and Canario, 2000; Tsai et al.,

2001), zebrafish (Kishida and Callard, 2001), fathead

minnow (Halm et al., 2001), and trout (Dalla Valle

et al., 2002).European eels show considerable differences of sexual

dimorphism; females gain five times and more body

weight than males. Sex ratios of eel are highly variable

according to environmental sites (D�Ancona, 1959;

Tesch, 1977), and under aquaculture conditions, the

majority of eels differentiate into males (Holmgren and

Mosegaard, 1996). The skewed sex ratio demonstrates

a strong influence of the environment on the processof sex determination in eel. The eel is therefore a good

candidate for the evaluation of aromatase role during

the labile period in a fish with a strong environment-de-

pendent sex determination mechanism.

To evaluate the importance of aromatase expression

during the labile period of sex determination, we iso-

lated and characterized the full length cDNA encoding

CYP19 in European eel. In addition, CYP19 mRNAlevels in the gonads and brains were analyzed, and cor-

related with sex and developmental stages. The existence

of a distinct CYP19 gene in the eel�s brain was examined

as well.

2. Materials and methods

2.1. Animals and treatment

European glass eels (Anguilla anguilla) caught during

their migration in February–March 1999 and April 2000

were purchased from UK (UK Glass). For dry-feed

training they were fed approximately 1 month with com-

mercial eel diet (Trovit, Hendrix, Italy: protein 42.5%,

fats 8%, and ash 11.5%) combined with decreasingamounts of cod raw. Elvers were kept at 23–26 �C, with2–3 changes of total water volume daily and gentle aer-

ation. The appropriate (see below) amount of aromatase

inhibitor (AI: Fadrozole, CGS16949A, Novartis, Sum-

mit, NJ) or estradiol (E2, Sigma–Aldrich, St. Louis,

MO) was dissolved in 99% ethanol. Food for the control

groups was prepared in the same way using ethanol

only. The food was then thoroughly mixed and left ina fume cupboard overnight to allow ethanol evapora-

tion, and then stored at �20 �C. Thawed portions were

supplied daily to the fish.

Glass eels (0.5g) or elvers (5–7g) were fed 300mg/kg

dry feed AI for 90 days, or 10mg/kg dry feed of E2 for

150 days, or 10mg/kg bw of E2 for 150 days followed by

300mg/kg AI treatment for 90 days. The feed was given

ad libitum twice per day.To increase the percentage of females, untreated glass

eels (0.5g) were kept at a low density (100 fish/tank)

during the labile period in tanks equipped with shelters

(Degani and Kushnirov, 1992). The macroscopic

classification of the gonadal sex (male, females, and

intersexes) was done according to Beullens et al.

(1997). In case of doubt, samples were fixed in 4% form-

aldehyde for 24h, then in 15% sucrose in phosphate buf-fer saline (PBS) for 24h, and frozen sections (5lm)

stained with methylene-blue were observed under the

microscope to verify the sex. Data for sex ratio of two

replicate tanks for each treatment were pooled by treat-

ments for 50 fish.

2.2. Isolation of total RNA

Ovaries and brains samples were collected from eels

at different developmental stages, or following E2 and

AI treatments. The samples were frozen in liquid nitro-

gen and stored at �80 �C until used for RNA extraction.

RNA for polymerase chain reaction (PCR) was isolated

from 100mg (or less) ovaries and brain samples by the

acid guanidinium thiocyanate–phenol–chloroform

extraction method (Chomczynski and Sacchi, 1987)using EZ-RNA (Biological Industries, Israel). Oli-

go(dT)-primed double stranded cDNA was synthesized

I. Tzchori et al. / General and Comparative Endocrinology 138 (2004) 271–280 273

using Promega cDNA synthesis system according to the

manufacturer�s recommendation.

2.3. Isolation and PCR amplification of CYP19 cDNA

fragment

To obtain a CYP19 cDNA fragment of European eel,

5 0 and 3 0 primers for PCR were designed from amino

acid sequences, which are highly conserved among spe-

cies. Ear-1 5 0 primer, 5 0-ATATTCGCTCAGAACC

AC-3 0 and Ear-2 3 0 primer, 5 0-CATACGCCCAACAT

TAAG-3 0 were designed. The positions at which they

hybridize are shown in Fig. 1. PCR was carried out in

25ll reaction mixture consisting of 10mM Tris–HCl,pH 8.3, 50lM MgCl2, 12.5lM of each primer, 1nM

each of dATP, dCTP, dGTP, and dTTP, 0.75U DyNA-

zyme II DNA polymerase (Finnzyme), and 10–100ng

cDNA. Thirty-five amplification cycles were carried

out under the following conditions: denaturation at

93 �C for 45s, annealing at 52 �C for 1min, and exten-

sion at 72 �C for 2min.

The PCR products were separated through 1% aga-rose gel and purified by using the QIAquick Gel Extrac-

tion kit (QIAGEN, Germany). The product was cloned

into the pGEM-T easy plasmid vector (Promega, Milan,

Italy) and sequenced using the BigDye Terminator Cycle

Sequencing Core kit (Applied Biosystem, Monza, Italy).

2.4. 5 0 and 3 0 RACE-PCR

To obtain the 5 0-end of the CYP19 sequence, three

gene specific antisenses, Ear-3R (5 0-GTCCACCAC

TGGATGATAGCGTAG-3 0), Ear-4R (5 0-CTTTCTT

GTCACCGATCACAGTGT-3 0), and Ear-5R (5 0-AC

CCTGAGGAACATCTGGTTGGAGA-3 0) were de-

signed to replicate regions in the open reading frame

of the CYP19 cDNA of the eel.

Fig. 1. Representative samples of specific RT-PCR products for

aromatase and b-actin in the gonads of European eel. The molecular

size marker (M) was the 100bp DNA ladder. The b-actin is a product

of 192bp and the aromatase is 145bp lengths.

Using the SMART RACE cDNA amplification kit

(BD Biosciences, Palo Alto), first strand synthesis was

carried out with 500ng of total RNA and 100nM of

Ear-3R. Two consecutive PCRs were performed, using

100nM of Ear-4R and Ear-5R in nested PCR. Amplifi-

cation by touchdown PCR was carried out for 30 cyclesof 30s at 94 �C, 5 cycles of 50s at 72 �C, and 25 cycles of

50s at 68 �C. PCR products were cloned, and four clones

containing the 5 0-end of European eel CYP19 were se-

quenced as described above. To obtain the 3 0-end of

the CYP19 cDNA sequences, a gene specific sense pri-

mer Ear-6R (5 0-AACAGCTGGCTGAGAAAGACA-

3 0) was designed. First strand synthesis and RT-PCR

were carried out using dT-anchor (Clontech). Amplifica-tion was performed using touchdown PCR as described

above, with range of Ta temperature from 50 to 60 �C.

2.5. Computer analysis

Sequence alignments and comparisons of homology

between various aromatase sequences were performed

using the Vector NTI Advance software (Informax).Phylogenetic trees were constructed by maximum-

parsimony analysis with the computer program PAUP

4 (b7). Bootstrap proportions were used to assess the

robustness of tree with 1000 bootstrap replications.

2.6. Real-time quantitative RT-PCR analysis

For the quantitative real-time PCR analysis of Ee-CYP19 mRNA expression levels, the LightCycler sys-

tem (Roche Molecular Biochemicals, Indianapolis) and

a LightCycler DNA master SYBR green I kit were used.

For hot start PCR, LightCycler FastStart DNA Master

SYBR green I containing the FastStart Taq DNA poly-

merase was used. Preliminary experiments were per-

formed to determine the optimal conditions for PCR

(annealing temperature and final Mg2+ concentration)on each pair of primers.

An aliquot of the RNA (1lg) was reversed tran-

scribed as described above and 1/100th of this reaction

was used in duplicate quantitative fluorescent PCR to

determine transcript abundance.

Primer sets were selected so that the amplified cDNA

would span two exon/intron boundaries of EeCYP19

gene, thus eliminating amplification of genomic DNA.The primer set for CYP19 was Ear-7 5 0-GAACC

CAGAGGTAGAGC-3 0 (forward) and Ear-8 5 0-ACCT

TGTAGCCTTCGAT-3 0 (reverse). The b-actin was used

as an internal control to normalize abundance, with pri-

mer set of 5 0-ACCACCTTCAACTCCATC-3 0 (for-

ward), 5 0-TCCTGCTTGCTAATCCAC-3 0 (reverse).

Each reaction (20ll) contained 2ll of the respective

cDNA dilution, primers at 0.2 mM, and Mg2+ at2mM. Amplification by touchdown PCR was carried

out for 30s at 94 �C, 5 cycles of 5s at 68 �C, 40 cycles


of 5s at 65 �C for the b-actin, and by 30s at 94 �C, 5 cy-

cles of 5s at 65 �C, 40 cycles of 5s at 63 �C for the

CYP19. Unspecific PCR products, e.g., primer dimers,

melt at the elevated temperature, the non-specific fluo-

rescence signal is eliminated, and accurate quantification

of the desired product is ensured (Fig. 1).To ensure the fidelity of mRNA extraction and re-

verse transcription, all samples were subjected to PCR

amplification with oligonucleotide primers specific for

the constitutively gene expressed b-actin. Transcript

abundance of the CYP19 was normalized to the abun-

dance of b-actin, and reported as a relative transcripts

abundance. Constructing a standard curve with serial

dilutions of known template concentration is essentialto quantify the concentration of unknown templates,

thus we used the serial dilution (1:1–1:108) of cDNA.

2.7. Southern blot

European eel genomic DNA was extracted from liver,

digested (10lg) with BglII or EcoRI, and fractionated

by electrophoresis on 0.8% agarose gel. Then it was blot-ted onto a nylon membrane (NYTRAN, Schleicher and

Schuell) and then incubated overnight at 42 �C in

hybridization buffer containing the 32P-labeled cDNA

probe, 6· SSC, 10· Denhard solution, 0.1% SDS, 50%

formamide, and 50lg/ml of calf thymus DNA. The

membrane was washed in 2· SSC, 0.5% SDS, then in

1· SSC, 0.1% SDS. After a final wash with 0.5· SSC,

0.1% SDS at 42 �C, the membrane was exposed on aPhosphorimager (Molecular Dynamics, CA) at room

temperature for 24h. The probe was a 192bp fragment

that coding for 64 amino acids of the European eel aro-

matase peptide.

2.8. Statistical analysis

The relative mRNA expression levels were calculatedfrom each set of quantified data. All data are shown as

means±SEM, although where appropriate they were

subject to logarithmic transformation before being ana-

lyzed by one way ANOVA followed by Tukey�s multiple

range test. A probability level of P<0.05 was used as the

criterion for significance of difference between groups.

The significance of difference in sex ratio between con-

trol and treated groups was tested by Fisher exact test.

3. Results

3.1. Isolation and sequence of European eel CYP19

cDNA

The gonadal form of EeCYP19 was cloned using RT-PCR and RACE-PCR. The full length of the open read-

ing frame (ORF) of CYP19 (EeCYP19) was 1539bp

encoding a protein of 513 amino acids (Fig. 2). This

cDNA sequence showed 97% identity to that of the A.

japonica. The EeCYP19 amino acid showed 97% iden-

tity with that of A. japonica, 59–66% with teleost gona-

dal isoforms, and 56–69% with teleost brain isoforms

(Figs. 3 and 4).Two other potential initiations signals situated 9 and

33bp downstream of the first ATG codon were

identified.

3.2. Tissue specific expression of EeCYP19 and sex ratio

Juvenile eels from the E2 treatment group displayed a

predominately female population (88%), while only a5% female presence was detected in the control un-

treated juvenile group at density of 1000 fish/1m3 (Table

1). Reducing the density of fish (100 fish/1m3) and the

social stress by providing shelters increased significantly

the female percentage to 36%.

The expression of EeCYP19 gene was investigated in

tissues of juvenile eels (0.5g), elvers (5–7g), adult males

(80–120g), and adult females (100–200g), using RT-PCR. The abundance of EeCYP19 transcripts indicated

that the gene was expressed in both the gonads and the

brain. Expression of EeCYP19 in the adult male�sgonads was 12-fold lower when compared to the adult

female group, and 30-fold lowers when compared to

the juvenile E2 treated group. In the control untreated

juvenile fish, transcripts were detected in only one

sample (1/11) (Fig. 5).The abundance of EeCYP19 transcripts in the brains

of elvers previously treated with E2, adult females,

adult males, and control were not significantly different

(Fig. 5).

3.3. Effects of aromatase inhibitor on mRNA levels and

sex ratio

The abundance of EeCYP19 transcripts after AI

treatments was quantified. EeCYP19 was not detected

in the gonads of AI treated fish and the treatment re-

sulted in a complete masculinization of all fish (Table 1).

EeCYP19 mRNA average levels did not differ be-

tween E2 and E2+AI treated fish due to the high vari-

ance, although only 50% (5/10) of the fish were

detected with mRNA levels in the E2+AI as comparedto 80% (8/10) in the E2 treated group (Fig. 6). In the

control untreated fish that were held under low density

conditions, the EeCYP19 mRNA levels were signifi-

cantly lower as compared to the two treatment groups,

and only 36% (4/11) of the fish had detectable EeCYP19

mRNA levels.

The abundance of EeCYP19 transcripts in the brains

was extremely high in the E2 treated group (Fig. 6).Treatment with AI resulted in a strong reduction of

the EeCYP19 mRNA expression in the E2+AI groups.

Fig. 2. The full length of nucleotide and the deduced amino acid sequences of the European eel aromatase form of CYP19 (EeCYP19). Open reading

frame of 1539bp encodes a protein of 513 residues. The numbers on the right refers to the position of the nucleotides and the amino acids. The

positions of primers used at real-time PCR and PCR are indicated with arrow under the sequence. The initiation codons (ATG) and the stop codon

(TAA) have been marked in bold font.


Moreover, the low-density control fish showed low

expression levels of EeCYP19 as well.

3.4. Southern blot analysis of eel genomic DNA

European eel DNA was digested by two restrictionenzymes (BglII and EcoRI) and hybridized with Ee-

CYP19 specific probe, as shown in Fig. 7. Hybridization

of the fractionated DNA to the labeled probe resulted in

a singled band corresponding to approximately 2.1kb

EeCYP19 cDNA.

4. Discussion

In this study, a cDNA encoding the cytochrome P450

aromatase (EeCYP19) was cloned and sequenced from

the ovary of the European eel, and the importance of

Fig. 3. Alignment of the amino acid sequence of European eel P450arom (CYP19) with that of the Japanese eel, tilapia (ovary and brain), medaka

(ovary and brain), goldfish (ovary and brain), fathead minnow (brain), stingray, chicken (ovary), and human placenta. The amino acid sequences

identical to the European eel CYP19 are marked by asterisks and dots.


Fig. 4. Phylogenetic tree of CYP19 amino acid. The tree was

constructed by using program PAUP 4. Comparisons were made with

the deduced amino acid sequences of CYP19 forms in chicken ovary,

human placenta, fathead minnow, goldfish (brain and ovary), tilapia

(brain and ovary), medaka (brain and ovary), and stingray. Length of

lines indicates genetic distance. The values are bootstrap probabilities

estimated by 1000 replications.

Table 1

Effects of treatments on percentage of females in European eel

Treatment n F (%)

High density (1000/1m3) 100 5ab

Low density (100/1m3) 50 36c

E2 50 88d

AI 50 0a

E2+AI 50 8b

Eels were randomly sampled from each group for macroscopic clas-

sification of the gonad according to Beullens et al. (1997). The groups

were high-density (1000fish/1m3) and low-density (100fish/1m3) tanks;

E2: fish fed estradiol for feminization for 150 days. AI: 300mg/kg bw

AI treatment for 90 days; E2+AI: estradiol treated group for 150 days

with 10mg/kg bw, followed by 300mg/kg bw AI treatment for 90 days.

Female�s percentages marked by the same letter do not differ signifi-

cantly from each other (p>0.05).


its expression and activity during sex determination was

demonstrated. The cloned cDNA of EeCYP19 from the

European eel gonad contains an open reading frame of

1539bp, encoding a deduced protein of 513 residues.

The predicted amino acid sequence shows 97% identity

with that of the Japanese eel. Interestingly, the compar-

ison of the EeCYP19 to other teleosts CYP19 gene re-

vealed that in some fish the higher identity was to thebrain form (medaka, tilapia, and trout), while in others

it was more similar to the ovarian form (goldfish, zebra-

fish). Two potential initiation sites (ATG) were found

downstream of the first ATG codon. These results

are similar to multiple initiation sites reported in other

teleosts (Dalla Valle et al., 2002; Halm et al., 2001; Ijiri

et al., 2003).

Quantitative real-time PCR analysis revealed that Ee-

CYP19 transcripts expressed in the gonads of females

were significantly higher than those reported for the

male counterparts. These differences are more evidentin juvenile eels previously subjected to E2 treatments

for feminization, whereas in the untreated juveniles

CYP19 expression, which was not detected. These re-

sults indicate a sexual differential expression of aroma-

tase in European eel gonads, favoring the female sex.

This trend is not unexpected considering the strong syn-

thesis of estrogen in the ovary, compared to limited

estrogen synthesis in testis.In contrast to the situation in the gonads, no signifi-

cant difference in expression levels was found in the

brain of eels at various developmental stages. It is

important to note that these values were determined in

eels that weigh more than 15g.; therefore, it is possible

that sexual differentiation may have already occurred

at this particular stage. In teleosts, similar studies aimed

to characterize the involvement of CYP19 gene/s expres-sion during sex differentiation indicate its complexity. In

the Nile tilapia (Oreochromis niloticus) (Kwon et al.,

2001) and in the European sea bass (Dicentrarchus lab-

rax) (Dalla Valle et al., 2002), high expression levels

of the gonadal form of CYP19 were found in the gonad

but not in the brain. In contrast, the initial differences in

the CYP19 expression in the zebrafish could be first de-

tected in the brain-form of CYP19 in the brain (Trantet al., 2001).

Treatment of juvenile eels with AI resulted in total

absence of aromatase expression. Since high levels of

CYP19 were found in the gonads after E2 treatment,

the decline in CYP19 levels in some fish after exposure

to AI in the combined treatment (E2+AI) can be due

to the inhibitory affect of AI. The heterogeneity of the

expression of this group can be due to the fact that somefish, which received E2 previously were already differen-

tiated into females, and therefore the gonads were no

longer sensitive to the AI treatments. The strong inhibi-

tion of CYP19 expression was found in the brain as well.

AI treatment of elvers, with or without previous E2

treatment, resulted in dramatic decreases in aromatase

expression in the brain of all treated fish.

The expression levels of EeCYP19 were 40-fold high-er in the E2 treated group compared to the AI treated

group. The fact that high levels of CYP19 were associ-

ated with high percentage of female, whereas AI treat-

ment causing full inhibition of CYP19 expression

resulted in the masculinization of all treated fish, indi-

cating an importance for high CYP19 levels during fe-

male differentiation. This is in line with the results of

(Kwon et al., 2002) in Nile tilapia, which showed thatlow aromatase activity during the crucial period of sex

differentiation always resulted in masculinization,

Fig. 5. Transcript abundance of aromatase (EeCYP19) gene expressed in male and female gonads (black bars) and brains (white bars) of European

eel. Transcript abundance of aromatase is normalized to that of b-actin. The number of individuals for each mean and SEM are presented. Mean

values marked by the same letter do not differ significantly from each other (p>0.05). The means values were derived from duplicate samples in a

single real-time PCR assay. Control: 15–25g untreated fish, E2: 15–25g young fish previously treated with 10mg/kg bw estradiol, adult male (80–

120g), and adult females (100–200g).

Fig. 6. Transcript abundance for aromatase (EeCYP19) gene expressed in gonads (black bars) and brains (white bars) of European eel after E2 and

AI treatment. Transcript abundance of aromatase was normalized to the abundance of b-actin. The number of individuals for each mean and the

SEM are presented. The number of individuals with detectable levels related to the total number of fish is shown in brackets. Mean values marked by

the same letter do not differ significantly from each other (p>0.05). The mean values were derived from duplicate samples in a single real-time PCR

assay. Control (positive): 15–25g untreated fish kept at low density, E2: 15–25g fish previously fed estradiol for feminization for 150 days. E2+AI:

estradiol treated group for 150 days with 10mg/kg bw, followed by 300mg/kg AI treatment for 90 days, AI: 300mg/kg AI treatment for 90 days.


regardless of temperature or genotype. In addition, fat-

head minnow exposed to AI showed decreased E2 levels,

whereas androgens levels increased (Ankley et al., 2002).

The involvement of estradiol in aromatase activity wasshown in Acanthopagrus schlegeli. Estradiol treatment

reduces the synthesis of testosterone, and increases aro-

matase enzyme activity (Chang and Lin, 1998). Results

obtained in protandrous species, indicate that estradiol

plays a direct role in sex reversal, by suppressing mascu-

linizing steroids and stimulating enzymes needed for

estrogen synthesis (Devlin and Nagahama, 2002).

The results of this work indicate that fadrozole, pre-

viously described as a competitive inhibitor of aroma-

tase activity (Steele et al., 1987), has a significant effect

in reducing the expression levels of CYP19, either di-rectly, or indirectly, via the reduction of E2 levels.

To date, it is not known if a second form of CYP19,

specific for the brain, exists in the eel. However, at-

tempts has been made during the course of this study,

to isolate a brain specific isoform by using degenerated

primers designed for the highly conserved sequences

of all known brain forms of aromatase in fish. This

Fig. 7. Southern blot analysis of European eel genomic DNA. DNA

prepared from eel liver was fractionated by electrophoresis on 0.8%

agarose gel after digestion with BglII and EcoRI. The fractionated

DNA was blotted onto NYTRAN membrane and hybridized with a32P-labeled cDNA probe coding for 64 amino acids of the eel

aromatase peptide. Sizes in kilobases are indicated in the right margin.


technique was unable to distinguish an extra-gonadal

isoform, revealing only a single form of CYP19, namely

the gonadal form. Furthermore, the results from South-

ern blot analysis show only a single band in the entire

genome. These results are consistent with those in thecartilaginous fish, the stingray. The transcripts from

the extra-gonadal form encodes a protein identical to

the ovarian form (Ijiri et al., 2000). Moreover, Northern

blot analysis detected only a single aromatase form in

the Japanese eel (Ijiri et al., 2003). The possible absence

of a second form of aromatase in eel may be explained

by a theory that duplication in the fish genome took

place after the Anguilliform fish diverged from the mainstock of the Euteleostei (Robinson-Rechavi et al., 2003).

This theory is supported by the sequence analysis of

EeCYP19, indicating a similarity to the brain form in

some fish and to the gonadal form in others.

In summary, we have cloned a CYP19 cDNA from

the ovary of the European eel. High amino acid identi-

ties were found with the gonadal form in some fish

and to the brain form in other fish. Transcripts ofEeCYP19 were found in both the ovary and brain tis-

sues; however, EeCYP19 levels were higher in ovarian

than in testicular tissues. Treatment with AI resulted

in a strong reduction of the EeCYP19 mRNA expres-

sion. No evidence could be provided for the presence

of an extra-gonadal (brain) form of aromatase in eel.

Acknowledgments

We thank Prof. Zvi Yaron and Dr. Hanna Rosenfeld

for helpful comments and critical reading the manu-

script. This work was supported by the Israeli Ministry

of Agriculture (Grant 99-0169-596), and by the Israeli

Ministry of Science, Culture and Sport (Grant 1296-

199). Fadrozole was kindly supplied by Novartis.

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