Environmental Gestagens Activate Fathead Minnow (Pimephales

Article pubs.acs.org/est

Environmental Gestagens Activate Fathead Minnow (Pimephales promelas) Nuclear Progesterone and Androgen Receptors in Vitro Laura E. Ellestad,† Mary Cardon,‡ Ian G. Chambers,† Jennifer L. Farmer,† Phillip Hartig,‡ Kyle Stevens,§ Daniel L. Villeneuve,§ Vickie Wilson,‡ and Edward F. Orlando*,† †

Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland 20742, United States US EPA, ORD-NHEERL, Reproductive Toxicology Division, Research Triangle Park, North Carolina 27711, United States § US EPA, Mid-Continent Ecology Division, Duluth, Minnesota 55804, United States ‡

S Supporting Information *

ABSTRACT: Gestagen is a collective term for endogenous and synthetic progesterone receptor (PR) ligands. In teleost fishes, 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S) are the predominant progestogens, whereas in other vertebrates the major progestogen is progesterone (P4). Progestins are components of human contraceptives and hormone replacement pharmaceuticals and, with P4, can enter the environment and alter fish and amphibian reproductive health. In this study, our primary objectives were to clone the fathead minnow (FHM) nuclear PR (nPR), to develop an in vitro assay for FHM nPR transactivation, and to screen eight gestagens for their ability to transactivate FHM nPR. We also investigated the ability of these gestagens to transactivate FHM androgen receptor (AR). Fish progestogens activated FHM nPR, with DHP being more potent than 20β-S. The progestin drospirenone and P4 transactivated the FHM nPR, whereas five progestins and P4 transactivated FHM AR, all at environmentally relevant concentrations. Progestins are designed to activate human PR, but older generation progestins have unwanted androgenic side effects in humans. In FHMs, several progestins proved to be strong agonists of AR. Here, we present the first mechanistic evidence that environmental gestagens can activate FHM nPR and AR, suggesting that gestagens may affect phenotype through nPR- and AR-mediated pathways.

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INTRODUCTION Fish are sensitive indicator organisms for effects of exposure to native and synthetic steroid hormones and other emerging chemicals of concern in the aquatic environment.1−4 Past research efforts have focused on effects of estrogenic and androgenic compounds on fish, wildlife, agricultural species, and humans. One of the most studied environmental estrogens is 17α-ethinylestradiol (EE2), a component of oral contraceptives.5 Gestagens are also present in contraceptives and hormone replacement therapeutics in quantities 5 to 100 times greater than EE2.6 The number of published studies investigating effects of exposure to environmental gestagens is small but growing, as this class of chemicals has come to the attention of researchers and regulators. Gestagens include natural progestogens (e.g., progesterone, P4) and synthetic progestins (e.g., drospirenone, DRO), and their effects are mediated through nuclear and membrane progesterone receptors (nPR and mPR, respectively). They can enter the aquatic environment through wastewater treatment plant effluent, as progestins from contraceptive and hormonal replacement therapies and as excreted progestogens and have been measured in the low ng/L to several hundred ng/L concentration in water (for review see ref 7). Similarly, excreted © 2014 American Chemical Society

P4 from cattle and other animals are found in agricultural runoff, as is melengestrol acetate (MGA), a progestin used as a growth promoter in beef cattle.8 Once present in waterways, environmental gestagens can potentially act as ligands for nPR, mPRs, and progesterone receptor membrane components (PGRMCs).9−11 In most vertebrates, the primary circulating progestogen is P4, whereas in teleosts 17,20β-dihydroxy-4-pregnen-3-one (DHP) or 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) are the primary progestogens. Final stages of gamete maturation, as well as early events in spermatogenesis, are controlled by DHP12−14 or 20β-S.15,16 Some excreted progestogens, along with androstenedione and prostaglandin F2α, function as pheromones that help coordinate reproductive behavior in fishes.17,18 Clearly, disruption of PR signaling by environmental gestagens could have important consequences on individual reproductive fitness and robustness of fish populations. Received: Revised: Accepted: Published: 8179

March 24, 2014 June 6, 2014 June 6, 2014 June 9, 2014 dx.doi.org/10.1021/es501428u | Environ. Sci. Technol. 2014, 48, 8179−8187

Environmental Science & Technology

Article

female FHM (∼6 month old; n = 6 per sex) obtained from a colony maintained at the US EPA Mid-Continental Ecology Division (Duluth, MN) were euthanized, and tissues were collected as described above. Husbandry and tissue collections were done in accordance with each facility’s Institutional Care and Use Committee guidelines. Cloning and Sequencing of FHM nPR. A traditional strategy employing reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) was used to clone the full length coding sequence of nPR. Oligonucleotide primers (Integrated DNA Technologies (IDT), Coralville, IA) used for cloning and sequencing are listed in Supporting Information, Table S1. Ovary tissue was homogenized with 1 mL of Trizol (Life Technologies), after which 0.2 mL chloroform was added, and the sample was shaken vigorously. After centrifugation, the aqueous phase was transferred to a clean microcentrifuge tube, mixed with an equal volume of 70% ethanol, and applied to an RNeasy Mini column (Qiagen, Valencia, CA) for extraction. Resulting total RNA was quantified using the Quant-iT RiboGreen RNA Quantitation reagent (Life Technologies), reverse transcription (RT) reactions were performed with the SMARTer RACE cDNA Amplification kit (Clontech, Mountain View, CA), and PCR amplification was conducted using GoTaq Green Master Mix (Promega, Madison, WI). Detailed methodology for PCR amplification, sequencing, sequence assembly, and cloning of the FHM nPR open reading frame into pCMV-Sport6.1 expression vector (Life Technologies) is provided in Supporting Information, Supplemental Material and Methods. Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). Total RNA was isolated from pituitaries, kidneys, and hearts using RNeasy Micro kits (Qiagen) and from remaining tissues with RNeasy Mini kits (Qiagen) and was quantified as described above. RNA extractions included an oncolumn DNase digestion and were conducted according to the manufacturer’s protocol with the following additions. Heart and muscle were digested with proteinase K (100 μg/mL) for 10 min at 55 °C after homogenization and prior to RNeasy column application. Ovary, liver, and brain tissue were extracted as described above. RT reactions (20 μL) were performed as described previously27,28 starting with 100 ng total RNA and were diluted to 25 μL prior to PCR analysis. Levels of nPR and ribosomal protein L8 (RPL8; accession no. AY919670) mRNA were quantified using primers (IDT) designed with Beacon Designer 7.0 (Bio-Rad, Hercules, CA) and are listed in Supporting Information, Supplemental Table S1. Amplification efficiencies were determined using CFX Manager Software 3.0 (Bio-Rad) by analyzing 2-fold serial dilutions of a cDNA template prepared from pools of total RNA (2 μg) extracted from ovary and testis and were calculated to be 95.7% for nPR and 95.5% for RPL8. PCR reactions (15 μL) were conducted in duplicate using iQ SYBR Green Supermix (Bio-Rad) containing 1 μL of diluted cDNA and 400 nM concentration of each primer in a CFX96 Real-Time Detection System (Bio-Rad). PCR cycling conditions were as follows: initial denaturation at 95 °C for 3 min followed by 40 cycles of 95 °C for 10 s and 58 °C for 1 min. Dissociation curve analysis was conducted to ensure that a single PCR product was amplified in each reaction but absent from no RT and no template controls. The amount of nPR mRNA was normalized to the amount of RPL8 mRNA in each sample and transformed using equations described previously.28 RPL8 was chosen as the normalization gene because it exhibited the least variation

Fathead minnows (FHM) are the predominant aquatic vertebrate toxicology model used by the Environmental Protection Agency (EPA). These fish are a native species with a broad distribution in North America and are known for their sensitivity to endocrine disrupting chemicals (EDCs),19 including environmental gestagens. The contraceptive progestins medroxyprogesterone acetate (MPA) and norethindrone (NOR) have been shown to have a low tendency to bioaccumulate in the common carp (Cyprinus carpio) and FHM.20,21 In contrast, levonorgestrel (LNG) has been measured in rainbow trout (Oncorhynchus mykiss) plasma at levels of 8.5−12 ng/mL when fish were exposed to wastewater treatment effluent containing ∼1 ng/L LNG, indicating that it can bioaccumulate.22 Importantly, FHM exposed to LNG or another contraceptive progestin, DRO, demonstrated a dose-dependent reduction in fecundity and a commensurate increase in ovarian apoptosis.23 Further, exposure of female FHM to LNG or the progestin gestodene (GES) inhibited reproduction and resulted in the expression of male secondary sex characteristics.24 This masculinization, in combination with known androgenic activity of several progestins commonly used in oral contraceptives, indicate that these compounds may also activate the androgen receptor (AR).6,25 Progestogens are critical regulators of vertebrate reproduction, and results of exposure studies have shown that gestagens negatively affect fecundity and sperm motility and can masculinize female fishes.7 Therefore, understanding the mechanism of action of these compounds and beginning to characterize their interaction with FHM nPR is critical. To this end, the objectives of the current study were (1) to clone fulllength nPR from FHM and assess its tissue distribution in adult fish; (2) to develop an in vitro assay for functional characterization of FHM nPR using fish progestogens; and (3) to use this assay, as well as an established assay for FHM AR transactivation,26 to evaluate the ability of environmental gestagens to activate these receptors.

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MATERIALS AND METHODS Reagents and Chemicals. Unless otherwise stated, chemicals were from Sigma-Aldrich (St. Louis, MO) and cell culture reagents and enzymes were from Life Technologies (Grand Island, NY). Kits and commercial reagents were used according to manufacturers’ instructions, unless otherwise noted. The following steroid hormones were purchased from Steraloids (Newport, RI): DHP, 20β-S, 17β-estradiol (E2), testosterone (T), 11-ketotestosterone (11-KT), P4, MGA, NOR, MPA, and LNG. The remaining steroids, including DRO, etonogestrel (ETO), GES, nomegestrol acetate (NOM), and dihydrotestosterone (DHT), were obtained from SigmaAldrich. For cell culture treatments, 100X (nPR assays) or 1000X (AR assays) stocks were prepared in either ethanol (DHP, 20β-S, E2, T, 11-KT, P4, DRO, ETO, GES, and NOM) or dimethyl sulfoxide (MGA, NOR, MPA, and LNG) and diluted to 10X (nPR assays) or 1X (AR assays) working stocks in medium immediately prior to treatment. Animals and Tissue Collection. For cloning nPR, adult female FHM (∼6 months old) obtained from a colony at the University of Maryland (College Park, MD) were euthanized in buffered tricaine methanesulfonate (MS-222; 200 mg/L). Ovaries were immediately removed, snap-frozen in liquid nitrogen, and stored at −80 °C until RNA was extracted. To investigate tissue distribution of FHM nPR, adult male and 8180

dx.doi.org/10.1021/es501428u | Environ. Sci. Technol. 2014, 48, 8179−8187


Article

relative to the tissue with the highest expression level (equal to 100%). Promoter activity data was expressed as fold induction over cells treated with vehicle. For half-maximal effective concentration (EC50) estimations, promoter activity from each dilution series was analyzed with a four-parameter logistic curve fit using Sigma Plot 12.0 (Systat Software Inc., San Jose, CA). Relative gene expression and promoter activity data were log2transformed prior to statistical analysis using SAS software (SAS Institute, Cary, NC). All data were analyzed by ANOVA using the MIXED models procedure in SAS with differences between groups determined by the test of least significant difference (PDIFF).

between tissues in a similar study characterizing FHM estrogen receptors FHM.29 Reverse Transcription PCR (RT-PCR). A pool of cDNA for each tissue collected from each sex was made from at least two replicate samples, and primers (Supporting Information, Supplemental Table S1) that amplify full-length nPR or a fragment of RPL8 were used to confirm tissue distribution by standard RT-PCR. Reactions were conducted using GoTaq Green Master Mix (Promega, Madison, WI) and contained 400 nM concentration of each primer and 1 μL template (cDNA or no RT, as appropriate). Cycling parameters were as follows: 95 °C for 3 min, 35 cycles of 95 °C for 45 s, 58 °C for 45 s, and 72 °C for 3 min, and a final extension at 72 °C for 7 min (nPR); or 95 °C for 3 min, 35 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 90 s, and a final extension at 72 °C for 5 min (RPL8). Products were visualized using agarose gel (1%) electrophoresis and ethidium bromide staining. In Vitro nPR and AR Transcriptional Activation Assays. COS-7 cells (American Type Culture Collection (ATCC), Manassas, VA) used for nPR assays were incubated in a 37.5 °C, 5% CO 2 atmosphere and maintained in DMEM supplemented with 10% fetal bovine serum (FBS; EquitechBio, Inc., Kerrville, TX). Prior to transfection, cells were plated (1.5 × 104/well) in white 96-well tissue culture plates (Greiner Bio-One, Monroe, NC) in growth medium overnight. The mouse mammary tumor virus-long terminal repeat (MMTVLTR) contains PR-inducible response elements30 and can be used as a transcriptional reporter when driving firefly luciferase (luc) expression. Cells were transfected with 100 ng of empty vector (pCMV-Sport6.1) or 100 ng of pCMV-nPR in combination with 100 ng of MMTV-LTR-Luc reporter construct (kindly provided by Dr. G. L. Hager, National Cancer Institute, Bethesda, MD) and 2 ng of pRL-SV40 normalization plasmid constitutively expressing renilla luciferase (Promega) using TransIT-LT1 Transfection Reagent (0.5 μL/well; Mirus Bio, Madison, WI) and Opti-MEM I medium. After 6 h, transfection medium was replaced with DMEM supplemented with charcoal-stripped FBS, and cells were allowed to recover overnight prior to treatment for 20 h with vehicle or indicated concentrations of each steroid. Cells were lysed, and firefly (reporter gene) and renilla (normalization gene) activities were determined with the Dual-Reporter Assay System (Promega) directly in the cell culture plate. Promoter activity in each sample was determined by dividing firefly luciferase activity by renilla luciferase activity. Plasmid pFAR-g26 containing the FHM AR expression cassette was used to generate Ad5FAR-g using standard techniques described previously.32 Ad/mLuc7 (kindly provided by Cary Weinberger, National Institute of Environmental Health Sciences, Research Triangle Park, NC) also contains MMTV-LTR-luc that acts as an inducible reporter for AR activity.31 Maintenance and transduction of CV-1 cells (ATCC)32 and the screening of gestagens for their ability to transcriptionally activate FHM AR have been described previously.33 For both assays, cell viability and toxicity effects were checked by visual inspection of cells in wells. In the FHM nPR assay, the intra-assay coefficient of variation (CV) was 9.1%, and the interassay CV was 14.5%. In the FHM AR assay, the intra-assay CV was 11.4% and the interassay CV was 22.4%. Data Analysis. For qRT-PCR, the normalized, transformed mRNA level of nPR in each sample was divided by the mean of the mRNA level for male kidney, such that data were expressed

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RESULTS FHM nPR Is Highly Homologous to Teleost nPR. The open reading frame of FHM nPR consists of 1,923 nucleotides that encode a 640-amino-acid protein containing typical steroid receptor domains (Supporting Information, Supplemental Figure S1A). Sequence comparisons between the predicted amino acid sequence of FHM nPR and those of several other vertebrates indicated that overall identity was highest with teleosts (54−79%), particularly goldfish and zebrafish. Two functional domains of FHM nPR, the DNA-binding domain (DBD; 88−97%) and ligand-binding domain (LBD; 65−92%), are the most highly conserved among species. Phylogenetic analysis of full-length nPR from various vertebrates indicated that FHM nPR clusters with two other cyprinids, goldfish and zebrafish, as well as other teleost species (Supporting Information, Supplemental Figure S1B). Tissue-Specific and Sexually Dimorphic Expression of FHM nPR. Using qRT-PCR, we evaluated mRNA expression levels of nPR in adult male and female FHM in a battery of tissues (Figure 1A). In both sexes, nPR mRNA was detected in all tissues and exhibited highest levels in pituitary, gonad, and brain. Interestingly, of all tissues examined, nPR mRNA was most strongly expressed in the male kidney, whereas expression was very low in female kidney (Figure 1A; P < 0.05). This tissue- and sex-specific expression pattern of nPR was confirmed using RT-PCR (Figure 1B). Tissue distribution of nPR in this species suggests it plays a major role in regulating reproduction in both sexes, while sexually dimorphic expression in the kidney suggests it may play a unique role in this tissue in males. Endogenous Progestogen Activation of FHM nPR. Functional activity of FHM nPR was evaluated by testing its ability to activate an MMTV-promoter-driven luciferase reporter construct in COS-7 cells transfected with an expression vector containing the coding sequence for FHM nPR along with the reporter construct and treated overnight with a wide range of doses of the teleost progestogens DHP and 20β-S. Although both steroids substantially increased MMTV promoter activity, DHP appeared the stronger agonist of the two. Activation of promoter activity occurred at 10−9 M DHP, while a 10-fold higher dose was required for activation by 20β-S (Figure 2A; P < 0.05). Other reproductive steroids, including E2, T, and 11-KT, were unable to transactivate FHM nPR (Figure 2B). Further, MMTV promoter activity was induced only in the presence of both the progestogen and the expression vector, indicating the increase in reporter activity was due to transactivation of FHM nPR by the steroid (Figure 2A,B). In order to confirm transactivation of FHM nPR by the two endogenous progestogens, as well as estimate EC50 values, cells 8181



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Figure 1. Expression levels of nuclear progesterone receptor (nPR) mRNA in adult male and female pituitary (Pi), kidney (Ki), heart (Ht), spleen (Sp), gill (Gi), liver (Li), muscle (Mu), intestine (In), brain (Br), testis (Te), and ovary (Ov) [gonad (Go) in panel A], as determined by (A) qRT-PCR and (B) standard RT-PCR. (A) Levels of mRNA in each sample were normalized to mRNA levels of ribosomal protein L8 (RPL8) and are expressed relative to male kidney (set to 100%). Values (mean + SE) without a common letter are significantly different (n = 4 fish/sex; P < 0.05). (B) Agarose gel picture depicting standard RT-PCR reactions for nPR and RPL8 in the same tissues listed. Control reactions containing no reverse transcriptase (NT) are shown.

were treated with a 2-fold dilution series of each steroid following transfection as described above. Transactivation properties of each compound were confirmed (Figure 2C,D; P < 0.05), and the EC50 value for DHP was estimated to be approximately 6-fold lower than that of 20β-S (Table 1). Using two endogenous fish progestogens, we have successfully developed an in vitro assay for functional characterization of FHM nPR that validates it as a bonafide PR. Importantly, this assay can be used to assess the capacity of different gestagens to activate its transcriptional activity. Gestagen Activation of FHM nPR and AR. The assay system described above was used to evaluate the ability of several known and potential environmental gestagens to bind to and activate FHM nPR. Nine gestagens were evaluated, including those that are used for reproductive management (P4) and growth promotion (MGA) in cattle operations,34,35 and ones that are common components of oral contraceptives, such as MPA, NOR, LNG, ETO, GES, DRO, and NOM.36,37 Of these, P4 and DRO increased MMTV promoter activity in the presence of FHM nPR at doses of 10−7 M and higher (Figure 3A,B; P < 0.05). The remaining compounds were unable to transactivate FHM nPR in our system (Supporting Information, Supplemental Figure S2). We repeated the assay using a 2-fold dilution series in order to estimate EC50 values for P4 and DRO. Again, both compounds increased transcriptional activity of FHM nPR in a concentration-dependent manner (Figure 3C,D; P < 0.05), and their EC50 values are given in Table 1. The EC50 value estimated for DRO was significantly higher than those estimated for DHP, 20β-S, and

Figure 2. Activation of fathead minnow nuclear progesterone receptor (FHM nPR) by endogenous circulating fish progestogens in COS-7 cells transfected with a PR-inducible MMTV-promoter-driven firefly luciferase reporter construct together with an empty expression vector or an expression vector for FHM nPR. (A) Cells (n = 3 independent experiments) were treated with ethanol (E) or indicated doses of 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S) for 20 h. (B) Cells (n = 4 independent experiments) were treated with E or 1 μM each of DHP, estradiol (E2), testosterone (T), or 11-ketotestosterone (11-KT) for 20 h. (C,D) Cells (n = 8 independent experiments) were transfected with the reporter construct along with FHM nPR and treated with a series of 2-fold dilutions of (C) DHP and (D) 20β-S to estimate halfmaximal effective concentration (EC50) values shown in Table 1. Promoter activity is expressed as fold induction over cells treated with ethanol within each vector. Values (mean ± SE) within each graph without a common letter are significantly different (P < 0.05).

P4 (P < 0.05), indicating it is a less potent agonist than fish progestogens and P4 in our system. Several older generation progestins used in oral contraceptives are 19-nortestosterone derivatives that exhibit androgenic activity.37 Therefore, we examined whether any of these gestagens could increase transcriptional activity of FHM AR. CV-1 cells were transduced with two replication deficient adenoviruses, one constitutively expressing FHM AR and the other containing the AR-inducible MMTV-promoter-driven luciferase reporter construct and then treated overnight with 8182



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Table 1. Half-Maximal Effective Concentration (EC50) Valuesa (nM) Estimated for Fathead Minnow Nuclear Progesterone Receptor (FHM nPR) and Androgen Receptor (FHM AR) in Response to Gestagen Treatment

Each value represents the mean ± SE of at least three determinations. If a value is missing, EC50 was not estimated for that receptor/gestagen combination. bEC50 values for endogenous teleost progestogens (DHP and 20β-S) and androgen (DHT) are shown for comparison. cValues within each receptor column with different letters (a, b, A, B) are significantly different (P < 0.05). a

varying amounts of each compound. While three compounds (MGA, MPA, and NOM) did not exhibit androgenic activity (Supporting Information, Supplemental Figure S3), all gestagens that are 19-nortestosterone derivatives (NOR, LNG, ETO, and GES) were potent activators of FHM AR (Figure 4A−D; P < 0.05). Both P4 and DRO were also able to transactivate FHM AR, though to a lesser extent (Figure 4E,F; P < 0.05). Estimation of the EC50 values for these compounds (Table 1) revealed that androgenic activity for the 19nortestosterone derivatives was at least as potent as that of DHT, a potent AR agonist. EC50 values for P4 and DRO were significantly higher, confirming they are less potent activators of FHM AR than androgenic gestagens and DHT. Taken together, these data indicate that some environmental gestagens can activate FHM nPR and AR, whereas older progestins activate only AR. Importantly, gestagens in aquatic ecosystems may interfere with the reproductive health of these fish through both nPR- and AR-mediated pathways.

Figure 3. Activation of fathead minnow nuclear progesterone receptor (FHM nPR) by progesterone (P4) and drospirenone (DRO) in COS7 cells transfected with a PR-inducible MMTV-promoter-driven firefly luciferase reporter construct together with an empty expression vector or an expression vector for FHM nPR. (A,B) Cells (n = 4 independent experiments) were treated with ethanol (E) or indicated doses of (A) P4 or (B) DRO for 20 h. (C,D) Cells (n = 8 independent experiments) were transfected with the reporter construct along with FHM nPR and treated with a series of dilutions of (C) P4 and (D) DRO to estimate half-maximal effective concentration (EC50) values given in Table 1. Promoter activity is expressed as fold induction over cells treated with ethanol within each vector. Values (mean ± SE) within each graph without a common letter are significantly different (P < 0.05).

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DISCUSSION In this study, we cloned FHM nPR, examined its sex- and tissue-specific expression in FHMs, developed an in vitro FHM nPR transcriptional activational assay, and screened a strategically chosen panel of environmental gestagens for activity in this assay and a previously developed assay for FHM AR transcriptional activity. We provided evidence that the biological activity of environmental gestagens may be mediated through both nPR and AR for some gestagens and through AR for the older generation progestins. FHM nPR Is Highly Homologous to Other Teleost nPRs. The successful sequencing of the full-length open reading frame for FHM nPR is important information in and of itself, as the complete coding sequence for nPR is known in only seven other teleost fishes, including African cichlid (Astatotilapia burtoni), Atlantic cod (Gadus morhua), Atlantic salmon (Salar salar), eel (Anguilla anguilla), goldfish (Carrasius

auratus), Japanese medaka (Oryzias latipes), and zebrafish (Danio rerio). As expected, there was a high percentage of amino acid identity between nPR sequence from other teleost fishes and that of FHM, with the two most similar species being the other cyprinids, goldfish and zebrafish.38,39 The LBD domain was much more conserved among fish species (79− 92%) than across vertebrates, and this difference in LBD identity may have important consequences for the biological 8183



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Figure 4. Gestagen activation of fathead minnow androgen receptor (FHM AR) in CV-1 cells transduced with an expression vector for FHM AR and an AR-inducible MMTV-promoter-driven firefly luciferase reporter. Cells (n = 3 independent experiments) were treated with vehicle [dimethyl sulfoxide (D) or ethanol (E)] or indicated doses of (A) norethindrone (NOR), (B) levonorgesterol (LNG), (C) etonogestrel (ETO), (D) gestodene (GES), (E) progesterone (P4), and (F) drospirenone (DRO). Promoter activity is expressed as fold induction over cells treated with vehicle. Values (mean ± SE) within each graph without a common letter are significantly different (P < 0.05). Data were used to estimate halfmaximal effective concentration (EC50) values shown in Table 1.

action of pharmaceutical progestins in exposed fish and other aquatic organisms, such as frogs, alligators, and turtles. Tissue-Specific and Sexually Dimorphic Expression of FHM nPR. An examination of tissue-specific expression of nPR mRNA showed that it is expressed broadly, with higher levels detected in brain, gonad, and pituitary in adult FHM of both sexes. This pattern agrees with what has been reported in zebrafish,38,39 Atlantic cod,40 and Atlantic salmon.41 The tissue with the highest expression of nPR mRNA was the male kidney. Interestingly, this expression was strongly sexually dimorphic, as it was almost absent in female kidneys. This pattern has been confirmed in a study examining ontogenic PR expression recently completed in our lab (unpublished data), and potential biological functions of male-specific kidney nPR expression and its potential alteration in gestagen exposed fish are being explored. Endogenous Progestogen Activation of FHM nPR. It is generally agreed that P4 is not the major teleost progestogen,42,43 and that DHP is the maturation-inducing substance (MIS) regulating gamete maturation in most teleosts,12,13,44 though in some members of the family Sciaenidae [spotted seatrout (Cynoscion nebulosus) and Atlantic croaker (Micropogonias undulatus)], it is 20β-S.15,16 In this study, it was found that DHP and 20β-S differentially transactivate FHM nPR, such that apparent binding affinity of DHP > 20β-S by approximately 5- to 10-fold. While the FHM MIS remains unidentified, our results indicate that DHP is the more potent agonist of nPR of the two fish progestogens and suggest that it

may be the MIS in this species, as has been reported for most teleosts and other cyprinids.45,46 Androstenedione, DHP and conjugated DHP excreted from the female cyprinid during the ovulatory cycle function as pheromones that inhibit and then stimulate male reproductive behavior and spermiogenesis, respectively.47,48 Although unknown in FHM, it is not unreasonable to hypothesize that excreted progestogens function in this species as they do in other cyprinids, and that this pheromonal signaling could be disrupted by environmental gestagens binding nPR, mPRs, PGRMCs, or AR in olfactory epithelia.47−49 Progestogens in fish may regulate the reproductive axis similarly as P4 in mammals, which is known to inhibit the gonadotropin releasing hormone (GnRH) surge and prevent ovulation.43,50 There is some evidence from in vitro studies with GnRH secreting GT1-7 cells51 and ex vivo cultures of hypothalamic tissues52 that mPRs may mediate inhibitory effects of progestogens on GnRH release in teleost fish.53 Given the central role that progestogens play in oocyte maturation, spermiogenesis, pheromonal signaling, and potential modulation of the hypothalamic−pituitary−gonadal axis through purported effects on GnRH-secreting neurons, environmental gestagens functioning through nPR, mPRs, or PGRMCs could profoundly influence the reproductive health of wild fish populations. Gestagen Activation of FHM nPR and AR. Progestins are used in oral contraceptives and in hormone replacement therapeutics.37,54 MGA is an anabolic gestagen administered to beef cattle in the U.S. to suppress estrus and promote growth 8184



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on feedlots.35 Researchers categorize human progestins into generations generally defined by the parent compound from which that generation of progestin was derived.37,54 First (I), second (II), and third (III) generations are based on 19nortestosterone, with the exception of MPA, which is based on 17-OH-progesterone. Examples of each generation include NOR (I), LNG (II), etonogestrel (III), and GES (III), all of which are relatively nonspecific and have androgenic effects in humans. DRO is a fourth (IV) generation progestin derived from the mineralocorticoid receptor antagonist spironolactone with little androgenic side effects. The most recently developed progestins, such as nestorone, NOM, and trimegestone, are derived from 19-norpregnane and produce negligible side effects since they are designed to be highly selective for human nPR,37,54 though they are not yet available in the U.S. Although exposure studies on fish are limited in number and species, the results of these and similar research on frogs clearly show that older generation progestins (I−III) are androgenic, whereas P4 and generation IV and newer progestins are not (for review see ref 7). Runnalls et al.24 have shown that yeast cells expressing human PR and AR have similar results to ours, but more progestins activated human PR. Binding characteristics of generation I−III progestins demonstrate unequivocally that they cross-react with human AR.37,54 Given the ability for progestins to bind both nPR and AR in humans and observed effects on fecundity and masculinization of female fish from progestin exposure studies, we thought it would be useful to examine transactivation properties of the same group of gestagens for FHM nPR and AR. We evaluated gestagens that represent progestin generations I−IV, a newer progestin (NOM), and P4 (Table 2). With the exception of GES (III) and NOM, these are commonly used in contraceptives and hormone replacement pharmaceuticals in the U.S. or, in the case of P4, have been measured in wastewater treatment plant effluent or animal agricultural runoff.49,55−58

Our results provide mechanistic evidence for how gestagens masculinize FHM exposed to older generation progestins. We have also shown that P4 and a newer generation progestin, DRO (IV), activate FHM nPR and are relatively weak FHM AR agonists. Spironolactone, the parent compound to DRO, is an AR antagonist in humans and an agonist in FHM and other fishes.59 To our knowledge, this is the first study demonstrating that gestagens can function through both nPR- and ARmediated pathways in fish, and our results suggest fish populations may be negatively affected by gestagen exposure. The nascent field of environmental gestagens now has a small, but impactful group of papers showing that gestagens affect reproductive physiology, development of secondary sex characteristics, and behavior; thus, they should garner increased attention of regulators and researchers alike.

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ASSOCIATED CONTENT

S Supporting Information *

Supplemental methods, figures, and table. This material is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 301-405-6386. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank J. Cavallin, M. Severson, and K. Jensen for their assistance and the Morris Animal Foundation for a grant to E.F.O. and L.E.E. (D12ZO-046). A special thank you to Dr. Peter Thomas for his encouragement and advice. The research described in this article has been reviewed by the U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

Table 2. Summary of Fathead Minnow Nuclear Progesterone Receptor (nPR) and Androgen Receptor (AR) Activation by Progestins Commonly Used in Oral Contraceptives

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a

Generations I−IV have been categorized by others.6 NOM is the most recently synthesized progestin used in this study, and its characterization as a “V” generation progestin is in quotes as it is the authors’ nomenclature. 8185



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Environmental Gestagens Activate Fathead Minnow (Pimephales

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