# Human and Zebrafish Nuclear Progesterone Receptors Are Differently Activated by Manifold Progestins

Clémentine Garoche, Selim Aït-Aïssa, Abdelhay Boulahtouf, Nicolas Creusot, Nathalie Hinfray, William Bourguet, Patrick Balaguer, François Brion

► To cite this version:

Clémentine Garoche, Selim Aït-Aïssa, Abdelhay Boulahtouf, Nicolas Creusot, Nathalie Hinfray, et al. Human and Zebrafish Nuclear Progesterone Receptors Are Differently Activated by Manifold Progestins. Environmental Science and Technology, 2020, 54 (15), pp.9510-9518. 10.1021/acs.est.0c02056. hal-04707325

HAL Id: hal-04707325

https://hal.science/hal-04707325v1

Submitted on 25 Sep 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et 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.

<figure><img src="image_1.png" /></figure>
1 Human and zebrafish nuclear progesterone receptors are differently activated by
2 progestins

3 Clémentine Garoche <sup>a</sup>, Selim Aït-Aïssa <sup>a,*</sup>, Abdelhay Boulahtouf <sup>b</sup>, Nicolas Creusot <sup>b</sup>,
4 Nathalie Hinfry <sup>a</sup>, William Bourget <sup>c</sup>, Patrick Balaguer <sup>b</sup>, François Brion <sup>a,*</sup>

5 <sup>a</sup> Institut National de l'Environnement Industriel et des Risques (INERIS), Unité
6 Écotoxicologie in vitro et in vivo, UMR-I 02-SEBIO, Parc ALATA, 60550 Verneuil-en-
7 Halatte, France

8 <sup>b</sup> Institut de Recherche en Cancérologie de Montpellier (IRCM), Inserm U1194, Université
9 Montpellier, Institut régional du Cancer de Montpellier (ICM), 34290 Montpellier, France

10 <sup>c</sup> Centre de Biochimie Structurale (CBS), Inserm, CNRS, Univ Montpellier, Montpellier,
11 France

12

13

14 * Corresponding authors:

15 E-mail: francois.brion@ineris.fr, phone +33(0)3 44 55 65 12

16 E-mail: selim.ait-aissa@ineris.fr, phone +33(0)3 44 55 65 11

17

18

19

20
21 Keywords: synthetic progestins; 17α,20β-dihydroxy-4-pregnen-3-one; human and zebrafish

22 nuclear progesterone receptor; luciferase reporter cell lines.
<figure><img src="image_2.png" /></figure>
26 Abstract

27 Environmental risk of natural and synthetic ligands of the nuclear progesterone receptor
28 (nPR) has been pointed out, however there is still a lack of mechanistic information regarding
29 their ability to interact with nuclear PR in aquatic species. To identify possible interspecies
30 differences, we assessed *in vitro* the ability of a broad range of progestins to transactivate
31 zebrafish (zf) and human (h) PRs, by using two established reporter cell lines, U2OS-zfPR
32 and HELN-hPR, respectively. Reference ligands highlighted some differences between the
33 two receptors. The reference human agonist ligands promegestone (R5020) and progesterone
34 (P4) induced luciferase activity in both cell lines in a concentration-dependent manner
35 whereas the natural zebrafish progestin 17α,20β-dihydroxy-4-pregnen-3-one (DHP) activated
36zfPR but not hPR. The potent human PR antagonist mifepristone (RU486) blocked PR-
37 induced luciferase in both cell models but with different potencies. In addition, a large set of
38 twenty-two synthetic progestins were screened on the two cell lines. Interestingly, all of the
39 tested compounds activated hPR in the HELN-hPR cell line whereas the majority of them
40 acted aszfPR antagonists in U2OS-zfPR. SuchzfPR-specific response was further confirmed
41 in zebrafish liver cells (ZFL). This study provides novel information regarding the activity of
42 a large set of progestins on human and zebrafish PR and highlights major interspecies
43 differences in their activity, which may result in differential effects of progestins between fish
44 and humans.
45
# 1. Introduction

Progestagenic sex steroids, also called progestins or progestogens, play crucial roles in development and reproduction in vertebrates, including teleosts. Progesterone (P4) is an endogenous biologically active progestin in mammals and fish. However, in most teleost species including zebrafish, other endogenous active progestins such as 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S) ¹ are also synthesized, P4 being considered as an intermediate. Progestins can exert their biological activity by acting through the nuclear progesterone receptor (nPR), a ligand-activated transcription factor that belongs to the steroid hormone receptor superfamily (Grim et al., 2016). In both mammals and fish, PRs are widely distributed in different reproductive and non-reproductive tissues, suggesting the widespread physiological effects of progestagenic hormones ². Indeed, the central role of nPR in mediating progesterone-regulated reproductive functions in diverse vertebrates, in both males and females, has been demonstrated in different vertebrate models, notably by using PR knockout (PRKO) mice ³ and more recently PRKO zebrafish ⁴,⁵.

During the last few years, several studies have documented the presence of progesterone and synthetic progestins in effluents of wastewater treatment plants (WWTPs) and surface waters, related to their production and consumption as pharmaceuticals in a diversity of medical applications such as contraception ⁶⁻¹⁰. Laboratory studies on aquatic models revealed the effects of natural and synthetic progestins on fish reproduction at environmentally relevant concentrations (i.e. in the ng/L range), thereby highlighting the risks they pose on aquatic species and aquatic ecosystems ¹¹,¹². Important steps forward have been recently made regarding the ability of some synthetic progestins to interfere with key endocrine molecular and cellular targets, such as human androgen receptor (AR)- and estrogen receptor (ER)-mediated signaling pathways and steroidogenesis, which could, at least partly, explain their
reproductive physiological effects in vivo ¹³⁻¹⁸. However, it is noticeable that much less
information is available regarding the ability of progesterone and synthetic progestins to
interact with fish nPR ¹⁹,²⁰ and subsequently alter nPR-regulated physiological processes ².
Because cross-species variations in nuclear receptor (NR) sequences can significantly
influence their structure, functioning and response to chemical ligands ²¹, there is a need for
characterizing the ability of environmentally occurring progestins to interact with fish nPR to
assess their environmental hazard to fish species.

The aim of this study was therefore i) to gain knowledge on the ability of a large set of (anti) 77 progestagenic compounds to transactivate the zebrafish nPR and ii) to compare these results 78 with their activity towards the human hPR (hPR, isoform B) to identify possible interspecies 79 differences. To this end, we first developed a novel in vitro luciferase-reporter-gene cell line 80 expressing the zebrafish PR, namely U2OS-zfPR. The activity of four natural or synthetic 81 PR-ligands used as reference substances, i.e. P4, DHP, promegestone (R5020), mifepristone 82 (RU486), as well as twenty-two pharmaceutical progestins representative of different 83 chemical classes were assessed in this newly established U2OS-zfPR cell line. All the 84 substances were also tested in an already established human cell line HELN-hPRB expressing 85 hPRB ²² for comparative purpose. 86
87

**2. Material and Methods**

88

**2.1. Chemicals and materials**

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

Materials for cell culture are from Life Technologies (Cergy-Pontoise, France). Luciferin (sodium salt) was purchased from Promega (Charbonnières, France). Chemical substances used in this study are presented in Table 1. 17β-estradiol (E2), testosterone (T), 11-ketotestosterone (11-KT), pregnenolone (P5), progesterone (P4), dydrogesterone (DYD), medroxyprogesterone (MEP), medroxyprogesterone acetate (MPA), megestrol acetate (MGA), chlormadinone acetate (CMA), cyproterone acetate (CPA), promegestone (R5020), nestorone (NES), nomegestrol acetate (NGA), ethisterone (ETH), ethynodiol diacetate (EDA), lynestrenol (LYN), norethindrone acetate (NEA), norethindrone (NET), tibolone (TIB), desogestrel (DSG), etonogestrel (ENG), gestodene (GES), levonorgestrel (LNG), norgestimate (NTE), norgestrel (NGL), drospirenone (DRO), mifepristone (RU486), 17α,20β-dihydroxy-4-pregnen-3-one (DHP), spironolactone (SPIRO) and canrenone (CAN) were obtained from Sigma-Aldrich Chemical Co. (St.Louis, MO). Gestonorone (GRN) was obtained from Toronto Research Chemicals (Toronto, Canada). Stock solutions of chemicals were prepared in dimethyl sulfoxide (DMSO) and stored at -20°C. Fresh dilutions of test chemicals in medium were prepared before each experiment.

104

**2.2. Reporter gene cell lines HELN-hPR and U2OS-zfPR**

105

106

107

108

109

110

111

To assess the ability of progestins to transactivate the human PR (hPR, isoform B) or the zebrafish PR (zfPR), two human luciferase-reporter-gene cell lines were established. The HELN-hPR cell line has already been described <sup>22</sup>. Briefly, HELN-hPR cells were obtained by stably transfection of HELN (HeLa ERE-luciferase-neomycin) cells <sup>23</sup> by a plasmid expressing hPR where the DNA binding domain was replaced by the hERα DNA binding domain (pSG5-hPR(ERα DBD)-puromycin. The U2OS-zfPR cell line was obtained by stable cotransfection of the plasmid ERE-Luciferase and an expression plasmid ofzfPR with the
hERα DNA binding domain ((pSG5-hPR(ERα DBD)-puromycin, supporting Information, Figure S1). Selection of HELN-hPRcells was made with 0.5 µg/mL puromycin and 1 mg/L G418 and selection of USO2-zfPR cells was made with 0.5 µg/mL puromycin..

Cell lines were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing phenol red, 1 g/L glucose, 5% fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 µg/mL of streptomycin. Culture medium was supplemented with 0.5 µg/mL puromycin and 1 mg/mL geneticin for HELN-hPR and 0.5 µg/mL puromycin for U2OS-zfPR cells. Cells were cultured in a 5% CO₂ humidified atmosphere at 37°C. Exposure was made in phenol red-free DMEM supplemented with 2% dextran-coated charcoal FBS (DCC), 100 units/mL of penicillin, and 100 µg/mL of streptomycin.

2.3. Reporter gene cell line ZFL-PR

Zebrafish liver cells ZFL cells have been stably cotransfected by the luciferase plasmid ARE₆-collagenase-Luciferase-hygromycine) and thezfPR expressing plasmid pSG5-zfPR-puromycin and grown in presence of 0.5 µg/mL puromycin and 0.25 mg/mL hygromycin during 3 weeks. Among the different resistant clones, the one which expresses luciferase in the most inductible manner (4 fold) was selected and called ZFL-PR.

The cells were grown in LDF medium (50% Leibovitz 15 culture medium L15, 35% DMEM
high glucose, and 15% Ham's-F12 medium) with 0.15 g/L sodium bicarbonate, 15 mM 4-(2-
hydroxy-ethyl)-1-piperazineethanesulfonic acid (HEPES), 0.01 mg/mL insulin, 50 ng/mL
epidermal growth factor (EGF), 5% fetal bovine serum (FBS), and 50 U/mL penicillin and
streptomycin antibiotics and supplemented with 5% vol/vol FBS. Cells were cultured in a 5%
CO₂ humidified atmosphere at 28°C. Exposure was made in LDF supplemented with 2%
dextran-coated charcoal FBS (DCC), 100 units/mL of penicillin, and 100 µg/mL of
streptomycin.
136

**2.4. In vitro transcriptional activation bioassays**

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

HELN-hPR and U2OS-zfPR cells were seeded in 96-wells white opaque culture plates (Greiner CellStar; Dutscher, Brumath, France) at a density of 7×10⁴ cells per well in 100 µL culture medium. After 24h, cells were exposed by adding serial dilutions of compounds or solvent control (DMSO; final concentration 0.1% v/v) in triplicates and incubated for 16h. For antagonist activity assessment, cells were exposed to different concentrations of the tested compounds and to a concentration of the reference compound that yield 50%-80% of the maximal response, i.e. R5020 at 10⁻⁹ M for HELN-hPR and DHP at 3×10⁻⁹ M for U2OS-zfPR. After 16h, medium was removed and replaced with 50 µL/well of medium containing 0.3 mM luciferin. Luminescence signal was monitored in living cells for 2 s per well (BioTek Synergy H4 Hybrid Microplate Reader). Results were expressed as the percentage of the maximum luciferase activity induced by R5020 at 3×10⁻⁸ M for HELN-hPRB cells and DHP at 3×10⁻⁸ M for U2OS-zfPR cells. Concentration-response curves were obtained with the Hill equation model using the Regtox 7.0.6 Microsoft Excel TM macro (http://www.normalesup.org/~vindimian/fr_index.html), from which effective (EC) and inhibitory concentrations (IC) were derived. For a given chemical, EC₅₀ is defined as the concentration inducing 50% of its maximal effect and IC₅₀ represents the concentration required for 50% inhibition. Relative Effect Potencies (REPs) were calculated by dividing the EC₅₀ of the reference compound by that of the tested compound.

155

156

157

158

159

Chemicals were tested in at least two independent experiments when inactive (i.e. maximal luciferase induction or inhibition below 20%) and three independent experiments when active (i.e. luciferase induction or inhibition above 20 %). Data are expressed as the percentage of the maximum luciferase activity induced by 3×10⁻⁸ M R5020 or 3×10⁻⁸ M DHP ± standard deviation (SD).
160

# 3. Results and discussion

161

## 3.1. hPR andzfPR transactivation by PR, ER and AR reference ligands

162

P4, R5020, RU486, and DHP were first tested to characterize the response of the established cell lines to reference PR ligands. Some marked differences were revealed in terms of transactivation efficacies and potencies depending of the cell model (Figure 1, Table 2).

163

164

165

In HELN-hPR, R5020 was the most efficient ligand with a full agonist response profile and a low EC₅₀ (in the sub-nM range) while it only partially induced luciferase activity in U2OS-zfPR. P4 was active in both cell models but was less efficient to induce luciferase activity as compared to R5020 in HELN-hPR and to DHP in U2OS-zfPR. For DHP, a major difference was noticed as it was the most active and efficient steroid in U2OS-zfPR but was unable to induce any luciferase activity in HELN-hPR (Figures 1A, 1B; Table 2). The higher efficacy of DHP to transactivate fish PR as compared to P4 confirms previous *in vitro* data using transactivation assays for teleost nPR including the zebrafish ², the rainbow fish ¹⁹ and the fathead minnow ²⁰. The species-specificity of DHP to inducezfPR and not hPR likely reflects its crucial role in reproductive endocrinology in most teleost species as maturation-inducing substance (MIS) while in mammals P4 is the endogenous progestagenic hormone. It is however noteworthy that DHP was able to inhibit R5020-induced hPR activity in HELN-hPR cells, but at a quite high concentration (i.e. IC₅₀ 576 nM, Table S1), suggesting that the fish progestin DHP is able to bind to the human nPR but with a much lower affinity than forzfPR.

166

167

168

169

170

171

172

173

174

175

176

177

178

179

A marked difference was also noticed regarding the efficacy of RU486 to antagonize human and zebrafish PRs (Figure 1C, D). In both cell lines, RU486 behaved as an antagonist ligand but with a 2000-fold higher efficacy in HELN-hPR than in U2OS-zfPR (Table 2), which could reflect a higher binding affinity of RU486 for hPR thanzfPR.

180

181

182
183

Importantly, none of the other steroidal hormones tested, *i.e.* 17$\beta$-estradiol, testosterone and 11-ketostestosterone were able to transactivate either hPR orzfPR (Figure S2). In addition, P4, R5020 and DHP were all inactive in HELN cells that express luciferase but no functional receptor (data not shown).

184

185

186

187

As the cellular context may influence the transcriptional activity of liganded nuclear receptors $^{24}$, the reference PR ligands were assessed in U2OS and HeLa cells that were transiently transfected with hPR andzfPR, respectively. The response profiles obtained in transient transfection experiments were very similar to that obtained with U2OS-zfPR and HELN-hPR stable cell lines (Figure S3). These results demonstrate that in our study the cellular context did not influence the biological response and the observed differences between the two cell lines were related to the different PRs.

188

189

190

191

192

193

194

195

196

Altogether, these data highlight that both models respond to reference PR ligands in a sensitive and PR-specific manner, revealing differential transactivation properties depending on the ligands and the origin species of the nPR.

197

## 3.2. Synthetic progestins transactivate hPR

198

199

200

201

202

203

204

205

206

207

The twenty-two pharmaceutical progestins (Table 1) were able to activate luciferase activity in a concentration-dependent manner in HELN-hPR (Figure 2A and 2B) with EC₅₀ values ranging from 0.14 nM for GES to 249 nM for MEP (Table 2). All the tested progestins, except LYN and MEP, were equally or more potent than the endogenous ligand P4, and five of them (*i.e.* MPA, NES, ENG, GES and LNG) had higher relative progestagenic potency than the prototypical synthetic ligand R5020. Interestingly, drospirenone, an analogue of spironolactone, had potent progestagenic activity in HELN-hPR (EC₅₀ 5 nM, Tables 2 and S1, Figure 2B), which was significantly higher than spironolactone and its metabolite canrenone that were previously described as hPR agonists ¹⁰ with EC₅₀ of 1008 nM and 657 nM, respectively (Figure S4).
208 Further characterization was achieved by coexposing progestins with R5020 1 nM to assess
209 hPR antagonistic activity. The full PR agonists (i.e. inducing 80 - 100% of maximum activity,
210 Figure 2A) were unable to inhibit R5020-induced luciferase (data not shown). Conversely, all
211 the progestins that partially induced luciferase activity (Figure 2B) were also able to inhibit
212 R5020-induced luciferase in a concentration-dependent manner to reach the activity level
213 obtained with the compound alone, which is the typical response profile of a partial agonistic
214 ligand (Table 2 and Table S1). For this second category, the calculated IC₅₀ values ranged
215 from 4.70 nM for MGA to 156 nM for EDA.

216 The progestagenic activities reported herein agree with EC50 values derived from in vitro cell-
217 based transactivation assays for 8 pharmaceuticals common to our study using either HELN-
218 hPRB 22 or U2OS-hPR cells 19 albeit the EC50 reported for tibolone in HG5LN-Gal4-hPR
219 cells was ten times higher than in our study 25.

220

221

**3.3. Progestins modulated zfPR activity but most of them were antagonists**

222 The *in vitro* response profiles of progestins were dramatically different when assessing zfPR
223 activity. In contrast to the results obtained on hPR, very few progestins induced zfPR activity
224 (Figure 2C). Only NES and DRO transactivated zfPR, with EC50 values of 1.4 nM and 14.1
225 nM, respectively (Table 2). In coexposure experiments, both compounds antagonized DHP-
226 induced luciferase activity to reach the level of activity obtained with the compound alone and
227 were therefore considered as partial zfPR agonists (Figure 2D, Tables 2 and S1). Interestingly,
228 the two compounds spironolactone (SPI) and its metabolite canrenone (CAN), were also able
229 to transactivate zfPR to induce luciferase activity in U2OS-zfPR by 10- and 2-fold higher
230 levels than in HELN-hPRB, respectively (Figure S4). Our results thus confirm the study of
231 Ellestad et al., 2014 who demonstrated that DRO activates fathead minnow PR *in vitro* and
232

newly reports an *in vitro* progestagenic activity of NES, SPI (the parent compound of DRO),
and CAN toward a fish PR.

233

234

None of the other pharmaceutical progestins were able to induce luciferase activity in U2OS-
zfPR at the concentrations tested (Table S1). Very few studies investigated the interaction of
progestins towards fish PR and the number of investigated progestins was limited to few of
them. Notwithstanding, the lack of progestagenic activity of progestins towardzfPR reported
herein agrees with available *in vitro* fish PR transactivation assays for the rainbowfish ¹⁹ and
the fathead minnow ²⁰. Therefore, our study confirms the lack of progestagenic activity of
some progestins and extends this knowledge to a broad range of pharmaceutical progestins in
another teleost fish species, the zebrafish. Hence, it suggests that the lack of progestagenic
activity of progestins is a common feature in teleost.

235

236

237

238

239

240

241

242

243

A major finding of our study was to demonstrate that all the progestins were able to fully
antagonize DHP-induced luciferase activity in a concentration-dependent manner in thezfPR
assay. The IC50 ranged from 14 nM for LNG to 1002 nM for DSG. Remarkably, RU486, a
potent antagonist compound on hPR (Figure 1C, Table 2), had a much weaker anti-
progestagenic activity in U2OS-zfPR, i.e. by 2000-fold, and was the least active anti-
progestagenic compound in U2OS-zfPR.

244

245

246

247

248

249

To ensure that the human cellular context did not influence the biological response measured
onzfPR in U2OS, we tested the interaction of selected ligands with the full lengthzfPR in a
fish hepatic cell context, the zebrafish liver cell line (ZFL). The ZFL cell line has been shown
to be metabolically competent possessing both phase I and phase II enzymes ²⁶,²⁷ and is a
relevant *in vitro* zebrafish model to study the interactions of EDCs with NR-regulated
signaling pathways such as AhR ²⁷ or ERs ²⁸⁻³⁰. In ZFL-zfPR cells, the reference agonists
DHP, R5020 and P4 were all active, with similar patterns in terms of both effective
concentrations and response intensity as compared to the U2OS-zfPR cells (Figure S5-A),

250

251

252

253

254

255

256
which validates the relevance of the established zebrafish cell model. RU486 had no agonistic activity but inhibited DHP-induced luciferase activity at high concentration, which reflects its weak anti-progestagenic activity observed in U2OS-zfPR (Figure S5-B). Most importantly, the ZFL-zfPR assay confirmed the lack of progestagenic activity of MPA and ENG while they antagonized DHP-induced luciferase (Figures S5-C, S5-D). It should be pointed out that the anti-progestagenic activities of MPA and ENG are less pronounced in the ZFL context compared to U2OS-zfPR. These differences might be attributed to the higher metabolic activity of the hepatic cell line compared to U2OS cells, which is important to consider given that progestins are known to be metabolized into actives metabolites possessing diverse endocrine properties <sup>17,31</sup>. Based on these data, it can be concluded that the *in vitro* anti-progestagenic activity of progestins was not specific to the cellular context, but on the contrary reflected the intrinsic properties of progestins onzfPR.

Altogether, the *in vitro* comparative data demonstrate that a great majority of the tested synthetic steroidal progestins have potent progestagenic activity towards hPR but anti-progestagenic activity towardszfPR (except DRO and NES).

## 3.4. Species-specificity of the nuclear progesterone receptor transactivation by progestins

We highlighted major differences regarding the progestagenic activity of progestins towards human and zebrafish PR. Previous studies investigating cross-species differences between mammalian and fish models for several nuclear receptors provided evidence that there exist differential binding affinity, transactivation properties and/or selectivity of ligands and environmental xenobiotics depending on the nuclear receptor studied and the origin species of NR<sup>28,32,33</sup>. Among them, the pregnane X receptor (PXR) showed the most significant differences in ligand specificity across species<sup>34,35</sup>. Our study further documents new evidence on such interspecies differences between human and fish for the PR. Comparative
analysis of the DBD/LBD amino acid sequences of human NRs and the corresponding
orthologs in fish could provide relevant insights into differential transactivation patterns
between human and fish <sup>35</sup>. For the PR, a single *prg* gene is transcribed in human into two
isoforms, PRA and PRB, by alternate initiation of transcription of two distinct promoters <sup>36</sup>
while in zebrafish, one *prg* gene coding for one PR have been cloned and characterized <sup>2</sup>. As
for other NRs, the DBD region of the PR is very well conserved between human and fish with
90% of sequence similarities while the LBD displayed more variation with only 67% of
similarities between amino acid sequences in human and zebrafish <sup>2,35,37</sup>. Similar differences
in amino acid sequences in the LBD domain between human and fish have been reported for
other NRs such as ERs and PPARs for which differences in transactivation properties by
environmental contaminants is documented <sup>38</sup>. However, none of these latter studies reported
such an ambivalent biological activity for a given substance, *i.e.* which behave as an agonist
in human and as an antagonist in fish as in our study for the progestins.

In order to explore the differential activity of PR ligands between h andzfPR, a structural
analysis and molecular modeling was done (Figure 3, Figure S6). It revealed several amino
acid differences within the ligand-binding pockets (LBPs) ofzfPR and hPR that are likely to
account for their ligand-binding specificities. Using the modeling and docking server EDMon
(http://edmon.cbs.cnrs.fr/) <sup>39</sup>, we generated homology models of the PR ligand-binding
domains (LBDs) of the two species in complex with RU486 or DHP, two ligands displaying
distinct species preferences (Table S1, Figure 1). A visual inspection of the h- andzf-models
reveals essentially five residue substitutions of amino acids in contact or in proximity to the
ligands (G722/C406, V760/L444, S793/P477, F794/I478, L887/V571). Interestingly, the
V760/L444 and L887/V571 compensatory mutations allow the preservation of the stabilizing
interaction between helix H5 and helix H11 (black dotted lines in Figure 3). In the human
receptor, V760 (H5) and L887 (H11) make moderate van der Waals interactions with both
307 ligands. In contrast, our model of thezfPR suggests tighter contacts of L444 (H5) and V571
308 (H11) with the ligands (magenta doted lines in Figure 3) which may account, at least in part,
309 for the higher affinity of DHP forzfPR. Another key substitution is the replacement of the
310 glycine residue G722 in the human receptor by cysteine C406 inzfPR. The additional carbon
311 (Cβ) and sulfur atoms are very likely to provide additional contact points with DHP, thereby
312 participating to increasing further the affinity of this ligand for thezfPR relative to its human
313 homolog (green doted lines in Figure 3D). In contrast, the side chain of C406 generates a
314 strong steric clash with the antagonist extension of RU486 (red asterisk in Figure 3B).
315 Because the position of the bulky RU486 is very much constrained by the amino acid
316 environment, it is very unlikely that the LBP can accommodate the cysteine side-chain
317 without a drastic loss in the binding affinity of RU486.

318 Overall, the comparison of the LBP and LBD sequences and modeling inform on the
319 differential affinity of investigated ligands between h andzfPR but is not informative enough
320 to explain the observed differences in term of activity. This should be further investigated, for
321 instance by using in depth analysis of the crystal structure of the purified human and zebrafish
322 PR.

323 **3.5. (Eco)toxicological relevance of these findings**

324 The observed differences between the *in vitro* progestagenic activities of the tested substances
325 on human and zebrafish PR raise the question of the relevance of the toxicological
326 information provided using mammalian models to predict possible hazard in other species
327 such as zebrafish. Based on our study, it seems that extrapolation of data from mammalian
328 pharmacology and toxicology to fish species may not be helpful. This point is particularly
329 important in the context of environmental hazard assessment of endocrine disrupting
330 substances. In this respect, our results argue for the development of species-specific strategies
to reliably inform on the endocrine mechanism, properties of environmental chemicals to fish,
such approaches being currently lacking from a regulatory point of view.

Progestins are now well-recognized as environmental contaminants occurring at low but
active concentrations, hence posing a risk to aquatic species such as fish <sup>11</sup>. The anti-
progestagenic activity reported for most of the investigated progestins as well as the
progestagenic activity for NES, DRO and the two spironolactone steroids, SPI and CAN on
zfPR, may have consequences on multiple physiological functions mediated by the PR
signaling pathway in exposed fish. An increasing number of studies reports multiple effects of
progestins in fish notably on reproductive functions with decreases in egg productions and/or
cessation of egg laying <sup>14</sup>. Although these *in vivo* effects are often interpreted with regard to
the other endocrine properties of progestins, notably their interaction with the androgen
receptor, the *in vitro* anti-progestagenic activities reported herein may also contribute to the
observed effects. Indeed, the inhibition of the DHP-inducedzfPR signaling agrees with recent
data in homozygous knockout zebrafish for PR females that are infertile due to ovulation
defects (anovulation), a physiological response directly attributed to the role of PR in
ovulation process <sup>4,5</sup>. Remarkably, the inability of females to ovulate, resulting in abdominal
swelling, has been observed in a wild population of gudgeon exposed to a pharmaceutical
manufacture effluent releasing synthetic steroids, including high concentrations of synthetic
progestins <sup>40</sup>. While the anti-progestagenic activity could provide a new molecular basis to
explain the inhibition of eggs released in exposed-female fish to progestins, direct
extrapolation of *in vitro* data to the *in vivo* situation should however consider
pharmacodynamics of progestins that are currently lacking for fish. Whatever, our data stress
the need for a better characterization of environmental contaminants towards fish NRs in
general, particularly fish PR and to further investigate the effects of progestins on key PR-
dependent physiological processes.
356

357

358

359 Acknowledgements

360 This research was supported by the ANR PROOFS "Occurrences and effects of

361 environmental ligands of progesterone receptor on fish reproduction and neurodevelopment"

362 (ANR-13-CESA-0006-03). Clémentine Garoche was supported by a doctoral fellowship from

363 the ANR PROOFS and the French Ministry of Ecology (grant P190 "Axe de Recherche

364 Ecotoxicologie" to INERIS).
366 (1) Nagahama, Y. 17 Alpha, 20 Beta-Dihydroxy-4-Pregnen-3-One, a Maturation-Inducing
367 Hormone in Fish Oocytes: Mechanisms of Synthesis and Action. *Steroids* **1997**, *62* (1),
368 190–196.

369 (2) Chen, S. X.; Bogerd, J.; García-López, A.; de Jonge, H.; de Waal, P. P.; Hong, W. S.;
370 Schulz, R. W. Molecular Cloning and Functional Characterization of a Zebrafish
371 Nuclear Progesterone Receptor. *Biol. Reprod.* **2010**, *82* (1), 171–181.
372 https://doi.org/10.1095/biolreprod.109.077644.

373 (3) Schneider, J. S.; Burgess, C.; Sleiter, N. C.; DonCarlos, L. L.; Lydon, J. P.; O'Malley,
374 B.; Levine, J. E. Enhanced Sexual Behaviors and Androgen Receptor
375 Immunoreactivity in the Male Progesterone Receptor Knockout Mouse. *Endocrinology
376* **2005**, *146* (10), 4340–4348. https://doi.org/10.1210/en.2005-0490.

377 (4) Tang, H.; Liu, Y.; Li, J.; Yin, Y.; Li, G.; Chen, Y.; Li, S.; Zhang, Y.; Lin, H.; Liu, X.;
378 et al. Gene Knockout of Nuclear Progesterone Receptor Provides Insights into the
379 Regulation of Ovulation by LH Signaling in Zebrafish. *Sci. Rep.* **2016**, *6*, 28545.
380 https://doi.org/10.1038/srep28545.

381 (5) Zhu, Y.; Liu, D.; Shaner, Z. C.; Chen, S.; Hong, W.; Stellwag, E. J. Nuclear Progestin
382 Receptor (Pgr) Knockouts in Zebrafish Demonstrate Role for Pgr in Ovulation but Not
383 in Rapid Non-Genomic Steroid Mediated Meiosis Resumption. *Front. Endocrinol.*
384 **(Lausanne)**. **2015**, *6* (MAR), 1–10. https://doi.org/10.3389/fendo.2015.00037.

385 (6) Africander, D.; Verhoog, N.; Hapgood, J. P. Molecular Mechanisms of Steroid
386 Receptor-Mediated Actions by Synthetic Progestins Used in HRT and Contraception.
387 *Steroids* **2011**, *76* (7), 636–652. https://doi.org/10.1016/j.steroids.2011.03.001.

388 (7) Besse, J. P.; Garric, J. Progestagens for Human Use, Exposure and Hazard Assessment
389 for the Aquatic Environment. *Environ. Pollut.* **2009**, *157* (12), 3485–3494.
390 https://doi.org/10.1016/j.envpol.2009.06.012.

391 (8) Sitruk-Ware, R. Pharmacological Profile of Progestins. *Maturitas* **2004**, *47* (4), 277–
392 283. https://doi.org/10.1016/j.maturitas.2004.01.001.

393 (9) Besse, J. P.; Garric, J. Human Pharmaceuticals in Surface Waters. Implementation of a
394 Prioritization Methodology and Application to the French Situation. *Toxicol. Lett.*
395 **2008**, *176* (2), 104–123. https://doi.org/10.1016/j.toxlet.2007.10.012.
396 (10) Creusot, N.; Aït-Aïssa, S.; Tapie, N.; Pardon, P.; Brion, F.; Sanchez, W.; Thybaud, E.;
397 Porcher, J.-M.; Budzinski, H. Identification of Synthetic Steroids in River Water
398 Downstream from Pharmaceutical Manufacture Discharges Based on a Bioanalytical
399 Approach and Passive Sampling. *Environ. Sci. Technol.* **2014**, *48* (7), 3649–3657.

400 (11) Fent, K. Progestins as Endocrine Disrupters in Aquatic Ecosystems: Concentrations, 401 Effects and Risk Assessment. *Environ. Int.* **2015**, *84*, 115–130.
402 https://doi.org/10.1016/j.envint.2015.06.012.

403 (12) Kumar, V.; Johnson, A. C.; Trubiroha, A.; Tumová, J.; Grabic, R.; Kloas, W.; Tanaka,
404 H.; Kroupová, H. K. Ecotoxicological Research : A Critical Review The Challenge
405 Presented by Progestins in Ecotoxicological Research : A Critical Review. **2015**.

406 (13) Svensson, J.; Fick, J.; Brandt, I.; Brunström, B. Environmental Concentrations of an
407 Androgenic Progestin Disrupts the Seasonal Breeding Cycle in Male Three-Spined
408 Stickleback (Gasterosteus Aculeatus). *Aquat. Toxicol.* **2014**, *147*, 84–91.
409 https://doi.org/10.1016/j.aquatox.2013.12.013.

410 (14) Paulos, P.; Runnalls, T. J.; Nallani, G.; La Point, T.; Scott, A. P.; Sumpter, J. P.;
411 Huggett, D. B. Reproductive Responses in Fathead Minnow and Japanese Medaka
412 Following Exposure to a Synthetic Progestin, Norethindrone. *Aquat. Toxicol.* **2010**, *99*
413 (2), 256–262. https://doi.org/10.1016/j.aquatox.2010.05.001.

414 (15) Svensson, J.; Fick, J.; Brandt, I.; Brunström, B. The Synthetic Progestin Levonorgestrel
415 Is a Potent Androgen in the Three-Spined Stickleback (Gasterosteus Aculeatus).
416 *Environ. Sci. Technol.* **2013**, *47* (4), 2043–2051.

417 (16) Brion, F.; Le Page, Y.; Piccini, B.; Cardoso, O.; Tong, S. K.; Chung, B. C.; Kah, O.
418 Screening Estrogenic Activities of Chemicals or Mixtures in Vivo Using Transgenic
419 (Cyp19a1b-GFP) Zebrafish Embryos. *PLoS One* **2012**, *7* (5), e36069.
420 https://doi.org/10.1371/journal.pone.0036069.

421 (17) Cano-Nicolau, J.; Garoche, C.; Hinfray, N.; Pellegrini, E.; Boujrad, N.; Pakdel, F.;
422 Kah, O.; Brion, F. Several Synthetic Progestins Disrupt the Glial Cell Specific-Brain
423 Aromatase Expression in Developing Zebra Fish. *Toxicol. Appl. Pharmacol.* **2016**, *305*.
424 https://doi.org/10.1016/j.taap.2016.05.019.

425 (18) Hinfray, N.; Tebby, C.; Garoche, C.; Piccini, B.; Bourgine, G.; Ait-Aissa, S.; Kah, O.;
426 Pakdel, F.; Brion, F. Additive Effects of Levonorgestrel and Ethinylestradiol on Brain
427 Aromatase (Cyp19a1b) in Zebrafish Specific in Vitro and in Vivo Bioassays. *Toxicol.*
428 *Appl. Pharmacol. 2016*, 307, 108–114. https://doi.org/10.1016/j.taap.2016.07.023.

429 (19) Bain, P. a.; Kumar, A.; Ogino, Y.; Iguchi, T. Nortestosterone-Derived Synthetic
430 Progestogens Do Not Activate the Progestogen Receptor of Murray–Darling
431 Rainbowfish (*Melanotaenia Fluviatilis*) but Are Potent Agonists of Androgen
432 Receptors Alpha and Beta. *Aquat. Toxicol.* **2015**, *163*, 97–101.
433 https://doi.org/10.1016/j.aquatox.2015.03.021.

434 (20) Ellestad, L. E.; Cardon, M.; Chambers, I. G.; Farmer, J. L.; Hartig, P.; Stevens, K.;
435 Villeneuve, D. L.; Wilson, V.; Orlando, E. F. Environmental Gestagens Activate
436 Fathead Minnow (Pimephales Promelas) Nuclear Progesterone and Androgen
437 Receptors in Vitro. *Environ. Sci. Technol.* **2014**, *48*, 8179–8187.
438 https://doi.org/10.1021/es501428u.

439 (21) Zhao, Y.; Castiglioni, S.; Fent, K. Synthetic Progestins Medroxyprogesterone Acetate
440 and Dydrogesterone and Their Binary Mixtures Adversely Affect Reproduction and
441 Lead to Histological and Transcriptional Alterations in Zebrafish (Danio Rerio).
442 *Environ. Sci. Technol.* **2015**, 150317142110007. https://doi.org/10.1021/es505575v.

443 (22) Bellet, V.; Hernandez-Raquet, G.; Dagnino, S.; Seree, L.; Pardon, P.; Bancon-Montiny,
444 C.; Fenet, H.; Creusot, N.; Ait-Aissa, S.; Cavailles, V.; et al. Occurrence of Androgens
445 in Sewage Treatment Plants Influents Is Associated with Antagonist Activities on
446 Other Steroid Receptors. *Water Res.* **2012**, *46* (6), 1912–1922.
447 https://doi.org/10.1016/j.watres.2012.01.013.

448 (23) Balaguer, P.; François, F.; Comunale, F.; Fenet, H.; Boussioux, A. M.; Pons, M.;
449 Nicolas, J. C.; Casellas, C. Reporter Cell Lines to Study the Estrogenic Effects of
450 Xenoestrogens. *Sci. Total Environ.* **1999**, *233* (1–3), 47–56.

451 (24) Kerdivel, G.; Habauzit, D.; Pakdel, F. Assessment and Molecular Actions of
452 Endocrine-Disrupting Chemicals That Interfere with Estrogen Receptor Pathways. *Int.
453 J. Endocrinol.* **2013**, *2013*, 501851. https://doi.org/10.1155/2013/501851.

454 (25) Escande, A.; Servant, N.; Rabenoelina, F.; Auzou, G.; Kloosterboer, H.; Cavailles, V.;
455 Balaguer, P.; Maudelonde, T. Regulation of Activities of Steroid Hormone Receptors
456 by Tibolone and Its Primary Metabolites. *J. Steroid Biochem. Mol. Biol.* **2009**, *116* (1–
457 2), 8–14. https://doi.org/10.1016/j.jsbmb.2009.03.008.
458 (26) Le Fol, V.; Aït-Aïssa, S.; Cabaton, N.; Dolo, L.; Grimaldi, M.; Balaguer, P.; Perdu, E.;
459 Debrauwer, L.; Brion, F.; Zalko, D. Cell-Specific Biotransformation of Benzophenone-
460 2 and Bisphenol-S in Zebrafish and Human in Vitro Models Used for Toxicity and
461 Estrogenicity Screening. *Environ. Sci. Technol.* **2015**, *49* (6), 3860–3868.
462 https://doi.org/10.1021/es505302c.

463 (27) Eide, M.; Rusten, M.; Male, R.; Jensen, K. H. M.; Goksøyr, A. A Characterization of
464 the ZFL Cell Line and Primary Hepatocytes as in Vitro Liver Cell Models for the
465 Zebrafish (Danio Rerio). *Aquat. Toxicol.* **2014**, *147*, 7–17.
466 https://doi.org/10.1016/j.aquatox.2013.11.023.

467 (28) Cosnefroy, A.; Brion, F.; Maillot-Maréchal, E.; Porcher, J.-M.; Pakdel, F.; Balaguer,
468 P.; Aït-Aïssa, S. Selective Activation of Zebrafish Estrogen Receptor Subtypes by
469 Chemicals by Using Stable Reporter Gene Assay Developed in a Zebrafish Liver Cell
470 Line. *Toxicol. Sci.* **2012**, *125* (2), 439–449. https://doi.org/10.1093/toxsci/kfr297.

471 (29) Le Fol, V.; Aït-Aïssa, S.; Sonavane, M.; Porcher, J.-M.; Balaguer, P.; Cravedi, J.-P.;
472 Zalko, D.; Brion, F. In Vitro and in Vivo Estrogenic Activity of BPA, BPF and BPS in
473 Zebrafish-Specific Assays. *Ecotoxicol. Environ. Saf.* **2017**, *142*, 150–156.
474 https://doi.org/10.1016/j.ecoenv.2017.04.009.

475 (30) Serra, H.; Scholze, M.; Altenburger, R.; Busch, W.; Budzinski, H.; Brion, F.; Aït-Aïssa, S. Combined Effects of Environmental Xeno-Estrogens within Multi-Component Mixtures: Comparison of in Vitro Human- and Zebrafish-Based Estrogenicity Bioassays. *Chemosphere* **2019**, *227*, 334–344.
476
477
478
479 https://doi.org/10.1016/j.chemosphere.2019.04.060.

480 (31) Enríquez, J.; Lemus, A. E.; Chimal-Monroy, J.; Arzate, H.; García, G. A.; Herrero, B.;
481 Larrea, F.; Pérez-Palacios, G. The Effects of Synthetic 19-Noprogestins on
482 Osteoblastic Cell Function Are Mediated by Their Non-Phenolic Reduced Metabolites.
483 *J. Endocrinol.* **2007**, *193* (3), 493–504. https://doi.org/10.1677/JOE-06-0038.

484 (32) Molina-Molina, J. M.; Escande, A.; Pillon, A.; Gomez, E.; Pakdel, F.; Cavaillès, V.;
485 Olea, N.; Aït-Aïssa, S.; Balaguer, P. Profiling of Benzophenone Derivatives Using Fish
486 and Human Estrogen Receptor-Specific in Vitro Bioassays. *Toxicol. Appl. Pharmacol.*
487 **2008**, *232* (3), 384–395. https://doi.org/10.1016/j.taap.2008.07.017.

488 (33) Hotchkiss, A. K.; Rider, C. V; Blystone, C. R.; Wilson, V. S.; Hartig, P. C.; Ankley, G.
489 T.; Foster, P. M.; Gray, C. L.; Gray, L. E. Fifteen Years after “Wingspread”--
490

Environmental Endocrine Disrupters and Human and Wildlife Health: Where We Are

491

Today and Where We Need to Go. *Toxicol. Sci.* **2008**, *105* (2), 235–259.

492

https://doi.org/10.1093/toxsci/kfn030.

493

(34) Ekins, S.; Reschly, E. J.; Hagey, L. R.; Krasowski, M. D. Evolution of Pharmacologic

494

Specificity in the Pregnane X Receptor. *BMC Evol. Biol.* **2008**, *8* (1), 103.

495

https://doi.org/10.1186/1471-2148-8-103.

496

(35) Zhao, Y.; Zhang, K.; Giesy, J. P.; Hu, J. Families of Nuclear Receptors in Vertebrate

497

Models: Characteristic and Comparative Toxicological Perspective. *Sci. Rep.* **2015**, *5*,

498

8554. https://doi.org/10.1038/srep08554.

499

(36) Giangrande, P.; McDonnell, D. The A and B Isoforms of the Human Progesterone

500

Receptor: Two Functionally Different Transcription Factors Encoded by a Single Gene.

501

*Recent Prog. Horm. Res.* **1999**, *54*, 291–314.

502

(37) Hanna, R. N.; Daly, S. C. J.; Pang, Y.; Anglade, I.; Kah, O.; Thomas, P.; Zhu, Y.

503

Characterization and Expression of the Nuclear Progestin Receptor in Zebrafish

504

Gonads and Brain. *Biol. Reprod.* **2010**, *82* (1), 112–122.

505

https://doi.org/10.1095/biolreprod.109.078527.

506

(38) Grimaldi, M.; Boulahtouf, A.; Delfosse, V. Reporter Cell Lines to Evaluate the

507

Selectivity of Chemicals for Human and Zebrafish Estrogen and Peroxysome

508

Proliferator Activated γ Receptors. **2015**, *9* (June), 1–9.

509

https://doi.org/10.3389/fnins.2015.00212.

510

(39) Schneider, M.; Pons, J.-L.; Bourguet, W.; Labesse, G. Towards Accurate High-

511

Throughput Ligand Affinity Prediction by Exploiting Structural Ensembles, Docking

512

Metrics and Ligand Similarity. *Bioinformatics* **2020**, *36* (1), 160–168.

513

https://doi.org/10.1093/bioinformatics/btz538.

514

(40) Sanchez, W.; Sremski, W.; Piccini, B.; Palluel, O.; Maillot-Maréchal, E.; Betoulle, S.;

515

Jaffal, A.; Aït-Aïssa, S.; Brion, F.; Thybaud, E.; et al. Adverse Effects in Wild Fish

516

Living Downstream from Pharmaceutical Manufacture Discharges. *Environ. Int.* **2011**,

517

37 (8), 1342–1348. https://doi.org/10.1016/j.envint.2011.06.002.

518
<table><caption>**Table 1:** Classification of natural hormones and natural and synthetic nPR ligands tested on HELN-hPRB and U2OS-zfPR cells.</caption><thead><tr><th>Classification</th><th>Compound</th><th>Abbreviation</th><th>Uses</th><th>Molecular weight (g/mol)</th><th>CAS</th><th>Structure</th></tr></thead><tbody><tr><td>Natural progestin in human and fish</td><td>Progesterone</td><td>P4</td><td>Clinical use</td><td>314.46</td><td>57-83-0</td><td></td></tr><tr><td>Natural progestin in fish</td><td>17 α,20 β-dihydroxy-4-pregnen-3-one</td><td>DHP</td><td></td><td>332.48</td><td>1662-06-2</td><td></td></tr><tr><td>Synthetic progestin derived from 19-norprogesterone</td><td>Promegestone</td><td>R5020</td><td>Clinical use</td><td>326.47</td><td>34184-77-5</td><td></td></tr><tr><td>PR antagonist</td><td>Mifepristone</td><td>RU486</td><td>Clinical use</td><td>429.60</td><td>84371-65-3</td><td></td></tr><tr><td>Synthetic progestin retroprogesterone, isomer of P4</td><td>Dydrogesterone</td><td>DYD</td><td>Clinical use</td><td>312.45</td><td>152-62-5</td><td></td></tr><tr><td rowspan="10">Progestins structurally related to progesterone</td><td rowspan="5">Derived from 17α-hydroxyprogesterone</td><td>Medroxyprogesterone</td><td>MEP</td><td>Clinical use</td><td>344.49</td><td>520-85-4</td><td></td></tr><tr><td>Medroxyprogesterone acetate</td><td>MPA</td><td>Clinical use</td><td>386.52</td><td>71-58-9</td><td></td></tr><tr><td>Megestrol acetate</td><td>MGA</td><td>Clinical use</td><td>384.51</td><td>595-33-5</td><td></td></tr><tr><td>Chlormadinone acetate</td><td>CMA</td><td>Clinical use</td><td>406.94</td><td>302-22-7</td><td></td></tr><tr><td>Cyproterone acetate</td><td>CPA</td><td>Clinical use</td><td>416.94</td><td>427-51-0</td><td></td></tr><tr><td rowspan="3">Derived from 19-norprogesterone</td><td>Promegestone</td><td>R5020</td><td>Clinical use</td><td>326.47</td><td>34184-77-5</td><td></td></tr><tr><td>Nestorone</td><td>NES</td><td>Clinical use</td><td>370.48</td><td>7759-35-5</td><td></td></tr><tr><td>Nomegestrol acetate</td><td>NGA</td><td>Clinical use</td><td>370.48</td><td>58652-20-3</td><td></td></tr><tr><td>Derived from 17α-hydroxy-19-norprogesterone</td><td>Gestonorone</td><td>GRN</td><td>Clinical use of gestonorone caproate</td><td>316.43</td><td>2137-18-0</td><td></td></tr></tbody></table>
<table><tbody><tr><td rowspan="10">Progestins structurally related to testosterone: derived from 19-nortestosterone</td><td rowspan="5">Estranes</td><td>Ethisterone</td><td>ETH</td><td>Stopped</td><td>312.45</td><td>434-03-7</td><td></td></tr><tr><td>Ethynodiol diacetate</td><td>EDA</td><td>Clinical use</td><td>384.51</td><td>297-76-7</td><td></td></tr><tr><td>Lynestrenol</td><td>LYN</td><td>Clinical use</td><td>284.44</td><td>52-76-6</td><td></td></tr><tr><td>Norethindrone acetate</td><td>NEA</td><td>Clinical use</td><td>340.46</td><td>51-98-9</td><td></td></tr><tr><td>Norethindrone</td><td>NET</td><td>Clinical use</td><td>298.42</td><td>68-22-4</td><td></td></tr><tr><td rowspan="5">Gonanes</td><td>Tibolone</td><td>TIB</td><td>Clinical use</td><td>312.4</td><td>5630-53-5</td><td></td></tr><tr><td>Desogestrel</td><td>DSG</td><td>Clinical use</td><td>310.47</td><td>54024-22-5</td><td></td></tr><tr><td>Etonogestrel</td><td>ENG</td><td>Clinical use, metabolite of desogestrel</td><td>324.45</td><td>54048-10-1</td><td></td></tr><tr><td>Gestodene</td><td>GES</td><td>Clinical use</td><td>310.43</td><td>60282-87-3</td><td></td></tr><tr><td>Levonorgestrel</td><td>LNG</td><td>Clinical use</td><td>312.45</td><td>797-63-7</td><td></td></tr><tr><td rowspan="2">Progestin structurally related to spironolactone</td><td rowspan="2"></td><td>Norgestimate</td><td>NTE</td><td>Clinical use</td><td>369.50</td><td>35189-28-7</td><td></td></tr><tr><td>Norgestrel</td><td>NGL</td><td>Clinical use</td><td>312.45</td><td>6533-00-2</td><td></td></tr><tr><td rowspan="3">Natural hormones and precursors</td><td rowspan="3"></td><td>Drospirenone</td><td>DRO</td><td>Clinical use</td><td>366.49</td><td>67392-87-4</td><td></td></tr><tr><td>Pregnenolone (progesterone precursor)</td><td>P5</td><td>Clinical use</td><td>316.48</td><td>145-13-1</td><td></td></tr><tr><td>17β-estradiol</td><td>E2</td><td>Clinical use</td><td>272.38</td><td>50-28-2</td><td></td></tr><tr><td></td><td></td><td>Testosterone</td><td>T</td><td>Clinical use</td><td>288,42</td><td>58-22-0</td><td></td></tr></tbody></table>

<figure><img src="image_3.png" /></figure>
<table><caption>**Table 2:** Calculated EC<sub>50</sub> and IC<sub>50</sub> (nM) in HELN-hPRB and U2OS-zfPR cells.</caption><thead><tr><th rowspan="2">Classification</th><th rowspan="2">Abb</th><th colspan="3">HELN-PRB</th><th colspan="3">U2OS-zfPR</th></tr><tr><th>EC<sub>50</sub> ± sd (nM)</th><th>max ± sd (%)</th><th>REP</th><th>IC<sub>50</sub> ± sd (nM)</th><th>EC<sub>50</sub> ± sd (nM)</th><th>max ± sd (%)</th><th>REP</th></tr></thead><tbody><tr><td rowspan="4">Reference compounds</td><td>P4</td><td>13.5 ± 7.14</td><td>70 ± 9</td><td>0.02</td><td>17.1 ± 15.7</td><td>1.97 ± 0.57</td><td>40 ± 7</td><td>0.95</td><td>2.09 ± 0.33</td></tr><tr><td>R5020</td><td>0.33 ± 0.10</td><td>100</td><td>1</td><td>-</td><td>1.93 ± 0.60</td><td>64 ± 11</td><td>0.97</td><td>-</td></tr><tr><td>DHP</td><td>ne</td><td></td><td></td><td>576 ± 319</td><td>1.87 ± 0.44</td><td>100</td><td>1</td><td>-</td></tr><tr><td>RU486</td><td>ne</td><td></td><td></td><td>1.04 ± 0.28</td><td>ne</td><td></td><td></td><td>2253 ± 292</td></tr><tr><td rowspan="20">Other progestins</td><td>DYD</td><td>8.03 ± 3.28</td><td>66 ± 13</td><td>0.04</td><td>14.5</td><td>ne</td><td></td><td></td><td>24.9 ± 6.38</td></tr><tr><td>MEP</td><td>249 ± 105</td><td>37 ± 8</td><td>0.001</td><td>39.6 ± 23.4</td><td>ne</td><td></td><td></td><td>91.4 ± 55.9</td></tr><tr><td>MPA</td><td>0.20 ± 0.05</td><td>68 ± 15</td><td>1.7</td><td>8.08 ± 5.74</td><td>ne</td><td></td><td></td><td>30.0 ± 7.86</td></tr><tr><td>MGA</td><td>0.57 ± 0.04</td><td>74 ± 14</td><td>0.58</td><td>4.70 ± 3.87</td><td>ne</td><td></td><td></td><td>95.2 ± 55.5</td></tr><tr><td>CMA</td><td>0.65 ± 0.07</td><td>84 ± 7</td><td>0.50</td><td>ne</td><td>ne</td><td></td><td></td><td>125 ± 22</td></tr><tr><td>CPA</td><td>1.15 ± 0.16</td><td>79 ± 0</td><td>0.29</td><td>ne</td><td>ne</td><td></td><td></td><td>244 ± 93.3</td></tr><tr><td>NES</td><td>0.25 ± 0.03</td><td>90 ± 5</td><td>1.34</td><td>ne</td><td>1.41 ± 0.41</td><td>40 ± 2</td><td>1.33</td><td>6.39 ± 4.00</td></tr><tr><td>NGA</td><td>0.60 ± 0.22</td><td>92 ± 14</td><td>0.55</td><td>ne</td><td>ne</td><td></td><td></td><td>17.8 ± 6.99</td></tr><tr><td>GRN</td><td>18.4 ± 5.11</td><td>50 ± 15</td><td>0.02</td><td>34.9 ± 15.0</td><td>ne</td><td></td><td></td><td>26.2 ± 11.3</td></tr><tr><td>ETH</td><td>6.20 ± 0.61</td><td>55 ± 12</td><td>0.05</td><td>14.1 ± 4.42</td><td>ne</td><td></td><td></td><td>69.5 ± 8.17</td></tr><tr><td>EDA</td><td>15.3 ± 4.80</td><td>62 ± 19</td><td>0.02</td><td>156 ± 117</td><td>ne</td><td></td><td></td><td>354 ± 295</td></tr><tr><td>LYN</td><td>25.7 ± 10.8</td><td>56 ± 2</td><td>0.01</td><td>61.9 ± 13.2</td><td>ne</td><td></td><td></td><td>503 ± 110</td></tr><tr><td>NEA</td><td>0.85 ± 0.57</td><td>90 ± 12</td><td>0.39</td><td>ne</td><td>ne</td><td></td><td></td><td>22.2 ± 0.55</td></tr><tr><td>NET</td><td>0.93 ± 0.27</td><td>83 ± 4</td><td>0.35</td><td>ne</td><td>ne</td><td></td><td></td><td>34.3 ± 1.66</td></tr><tr><td>TIB</td><td>9.97 ± 1.33</td><td>85 ± 6</td><td>0.03</td><td>ne</td><td>ne</td><td></td><td></td><td>146</td></tr><tr><td>DSG</td><td>14.1 ± 6.80</td><td>72 ± 24</td><td>0.02</td><td>76.0 ± 51.2</td><td>ne</td><td></td><td></td><td>1002</td></tr><tr><td>ENG</td><td>0.18 ± 0.04</td><td>94 ± 7</td><td>1.77</td><td>ne</td><td>ne</td><td></td><td></td><td>148 ± 98</td></tr><tr><td>GES</td><td>0.14 ± 0.05</td><td>95 ± 5</td><td>2.43</td><td>ne</td><td>ne</td><td></td><td></td><td>15.2 ± 16.2</td></tr><tr><td>LNG</td><td>0.16 ± 0.04</td><td>96 ± 6</td><td>2.08</td><td>ne</td><td>ne</td><td></td><td></td><td>14.0 ± 7.41</td></tr><tr><td>NTE</td><td>1.11 ± 0.26</td><td>78 ± 10</td><td>0.30</td><td>ne</td><td>ne</td><td></td><td></td><td>77.4 ± 4.16</td></tr><tr><td>NGL</td><td>0.47 ± 0.12</td><td>101 ± 1</td><td>0.70</td><td>ne</td><td>ne</td><td></td><td></td><td>43.6 ± 17.2</td></tr><tr><td>DRO</td><td>5.72 ± 1.38</td><td>60 ± 14</td><td>0.06</td><td>58.0 ± 66.1</td><td>14.1 ± 4.49</td><td>30 ± 6</td><td>0.13</td><td>16.1 ± 1.44</td></tr></tbody></table>

REP: Relative potency in HELN-hPRB cells (EC<sub>50</sub>(R5020)/EC<sub>50</sub>(chemical)) and U2OS-zfPR cells (EC<sub>50</sub>(DHP)/EC<sub>50</sub>(chemical)). Standard deviations (sd); ne: no effect.
<figure><img src="image_4.png" /><figcaption>**Figure 1:** Dose-response curves of the activity of reference ligands in HELN-hPRB and U2OS-zfPR cells expressed as the percentage of the maximum luciferase activity induced by 3x10⁻⁸ M R5020 (A,C) or 3x10⁻⁸ M DHP (B,D). P4: progesterone; DHP: 17α,20β-dihydroxy-4-pregnen-3-one; R5020: promegestone, RU486: mifepristone. Error bars represent standard deviation.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>(C) Agonists on U2OS-zfPR</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>(B) Partial agonists on HELN-hPRB</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>(D) Antagonists on U2OS-zfPR</figcaption></figure>

<figure><img src="image_8.png" /></figure>

**Figure 2:** Dose-response curves of the activity of progestins in HELN-hPRB and U2OS-zfPR cells expressed as the percentage of the maximum luciferase activity induced by 3x10⁻⁸ M R5020 (A, B) or 3x10⁻⁸ M DHP (C, D). P4: progesterone; DHP: 17α,20β-dihydroxy-4-pregnen-3-one; R5020: promegestone, P5: pregnenolone; DYD: dydrogesterone; MEP: medroxyprogesterone; MPA: medroxyprogesterone acetate; MGA: megestrol acetate; CMA: chlormadinone acetate; CPA: cyproterone acetate; NES: nestorone; NGA: nomegestrol acetate; ETH: ethisterone; EDA: ethynodiol diacetate; LYN: lynestrenol; NEA: norethindrone acetate; NET: norethindrone; TIB: tibolone; DSG: desogestrel; ENG: etonogestrel; GES: gestodene; LNG: levonorgestrel; NTE: norgestimate; NGL: norgestrel; DRO: drospirenone; GRN: gestonorone. Error bars represent standard deviation (SD, n=3 replicates per concentration). All curves for individual compounds are presented separately in Supplementary Materials, Table S1.
<figure><img src="image_9.png" /></figure>

**Figure 3:** Structural analysis of species-specificity for RU486 and DHP. Structure (A), PDB code 2w8y, and docking model (B) of RU486 bound to human and zebrafish PR, respectively. (C, D) Docking models of DHP bound to human and zebrafish PR, respectively. The ligands and residues discussed in the main text are shown as sticks. The structural elements of PR LBD are shown in grey and labelled. The dashed lines denote the interactions described in the text. The red asterisk denotes a steric clash between C406 and RU486. Docking models were generated using the server EDMon (http://edmon.cbs.cnrs.fr) and the default parameters. Figures were prepared with PyMOL (http://pymol.org/).
**Supporting information**

**Human and zebrafish nuclear progesterone receptors are differently activated by
progestins**

Clémentine Garoche, Selim Aït-Aïssa, Abdelhay Boulahtouf, Nicolas Creusot, William
Bourget, Patrick Balaguer, François Brion

**Table of content**

**Figure S1:** Constructions of the cell lines HELN-hPRB (A) and U2OS-zfPR (B).

**Table S1:** Typical concentration-response curves of the activity of progestins in HELN-hPRB and U2OS-zfPR cells.

**Figure S2:** HELN-hPRB cells (A) and U2OS-zfPR (B) cells were treated with indicated concentrations of 17β-estradiol (E2), testosterone (T) and 11-Ketotestosterone (11-KT).

**Figure S3:** Concentration-response curves of the activity of progestins in U2OS cells expressing hPR (A) and HeLa cells expressingzfPR (B).

**Figure S4:** Activity of spironolactone and canrenone towards h andzfPR.

**Figure S5:** Activity of progestins in ZFL-zfPR cells.

**Figure S6:** Amino acids sequence alignment of the human (hPR-B) and zebrafish (zfPR) progesterone receptor.
<figure><img src="image_10.png" /><figcaption>(B) U2OS-zfPR</figcaption></figure>

<figure><img src="image_11.png" /></figure>

**Figure S1:** Constructions of the cell lines HELN-hPRB (A) and U2OS-zfPR (B). Each contains six domains. Domain A/B: activation factor; domain C: human estrogen receptor hERα DNA binding domain (C251-M316); domain D: hinge; domain E: hPR-B (A) orzfPR (B) ligand binding domain.
<figure><img src="image_12.png" /><figcaption>**Table S1: Typical concentration-response curves of the activity of progestins in HELN-hPRB and U2OS-zfPR cells.** Results are expressed as percentage of the maximum luciferase activity induced by $3×10^{-8}$ M R5020 in HELN-hPRB or $3×10^{-8}$ M DHP in U2OS-zfPR. Cells were treated with progestins alone (black circles) or in co-exposure with R5020 or DHP at concentrations inducing 80% of the maximum luciferase activity ($10^{-8}$ M R5020 and $3×10^{-9}$ M respectively) (white circles). Error bars represent standard deviation.</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>Table S1 (continued)</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>**Figure S2:** HELN-hPRB cells (A) and U2OS-zfPR (B) cells were treated with indicated concentrations of 17β-estradiol (E2), testosterone (T) and 11-Ketotestosterone (11-KT). Results are expressed as the percentage of the maximum luciferase activity induced by 3x10⁻⁸ M R5020 (A) or 3x10⁻⁸ M DHP (B). Error bars represent standard deviation. The absence of luciferase activity by the steroidal hormones indicates the specificity of response of the cell models.</figcaption></figure>
(A) U2OS-hPRB

<figure><img src="image_15.png" /></figure>

(B) HeLa-zfPR

<figure><img src="image_16.png" /></figure>

**Figure S3: Concentration-response curves of the activity of progestins in U2OS cells expressing hPR (A) and HeLa cells expressingzfPR (B).** The results are expressed as the percentage of the maximum luciferase activity induced by 3x10⁻⁸ M R5020 (A) or 3x10⁻⁸ M DHP (B). P4: progesterone; DHP: 17α,20β-dihydroxy-4-pregnen-3-one; R5020: promegestone, MPA: medroxyprogesterone acetate; ENG: etonogestrel; RU486: mifepristone. Error bars represent standard deviation. U2OS cells were transiently cotransfected by a plasmid expressing ERE-luciferase and a second one expressing the hPR with the EhRα DNA binding domain. HeLa cells were transiently cotransfected by a plasmid expressing ERE-luciferase and a second one expressing thezfPR with the hERα DNA binding domain.
<figure><img src="image_17.png" /><figcaption>(C) Canrenone on hPR</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>(B) Spironolactone onzfPR</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>(D) Canrenone onzfPR</figcaption></figure>

<figure><img src="image_20.png" /></figure>

**Figure S4:** Activity of spironolactone and canrenone towards h andzfPR. Typical concentration-response curves of the activity of spironolactone and canrenone in HELN-hPRB and U2OS-zfPR cells expressed as percentage of the maximum luciferase activity induced by 3×10⁻⁸ M R5020 in HELN-hPRB or 3×10⁻⁸ M DHP in U2OS-zfPR. Cells were treated with chemical substances alone (black circles) or in coexposure with R5020 or DHP at concentrations inducing 80% of the maximum luciferase activity (10⁻⁸ M R5020 and 3×10⁻⁹ M respectively) (white circles). Error bars represent standard deviation.
<figure><img src="image_21.png" /><figcaption>**Figure S5: Activity of progestins in ZFL-zfPR cells.** Results are expressed as the percentage of the maximum luciferase activity induced by 3x10⁻⁸ M DHP. P4: progesterone; DHP: 17α,20β-dihydroxy-4-pregnen-3-one; R5020: promegestone, MPA: medroxyprogesterone acetate; ENG: etonogestrel; RU486: mifepristone. Coexposure are made with DHP at a concentration inducing 80% of the maximum luciferase activity (3x10⁻⁹ M). Error bars represent standard deviation. Letters correspond to significant differences between groups after t-test Student, *** p<0.001.</figcaption></figure>
hPRB (V633-K933)
zfPR (M317-K617)

VLGGRKFKKFNKVRVVRALDAVALPQPVGVPNESQALSQRFTFSPGQDIQLIPPLINLLMS
MLGGRKMKRFAGLKVMGLSPSLMFQSPLSLLTDGQTLSLPCMISAMRELQLSPQMISILEN

IEPDVIYAGHDNTKPDTSSSLLTSLNQLGERQLLSVVKWSKSLPGFRNLHIDDQITLIQYS
IEPQVVYSGYDNTQPEVPHLLLNSLNRLCERQLLLWIVRWSKSLPGFRNLHINDQMTLIQYS

WMSLMVFGLGWRSYKHVSGOMLYFAPDLILNEORMKESSFYSLCLTMWQIPQEFVKLQVSQ
WMGLMLFSLGWRTFQNVTPDYLYFAPDLVLSNDQLRRSPIYDLCLAMQFVPQEFANLQVTK

EEFLCMKVLLLLNTIPLEGLRSQTQFEEMRSSYIRELIKAIGLRQKGVVSSSQRFYQLTKL
EEFLCMKALMLLNTVPLEGLKSQTQFDEMRQNYICELSKAIQLKEKGVVASSQRFYHLTKL

LDNLHDLVKQLHLYCLNTFIQSRALSVEFPEMMSEVIAAQLPKILAGMVKPLLFHKK
MDNMHEIVKKVNLYCLTTFIQADAMKVEFPEMMTEVIASQLPKVLAGMVKPLMFHHK
Figure S6. Amino acids sequence alignment of the human (hPR-B) and zebrafish (zfPR) progesterone receptor ligand binding domain.

The five residue substitutions of amino acids in contact or in proximity to the ligands (G722/C406, V760/L444, S793/P477, F794/I478, L887/V571) are framed.