Dr Nathalie Hinfray
email: nathalie.hinfray@ineris.fr

Frédéric Sohm

Morgane Caulier

Edith Chadili

Benjamin Piccini

Camille Torchy

Jean-Marc Porcher

Yann Guiguen

Dr François Brion
email: francois.brion@ineris.fr

Hinfray Nathalie

Sohm Frédéric

Caulier Morgane

Chadili Edith

Piccini Benjamin

Torchy Camille

Porcher Jean-Marc

Guiguen Yann

Brion François

Dynamic and differential expression of the gonadal aromatase during the process of sexual differentiation in a novel transgenic cyp19a1a -eGFP zebrafish line

Keywords: gonadal aromatase, transgenic zebrafish, sexual differentiation

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Despite all the interest granted to zebrafish (Danio rerio) in the last few decades, the developmental genetic mechanisms controlling sexual fate in this species remain unclear (for review see [START_REF] Liew | Zebrafish sex: a complicated affair[END_REF][START_REF] Nagabhushana | Finding clues to the riddle of sex determination in zebrafish[END_REF][START_REF] Wilson | Wild sex in zebrafish: loss of the natural sex determinant in domesticated strains[END_REF]. In natural wild strains of zebrafish, a strong sex determinant located at the end of the long (right) arm of chromosome 4 was recently discovered and this determinant is necessary but not sufficient for female development [START_REF] Wilson | Wild sex in zebrafish: loss of the natural sex determinant in domesticated strains[END_REF]. In contrast, laboratory strains most often lack this sex determination system [START_REF] Wilson | Wild sex in zebrafish: loss of the natural sex determinant in domesticated strains[END_REF]. Analysis of the available literature suggests that sex in those laboratory zebrafish strains would results of a complex interaction between genome (a polygenic sex determination system), primordial germ cells and environmental factors along with epigenetic factors possibly playing a role in mediating these processes [START_REF] Liew | Zebrafish sex: a complicated affair[END_REF][START_REF] Luzio | Screening and identification of potential sex-associated sequences in Danio rerio[END_REF][START_REF] Nagabhushana | Finding clues to the riddle of sex determination in zebrafish[END_REF].

During the sexual differentiation process, all male zebrafish develop a "juvenile ovary" which is later degenerated ("ovary-to-testis" transforming gonad) and transformed into a testis [START_REF] Maack | Morphological development of the gonads in zebrafish[END_REF][START_REF] Takahashi | Juvenile hermaphroditism in the Zebrafish, Brachydanio rerio[END_REF]. While the identity of the gene(s) triggering sex determination/differentiation in zebrafish is not known at present, one common conserved factor in teleost sexual differentiation process is the involvement of estrogens-to-androgens ratios and P450 aromatase, the enzyme responsible for estrogens synthesis (for review see [START_REF] Guiguen | Ovarian aromatase and estrogens: A pivotal role for gonadal sex differentiation and sex change in fish[END_REF]. In this regard, it is well-established that exposure to estrogens during the process of sexual differentiation resulted in a female biased sex-ratios at the adult stage (Baumann et al., 2014b;[START_REF] Brion | Impacts of 17[beta]-estradiol, including environmentally relevant concentrations, on reproduction after exposure during embryo-larval-, juvenile-and adult-life stages in zebrafish (Danio rerio)[END_REF][START_REF] Holbech | Detection of endocrine disrupters: Evaluation of a Fish Sexual Development Test (FSDT)[END_REF][START_REF] Maack | Life-stage-dependent sensitivity of zebrafish (Danio rerio) to estrogen exposure[END_REF], while androgen and aromatase inhibitor exposures led to male biased sex-ratios (Baumann et al., 2014a;[START_REF] Fenske | Aromatase modulation alters gonadal differentiation in developing zebrafish (Danio rerio)[END_REF][START_REF] Mcallister | Early life exposure to environmental levels of the aromatase inhibitor tributyltin causes masculinisation and irreversible sperm damage in zebrafish (Danio rerio)[END_REF][START_REF] Morthorst | Trenbolone causes irreversible masculinization of zebrafish at environmentally relevant concentrations[END_REF][START_REF] Uchida | An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sex-reversal[END_REF].

In most teleost fish, including zebrafish, the aromatase gene was duplicated giving rise to two different genes named cyp19a1a and cyp19a1b [START_REF] Blazquez | Cloning, sequence analysis, tissue distribution, and sex-specific expression of the neural form of P450 aromatase in juvenile sea bass (Dicentrarchus labrax)[END_REF][START_REF] Chang | Two types of aromatase with different encoding genes, tissue distribution and developmental expression in Nile tilapia (Oreochromis niloticus)[END_REF]Chiang et al., 2001a;Chiang et al., 2001b;Dalla Valle et al., 2002a;Dalla Valle et al., 2002b;[START_REF] Kobayashi | Characterization of two types of cytochrome P450 aromatase in the serial-sex changing gobiid fish, Trimma okinawae[END_REF][START_REF] Tchoudakova | Identification of multiple CYP19 genes encoding different cytochrome P450 aromatase isozymes in brain and ovary[END_REF]. In zebrafish, the cyp19a1b gene is mainly expressed in the brain, specifically in radial glial cells, of both male and female zebrafish and is called the "brain aromatase" (Chiang et al., 2001a;[START_REF] Menuet | Expression and estrogen-dependent regulation of the zebrafish brain aromatase gene[END_REF][START_REF] Pellegrini | Identification of aromatase-positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish[END_REF][START_REF] Sawyer | Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish: Gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation[END_REF]. The cyp19a1a gene is mainly expressed in the gonads, especially the ovary, and is also called the "gonadal aromatase" or "ovarian aromatase" (Chiang et al., 2001a;[START_REF] Sawyer | Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish: Gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation[END_REF]. Current studies do support a decisive role of cyp19a1a gene in the phenotypic sex orientation of the zebrafish gonads. For instance, in vas::egfp transgenic zebrafish, cyp19a1a transcript levels clearly decreased in gonads undergoing subsequent testicular differentiation [START_REF] Wang | Anti-Mullerian hormone and 11 beta-hydroxylase show reciprocal expression to that of aromatase in the transforming gonad of zebrafish males[END_REF]. Moreover, by producing loss-of-function mutations in zebrafish, [START_REF] Dranow | Bmp15 Is an Oocyte-Produced Signal Required for Maintenance of the Adult Female Sexual Phenotype in Zebrafish[END_REF] recently confirmed that Cyp19a1a is necessary for female sexual development in this species. Altogether, these studies support a critical role of Cyp19a1a in the ovarian differentiation and in the maintenance of the ovarian condition.

Aromatase expression and/or activities have been shown to be disrupted by environmental endocrine disrupting chemicals (EDCs) (Hinfray et al., 2006b;[START_REF] Sanderson | Induction and Inhibition of Aromatase (CYP19) Activity by Various Classes of Pesticides in H295R Human Adrenocortical Carcinoma Cells[END_REF]. For instance, several contaminants belonging to diverse class of chemical families can induce or inhibit aromatase in fish leading to biased sex ratios and/or disruption of reproductive outputs [START_REF] Fenske | Aromatase modulation alters gonadal differentiation in developing zebrafish (Danio rerio)[END_REF][START_REF] Kitano | Aromatase inhibitor and 17alphamethyltestosterone cause sex-reversal from genetical females to phenotypic males and suppression of P450 aromatase gene expression in Japanese flounder (Paralichthys olivaceus)[END_REF][START_REF] Skolness | Propiconazole Inhibits Steroidogenesis and Reproduction in the Fathead Minnow (Pimephales promelas)[END_REF][START_REF] Sun | Toxicity of the aromatase inhibitor letrozole to Japanese medaka (Oryzias latipes) eggs, larvae and breeding adults[END_REF][START_REF] Uchida | An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sex-reversal[END_REF]. In an attempt to further study the spatio-temporal expression and perturbation of aromatase A expression by EDCs, transgenic fish expressing a fluorescent reporter under the control of the cyp19a1a promoter would therefore be relevant biological models. In the present study, we developed and characterized a novel transgenic zebrafish line expressing green fluorescent protein (GFP) under the control of the zebrafish cyp19a1a promoter. Herein we report the expression and/or localization of cyp19a1a gene in adult zebrafish gonads both in control and in E2-exposed fish. Through fluorescence image analysis of GFP, the spatio-temporal expression of cyp19a1a was analyzed and semi-quantified along the process of histological sexual differentiation of the zebrafish and we confirmed that the downregulation of cyp19a1a gene expression is likely responsible for the testicular differentiation in zebrafish. The potential use of this novel transgenic zebrafish line for EDCs studies is also discussed.

Materials and Methods

cyp19a1a-eGFP transgenic zebrafish line

2.1.1. Extraction of genomic DNA cyp19a1a promoter was isolated from genomic DNA of AB strain zebrafish. Genomic DNA was retrieved using a proteinase K protocol. Briefly, 30 hatched larvae were euthanized by an overdose of ethyl 3-aminobenzoate methanesulfonate (MS-222, Sigma-Aldrich, Lyon, France) and incubated in 250 µL solution of Proteinase K, (Merck, 10 mg/mL) overnight at 56 o C. The solution was completed with 83.3 µL of saturated NaCl solution (Sigma, 350 g/L) and centrifuged (18,000 g, 20 minutes). The liquid phase (250 µL) was retrieved, precipitated using iso-propanol and washed twice in 70 % ethanol. The pellet was resuspended in 30 µL Tris buffer (Sigma, 10 mM, pH 8) by incubation at 65 o C for one hour and subsequently stored at 4 o C.

Transgenic vector design

We made use of transgenesis vector designed by AMAGEN as 'all fish species' transgenesis carriers. The sequences and important features of the cloning and transgenesis vectors are provided in Supplemental figures 1 to 4. Briefly, the vectors were designed upon the multiple Gateway® cloning system described in [START_REF] Roure | A Multicassette Gateway Vector Set for High Throughput and Comparative Analyses in Ciona and Vertebrate Embryos[END_REF] and carried 2 expression cassettes. The first cassette is an enhanced Cyan Fluorescent Protein (eCFP) coding sequence under the control of the zebrafish betacrystallin-b1 promoter (CrystBB1 cassette) which allows to follow the presence of the transgene in zebrafish from 48 hours post-fertilisation (hpf) onward [START_REF] Emelyanov | Mifepristone-inducible LexPR system to drive and control gene expression in transgenic zebrafish[END_REF]. The second expression cassette allows to clone a promoter of interest (cyp19a1a in the present study) in front of an enhanced GFP (eGFP) reporter coding sequence. The insertion of the promoter is realised with a Gateway® Cloning kit using the attP3 and attP5 sites of the pEXP-GFP35H vector [START_REF] Roure | A Multicassette Gateway Vector Set for High Throughput and Comparative Analyses in Ciona and Vertebrate Embryos[END_REF]. The two cassettes are separated by a NOS terminator in order to reinforce transcriptional arrest after the CrystBB1 cassette [START_REF] Emelyanov | Mifepristone-inducible LexPR system to drive and control gene expression in transgenic zebrafish[END_REF]. The transgenesis is realised through Involin [START_REF] Thermes | I-SceI meganuclease mediates highly efficient transgenesis in fish[END_REF] using the two I-SceI sites present on each side of the transgene.

PCR amplification of the promoter

The 7.8 kb region upstream of the start codon of cyp19a1a was used as promoter to drive GFP expression. The PCR was designed in order to use the initiation codon and the Kozak consensus translation start of cyp19a1a fused to the eGFP coding sequence. The promoter was inserted using Gateway® cloning system (InVitrogen) as described in [START_REF] Roure | A Multicassette Gateway Vector Set for High Throughput and Comparative Analyses in Ciona and Vertebrate Embryos[END_REF] into the pDONR35H (Supplemental figure 1). Briefly, a first fragment (8233 bp) was amplified from genomic DNA. We used the primers Cyp19a1a_Fwd and Cyp19a1a_Rev (Supplemental table 1) -11 min 30 sec). This reaction yielded several products. A second reaction was used to improve specificity and insert the attB3 and attB5 Gateway recombination sites necessary for the cloning. The first PCR was diluted to the 10 th and 2 µL were used as template of the nested PCR reaction. The primers Cyp19a1a_B3 and Cyp19a1a_B5 (Supplemental table 1) allowed to insert attB3 and attB5 recombination sites respectively at the 5' and 3' end of the promoter. The PCR conditions were identical to the first round except hybridization temperature, which was lowered to 47 o C. This reaction yielded a unique 7897 bp fragment (7839 bp of promoter and 58 bp of linkers). The attB3-attB5 tagged PCR fragment was purified using QIA quick PCR purification kit (Qiagen) according to manufacturer instructions. 50 femtomole of the PCR product and the vector pDONR35H (Supplemental figure 1) were used to carry out the cloning reaction using the BP clonase II kit (InVitrogen) to generate the pENT24-3 (Supplemental figure 2). The entry vector pENT24-3 was then recombined with pDEST35H (Supplemental figure 3) to generate the transgenesis vector pEXP24-3 (Supplemental figure 4). Both the pENT24-3 and the pEXP24-3 were fully sequenced (the sequences are provided in the supplemental figure 3 and 4).

Generation of transgenic cyp19a1a-GFP zebrafish

Transgenic zebrafish were generated by injecting the pEXP24-3 expression vector into one cell stage zebrafish eggs. Briefly, a solution containing pEXP24-3 maxiprep (15 ng/µL final) and I-SceI enzyme (Roche, 1U/µL final) was injected into one cell stage eggs using borosilicate glass microcapillaries (GC100-10, Harvard Apparatus Ltd, UK), a femtojet injector (Eppendorf, Germany), a binocular loupe (Stemi Zv6, Zeiss, Germany) and Narishige (Japan) micromanipulator. In total, 789 eggs were injected in 3 sessions. At 48 hpf, the embryos were sorted according to eCFP expression in the lens under an epifluorescence dissecting microscope (MZFLIII Leica). Embryos which displayed differential expression from one eye to the other, strong mosaicism, weak or absence of CFP/GFP expression were eliminated. Roughly 20% of the injected eggs were selected for rearing (a batch of 77 mild expressing embryos and a batch of 90 embryos displaying strong expression). After a rearing period of 4 months, 12 female and 4 male F0 adult fish were tested for their ability to transmit the transgene to their progeny. Among those, 4 females and 1 male transmitted the transgene to their progeny (Table 1). Progeny from each of these founders were further analysed for expression of GFP in the gonads. Two of the 5 lines did not express GFP at all. Each of the 3 other lines did express GFP according to a cyp19a1a compatible pattern albeit at levels varying from one line to the other (data not shown). The progeny of female 4 was selected as the most appropriate to our purpose (highest GFP expression intensity) and all further analysis were carried out using the progeny of this animal. The term cyp19a1a-eGFP thereafter refers exclusively to the lineage of this female 4.

Zebrafish maintenance and reproduction

Both transgenic (cyp19a1a-eGFP) and wild type (AB strain) zebrafish were raised in our laboratory facility at INERIS (Institut National de l'Environnement Industriel et des Risques, Verneuilen-Halatte, France). Zebrafish were maintained in 3.5 L aquaria in a recirculation system (Zebtec, Tecniplast, Buguggiate, Italy) on a 14:10 light:dark cycle at a temperature of 27.0 ± 2.0°C. Animal handling and experimentations were in accordance with the EU Directive 2010/63/EU for animal experiments.

Transgenic cyp19a1a-eGFP zebrafish were crossed with wild type zebrafish (AB strain) and fertilized eggs were harvested. Eggs were disinfected 5 min in water supplemented with 0.1% of commercial bleach (2.6% of sodium hypochlorite). The CFP positive eggs/larvae were maintained in semi-static conditions until 8 days post fertilization (dpf) and then transferred in 3.5 L aquaria of the Zebtec system (around 25 larvae per liter) to grow.

To obtain the transgene transmission rate in the cyp19a1a-eGFP zebrafish line, 6 pairs of breeders each consisting of one transgenic and one wild type zebrafish, were allowed to reproduce.

Transmission rate was determined by calculating the ratio of transgenic eggs over wild type eggs for each pair of breeders.

GFP fluorescence imaging and semi-quantification in the entire gonads of mature zebrafish

Mature cyp19a1a-eGFP transgenic zebrafish were euthanized in ice-cold water [START_REF] Strykowski | Effectiveness of recommended euthanasia methods in larval zebrafish (Danio rerio)[END_REF][START_REF] Wilson | Evaluation of rapid cooling and tricaine methanesulfonate (MS222) as methods of euthanasia in zebrafish (Danio rerio)[END_REF] before sectioning the spinal cord. The abdominal cavity was open to remove gut, liver, spleen and bile vesicle. Gonads were photographed with a Discovery V8 stereomicroscope equipped with an AxioCam Mrm camera (Carl Zeiss GmbH, Göttingen, Germany). GFP fluorescence in the entire gonads was then photographed and semi-quantified using a fluorescence imaging system IVIS Lumina II (Perkin Elmer, Courtaboeuf, France). All the pictures were taken in the same conditions (zoom, time of exposure, binning, F-Stop, excitation filter and emission filter). GFP fluorescence levels in the entire gonads were expressed as a measure of the photons incidence on the CCD camera (counts) and normalized by the area of the entire gonads (in squared millimeters, mm 2 ). For each sex, 6 individuals were analyzed.

Zebrafish exposure to estradiol and GFP fluorescence measurement

Mature female cyp19a1a-eGFP zebrafish were exposed to 10 nM E2 (Sigma-Aldrich) or to solvent alone (dimethyl sulfoxide (DMSO), 0.00001%; Sigma-Aldrich, Lyon, France) during 7 days.

Fish were exposed under semi-static conditions in 4 L tanks with 100% water renewal every day (temperature: 28 ± 1°C; pH: 8.0 ± 0.2; dissolved oxygen: 6.6 ± 0.3 mg/l; conductivity: 275 ± 43 mS/cm). Exposure was performed in one tank per condition, each containing four female fish. At the end of exposure, zebrafish were euthanized and gonads were removed and fixed 48h in 4% paraformaldehyde in saline phosphate buffer (PBS) at 4°C. After rinsing in PBS, samples were successively transferred to 10%, 20% and 30% sucrose-PBS solutions at 4°C and finally embedded in Neg-50 cryo-compound (Microm Microtech, France). Frozen sections of gonads were performed at 12 µm thick. Slides were rinsed in PBS to remove Neg-50 cryo-compound and coverslipped using a fluorescence mounting medium (DAKO, France).

GFP fluorescence was observed directly on cryo-sections using a Zeiss AxioImager Z1 fluorescence microscope equipped with an AxioCam Mrm camera (Carl Zeiss GmbH, Göttingen, Germany). Micrographs were taken using the Axiovision Imaging software. Semi-quantification of the fluorescence of GFP of DMSO-and E2-exposed fish ovaries was performed on micrographs taken with the same magnification (x2.5 objective) and time of exposure for all individuals. For each experimental condition, 4 fish were used and 4 micrographs were analyzed per fish. Fluorescence was measured as integrated density (IntDen, ImageJ software), i.e. the sum of the grey values of all the pixels in the region of interest. The IntDen values were then normalized to the total area of tissue on the micrograph.

Fluorescence immunohistochemistry

cyp19a1a-eGFP transgenic/wild type AB zebrafish were euthanized and gonads were removed and fixed in modified Davidson's fixative (22% v/v formaldehyde 37-40%; 11.5% v/v glacial acetic acid; 33% v/v ethyl alcohol 95%; 33.5% v/v distilled water) for 48 hours at room temperature.

After fixation, samples were dehydrated in ethanol, cleared in toluene and embedded in paraffin, according to conventional procedures. Zebrafish gonads were sectioned at 5 µm.

Immunohistochemistry was then carried out using an In situ Pro VSI robotic station (Intavis, Köln, Germany, distributed in France by Proteigene) as previously described in [START_REF] Hinfray | Cyp17a1 and Cyp19a1 in the zebrafish testis are differentially affected by oestradiol[END_REF].

For GFP localization, a primary antibody produced in goat was used at a dilution of 1:600 (polyclonal goat anti-GFP, Abcam, Paris, France, reference number: ab5450). For Cyp19a1a localization, a primary antibody produced in rabbit was used at a dilution of 1:600 (polyclonal rabbit anti-zfCyp19a1a, AnaSpec, Fremont, USA, distributed in France by Tebu-Bio, reference number: 55474s). For colocalization experiment, slides were incubated with a mix of these two antibodies at a dilution of 1:600.

These primary antibodies have been already used successfully in zebrafish [START_REF] Hinfray | Cyp17a1 and Cyp19a1 in the zebrafish testis are differentially affected by oestradiol[END_REF][START_REF] Klein | Neuron navigator 3a regulates liver organogenesis during zebrafish embryogenesis[END_REF]. Slides were then incubated with a donkey anti-goat antibody coupled to AlexaFluor® 488 or a mix of donkey anti-goat coupled to AlexaFluor® 488 and donkey anti-rabbit coupled to AlexaFluor® 594 antibodies (Invitrogen, Praisley, UK) at a dilution of 1:200. Nuclei were revealed with Hoechst 33342. The specificity of the immunoreactivity was controlled by processing adjacent sections without primary antibody or with only one primary antibody and the two secondary antibodies. The specificity of the anti-GFP antibody was controlled by performing immunohistochemistry on wild type zebrafish gonad sections. The specificity of the anti-Cyp19a1a antibody was also controlled by performing immunohistochemistry with the anti-Cyp19a1a antibody pre-incubated with 100µg/ml of the synthetic peptide. In these negative controls, no cross-reactivity was seen (Supplemental Figure 5).

GFP fluorescence was observed and photographed using an FV1000 confocal microscope (Olympus, Rungis, France). Acquisition parameters (time of exposure, laser intensity…) were optimized for each micrograph independently.

Gonadal GFP fluorescence during the process of sexual differentiation

cyp19a1a-eGFP transgenic zebrafish larvae were sampled at 20, 30, 35 and 40 dpf (n = 8 at 20 dpf and 15 at the other sampling time). The larvae were euthanized with an overdose of MS-222.

Entire larvae (20 dpf) or larvae without their heads (30, 35, 40 dpf) were fixed 48h in 4%

paraformaldehyde in PBS at 4°C. After rinsing in PBS, larvae were successively transferred to 10%, 20% and 30% sucrose-PBS solutions at 4°C, embedded in Neg-50 cryo-compound and sectioned transversally at 12 µm thick. Slides were rinsed in PBS and coverslipped using the DAKO fluorescence mounting medium.

GFP fluorescence was observed directly on cryo-sections using a Zeiss AxioImager Z1 fluorescence microscope equipped with an AxioCam Mrm camera and combined with an ApoTome (Carl Zeiss GmbH, Göttingen, Germany). Illustration micrographs were taken using the ApoTome system and Axiovision Imaging software. Relative quantification of the fluorescence of GFP of the larvae gonads was performed on micrographs taken without the ApoTome system but with the same magnification (x10 objective) and time of exposure for all individuals whatever the sex and stage of development. For each larva, 3 to 4 micrographs of the entire gonads were analyzed. GFP fluorescence was measured as integrated density (IntDen, ImageJ software).

Whole Mount In Situ Hybridization (WMISH) on early stage zebrafish

Wild type AB zebrafish larvae were sampled at 20 dpf, euthanized with an overdose of MS-222 and fixed in 4% paraformaldehyde (24 hours, 4°C). After fixation, larvae were rinced 3 times (PBS-Tween 20 0.1%), progressively dehydrated in methanol and stored in methanol 100% until WMISH.

RNA probes were produced from PCR product obtained by following amplification of the IRBOp991C1282D clone (Source Bioscience, UK) containing the zebrafish cyp19a1a cDNA. Colonies containing plasmid were isolated with 50 µg/ml ampicillin. Plasmids were purified using PureLink® Quick Plasmid Miniprep kit (ThermoFisher Scientific) according to manufacturer's protocol. PCR amplification was carried out on 2 ng of plasmid as follow: 1 cycle at 94°C for 1 min, 35 cycles at (94°C for 40 sec, 46°c for 40 sec and 72°C for 2 min), and 1 cycle at 72°C for 10 min; using the following primers 5'-TAA TAC GAC TCA CTA TAG GG-3' and 5'-CAA TTA ACC CTC ACT AAA GG-3'.

Ten nanograms of the resulting PCR product was then used as template for digoxygenin-labeled RNA probes synthesis using DIG RNA labeling mix (Roche, France) and T3 or T7 RNA polymerase (Roche, France) following standard protocols. Labeled RNA probes were then purified using Micro Bio-Spin 30

Columns (BioRad, France) and stored at -80°C until use.

WMISH was carried out using an In Situ Pro (Intavis) robotic station as described in [START_REF] Yano | The duplucated Rainbow Trout (Oncorhynchus mykiss) T-box transcription factors 1, tbx1a and tbx1b, are up-regulated during testicular development[END_REF] with minor modifications. Briefly, 20 dpf zebrafish larvae were rehydrated, permeabilized by proteinase K treatment (25 µg/ml, 35 min, room temperature), and post-fixed (4% paraformaldehyde and 0.8% glutaraldehyde, 20 min). Pre-hybridization and hybridization medium contained 50% formamide, 5X SCC, 0.1% Tween 20, 0.005% heparin, 0.1 mg/ml tRNA. Hybridization was carried out at 37°C for 16 hours. After post-hybridization washes, larvae were incubated in blocking buffer for 1 hour (Triton 1%, Tween 20 0.2%, 2% serum/PBS) before addition of the alkaline phosphatase coupled anti-digoxygenin antibody (1:2,000, Roche Diagnostics Corp) for 6 hours. After washes, color reaction was performed in the presence of NBT/BCIP (Roche). After WMISH, larvae were dehydrated and embedded in paraffin according to conventional procedures, and sectioned at 5 µm.

Statistics

To compare mean fluorescence data (female vs male, DMSO vs E2 and GFP expression in the different sexes and stages of development), a non-parametric Wilcoxon's test was performed, where it was possible, using RTM (R software, R development Core Team). Differences between groups were considered to be significant if p < 0.05 (*) and p < 0.01 (**).

Results

Transmission of the transgene in the cyp19a1a-eGFP transgenic zebrafish line

Adult cyp19a1a-eGFP heterozygote transgenic zebrafish (N=3 males and 3 females) were allowed to reproduce with wild type AB-strain zebrafish to determine the transmission rate of the transgene from generation 3 to generation 4. Transgenic larvae were identified by the presence of CFP fluorescence in the eyes. Transmission of the transgene occurred for the 6 transgenic fish and the transmission rate was about 50% for each breeder fish (Table 2) suggesting a unique insertion of the transgene in these transgenic fish. Besides the unique insertion of the transgene, this cyp19a1a-eGFP line also displays interesting features such as being a stable transgenic line with the possibility to select transgenic eggs as soon as 48 hpf.

GFP expression, localization and regulation in the cyp19a1a-eGFP transgenic zebrafish line

The expression of GFP in mature zebrafish gonads was determined by fluorescence imaging and relative quantification (Figure 1). In all male and female fish studied, GFP expression was higher in ovaries than in testis (Figure 1A, B) with a 9-fold higher expression in ovaries compared to testis (Figure 1B). To assess whether GFP is indeed expressed in Cyp19a1a-expressing cells, sections of cyp19a1a-eGFP transgenic fish gonads were immunolabeled both with anti-GFP and anti-Cyp19a1a

antibodies. In both mature ovary and testis, a perfect co-localization of GFP and Cyp19a1a proteins was observed (Figure 3). In a previous study, we showed that exposure to 10nM of E2 for 7 days inhibited cyp19a1a gene expression in wild type zebrafish ovary (Hinfray et al., 2006a). In the present study, GFP fluorescence in adult cyp19a1a-eGFP transgenic zebrafish ovaries was also inhibited by exposure to E2 (10nM, 7 days) (Figure 4), indicating that GFP expression regulation by E2 in transgenic zebrafish is similar to that of cyp19a1a gene in wild type zebrafish.

Spatio-temporal expression of GFP in cyp19a1a-eGFP transgenic zebrafish gonads during development

The spatio-temporal expression of cyp19a1a gene was studied during the process of sexual differentiation using GFP fluorescence imaging and semi-quantification in gonad sections of transgenic zebrafish between 20 and 40 dpf (Figure 5, 6 and 7). A slight expression of GFP was observed in all immature gonads of 20 dpf-old zebrafish and tends to increase in 30 and 35 dpf-old zebrafish immature gonads (Figure 5). In immature gonads of 20 dpf cyp19a1a-eGFP transgenic zebrafish, GFP and Cyp19a1a expression was localized in the cytoplasm of all cell types (Figure 6 A, B). At the same stage of development, cyp19a1a mRNA was also localized in the cytoplasm of all cell types of wild type zebrafish gonads (Figure 6 C). In females (from 30 dpf), GFP expression was higher than in immature gonads and its expression increased to reach high GFP intensity in well-differentiated ovaries of 40 dpf-old zebrafish (Figure 5). At this stage, GFP was mainly observed in the cytoplasm of peri-follicular cells but a slight fluorescence was also noticed in the cytoplasm of oocytes (Figure 7).

On the contrary, males consistently displayed low GFP expression as compared to females whatever their stage of development, resulting in a clear dimorphic expression between both sexes (Figure 5).

At 40 dpf, GFP was localized in the cytoplasm of several isolated cells distributed in the entire testis (Figure 7). Interestingly, gonads of fish that undergo ovary-to-testis transition (35 and 40 dpf)

presented GFP levels similar to males or intermediate between females and males (Figure 5). In this ovary-to-testis transition gonads, GFP is expressed in cells at the periphery of the gonad while no GFP was found in the peri-follicular cells surrounding remaining oocytes (Figure 7).

Discussion

The cyp19a1a gene is expressed both in female and male gonads

In the present study, we established a novel and useful transgenic line of zebrafish expressing GFP under the control of the zebrafish cyp19a1a gene promoter. In these transgenic animals, GFP was expressed in both male and female gonads with a 9-fold higher fluorescence level in ovaries, which is consistent with the crucial production of estrogens in females with important roles in oocyte growth and hepatic vitellogenin synthesis [START_REF] Nagahama | Endocrine regulation of gametogenesis in fish[END_REF]. This dimorphic GFP expression is in agreement with the higher expression of cyp19a1a gene [START_REF] Fenske | Aromatase modulation alters gonadal differentiation in developing zebrafish (Danio rerio)[END_REF][START_REF] Sawyer | Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish: Gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation[END_REF][START_REF] Trant | Developmental expression of cytochrome P450 aromatase genes (CYP19a and CYP19b) in zebrafish fry (Danio rerio)[END_REF] and Cyp19a1a protein [START_REF] Caulier | Localization of steroidogenic enzymes and Foxl2a in the gonads of mature zebrafish (Danio rerio)[END_REF] measured in wild type zebrafish ovaries compared to testis as well as with the higher circulating concentrations of estradiol in female zebrafish [START_REF] Deng | Chronic exposure to environmental levels of tribromophenol impairs zebrafish reproduction[END_REF][START_REF] Ji | Effects of Bisphenol S Exposure on Endocrine Functions and Reproduction of Zebrafish[END_REF][START_REF] Liu | Effects of TDCPP or TPP on gene transcriptions and hormones of HPG axis, and their consequences on reproduction in adult zebrafish (Danio rerio)[END_REF][START_REF] Liu | Endocrine disruption potentials of organophosphate flame retardants and related mechanisms in H295R and MVLN cell lines and in zebrafish[END_REF][START_REF] Ma | Disruption of endocrine function in in vitro H295R cell-based and in in vivo assay in zebrafish by 2,4dichlorophenol[END_REF][START_REF] Yu | Endocrine disruption and reproduction impairment in zebrafish after long-term exposure to DE-71[END_REF].

In ovaries, GFP was detected in the cytoplasm of peri-follicular cells (thecal and granulosa cells) surrounding both previtellogenic and vitellogenic oocytes with a stronger expression around vitellogenic oocytes. This pattern of expression is similar to the wild type pattern of cyp19a1a gene and its protein in zebrafish ovaries [START_REF] Caulier | Localization of steroidogenic enzymes and Foxl2a in the gonads of mature zebrafish (Danio rerio)[END_REF][START_REF] Goto-Kazeto | Localization and expression of aromatase mRNA in adult zebrafish[END_REF][START_REF] Rodriguez-Mari | Characterization and expression pattern of zebrafish anti-Mullerian hormone (amh) relative to sox9a, sox9b, and cyp19a1a, during gonad development[END_REF][START_REF] Wang | Anti-Mullerian hormone and 11 beta-hydroxylase show reciprocal expression to that of aromatase in the transforming gonad of zebrafish males[END_REF] and to another recently developed cyp19a1a-EGFP transgenic zebrafish [START_REF] Dranow | Bmp15 Is an Oocyte-Produced Signal Required for Maintenance of the Adult Female Sexual Phenotype in Zebrafish[END_REF]. A slight GFP fluorescence was also measured in the cytoplasm of oocytes. In other fish species such as medaka, rainbow trout, air-breathing catfish or gilthead seabream, cyp19a1a gene or its protein product have already been detected in the germinal compartment, i.e. the ooplasm [START_REF] Gohin | Aromatase is expressed and active in the rainbow trout oocyte during final oocyte maturation[END_REF][START_REF] Park | Fluorescence in situ hybridization techniques (FISH) to detect changes in CYP19a gene expression of Japanese medaka (Oryzias latipes)[END_REF][START_REF] Raghuveer | Dimorphic expression of various transcription factor and steroidogenic enzyme genes during gonadal ontogeny in the air-breathing catfish, Clarias gariepinus[END_REF][START_REF] Zapater | Piscine folliclestimulating hormone triggers progestin production in gilthead seabream primary ovarian follicles[END_REF], and proved to be functional in rainbow trout late vitellogenic oocytes [START_REF] Gohin | Aromatase is expressed and active in the rainbow trout oocyte during final oocyte maturation[END_REF].

In zebrafish, however, due to the presence of Cyp19a1b protein in the ooplasm [START_REF] Caulier | Localization of steroidogenic enzymes and Foxl2a in the gonads of mature zebrafish (Danio rerio)[END_REF], the specific functionality of Cyp19a1a protein remains difficult to demonstrate in this compartment.

In testis, both Leydig cells and germ cells expressed GFP. In wild type zebrafish testis, while cyp19a1a gene expression has been measured, no mRNA localization was achieved (Hinfray et al., 2006a;[START_REF] Rodriguez-Mari | Characterization and expression pattern of zebrafish anti-Mullerian hormone (amh) relative to sox9a, sox9b, and cyp19a1a, during gonad development[END_REF][START_REF] Sawyer | Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish: Gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation[END_REF][START_REF] Wang | Anti-Mullerian hormone and 11 beta-hydroxylase show reciprocal expression to that of aromatase in the transforming gonad of zebrafish males[END_REF]. Nevertheless, this GFP localization to Leydig and germ cells is consistent with our previous studies showing the same pattern of localization of Cyp19a1a protein in wild type zebrafish testis [START_REF] Caulier | Localization of steroidogenic enzymes and Foxl2a in the gonads of mature zebrafish (Danio rerio)[END_REF][START_REF] Hinfray | Cyp17a1 and Cyp19a1 in the zebrafish testis are differentially affected by oestradiol[END_REF]. In other teleosts, cyp19a1a gene and/or protein have been detected in both Leydig cells [START_REF] Guiguen | Ovarian aromatase and estrogens: A pivotal role for gonadal sex differentiation and sex change in fish[END_REF][START_REF] Kobayashi | Characterization of two types of cytochrome P450 aromatase in the serial-sex changing gobiid fish, Trimma okinawae[END_REF][START_REF] Kotula-Balak | Aromatase expression in testes of XY, YY, and XX rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Vinas | Stage-specific gene expression during fish spermatogenesis as determined by laser-capture microdissection and quantitative-PCR in sea bass (Dicentrarchus labrax) gonads[END_REF][START_REF] Von Schalburg | Sex-specific expression, synthesis and localization of aromatase regulators in one-year-old Atlantic salmon ovaries and testes[END_REF] and germ cells [START_REF] Kotula-Balak | Aromatase expression in testes of XY, YY, and XX rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Vinas | Stage-specific gene expression during fish spermatogenesis as determined by laser-capture microdissection and quantitative-PCR in sea bass (Dicentrarchus labrax) gonads[END_REF][START_REF] Von Schalburg | Sex-specific expression, synthesis and localization of aromatase regulators in one-year-old Atlantic salmon ovaries and testes[END_REF] of the testis. All these data argue for a role of estrogens in fish testis that might be linked to the involvement of estradiol in spermatogonial stem cell renewal in fish [START_REF] Miura | Estradiol-17[beta] Stimulates the Renewal of Spermatogonial Stem Cells in Males[END_REF][START_REF] Miura | Complementary deoxyribonucleic acid cloning of spermatogonial stem cell renewal factor[END_REF].

Altogether, expression of GFP in the cyp19a1a-eGFP transgenic zebrafish is consistent with the expression of endogenous cyp19a1a gene expression in the gonads of wild type zebrafish. The presence of the CrystBB1-CFP reporter allows for early selection of transgenic eggs and does not seem to disturb the expression of the cyp19a1a promoter. These combine to make of this line a useful and powerful tool to monitor cyp19a1a expression and determine its precise role in different physiological processes.

Down-regulation of gonadal cyp19a1a expression is likely responsible for testicular differentiation

In the present study, the cyp19a1a-eGFP transgenic zebrafish was used to analyze the spatiotemporal expression of the cyp19a1a gene in individual gonads during the sexual differentiation process. At the early stages (from 20 dpf to 35 dpf), a slight GFP fluorescence was detected in all cell types of all immature gonads. At 20 dpf, cyp19a1a mRNA and protein were also localized in the cytoplasm of all cell types of zebrafish gonads. This early expression of the cyp19a1a gene has already been evidenced in wild type zebrafish trunks by RT-PCR [START_REF] Fenske | Aromatase modulation alters gonadal differentiation in developing zebrafish (Danio rerio)[END_REF][START_REF] Wang | Anti-Mullerian hormone and 11 beta-hydroxylase show reciprocal expression to that of aromatase in the transforming gonad of zebrafish males[END_REF] and individual gonads by in situ hybridization [START_REF] Rodriguez-Mari | Characterization and expression pattern of zebrafish anti-Mullerian hormone (amh) relative to sox9a, sox9b, and cyp19a1a, during gonad development[END_REF], and also in gonads of the TgBAC(cyp19a1a:EGFP) uc44 zebrafish of 13 dpf [START_REF] Dranow | Bmp15 Is an Oocyte-Produced Signal Required for Maintenance of the Adult Female Sexual Phenotype in Zebrafish[END_REF]. However, contrary to the localization of cyp19a1a mRNA by in situ hybridization which has been shown to be restricted to some regions of the immature gonads [START_REF] Rodriguez-Mari | Characterization and expression pattern of zebrafish anti-Mullerian hormone (amh) relative to sox9a, sox9b, and cyp19a1a, during gonad development[END_REF], a wider distribution of GFP and Cyp19a1a protein within the cyp19a1a-eGFP gonads and of cyp19a1a mRNA within the wild type gonads is reported herein. The rationale behind this discrepancy is not known at present and deserves further study.

In cyp19a1a-eGFP female ovaries, GFP was mainly localized to the cytoplasm of peri-follicular cells which is consistent with the localization of cyp19a1a mRNA in wild type zebrafish ovaries [START_REF] Rodriguez-Mari | Characterization and expression pattern of zebrafish anti-Mullerian hormone (amh) relative to sox9a, sox9b, and cyp19a1a, during gonad development[END_REF][START_REF] Wang | Anti-Mullerian hormone and 11 beta-hydroxylase show reciprocal expression to that of aromatase in the transforming gonad of zebrafish males[END_REF]. In ovaries, GFP fluorescence was higher than in immature gonads and increased during ovarian development in relation with the key role of cyp19a1a in ovarian differentiation in fish [START_REF] Guiguen | Ovarian aromatase and estrogens: A pivotal role for gonadal sex differentiation and sex change in fish[END_REF]. In gonads of cyp19a1a-eGFP fish that undergo ovary-to-testis transition, GFP fluorescence levels were intermediate between females and males or similar to males. This pattern of gender expression of GFP is similar to the pattern of cyp19a1a gene expression in wild type zebrafish trunks/gonads [START_REF] Rodriguez-Mari | Characterization and expression pattern of zebrafish anti-Mullerian hormone (amh) relative to sox9a, sox9b, and cyp19a1a, during gonad development[END_REF][START_REF] Wang | Anti-Mullerian hormone and 11 beta-hydroxylase show reciprocal expression to that of aromatase in the transforming gonad of zebrafish males[END_REF]. The results of the present study suggest that cyp19a1a expression is necessary for the maintenance of the ovarian fate and that the down-regulation of cyp19a1a expression is likely responsible for testicular differentiation. Estrogens depletion may be one of the reasons leading to the degeneration of ovary during gonadal transformation in males as previously suggested by Wang and collaborators (2007). Recent studies in zebrafish but also tilapia and common carp showed that exposure of adult fish to aromatase inhibitors could lead to the transdifferentiation of fully differentiated ovaries into testis [START_REF] Ogawa | Post-sex differentiational' sex reversal in the feamle common carp (Cyprinus carpio)[END_REF][START_REF] Sun | Transdifferentiation of differentiated ovary into functional testis by long-term treatment of aromatase inhibitor in Nile tilapia[END_REF][START_REF] Takatsu | Induction of Female-to-Male Sex Change in Adult Zebrafish by Aromatase Inhibitor Treatment[END_REF]. Moreover, in tilapia, this transdifferentiation could be rescued by exogenous estradiol supplementation [START_REF] Sun | Transdifferentiation of differentiated ovary into functional testis by long-term treatment of aromatase inhibitor in Nile tilapia[END_REF], confirming the importance of estrogens in ovarian maintenance.

Estrogens in fish are produced by two different aromatase proteins, i.e. Cyp19a1a and Cyp19a1b. While some results suggested a bimodal expression of cyp19a1b gene in the entire zebrafish larvae during the sexual differentiation process [START_REF] Trant | Developmental expression of cytochrome P450 aromatase genes (CYP19a and CYP19b) in zebrafish fry (Danio rerio)[END_REF], more detailed studies established that cyp19a1b gene and protein are not expressed in a sexually dimorphic manner in the zebrafish larvae heads/brains during gonadal differentiation [START_REF] Kallivretaki | The zebrafish, brain-specific, aromatase cyp19a2 is neither expressed nor distributed in a sexually dimorphic manner during sexual differentiation[END_REF][START_REF] Vosges | 17alpha-ethinylestradiol disrupts the ontogeny of the forebrain GnRH system and the expression of brain aromatase during early development of zebrafish[END_REF]. Moreover, a recent study showed no defects in the sexual differentiation process of cyp19a1b loss-of-function mutant zebrafish while cyp19a1a loss-of-function mutant zebrafish all developed as males [START_REF] Dranow | Bmp15 Is an Oocyte-Produced Signal Required for Maintenance of the Adult Female Sexual Phenotype in Zebrafish[END_REF]. Current studies thus do not support a decisive role of Cyp19a1b in the phenotypic sex orientation of the zebrafish gonads. Altogether, these observations rather support earlier studies [START_REF] Fenske | Aromatase modulation alters gonadal differentiation in developing zebrafish (Danio rerio)[END_REF][START_REF] Mcallister | Early life exposure to environmental levels of the aromatase inhibitor tributyltin causes masculinisation and irreversible sperm damage in zebrafish (Danio rerio)[END_REF][START_REF] Uchida | An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sex-reversal[END_REF][START_REF] Wang | Anti-Mullerian hormone and 11 beta-hydroxylase show reciprocal expression to that of aromatase in the transforming gonad of zebrafish males[END_REF] that favor the hypothesis of Cyp19a1a being the rate-limiting step in female sexual development.

Regarding males, GFP expression was detectable in the present study in male gonads of the cyp19a1a-eGFP transgenic fish and this persisted during male development contrary to the absence of mRNA detection by in situ hybridization [START_REF] Rodriguez-Mari | Characterization and expression pattern of zebrafish anti-Mullerian hormone (amh) relative to sox9a, sox9b, and cyp19a1a, during gonad development[END_REF]. The presence of GFP in testis is relevant regarding the known cyp19a1a gene expression in male gonads both at the adult stage (Hinfray et al., 2006a;[START_REF] Sawyer | Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish: Gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation[END_REF] and during development [START_REF] Wang | Anti-Mullerian hormone and 11 beta-hydroxylase show reciprocal expression to that of aromatase in the transforming gonad of zebrafish males[END_REF].

Estrogen receptors have also been localized in the testis of many fish including zebrafish [START_REF] Bouma | Estrogen receptor-alpha protein localization in the testis of the rainbow trout (Oncorhynchus mykiss) during different stages of the reproductive cycle[END_REF][START_REF] Chandrasekar | Levels of 17betaestradiol receptors expressed in embryonic and adult zebrafish following in vivo treatment of natural or synthetic ligands[END_REF][START_REF] Menuet | Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions[END_REF][START_REF] Vinas | Stage-specific gene expression during fish spermatogenesis as determined by laser-capture microdissection and quantitative-PCR in sea bass (Dicentrarchus labrax) gonads[END_REF][START_REF] Wu | Localization of estrogen receptor alpha and beta RNA in germinal and nongerminal epithelia of the channel catfish testis[END_REF]. Altogether, these data suggest a role of estrogens in testis development, steroid production and spermatogenesis. Indeed, evidences indicate that spermatogonial stem cell renewal is regulated by estradiol [START_REF] Miura | Estradiol-17[beta] Stimulates the Renewal of Spermatogonial Stem Cells in Males[END_REF][START_REF] Miura | Complementary deoxyribonucleic acid cloning of spermatogonial stem cell renewal factor[END_REF].

cyp19a1a-eGFP transgenic zebrafish as a reliable model to investigate reproductive functions and their perturbations by EDCs

During the recent years, an increasing interest has emerged as regards in vivo transgenic fish models to assess environmental hazard of chemicals notably EDCs. Several transgenic fish models have been developed to study the effects of chemicals acting on the endocrine system through nuclear receptor pathways such as estrogen receptors [START_REF] Gorelick | Visualization of estrogen receptor transcriptional activation in zebrafish[END_REF][START_REF] Green | High-Content and Semi-Automated Quantification of Responses to Estrogenic Chemicals Using a Novel Translucent Transgenic Zebrafish[END_REF][START_REF] Legler | Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity[END_REF] or glucocorticoid receptor [START_REF] Weger | A chemical screening procedure for glucocorticoid signaling with a zebrafish larva luciferase reporter system[END_REF] and disrupting the tissuespecific expression of hormone-regulated genes in the brain [START_REF] Brion | Screening estrogenic activities of chemicals or mixtures in vivo using transgenic (cyp19a1b-GFP) zebrafish embryos[END_REF] or in the liver [START_REF] Kurauchi | In vivo visual reporter system for detection of estrogen-like substances by transgenic medaka[END_REF][START_REF] Sébillot | Rapid Fluorescent Detection of (Anti)androgens with spiggin-gfp Medaka[END_REF][START_REF] Spirhanzlova | Oestrogen reporter transgenic medaka for non-invasive evaluation of aromatase activity[END_REF]. Mechanism-based in vivo fish bioassays that use transgenic fish were recently developed and have been shown to be very useful tools to screen EDCs alone or in combination [START_REF] Brion | Screening estrogenic activities of chemicals or mixtures in vivo using transgenic (cyp19a1b-GFP) zebrafish embryos[END_REF][START_REF] Cano-Nicolau | Several synthetic progestins disrupt the glial cell specific-brain aromatase expression in developing zebrafish[END_REF][START_REF] Gorelick | Visualization of estrogen receptor transcriptional activation in zebrafish[END_REF][START_REF] Green | High-Content and Semi-Automated Quantification of Responses to Estrogenic Chemicals Using a Novel Translucent Transgenic Zebrafish[END_REF][START_REF] Hinfray | Additive effects of levonorgestrel and ethinylestradiol on brain aromatase (cyp19a1b) in zebrafish specific in vitro and in vivo bioassays[END_REF], some of them being under validation at the OECD level (i.e. EASZY assay that uses cyp19a1b-GFP zebrafish embryos). The usefulness of the newly developed cyp19a1a-eGFP line for studies on environmental effects of EDCs need to be further investigated. However, we demonstrated that this model has favorable and interesting features such as being a stable transgenic line, possessing only one transgene insertion with the possibility of inheritance of the transgene from both males and females making this line easier to maintain. Further, in adult cyp19a1a-eGFP transgenic zebrafish ovaries, pharmacological treatment with E2 resulted in inhibition of GFP fluorescence in a similar manner to cyp19a1a gene expression in wild type zebrafish ovaries (Hinfray et al., 2006a). Furthermore, the clear involvement of "ovarian" aromatase in the processes of reproduction and sexual differentiation in fish makes this transgenic zebrafish line a powerful model for studies onto these physiological processes and their perturbations by EDCs.

Currently, at OECD, several test guidelines for screening and testing of endocrine active substances have been adapted in fish to detect compounds acting as aromatase inhibitors (Test Guideline 229/230) and affecting the sex development of the gonads and/or the sex-ratio (Test Guideline 234).

The use of cyp19a1a-eGFP line for these assays should allow the relative quantification of GFP in gonads through image analysis thus providing additional mechanistic insight on aromatase that are currently lacking in OECD test guidelines. To do so, it would however be advisable to (i) evaluate the sensitivity of the cyp19a1a-eGFP zebrafish line to estrogens and also to other EDCs such as aromatase modulating compounds to refine actual mechanistic based assays, and (ii) improve the in vivo measurement of GFP in developing and adult fish in a non-invasive way to assess the timedependent effect of chemicals on GFP without increasing the number of fish. This could be notably achieved by cross-breeding our transgenic fish with the casper mutant zebrafish that do not develop pigments [START_REF] White | Transparent adult zebrafish as a tool for in vivo transplantation analysis[END_REF]. Altogether, the time-and concentration-dependent measurement of GFP in live transgenic cyp19a1a-eGFP together with other "classical" endpoints (e.g., vitellogenin concentrations, gonadal histology) would usefully bring complementary comprehensive data for the refinement of Adverse Outcome Pathways (AOPs) and the development of quantitative AOPs thereby increasing the relevance of such model and bioassays for advanced assessment of EDCs in fish.

Conclusions

In the present study, we successfully established a stable transgenic zebrafish line expressing GFP under the control of zebrafish cyp19a1a gene promoter. In the cyp19a1a-eGFP zebrafish line, GFP is expressed, localized and down-regulated by estradiol in the same way as cyp19a1a gene in wild type zebrafish showing that GFP can be used reliably to monitor cyp19a1a expression. In the cyp19a1a-eGFP transgenic line, our results confirm that cyp19a1a is expressed early during development, before the histological differentiation of the gonads, and that the down-regulation of cyp19a1a expression is likely responsible for testicular differentiation. Moreover, we show that although cyp19a1a expression exhibits a clear dimorphic expression pattern in gonads during sexual differentiation, its expression persists whatever the sex suggesting that estradiol synthesis is important for gonadal development including for testis where the expression was low but significant. In the end, monitoring the expression of GFP in control and exposed-fish will help determine the sensitivity of this transgenic line to EDCs and its relevance to refine mechanistic based-assays for the study of EDCs. In fine, this transgenic zebrafish line is a useful tool to study physiological processes such as reproduction and sexual differentiation, and their potential perturbations by EDCs.

  in a 50 µL PCR mix (1 µL of genomic DNA (14 ng), KAPA Long Range HotStart DNA Polymerase (KAPA Biosystems, 1.25 U), 5x Kapa Long range buffer (1 µL), dNTPs (0.3 mM each), MgCl2 (1.75 mM) and 0.5 µM of each primer) to run the first PCR: 1x(94 o C -3 sec); 35x(94 o C -25 sec; 49 o C -15 sec; 68 o C -11 min 30 sec); 1x(72 o C

Figure 1 :

 1 Figure 1: Relative quantification of GFP fluorescence in mature zebrafish gonads of the cyp19a1a-GFP transgenic line. (A) Imaging of GFP fluorescence (green) in the entire gonads using a fluorescence imaging system IVIS Lumina II from Perkin Elmer (Top) and a Discovery V8 stereomicroscope from Carl Zeiss (Bottom). Scale bars = 50 μm (B) Relative quantification of GFP fluorescence in the entire gonads of females (N=6) and males (N=6) with the IVIS Lumina II system. Results are expressed as the median value (bars in the middle of the box), upper quartile (upper edge of the box) and lower quartile (lower edge of the box). Different letters indicate a statistically significant difference between the groups (Wilcoxon's Test, p<0.05).

Figure 2 :

 2 Figure 2: GFP fluorescence localization in zebrafish gonads of the cyp19a1a-GFP transgenic line. In 55 days post fertilization (dpf) zebrafish, GFP was localized to the cytoplasm of peri-follicular cells surrounding pre-vitellogenic oocytes (A,B). In mature ovary of adult zebrafish, GFP is mainly immunolocalized in the cytoplasm of peri-follicular cells (granulosa and thecal cells) surrounding vitellogenic oocytes and some pre-vitellogenic oocytes (C-G). (D) Enlargement of peri-follicular cells from micrograph C. (F) Enlargement of peri-follicular cells from micrograph E. In mature testis, GFP localized to the cytoplasm of both Leydig cells and germ cells (H). Imaging was realized with an Olympus FV1000 confocal microscope. Acquisition parameters were optimized for each micrograph independently. Nuclei are in blue. PO: pre-vitellogenic oocyte; VO: vitellogenic oocyte; LC: Leydig cells; SC: spermatocytes; SG: spermatogonia; ST: spermatids; SZ: spermatozoa. White bars: 50 μm; Red bar: 20 μm; Yellow bars: 10 μm.

Figure 3 :

 3 Figure 3: Co-localization of GFP and Cyp19a1a in mature zebrafish gonads of the cyp19a1a-GFP transgenic line. GFP (green) and Cyp19a1a (Red) proteins were immunolocalized both in ovaries (Top) and testis (Bottom). The merge of the two is showed in yellow. Nuclei are in blue. Imaging was realized with an Olympus confocal microscope FV1000. Acquisition parameters were optimized for each sex independently. White bars: 50 μm.

Figure 4 :

 4 Figure 4: Relative quantification of GFP fluorescence in mature cyp19a1a-GFP transgenic zebrafish ovaries after a 7-days exposure to 10 nM of Estradiol (E2) or solvent (DMSO). Results are expressed as the median value (bars in the middle of the box), upper quartile (upper edge of the box) and lower quartile (lower edge of the box). For each individual (n=4 individuals / condition), four micrographs of ovarian sections were analyzed. Different letters indicate a statistically significant difference between the groups (Wilcoxon's Test, p<0.05).

Figure 5 :

 5 Figure 5: Relative quantification of GFP fluorescence in gonads of cyp19a1a-GFP transgenic zebrafish during development. Results are expressed as mean ± S.E.M. Number of fish analyzed is indicated in the bars of the histogram and 3-4 sections were analyzed for each fish. Different letters indicate statistically significant differences across development for one sex (Wilcoxon's test, p<0.05). * and ** indicate statistically significant differences between sexes within one stage of development (Wilcoxon's test, * p<0.05, ** p<0.01). dpf: days post fertilization.

Figure 6 :

 6 Figure 6: Localization of GFP and cyp19a1a protein and mRNA in 20 dpf gonads of zebrafish. A:Imaging of GFP fluorescence (green) was realized directly on cryo-sections of cyp19a1a-GFP transgenic zebrafish gonads. B: Cyp19a1a (Red) proteins were immunolocalized using the anti-Cyp19a1a antibody in cyp19a1a-GFP transgenic zebrafish gonads. C: cyp19a1a mRNA was localized by whole mount in situ hybridization in wild type zebrafish gonads. Fluorescence imaging was realized using a Zeiss AxioImager Z1 fluorescence microscope equipped with a MRm5 camera combined with an ApoTome. Nuclei are in blue. Acquisition parameters were optimized for each micrograph independently. Transmitted light imaging was performed using a Nikon 90i microscope equipped with a Nikon DS Ri1 camera. dpf: days post fertilization. Scale bars: 20 μm.

Figure 7 :

 7 Figure 7: Localization of GFP fluorescence in 40 dpf gonads of cyp19a1a-GFP transgenic zebrafish. Imaging of GFP fluorescence (green) was realized directly on cryo-sections using a Zeiss AxioImager Z1 fluorescence microscope equipped with an MRm5 camera combined with an ApoTome. Acquisition parameters were optimized for each micrograph independently. Nuclei are in blue. dpf: days post fertilization. Scale bars: 50 μm.

Table 1 : Transmission of the transgene from the five cyp19a1a-eGFP founders from generation 0 (F0) to generation 1 (F1)

 1

	ID and sex of the	Total number of	Number	of	%	positive
	transgenic	fertilized eggs	transgenic		progeny
	founders (F0)		embryos (F1)
	1 / female	339	149		44
	2 / female	51	11		22
	3 / female	103	26		25
	4 / female	383	80		21
	5 / male	234	88		38

Table 2 : Transmission rate of the transgene from generation 3 (F3) to generation 4 (F4) in the cyp19a1a

 2

	ID and sex of the	Total number of	Number of transgenic	Transmission rate
	transgenic fish (F3)	fertilized eggs	embryos (F4)	(%)
	1 / female	388	196	50.5
	2 / female	187	87	46.5
	3 / female	216	104	48.1
	4 / male	203	93	45.8
	5 / male	183	87	47.5
	6 / male	123	64	52.0

-GFP transgenic zebrafish line. 6 transgenic zebrafish were cross-breaded with wild type AB zebrafish.

Acknowledgements

This work was supported by the French National Ministry of Ecology and Sustainable Development [Program 190 ECOT AP 2013-2016; NEMO project and Program 181-DRC 50] and by the French National Research Program for Environmental and Occupational Health of Anses [AIDEZ project n° EST-2016/1/189]. AMAGEN received the support of the French National Research Agency (ANR) "Investissement d'Avenir" program [TEFOR ANR-II-INSB-0014]. The authors wish to acknowledge the technical support of: S. Bruneau and A. Heuzé for the design and production of transgenesis vectors; Z. Radev and J. Edouard for the promoter cloning and transgenic generation; M. Simion and L. Legendre for zebrafish husbandry.

Declaration of interests

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

SUPPLEMENTARY MATERIALS

Supplemental Table 1: Sequence of the primers used to clone and verify the promoter of Cyp19a1a. The underlined sequences in Cyp19a1a_B3 and Cyp19a1a_B5 primers correspond respectively to the attB3 and attB5 sites inserted during the second round of nested PCR. Those recombination sites are necessary for the cloning by Gateway(R) recombination.

Used for

Primer name Sequence 5: Specificity controls of anti-GFP and anti-Cyp19a1a antibodies to zebrafish gonads. GFP (green, A-D) protein was immunolocalized both in testis (Left) and ovary (Right) of mature zebrafish. A: testis of a cyp19a1a-GFP transgenic zebrafish showing a labeling in Leydig and germ cells. B: ovary of a cyp19a1a-GFP transgenic zebrafish showing labeling in perifollicular cells. C: testis of a wild type zebrafish showing a very slight green fluorescence background. D: ovary of a wild type zebrafish showing no labeling. Cyp19a1a (E-H) protein was immunolocalized in testis (Left) and ovary (Right) of zebrafish. Cyp19a1a specific labeling is detected in the Leydig and germ cells of the testis (E) and in the peri-follicular cells of the oocytes (F). When this anti-Cyp19a1a antibody is preincubated with the synthetic peptide (100 μg/ml), no labelling is observed in the testis (G) and only a slight unspecific labeling is observed in ooplasm of oocytes (H). Without any primary antibody (secondary antibody: AlexaFluor® 594), no labeling was observed neither in the testis (I) nor in the ovary (J). Nuclei are in blue. Imaging was realized with a Zeiss AxioImager Z1 fluorescence microscope equipped with an MRm5 camera combined with an ApoTome. Acquisition parameters were the same for all GFP photomicrographs and for all Cyp19a1a photomicrographs. Scale bars: 20 μm.