Joel Cano-Nicolau

Clémentine Garoche

Nathalie Hinfray

Elisabeth Pellegrini

Noureddine Boujrad

Farzad Pakdel

Olivier Kah
email: oliver.kah@univ-rennes1.fr

François Brion
email: francois.brion@ineris.fr

Several synthetic progestins disrupt the glial cell specific-brain aromatase expression in developing zebra fish

Keywords: progestins, brain aromatase, radial glial cells, cyp19a1b-GFP, zebrafish

The effects of some progestins on fish reproduction have been recently reported revealing the hazard of this class of steroidal pharmaceuticals. However, their effects at the central nervous system level have been poorly studied until now. Notwithstanding, progesterone, although still widely considered primarily a sex hormone, is an important agent affecting many central nervous system functions. Herein, we investigated the effects of a large set of synthetic ligands of the nuclear progesterone receptor on the glial-specific expression of the zebrafish brain aromatase (cyp19a1b) using zebrafish mechanism-based assays. Progesterone and 24 progestins were first screened on transgenic cyp19a1b-GFP zebrafish embryos. We showed that progesterone, dydrogesterone, drospirenone and all the progesterone-derived progestins had no effect on GFP expression. Conversely, all progestins derived from 19-nortesterone induced GFP in a concentration-dependent manner with EC 50 ranging from the low nM range to hundreds nM.

The 19-nortestosterone derived progestins levonorgestrel (LNG) and norethindrone (NET) were further tested in a radial glial cell context using U251-MG cells co-transfected with zebrafish ER subtypes (zfERα, zfERβ1 or zfERβ2) and cyp19a1b promoter linked to luciferase. Progesterone had no effect on luciferase activity while NET and LNG induced luciferase activity that was blocked by ICI 182,780. Zebrafish-ERs competition assays showed that NET and LNG were unable to bind to ERs, suggesting that the effects of these compounds on cyp19a1b require metabolic activation prior to elicit estrogenic activity.

Overall, we demonstrate that 19-nortestosterone derived progestins elicit estrogenic activity by inducing cyp19a1b expression in radial glial cells. Given the crucial role of radial glial cells and neuro-estrogens in early development of brain, the consequences of exposure of fish to these compounds require further investigation.

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Introduction

During the past twenty years, numerous studies have examined the effects of estrogenic compounds because these substances were among the first identified as endocrine disrupting compounds (EDCs) responsible for the feminization of wild populations of fish observed worldwide [START_REF] Sumpter | Endocrine disrupters in the aquatic environment: An overview[END_REF]. Among the most studied xeno-estrogens, a particular attention has been paid to 17α-ethinylestradiol (EE2), a potent synthetic steroidal estrogen used as pharmaceuticals in contraceptive pills. From these multiple studies, the mechanism of action and the adverse effects of EE2 on reproductive fitness of aquatic species have been largely

documented (e.g., [START_REF] Nash | Long-term exposure to environmental concentrations of the pharmaceutical ethynylestradiol causes reproductive failure in fish[END_REF][START_REF] Kidd | Collapse of a fish population after exposure to a synthetic estrogen[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][START_REF] Frye | Endocrine disrupters: a review of some sources, effects, and mechanisms of actions on behaviour and neuroendocrine systems[END_REF][START_REF] Soffker | Endocrine disrupting chemicals and sexual behaviors in fish-a critical review on effects and possible consequences[END_REF], which permitted the derivation of Environmental Quality Standard (EQS) for freshwater community (E.C., 2011) and the inscription of EE2 on the watch list of the Water Framework Directive.

In addition to (xeno)-estrogens, there is now evidence of the occurrence of other natural and synthetic steroids released from urban, hospital or industrial waste water treatment plants (WWTPs) effluents [START_REF] Van Der Linden | Detection of multiple hormonal activities in wastewater effluents and surface water, using a panel of steroid receptor CALUX bioassays[END_REF][START_REF] Chang | Occurrence of androgens and progestogens in wastewater treatment plants and receiving river waters: comparison to estrogens[END_REF]. In a recent study, the release of hormonally active pharmaceuticals from an industrial WWTP effluent has been associated with altered reproductive physiological functions such as abnormal vitellogenin synthesis, intersex and altered sex-ratio in wild population of gudgeon [START_REF] Sanchez | Adverse effects in wild fish living downstream from pharmaceutical manufacture discharges[END_REF][START_REF] Creusot | Identification of synthetic steroids in river water downstream from pharmaceutical manufacture discharges based on a bioanalytical approach and passive sampling[END_REF]; thereby stressing the need to increase our knowledge of the effects of steroidal pharmaceuticals on aquatic organisms and ecosystems [START_REF] Creusot | Identification of synthetic steroids in river water downstream from pharmaceutical manufacture discharges based on a bioanalytical approach and passive sampling[END_REF][START_REF] Fent | Progestins as endocrine disrupters in aquatic ecosystems: Concentrations, effects and risk assessment[END_REF].

Among steroidal pharmaceuticals, several authors have pointed out the hazard and risks posed by synthetic progestogens on aquatic species [START_REF] Sumpter | Endocrine disrupters in the aquatic environment: An overview[END_REF][START_REF] Besse | Progestagens for human use, exposure and hazard assessment for the aquatic environment[END_REF], Runnalls et al., 2010[START_REF] Fent | Progestins as endocrine disrupters in aquatic ecosystems: Concentrations, effects and risk assessment[END_REF]. Progestagens, also called gestagens or progestins are widely used, not only in the formulation of contraceptive pills, but also in various medical

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applications such as hormone replacement therapy, secondary amenorrhea, dysfunctional uterine bleeding, endometriosis, and in the treatment of several cancers. A large number of progestins that are used as active ingredients of contraceptive pills are used either alone or in combination with estrogens. Part of them are 19-norprogesterone derivatives, often termed pure progestational molecules since they act only through progesterone receptor [START_REF] Sitruk-Ware | Pharmacological profile of progestins[END_REF]. This progesterone-derived group includes notably medroxy-progesterone, medroxyprogesterone acetate and nestorone. A second group, referred to as testosterone-derivatives (19-Nortestosterone), includes estranes that have a methyl group at the C13 position and gonanes that do not. Among estranes figure ethisterone, norethindrone (NET; also known as norethisterone), norethindrone acetate, ethynodiol-diacetate, tibolone and etonogestrel.

Gonanes include compounds such as levonorgestrel, desogestrel, gestodene and norgestimate [START_REF] Kumar | The challenge presented by progestins in ecotoxicological research: a critical review[END_REF][START_REF] Sitruk-Ware | Pharmacological profile of progestins[END_REF]. Finally, new molecules have been produced recently, notably drospirenone, a derivative of spironolactone and an aldosterone antagonist [START_REF] Kumar | The challenge presented by progestins in ecotoxicological research: a critical review[END_REF][START_REF] Sitruk-Ware | Pharmacological profile of progestins[END_REF].

The presence of progestins in aquatic environments is much less documented as compared to xeno-estrogens and available data are limited to only few of them [START_REF] Kumar | The challenge presented by progestins in ecotoxicological research: a critical review[END_REF][START_REF] Fent | Progestins as endocrine disrupters in aquatic ecosystems: Concentrations, effects and risk assessment[END_REF]. Furthermore, it is noteworthy that nothing is known about the potential occurrence of their transformation products issued from biotic and/or abiotic process in aquatic environment. Notwithstanding, various studies reported that some progestins can be detected in industrial and urban WWTPs influents and effluents or surface waters at concentration ranging from low nanograms per liter up to 50 ng/L while in animal farm waste and runoff, their concentrations can reach high levels up to μg/L [START_REF] Chang | Occurrence of androgens and progestogens in wastewater treatment plants and receiving river waters: comparison to estrogens[END_REF][START_REF] Labadie | Development of an analytical procedure for determination of selected estrogens and progestagens in water samples[END_REF]Budzinski, 2005, Creusot et al., 2014, for reviews see Orlando and Ellstad, 2014, Kumar et al., 2015and Fent, 2015). Importantly, recent research performed on different fish and frog vertebrate models documented that some synthetic progestins disrupted expression of

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hormono-regulated genes and altered reproductive outputs at environmentally relevant concentrations [START_REF] Bluthgen | Effects of low concentrations of the antiprogestin mifepristone (RU486) in adults and embryos of zebrafish (Danio rerio): 1. Reproductive and early developmental effects[END_REF][START_REF] Han | The synthetic progestin megestrol acetate adversely affects zebrafish reproduction[END_REF][START_REF] Liang | Long-term exposure to environmentally relevant concentrations of progesterone and norgestrel affects sex differentiation in zebrafish (Danio rerio)[END_REF][START_REF] Overturf | Levonorgestrel exposure to fathead minnows (Pimephales promelas) alters survival, growth, steroidogenic gene expression and hormone production[END_REF][START_REF] Safholm | Mixture effects of levonorgestrel and ethinylestradiol: estrogenic biomarkers and hormone receptor mRNA expression during sexual programming[END_REF][START_REF] Zucchi | Transcriptional and physiological responses induced by binary mixtures of drospirenone and progesterone in zebrafish (Danio rerio)[END_REF].

Up to now, studies on the environmental effects of progestins in aquatic species are limited to only few progestins. They mainly focused on peripheral organs, such as liver and gonads, in mature fish and pointed out the risk posed by these substances on fish reproduction [START_REF] Zeilinger | Effects of synthetic gestagens on fish reproduction[END_REF][START_REF] Paulos | Reproductive responses in fathead minnow and Japanese medaka following exposure to a synthetic progestin[END_REF][START_REF] Runnalls | Several synthetic progestins with different potencies adversely affect reproduction of fish[END_REF]. Conversely, investigations on the effects of progesterone and progestins at early developmental stages of fish at the central nervous system are scarce. Notwithstanding, significant dysregulation of various genes in the brain of female zebrafish following exposure to drospirenone, a spironolactone derivative, and progesterone have been recently reported [START_REF] Zucchi | Progesterone alters global transcription profiles at environmental concentrations in brain and ovary of female zebrafish (Danio rerio)[END_REF][START_REF] Zucchi | Transcriptional and physiological responses induced by binary mixtures of drospirenone and progesterone in zebrafish (Danio rerio)[END_REF], Zhao et al., 2015). However, the underlying mechanisms are still poorly understood. The mechanisms by which progesterone acts in the brain are not fully defined; however in mammals progesterone is known to regulate signals in the brain involving sexually responsive behavior and also to affect gene expression in areas of the brain not involved in sexual behavior [START_REF] Graham | Physiological Action of Progesterone in Target Tissues[END_REF]. Nuclear progesterone receptor (nPR) are strongly expressed and exhibit a very large distribution within the brain, suggesting that the central actions of progestagens in fish probably extend beyond reproductive functions possibly including neurogenesis, neuronal plasticity, and/or neuroprotection (Diotel et al., 2011a). Recent data in zebrafish clearly show that nuclear progesterone receptor (nPR) was found to be highly expressed in the preoptic region of the hypothalamus (Hannah et al., 2010). nPR is expressed both neurons and radial glial cells (Diotel et al., 2011b) and the stronger expression of nPR in estrogen-synthesizing radial glial cells suggests a key role of steroids and a probably local action of neurosteroids on radial glial cells activity (Diotel et al., 2011b).

In zebrafish, nuclear progesterone receptor (nPR) was found to be highly expressed in the preoptic region of the hypothalamus (Hannah et al., 2010), but the effects of progestins through nPR would be minimal given that several progestins seems unable to activate nPR in

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different fish species [START_REF] Bain | Nortestosterone-derived synthetic progestogens do not activate the progestogen receptor of Murray-Darling rainbowfish (Melanotaenia fluviatilis) but are potent agonists of androgen receptors alpha and beta[END_REF][START_REF] Ellestad | Environmental gestagens activate fathead minnow (Pimephales promelas) nuclear progesterone and androgen receptors in vitro[END_REF]. Conversely, they may act through other pathways as they can interfere with multiple nuclear steroid receptors (e.g., androgen, glucocorticoid, mineralocorticoid and estrogen receptors) potentially inducing multiple hormonal activities [START_REF] Besse | Progestagens for human use, exposure and hazard assessment for the aquatic environment[END_REF]Garric, 2009, Fent, 2015).

In light of this context, this work aimed to assess the effects of progesterone (P4) and a large set of (anti-)progestins on the brain aromatase in zebrafish embryos, a commonly recognized alternative model in (eco)toxicology (Strahle et al., 2012). Brain aromatase, encoded by the cyp19a1b gene, is the enzyme responsible for the biosynthesis of neuroestrogens. We have previously shown that cyp19a1b expression is strongly stimulated by estrogens and that the estrogen-dependent cyp19a1b up-regulation requires the presence of functional estrogen receptors (ERs) and a glial X factor [START_REF] Menuet | Expression and estrogen-dependent regulation of the zebrafish brain aromatase gene[END_REF][START_REF] Page | Characterization of a cis-acting element involved in cell-specific expression of the zebrafish brain aromatase gene[END_REF]. Furthermore, we showed that expression of brain aromatase is restricted to radial glial cells of embryonic and adult zebrafish [START_REF] Menuet | Expression and estrogen-dependent regulation of the zebrafish brain aromatase gene[END_REF][START_REF] Tong | A cyp19a1b-gfp (aromatase B) transgenic zebrafish line that expresses GFP in radial glial cells[END_REF].

In the present work, the concentration-dependent effects of P4 and 24 (anti-)progestins on brain aromatase were quantified using the EASZY assay, a mechanism-based assay in tg(cyp19a1b-GFP) zebrafish embryos, that detects substances acting through estrogen receptors [START_REF] Brion | Screening estrogenic activities of chemicals or mixtures in vivo using transgenic (cyp19a1b-GFP) zebrafish embryos[END_REF]. Among the progestins tested, the molecular mode of action of two of them, levonorgestrel (LNG) and norethindrone (NET) were then further studied in the ER negative glial cell line U251-MG co-transfected with estrogen receptor zfERα, zfERβ1, or zfERβ2 [START_REF] Menuet | Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions[END_REF] together with a reporter luciferase gene linked to 500pb of the proximal promoter of zf cyp19a1b (Le [START_REF] Page | Assessment of xenoestrogens using three distinct estrogen receptors and the zebrafish brain aromatase gene in a highly responsive glial cell system[END_REF].

Herein, we reported that all the 19 nor-testosterone derivatives induced an early developmental effect in the developing brain of zebrafish by targeting radial glial cells and inducing brain aromatase through an ER-dependent mechanism at concentrations ranging from the low nM to hundreds nM. The estrogenic effect likely required metabolic activation

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of the parent compounds into estrogenic metabolites as they do not bind to zfERs. The (eco)toxicological relevance of this findings is discussed as regards the potential impact of progestins on fish on (neuro)-development.

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Material and Methods

Chemicals

Animals and Exposures to EDCs: EASZY assay

Fertilized tg(cyp19a1b-GFP) zebrafish eggs were exposed to chemicals or to solvent control (DMSO; 0.01% v/v). Each experimental group consisted of 20 embryos exposed in 25 ml of water. Embryos were kept in an incubator at 28°C, under semi-static conditions.

Exposures were performed from 0 to 4 days post-fertilization (dpf). At the end of the exposure period, 4-dpf old zebrafish were processed for fluorescence measurement by in vivo imaging using wide-field fluorescence microscopy. Each chemical was tested at least twice.

In Vivo Imaging: Wide-field Fluorescence Microscopy

Live tg(cyp19a1b-GFP) embryos were observed in dorsal view and each was

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photographed using a Zeiss AxioImager.Z1 fluorescence microscope equipped with a AxioCam Mrm camera (Zeiss GmbH, Göttingen, Germany). Only the head was photographed using a X10 objective, with a 134 ms exposure time at maximal intensity. Photographs were analyzed using the Zen software and fluorescence quantification was realized using the ImageJ software. For each picture, the integrated density was measured, i.e. the sum of the gray-values of all the pixels within the region of interest. A gray-value of 290 was defined as background value.

Plasmid constructs.

The zfER-α, zfER-β1, and zfER-β2 expression vectors correspond to TOPO-pcDNA3 expression vector (Invitrogen, San Diego, CA, USA), containing the coding regions of each cDNA receptor sequence as previously described [START_REF] Menuet | Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions[END_REF]. The cyp19a1bluciferase reporter gene consists of 500pb of the proximal promoter region of zebrafish cyp19a1b containing an estrogen response element (ERE), coupled to the luciferase reporter gene, as previously described [START_REF] Menuet | Expression and estrogen-dependent regulation of the zebrafish brain aromatase gene[END_REF].

In vitro transcription/translation of zfERs

To synthesize zfER proteins, we performed an in vitro translation reaction using the TNT Quick Coupled Transcription/Translation Systems kit (Promega) and adding 1 μg of each ER expression vector. The transcend Non-Radioactive Translation Detection Systems kit (Promega, Madison, Wisconsin, USA) was used to visualize all three receptors.

Cell culture and transfection

Cell culture and transfection were performed according to [START_REF] Page | Assessment of xenoestrogens using three distinct estrogen receptors and the zebrafish brain aromatase gene in a highly responsive glial cell system[END_REF]. In brief, U251-MG cells were maintained at 37°C in 5% CO2 atmosphere in phenol red-free Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 2,5% non-steroid fetal calf serum (Biowest, Nuaillé, France). The medium was also supplemented with 1mM Na-

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Pyruvate (Life Technologies, Saint Aubin, France), 4nM L-glutamine, and the antibiotics penicillin (20 U/mL), streptomycin (20 μg/mL), and amphotericin B (50 ng/mL) (Gibco, Carlsbad, CA, USA). For transfection, cells were plated in 24-well plates at a density of 0.25×10 5 cells/ml. In each well, 25 ng of expression vector, 25 ng of cytomegalovirus-βgalactosidase plasmid and 150 ng of luciferase reporter construct were transfected using jetPEI transfection reagent (Polyplus, Transfection, Illkirch, France). After one night, medium was replaced with fresh DMEM with xenoestrogen or vehicle. Luciferase activity was measured 24 hours later, using the luciferase assay system (Promega, Madison, WI, USA). βgalactosidase activity was used to normalize transfections efficiency in all experiments. Each experiment was performed in triplicate.

Estradiol receptor binding studies

The binding properties of LNG and NET to all three zfERs were monitored by radioactive competition assay [START_REF] Blair | The estrogen receptor relative binding affinities of 188 natural and xenochemicals: structural diversity of ligands[END_REF]. Cytosol aliquots of zfERs were incubated with 10 -9 M

[ 3 H]-E2 at 4°C overnight, in the absence or presence of increasing concentrations of radioinert E2 (10 -11 M; 10 -10 M, 10 -9 M, 10 -8 M, 10 -7 M) and both synthetic progestagens LNG and NET (10 -11 M; 10 -10 M, 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M). The relative binding affinities of the compounds for cytosolic zfER were evaluated by their capability to displace [ 3 H]-E2 from the zfER binding site. The results were expressed as a percentage of displaced [ 3 H]-E2 binding.

The 10 -7 M E2 contained a 100-fold excess of radioinert E2 compared to [ 3 H]-E2 and thus represents the non-specific binding [START_REF] Blair | The estrogen receptor relative binding affinities of 188 natural and xenochemicals: structural diversity of ligands[END_REF].

Data Analysis

Chemicals were tested in at least two independent experiments. Data are expressed as a mean fold induction above solvent control +/-standard error of the mean (SEM).

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Concentration-response curves were modeled using the Regtox 7.0.6 Microsoft Excel TM macro (http://www.normalesup.org/~vindimian/fr_index.html), which uses the Hill equation model and allows calculation of EC 50 . For a given chemical, EC 50 was defined as the concentration inducing 50% of its maximal effect. Data were first checked for normality using Shapiro-Wilk test, then homogeneity of variances was checked using Fligner-Killeen test. An ANOVA one-way test was used for the analysis of the variance, followed by Tukey HSD post-hoc test. When the data were not normal, they were log-transformed. If the data were still not normal data or the variances non-homogenous, Kruskal-Wallis test was used for the analysis of the variance. All analyses were conducted in R (R development Core Team, 2015).

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Results

Effects of progestins on GFP expression in transgenic cyp19a1b-GFP zebrafish embryos (EASZY assay).

The concentration-dependent-responses of E2, T, P5 as well as P4 and synthetic nPR agonists and antagonist ligands were first established on tg cyp19a1b-GFP zebrafish embryos assay. For each compound the effects were quantified using in vivo imaging and ECs were derived for active compounds (Table 2, Figure S1). As expected, E2 and T induced GFP expression in a concentration-dependent manner. P5 also significantly up-regulated GFP expression while P4 and its isomer DYD, as well as progestins structurally related to progesterone showed no effect on GFP expression in the radial glial cells of zebrafish (figure S1). This includes the compounds derived from 17α-hydroxyprogesterone (MEP, MPA, MGA, CMA and CPA), the compounds derived from 19-norprogesterone (R5020, NES and NGA) and GRN derived from 17α-hydroxy-19-norprogesterone (Table 2, Figure S1).

Conversely, the progestins derived from 19-nortestosterone ETH, EDA, LYN, NEA, NET, TIB, ENG, GES, LNG, NTE, and NGL induced the expression of GFP in the radial glial cells of zebrafish (Figure 1). Each of these compounds induced GFP in a concentration-dependent manner (figure S1), with EC 50 calculated between 0.3 nM for the most active compound (TIB) and 222 nM for the less active (GES) (Table 2). The 19-nortestosterone derived progestin DSG and the nPR antagonist RU486 showed a weak estrogenic effect at the highest tested concentration (10 -6 M) (Figure 1, Figure S1). The progestin derived from spironolactone DRO did not induce GFP expression compared to the solvent control.

Mode of action of LNG and NET using U251-MG cell lines

In order to get further insights on the mechanisms underlying those estrogenic effects, we used an in vitro assay previously developed in our laboratory using the ER negative human astrocyte cell line U251-MG (Le [START_REF] Page | Assessment of xenoestrogens using three distinct estrogen receptors and the zebrafish brain aromatase gene in a highly responsive glial cell system[END_REF][START_REF] Menuet | Expression and estrogen-dependent regulation of the zebrafish brain aromatase gene[END_REF]. To study the

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respective role of each ZfER, cells were co-transfected with one of the three subtypes (zfERα, zfERβ1, or zfERβ2) and the luciferase gene under the control of cyp19a1b promoter. The cells were treated for 24 hours with the different compounds. Figure 2 shows that, as expected, E2 (10 -8 M) used as a positive control caused a strong induction with the three receptors. As documented before (Le [START_REF] Page | Assessment of xenoestrogens using three distinct estrogen receptors and the zebrafish brain aromatase gene in a highly responsive glial cell system[END_REF], the fold induction was much stronger with zfERα (25 to 35 times) than with zfERβ1 (10 to 15), or zfERβ2 (15 to 20). In contrast,

P4 at concentrations of 10 -9 M to 10 -6 M had absolutely no effect with zfERs (Figure 2A-F), confirming the results obtained in vivo. Figure (3A-C) shows that NET causes luciferase induction at 10 -7 M and 10 -6 M, however at the concentration of 10 -7 M, the effect was significant only with zfERβ2. In the context of the U251-MG cells, these effects were completely blocked by ICI 182,780 (10 -6 M), which had no effect by itself. It should be noted that the maximal induction caused by NET on zfERβ2 is within the same range than that observed with zfERα (around 12 times). LNG also increased luciferase expression but only at 10 -6 M and the maximal induction was much lower (around 2-3 times) than with NET (Figures 4A-C). In the same way these effects were fully blocked by ICI 182,780 at 10 -6 M (Figures 4D-F).

Binding activity of NET and LNG on the three zfERs

In order to investigate whether the estrogenic activities of NET and LNG in vivo and in vitro were due to a direct activation of zfER, we monitored the binding properties of these compounds to the three zfER expressed in vitro. Figure 5A-C shows that, in contrast to E2 that started displacing [ 3 H]-E2 binding at 10 -10 M (zfERβ2) or 10 -9 M (zfERα and zfERβ1),

both progestins NET and LNG did not yield any displacement at a concentration of 10 -6 M.

Only concentrations of 10 -5 M or 10 -4 M were effective in reducing [ 3 H]-E2 (data not shown).

Effects of the 5α-reductase inhibitor finasteride and 3ß-5α-hydroxysteroid dehydrogenase inhibitor trilostan on the estrogenic effects of LNG and NET

Because progestins showed no binding to zfERs at relevant concentrations that exhibited in vivo and in vivo estrogenic activity, we investigated whether the estrogenic effects could be due to metabolization of these compounds by 5α-reductase and/or 3ß-5α-hydroxysteroid dehydrogenase as shown in mammals. Exposure of zebrafish to finasteride and trilostane alone had no effect on cyp19a1b expression neither in vivo nor in vitro (figures S2-S5). Finasteride and trilostane at a concentration of 10 -6 M failed to prevent the GFP induction caused by NET and LNG in tg(cyp19a1b-GFP) embryos (figures S2-S4 respectively). Similarly, finasteride and trilostane had no or very little effect on LNG-and NET-induced cyp19a1b-luciferase in the U251-MG cells, (Figure S3-S5). Surprisingly, finasteride caused a significant reduction of the NET luciferase induction, but only with the zfERß1.

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Discussion

All 19-nortestosterone derived progestins up-regulate brain aromatase in an ER-dependent manner

We used the EASZY assay as a screening tool to establish concentration-response curves for 4 natural steroids and 24 (anti-)progestins on cyp19a1b expression, hence covering all chemical classes to which progestins belong to. Among all the tested compounds, 17βestradiol (E2) was, as expected, the most active steroid on brain aromatase while testosterone exhibited a weaker estrogenic activity that likely results from its active aromatization into estradiol [START_REF] Mouriec | Androgens upregulate cyp19a1b (aromatase B) gene expression in the brain of zebrafish (Danio rerio) through estrogen receptors[END_REF][START_REF] Brion | Screening estrogenic activities of chemicals or mixtures in vivo using transgenic (cyp19a1b-GFP) zebrafish embryos[END_REF]. Pregnenolone, a precursor of steroid hormones that has been already detected in aquatic environment [START_REF] Creusot | Identification of synthetic steroids in river water downstream from pharmaceutical manufacture discharges based on a bioanalytical approach and passive sampling[END_REF],

showed an estrogenic activity which could reflect either its capacity to activate ER as shown in human cells [START_REF] Lemmen | Detection of oestrogenic activity of steroids present during mammalian gestation using oestrogen receptor alpha-and oestrogen receptor beta-specific in vitro assays[END_REF][START_REF] Creusot | Identification of synthetic steroids in river water downstream from pharmaceutical manufacture discharges based on a bioanalytical approach and passive sampling[END_REF] or its biotransformation by steroidogenic enzymes that are expressed in the brain of zebrafish (Diotel et al., 2011c).

Interestingly, the in vivo relative estrogenic potency of pregnenolone we report herein is much higher than previously reported in vitro in human cells [START_REF] Lemmen | Detection of oestrogenic activity of steroids present during mammalian gestation using oestrogen receptor alpha-and oestrogen receptor beta-specific in vitro assays[END_REF][START_REF] Creusot | Identification of synthetic steroids in river water downstream from pharmaceutical manufacture discharges based on a bioanalytical approach and passive sampling[END_REF].

Progesterone, the natural ligand of the nPR, exhibited no effect on cyp19a1b in radial glial cell context neither in vivo nor in vitro in U-251 MG cells (Figure S1, Figure 2), thus confirming the lack of transactivation activity of P4 in CHO cells co-transfected with ERE-TK-Luc and zebrafish ER subtypes [START_REF] Menuet | Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions[END_REF]. Similarly, we found that all the progesterone-derived progestins do not exhibit any estrogenic activity similar to what is known in mammals [START_REF] Botella | Lack of estrogenic potential of progesterone-or 19-nor-progesterone-derived progestins as opposed to testosterone or 19-nor-testosterone derivatives on endometrial Ishikawa cells[END_REF]. Interestingly, the anti-progestin mifepristone significantly induced GFP expression in embryos in an ER-dependent manner but only at high concentrations.

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In contrast to progesterone-derived progestins, our data show that all testosteronederived progestins are able to affect cyp19a1b-GFP expression in vivo in a concentrationdependent manner. We thus confirmed the effect of LNG and NET on brain aromatase [START_REF] Brion | Screening estrogenic activities of chemicals or mixtures in vivo using transgenic (cyp19a1b-GFP) zebrafish embryos[END_REF] and extended these finding to 11 other progestins (ETH, EDA, LYN, NEA, TIB, DSG, ENG, GES, NTE, NGL, RU486) among which 9 had never been studied before in zebrafish embryos.

All active compounds induced full concentration-dependent responses with maximal inductions of GFP that often exceeded those measured in zebrafish exposed to E2 or EE2 (Figure S1, [START_REF] Brion | Screening estrogenic activities of chemicals or mixtures in vivo using transgenic (cyp19a1b-GFP) zebrafish embryos[END_REF]. Overall, estranes were the most active compounds as compared to gonanes with the exception of ENG which exhibited a similar activity as ETH.

All were active at concentrations in the sub nM range to hundreds nM. It is noteworthy that the estrogenic potency of tibolone was more than 5-fold higher than that of E2, while other progestins such as norethindrone acetate and norethindrone had estrogenic activities much closer to that of E2, highlighting the strong estrogenic activity of these compounds in the brain of zebrafish embryos.

LNG and NET exhibit estrogenic effects in the U251-MG cell line transfected with ZfERs but do not bind to ZfERs

In order to get further insights into the mechanisms underlying the estrogenic effects of the 19-nortestosterone derivatives, we focused on NET and LNG. Our in vivo data using the EASZY assay showed that NET is very active with an EC 50 in the nM range, while LNG is less potent. This is confirmed by the in vitro assay using the U-251 MG cells indicating that both NET and LNG can induce transactivation of the cyp19a1b-luciferase reporter in the presence of one of the three zefrafish ER. However, with the three zebrafish ER, NET is much more efficient than LNG, eliciting a response with a concentration of 10 -7 M and a maximal

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fold induction 3 to 5 times higher. Thus, these in vitro data confirms the data obtained in vivo.

In all cases, ICI 182,780 blocked these effects, indicating that they are ER-mediated.

However, binding assays showed that NET and LNG were not able to displace [ 3 H]-E2 binding on the three receptors, except only at very high concentrations (superior to 10 -6 M).

Interestingly, although NET is about ten times more efficient than LNG in vivo and in U-251 MG cells, it exhibits a much lower capacity than LNG in displacing [ 3 H]-E2. This strongly suggests that the in vitro and in vivo effects of NET and LNG in the U-251 MG and tg cyp19a1b-GFP on cyp19a1b expression do not involve binding of the parent compounds to zfERs but their estrogenic effects are likely to be due to metabolization into estrogenic metabolites.

In mammalian target tissues, in particular in bones, NET was shown to be biotransformed by 5α-reductase into 5α-NET, which can be further metabolized in 3α,5αtetrahydro-NET (3α,5α-NET) and 3β,5α-tetrahydro-NET (3β,5α-NET) by 3α-5αhydroxysteroid and 3ß-5α-hydroxysteroid deshydrogenases, respectively [START_REF] Enriquez | The effects of synthetic 19-norprogestins on osteoblastic cell function are mediated by their non-phenolic reduced metabolites[END_REF][START_REF] Garcia-Becerra | Synthetic 19-nortestosterone derivatives as estrogen receptor alpha subtype-selective ligands induce similar receptor conformational changes and steroid receptor coactivator recruitment than natural estrogens[END_REF][START_REF] Lemus | Bioconversion of norethisterone, a progesterone receptor agonist into estrogen receptor agonists in osteoblastic cells[END_REF]. In rat cultured osteoblastic cells, these reduced derivatives of NET induced significant effects on rat osteoblast proliferation and differentiation, similar to estradiol [START_REF] Lemus | Bioconversion of norethisterone, a progesterone receptor agonist into estrogen receptor agonists in osteoblastic cells[END_REF]. However, these effects were completely blocked by finasteride, a type II and III 5α-reductase inhibitor. Finasteride has a molecular weight of 372 g/mol, is lipophilic [START_REF] Azeem | Microemulsions as a surrogate carrier for dermal drug delivery[END_REF] and has recently been shown to be active in teleost fishes [START_REF] Lee | Evaluation of the 5alpha-reductase inhibitor finasteride on reproduction and gonadal development in medaka, Oryzias latipes[END_REF]. Because 5α-reductase is abundant in the brain of fish (Diotel et al., 2011a;[START_REF] Pasmanik | Changes in brain aromatase and 5 alpha-reductase activities correlate significantly with seasonal reproductive cycles in goldfish (Carassius auratus)[END_REF], we attempted to block the effects of NET or LNG with this compound. In vivo, finasteride alone had no visible effect on the development of embryos. In combination with estradiol, finasteride had no effect on the estradiol-induced GFP expression, and in association with NET or LNG, the 5α-reductase inhibitor did not affect at all the estrogenic activity of these two progestagens. Because it is

A C C E P T E D M A N U S C R I P T
possible that finasteride, despite its small size and lipophilic nature, did not penetrate into the embryos, we repeated this experiment in vitro in the U-251 MG cells, since 5α-reductase is known for being expressed in astrocytes [START_REF] Bruzzone | Immunohistochemical localization and biological activity of 3beta-hydroxysteroid dehydrogenase and 5alpha-reductase in the brain of the frog, Rana esculenta, during development[END_REF][START_REF] Kiyokage | Localization of 5alphareductase in the rat main olfactory bulb[END_REF].

Finasteride did not affect the luciferase induction caused by NET or LNG in these cells.

Trilostane, an inhibitor of 3ß-5α-hydroxysteroid deshydrogenases that has been demonstrated to be active in medaka fish [START_REF] Sun | Effects of trilostane and fipronil on the reproductive axis in an early life stage of the Japanese medaka (Oryzias latipes)[END_REF] did not affect either the in vivo or the in vitro estrogenic activities of NET or LNG. The underlying metabolic pathways through which these compounds are metabolized into estrogenic metabolites remain to be identified in fish.

(Eco)toxicological relevance of progestin-induced cyp19a1b expression during embryogenesis.

To our knowledge, our study is the first to provide an extensive characterization of the effects of progestins on an estrogen-dependent gene in brain of fish and to demonstrate that more than half of the nPR ligands tested (i.e., 13 out of 25) act in a similar way to 17βestradiol. Even though progestin concentrations used to trigger induction of aromatase in the short-term EASZY assay are higher than those found in effluent and surface waters, it should be stressed that due to the hydrophobic nature of progestins with log Kows between 3.1 and 5.4 they tend to accumulate in aquatic organisms [START_REF] Kumar | The challenge presented by progestins in ecotoxicological research: a critical review[END_REF][START_REF] Fent | Progestins as endocrine disrupters in aquatic ecosystems: Concentrations, effects and risk assessment[END_REF].

Furthermore wild fish are very likely exposed to mixtures of progestins together with other EDCs, some of which act as estrogens. Investigating the combined effects of progestins with estrogens on cyp19a1b expression thus appears relevant to further assess the hazard of these environmental contaminants (Hinfray et al., under revision).

Previous studies reported effects of P4 and some of the (anti-)progestins tested in this study (i.e., DYD, MPA, NET, LNG, NGL, DRO and RU486) on the transcriptional activity of multiple genes (e.g., steroid hormone receptors, steroidogenic enzymes or circadian rhythm

A C C E P T E D M A N U S C R I P T
genes) during zebrafish embryogenesis that may affect brain and sex development [START_REF] Zucchi | Progestins and antiprogestins affect gene expression in early development in zebrafish (Danio rerio) at environmental concentrations[END_REF][START_REF] Liang | Long-term exposure to environmentally relevant concentrations of progesterone and norgestrel affects sex differentiation in zebrafish (Danio rerio)[END_REF], Zhao et al., 2015, for a review see [START_REF] Fent | Progestins as endocrine disrupters in aquatic ecosystems: Concentrations, effects and risk assessment[END_REF]Kumar et al., 2015). Herein, the effects of 17 progestins on zebrafish embryos were newly assessed. By means of fluorescence imaging in progestins exposed-transgenic cyp19a1b-GFP embryos, we further describe the negative effect of 13 synthetic (anti)progestins on the brain of fish by demonstrating their capacity to target radial glial cells and to induce brain aromatase expression in an ER-dependent manner. The spatio-temporal expression of ERs subtypes have been well-described during zebrafish embryogenesis revealing that ERβ subtypes and brain aromatase are functionally co-expressed very early during brain development [START_REF] Mouriec | Androgens upregulate cyp19a1b (aromatase B) gene expression in the brain of zebrafish (Danio rerio) through estrogen receptors[END_REF]. Our data thus demonstrate that several synthetic progestins have the potential to interfere with ER signaling in developing fish. It is important to recall here that progestins are known to act on multiple nuclear receptors and it would therefore be advisable to further document their potential effects on other NR-regulated pathways in developing brain of fish.

Among those, activation of fathead minnow (Pimephales promelas) AR by progestins has been recently pointed out [START_REF] Ellestad | Environmental gestagens activate fathead minnow (Pimephales promelas) nuclear progesterone and androgen receptors in vitro[END_REF]. However, no information is currently available in zebrafish. Furthermore, as compared to ER, much less information is available on AR signaling pathway during embryogenesis, even though AR was found to be expressed in discrete regions of the brain (Gorelick et al., 2008) and AR-responsive genes have recently been described (Fetter et al., 2015).

An important outcome of this study relies on the fact that progestins act on radial glial cells that play a crucial role in neurogenesis and brain repair as they are brain stem cells in developing and adult fish [START_REF] Lam | gfap and nestin reporter lines reveal characteristics of neural progenitors in the adult zebrafish brain[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] Zupanc | Adult neurogenesis and neuronal regeneration in the brain of teleost fish[END_REF] giving birth to neurons [START_REF] Diotel | Effects of estradiol in adult neurogenesis and brain repair in zebrafish[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]. It is known that estrogens in adult zebrafish and larvae affect neurogenesis by modulating the neurogenic activity of radial glial cells [START_REF] Diotel | Effects of estradiol in adult neurogenesis and brain repair in zebrafish[END_REF]. Consequences of early exposure to environmental progestins on A C C E P T E D M A N U S C R I P T brain development are unknown, but given the estrogenic activity that we report for the majority of progestins studied, it would therefore be advisable to further document their effects on cell proliferation, survival and migration in brain.

Conclusion

This study provides an extensive toxicological characterization of a large number of currently used (anti-)progestin pharmaceuticals as regard their estrogenic activity. We demonstrate that a majority of them have the capacity to target radial glial cells and to induce brain aromatase expression in zebrafish embryos in an ER-dependent manner. This work thus provides new information on the effects of progestins in fish at the brain level that deserve further study.

Figure 1 :

 1 Figure captions

Figure 2 .

 2 Figure 2. Luciferase expression after progesterone (P4) treatment. U251-MG cells were transfected

Figure 3 .Figure 4 .

 34 Figure 3. Luciferase fold induction after norethindrone (NET) treatment. U251-MG glial cells were

Figure 5 .

 5 Figure 5. Binding properties of NET and LNG with zfERs. zfERs proteins synthetized and

  Fig. 3

  Fig. 4

Table 1 :

 1 Classification of some natural hormones and natural and synthetic nPR ligands.

		Classification	Compound	Abbreviatio n	Uses	Molecula (g/mol) r weight	CAS	Structure
	Natural estrogen	17β-estradiol	E2	Clinical use	272.38	50-28-2
	Natural androgen	Testosterone	T	Clinical use	288,42	58-22-0
	Natural hormone	Pregnenolone (progesterone precursor)	P5	Clinical use	316.48	145-13-1
	Endogenous progestin in human and fish	Progesterone	P4	Clinical use	314.46	57-83-0
	Synthetic progestin retroprogesterone	Dydrogesterone	DYD	Clinical use	312.45	152-62-5
			Medroxyprogestero ne	MEP	Clinical use	344.49	520-85-4
	Progestins structurally related to progesterone	Derived from 17α-hydroxyprogestero ne Derived from 19-norprogesterone	Medroxyprogestero ne acetate Megestrol acetate Chlormadinone acetate Cyproterone acetate CPA MPA MGA CMA Promegestone R5020 Nestorone NES Nomegestrol acetate NGA	Clinical use Clinical use Clinical use Clinical use Clinical use Clinical use Clinical use Clinical	386.52 384.51 406.94 416.94 326.47 370.48 370.48	71-58-9 595-33-5 302-22-7 427-51-0 34184 -77-5 7759-35-5 58652 -20-3
		Derived from 17α-hydroxy-19-norprogesterone	Gestonorone	GRN	e caproate use of gestonoron	316.43	2137-18-0

Progestins structurally related to testosterone: derived from 19- nortestosterone

				Clinical
				use,
		Etonogestrel	ENG	metabolite of	324.45	54048 -10-1
				desogestre
				l
	Progestin structurally related to spironolactone PR antagonist	Gestodene Levonorgestrel Norgestimate Norgestrel Drospirenone Mifepristone	GES LNG NTE NGL DRO RU486 M A N U S C R I P T Clinical use 310.43 Clinical use 312.45 Clinical use 369.50 Clinical use 312.45 Clinical use 366.49 Clinical use 429.60	60282 -87-3 797-63-7 35189 -28-7 6533-00-2 67392 -87-4 84371 -65-3
	A C C E P T E D
		Ethisterone	ETH	Stopped	312.45	434-03-7
		Ethynodiol diacetate	EDA	Clinical use	384.51	297-76-7
	Estranes	Lynestrenol Norethindrone acetate	LYN NEA	Clinical use Clinical use	284.44 340.46	52-76-6 51-98-9
		Norethindrone	NET	Clinical use	298.42	68-22-4
		Tibolone	TIB	Clinical use	312.4	5630-53-5
	Gonanes	Desogestrel	DSG	Clinical use	310.47	54024 -22-5

Table 2 :

 2 Effects of some natural hormones and synthetic progestins on the EASZY assay, calculated effective concentrations (EC 20 and EC 50 expressed in nM) and relative estrogenic potency (REP) for active compounds. +: GFP induction, ne: no effect, nc: not calculated.

		Classification	Compound	Abbrevia tion	Effect in EASZY	EC 20 (nM)	EC 50 (nM)	REP
	Natural estrogen	17β-estradiol	E2	+	0.62	1.7	1
	Natural androgen	Testosterone	T	+	337	505	0.003
				Pregnenolone
	Natural hormone	(progesterone	P5	+	105	826	0.002
				precursor)
	Endogenous progestin in human and fish	Progesterone	P4	ne	-	-	-
	Synthetic progestin retroprogesterone	Dydrogesterone	DYD	ne	-	-	-
				Medroxyprogesterone MEP	ne	-	-	-
	Progestins structurally related to	progesterone	Derived from 17α-hydroxyprogest erone Derived from 19-norprogesterone Derived from 17α-hydroxy-19-	Medroxyprogesterone acetate Megestrol acetate Chlormadinone acetate Cyproterone acetate Promegestone Nestorone Nomegestrol acetate Gestonorone	MPA MGA CMA CPA R5020 NES NGA GRN	ne ne ne ne ne ne ne ne	--------	--------	--------
			norprogesterone
	Progestins

structurally related to testosterone: derived from 19- nortestosterone

  induction of GFP in tg cyp19a1b-GFP embryos only at 10 -6 M.

		Ethisterone	ETH	+	5.5	40	0.04
		Ethynodiol diacetate	EDA	+	34	53	0.03
		Lynestrenol	LYN	+	3.5	16	0.1
	Estranes	Norethindrone acetate	NEA	+	0.4	2.4	0.7
		Norethindrone	NET	+	1	4.0	0.4
		Tibolone	TIB	+	0.1	0.3	5.7
		Desogestrel	DSG	+ *	nc	nc	nc
		Etonogestrel	ENG	+	7	39	0.04
	Gonanes	Gestodene Levonorgestrel	GES LNG	+ +	41 39	222 89	0.008 0.02
		Norgestimate	NTE	+	65	126	0.01
		Norgestrel	NGL	+	73	184	0.009
	Progestin structurally related to spironolactone	Drospirenone	DRO	ne	-	-	-
	PR antagonist	Mifepristone	RU486	+ *	nc	nc	nc

*

Acknowledgements.

The authors wish to express their sincere thanks to the anonymous reviewers for their positive and constructive comments. This research was supported by the ANR PROOFS "Occurrences and effects of environmental ligands of progesterone receptor on fish reproduction and neuro-development" (ANR-13-CESA-0006-03). Clémentine Garoche was supported by a doctoral fellowship from the ANR PROOFS and the French Ministry of Ecology (P190 "Axe de Recherche Ecotoxicologie"). The authors acknowledge the staff of the zebrafish Biosit facility (LPGP, INRA, Rennes) and Benjamin Piccini (ECOT unit, INERIS, Verneuil-en-Halatte) for his help in performing zebrafish exposures.

Effect of progestins should be further investigated at the brain level


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  Chronic exposure to soil salinity in terrestrial species: Does plasticity and underlying physiology differ among specialized ground-dwelling spiders? David Renault, Charlène Puzin, Natacha Foucreau, Alain Bouchereau, Julien Pétillon To cite this version: David Renault, Charlène Puzin, Natacha Foucreau, Alain Bouchereau, Julien Pétillon. Chronic exposure to soil salinity in terrestrial species: Does plasticity and underlying physiology differ among specialized ground-dwelling spiders?. Journal of Insect Physiology, 2016, 90, pp.49-58. 10.1016/j.jinsphys.2016.05.005. hal-01371663

Table 1

 1

	. Mean (± 3 % confidence limit) hemolymph concentrations (pmole ion /mg of dry
	hemolymph mass) of sodium; magnesium and potassium, [Na + ], [Mg 2+ ] and [K + ],
	respectively, in Pardosa saltans, Pardosa purbeckensis and Arctosa fulvolineata) after one
	day acclimation at null salinity at 20°C.

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