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Department of Biology, East Carolina University, 1000 E. 5th Street, Greenville, North Carolina 27858-4553, USA
¹ Marine Science Institute, University of Texas at Austin, 750 Channelview Drive, Port Aransas, Texas 78373, USA

(Requests for offprints should be addressed to Y Zhu; Email: zhuy{at}mail.ecu.edu)

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recently, a unique family of membrane progestin receptors (mPR, mPRß, and mPR) was identified, which may be responsible for mediating rapid, nongenomic actions of progestins in a variety of target tissues. In this study, the mPR and mPRß isoforms from zebrafish were shown to be rapidly and specifically activated by the maturation-inducing steroid (MIS) of this species, 4-pregnen-17,20ß-diol-3-one (17,20ß-DHP). The zebrafish mPR and a previously uncharacterized mPRß isoform were stably expressed in nuclear progesterone receptor-deficient mammalian breast cancer cells, MDA-MB-231. Expression and surface localization of the receptors were verified by flow cytometry, biotin surface labeling, and Western blotting. Plasma membrane proteins from mPR- or mPRß-transfected cells showed high affinity (mPR, K_d 7 nM; mPRß, K_d 12 nM), saturable, displaceable, single-binding sites specific for 17,20ß-DHP, whereas negligible specific 17,20ß-DHP binding was observed in nontransfected cells. Progestin treatment caused significant activation of mitogen-activated protein kinase (MAPK) within 5 min in cells transfected with either of the receptors as measured by western blotting and flow cytometry. The rank order of the potencies of several progestins in activating MAPK via mPR and mPRß was the same (17,20ß-DHP>progesterone >4-pregnen-17,20ß,21-triol-3-one). Interestingly, the MIS in zebrafish, 17,20ß-DHP, was also the most potent inhibitor, among the progestins tested, of adenylyl cyclase activity in cells transfected with either of the receptors. This progestin significantly decreased cAMP levels in both mPR- and mPRß-transfected cells in a dose-responsive and time-dependent manner. In addition, signaling of the zebrafish mPR was blocked by pertussis toxin, implying activation of a G_i protein, while sensitivity to pertussis or cholera toxin was not shown with mPRß-mediated signaling, possibly indicating that this receptor activates a different pertussis toxin-insensitive G protein. The results of this study suggest that zebrafish mPR and mPRß signal similarly upon progestin binding resulting in rapid activation of MAPK and downregulation of adenylyl cyclase activity.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Steroids have been known to exert nonclassical steroid actions, not involving genomic mechanisms, for over 30 years (Pietras & Szego 1975, Kostellow et al. 1980). All major classes of steroids have been shown to exert rapid nongenomic actions (Norman et al. 2004). Some specific nongenomic actions of progestins identified thus far include induction of the acrosomal reaction in sperm (Blackmore et al. 1990, Sabeur et al. 1996, Baldi et al. 1998, Luconi et al. 2004), an increase in sperm motility (Thomas et al. 2005), modulation of gonadotropin-releasing hormone discharge in the brain (Majewska et al. 1986, Calogero et al. 1998, Sim et al. 2001), and induction of oocyte maturation in fish and amphibian species (Kostellow et al. 1980, Patiño & Thomas 1990a, Ferrell 1999). Moreover, steroid-binding moieties with the characteristics of progestin receptors have been detected by radioreceptor assays on plasma membranes prepared from several of these progestin target tissues (Patiño & Thomas 1990b, Blackmore & Lattanzio 1991, Falkenstein et al. 1999). Although these studies have provided strong evidence for the existence of rapid, nongenomic progestin actions, and specific membrane receptors through which they can act, many details of this nonclassical steroid mechanism remain unclear. For example, the precise molecular structures of the progestin membrane receptors and their mechanisms of action are unresolved (Losel et al. 2003, Norman et al. 2004).

Recently, a novel family of membrane progestin receptors (mPR, mPRß, and mPR) has been identified, which may be responsible for mediating some of these rapid nongenomic progestin actions (Zhu et al. 2003a, 2003b). The mPRs are well conserved across a broad range of vertebrate species from fish to humans, and have similar structures and progestin-binding activities (Zhu et al. 2003a, 2003b). Recombinant mPR proteins from seatrout, as well as mPR, mPRß, and mPR proteins from human and mouse have been shown to specifically bind progestins (Zhu et al. 2003a, 2003b). In addition, the mPRs have G protein coupled receptor (GPCR)-like structures, including seven transmembrane domains, N-terminal glycosylation sites, and conserved cysteine residues for disulfide bonding. Despite their similarities to GPCRs, the mPRs have been grouped in a unique receptor class called the progestin and adiponectin receptor (PAQR) family, which is based on seven trans-membrane domains and an uncharacterized UPF0073 motif (Fernandes et al. 2005, Tang et al. 2005). It is generally accepted that all GPCRs have seven transmembrane domains with the N-terminus on the outside of the cell and C-terminus on the inside of the cell. However, Tang et al.(2005) have proposed an extracellular C-terminus topology of the PAQR superfamily based on a computer predication. As a result of this unique receptor classification and topology predication, it is difficult to assess whether all members of the receptor family signal and function through typical GPCR pathways.

The spotted seatrout mPR was the first mPR to be characterized, and evidence was obtained for its involvement in oocyte maturation in this species (Zhu et al. 2003a). The seatrout mPR is localized on the oocyte membrane, its expression in late stage oocytes increases prior to oocyte maturation, and is hormonally upregulated by gonadotropin during oocyte ‘priming’ (Zhu et al. 2003a). Similar findings have been obtained with the goldfish mPR, including oocyte membrane localization, inhibition of oocyte maturation by antisense microinjections, and a correlation between receptor protein levels and the ability of the maturation-inducing steroid (MIS) to induce oocyte maturation (Tokumoto et al. 2006).

The signaling of seatrout mPR is also consistent with previous reports on the intracellular-signaling pathways activated by progestins during oocyte maturation in fish (Nagahama 1997, Thomas et al. 2002, Pace & Thomas 2005). Activation of recombinant seatrout mPR in transfected human breast cancer cells (MDA-MB-231) by the known MIS of this species, 4-pregnen-17,20ß3,21-triol-3-one (20ß-S), causes stimulation of the mitogen-activated protein kinase (MAPK) cascade and inhibition of cAMP production through a pertussis toxin-sensitive, inhibitory G-protein (G_i) pathway (Zhu et al. 2003a). These findings support the hypothesis that mPR acts as an intermediary in MIS induction of oocyte maturation in teleost fish. Recent results in human myometrial cells have shown that both mPR and mPRß are involved in downregulation of adenylyl cyclase activity through a pertussis toxin-sensitive G_i pathway (Karteris et al. 2006). However, only mPR appears to mediate progesterone phosphorylation of the myosin light chain through activation of p38MAPK and downregulate expression of the nuclear receptor co-activator, SRC2, in these myometrial cells (Karteris et al. 2006). Therefore, the role of mPRß in nongenomic signaling and its physiological functions still remain to be elucidated.

Zebrafish may be an excellent model to compare the functions and signaling mechanisms of different mPRs, since mPR and mPRß are co-expressed in the ovary and brain (Kazeto et al. 2005, Kohli et al. 2005). However, it is not known if mPRß can mediate similar signaling as the mPR in zebrafish and has similar functions. In this study, we examine the ability of various steroids to bind to the previously uncharacterized zebrafish mPR and mPRß receptors and rapidly activate the MAPK and cAMP pathways in stably transfected, nuclear progesterone receptor-deficient human cell lines. Our findings show that progestins bind to the mPR and mPRß specifically, and among the progestins tested, the MIS in zebrafish causes the greatest activation of MAPK and cAMP pathways through both mPR and mPRß, suggesting these receptors have similar roles in mediating rapid progestin nongenomic actions.

	Materials and Methods

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Plasmid constructs

Zebrafish mPR and mPRß cDNAs, obtained previously (Zhu et al. 2003b), were inserted into mammalian expression vectors containing a CMV promoter and two C-terminal tags, V5 and His. The coding region of the mPR was amplified by PCR with primers for removal of the stop codon and containing BamHI and EcoRI enzyme-cutting sites for insertion into the mammalian expression vector pcDNA3.1/V5–His A vector (Invitrogen). The primers used to amplify mPR cDNA were 5'-AATGGATCCTCACCATGG-3' (forward) and 5'-CCACGACATGAATTCCTG-3' (reverse). The mPRß-coding region was amplified with primers for removal of the stop codon and the CACC TOPO recognition site in the pcDNA TOPO vector (Invitrogen). The primers used to amplify mPRß cDNA were 5'-CACCATGTCAAGTGGAGT-3' (forward) and 5'-TCAGTCTTTTTTCCTCACCTG-3' (reverse). The PCR products for both receptors were purified by electrophoresis on a 1% agarose gel, extracted with a gel prep kit (Qiagen), ligated with vector, and transformed into chemically competent Top 10 Escherichia coli cells following the manufacturer’s instructions (Invitrogen). Transfected E. coli were spread on ampicillin-coated plates and grown overnight. Resistant colonies were then selected, regrown overnight, and the plasmid was purified by a MiniElute gel extraction kit (Qiagen). Constructs were verified for proper sequence by DNA sequencing using the BigDye terminator mix. The verified plasmid stocks were grown overnight and purified by alkaline lysis and PEG precipitation as described in Current Protocols in Molecular Biology (Ausubel et al. 2000).

Cell culture and transfection

Human MDA-MB-231 breast carcinoma cells (American Type Culture Collection, Manassas, VA, USA) were used to express the recombinant mPR and mPRß. The cells were cultured and maintained at 37 °C with 5% CO₂ in Dulbeco’s modified Eagle’s medium/F-12 media without phenol red (Sigma) containing 10% fetal bovine serum (Gibco). Media were changed every 2 days and cells were split among three plates when 90% confluent. Purified vector constructs of mPR and mPRß were then transfected into the cells using Lipofectamine reagent (Invitrogen) following the manufacturer’s instructions. Two days after transfection, plasmid-expressing cells were selected using 1000 µg/ml G418 (Research Products International, Mt Prospect, IL, USA). Resistant colonies were then isolated and propagated with 500 µg/ml G418 in order to produce stably transfected cell lines.

Western blotting

Expression of mPR and mPRß in the stably transfected cell lines was confirmed by Western blotting using a previously developed polyclonal antibody developed for the N-terminal of seatrout mPR (F1N2; Zhu et al. 2003a), which cross-reacts with both zebrafish mPR and mPRß cells. The corresponding N-terminal region of zebrafish mPR (variable residues, VSDVPWVFRESHIITGYRP) shares 84% identity with the peptide antigen (VSDVPWVFRERHILTGYRQ) used for developing seatrout mPR N-terminal antibodies, whereas the corresponding N-terminal region of zebrafish mPRß (ASEVPSLFREPYILSGYRP) shares 58% identity with the peptide. Samples were collected by scraping the cells at 4 °C into PBS containing 0.1% protease inhibitor cocktail (Sigma). Ovary samples were collected by cervical dislocation followed by immediate removal and transfer of the ovary into PBS. Samples were sonicated with ten short bursts on a sonicator (Sonic Dismembrator, Fisher Scientific, Pittsburgh, PA, USA), the homogenate was centrifuged for 10 min at 20 000 g, and the resulting pellets were resuspended in lysis buffer containing 150 mM NaCl, 10 mM Tris–HCl (pH 7.4), 1% NP-40 (Calbiochem, San Diego, CA, USA), and 0.1% protease inhibitor cocktail (Sigma), and incubated for 30 min at 4 °C in order to solubilize membrane proteins. Samples were then spun for 10 min at 20 000 g to remove undissolved material. The remaining supernatant was mixed with equal amounts of 2xSDS sample buffer (0.125 M Tris–HCl (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol), boiled for 5 min and then cooled on ice.

For western-blot analysis, 20 µg (MAPK assays) and 50 µg (mPR expression assays) total protein estimated by the Bradford assay (Bio-Rad), were loaded onto a SDS/12% PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat milk in TBST (50 mM Tris, 100 mM NaCl, 0.1% Tween 20 (pH 7.4)) for 1 h. The membrane was further incubated with the mPR antibody (1:5000 dilution) or extracellular signal-related kinase (ERK) and phosphorylated ERK antibodies overnight (Cell Signaling, Beverly, MA, USA), followed by four 5-min TBST washes, incubated for 1 h with horseradish peroxidase conjugated to goat anti-rabbit antibody (1:2000 dilution; Cell Signaling), and finally washed three times for 5 min with TBST. The blots were then developed using Super Signal West Extended Dura Substrate (Pierce, Rockford, IL, USA) and visualized using a Fluor Chem 8900 imaging station (Alpha Innotech, San Leandro, CA, USA).

Cell surface biotinylation and pull-down assay

Surface expression of the receptor was also verified by biotin labeling of surface proteins with EZ-Link Sulfo-NHS-LC-Biotin (Pierce), followed by immunoprecipitation with immobilized streptavidin (Pierce) and western blotting with the seatrout mPR-specific antibody. Cells were grown for at least 48 h until they were 90% confluent in 25 cm² cell culture-treated flasks and then washed three times with PBS (pH 8). The cells were then incubated with 1 mg/ml sulfo-NHS-LC biotin (Pierce) for 30 min at 4 °C. The reaction was quenched by washing the cells twice with 10 ml ice-cold PBS containing 50 mM Tris–HCl (pH 7.5). Cell samples were removed by the addition of lysis buffer containing 150 mM NaCl, 10 mM Tris–HCl (pH 7.5), 1% NP-40 (Calbiochem), and 0.1% protease inhibitor cocktail (Sigma) directly to the plates. The samples were solubilized for 30 min at 4 °C with shaking and then centrifuged for 10 min at 20 000 g to remove undissolved material. The biotinylated proteins were purified by incubating supernatants with 200 µl immobilized strepavidin–agarose slurry (Pierce) at 4 °C overnight with continuous mixing. Beads were then washed three times with 1 ml 0.1% wash buffer containing 0.1% NP40, 150 mM NaCl, and 20 mM Tris–HCl (pH 7.5), and then biotinylated proteins were eluted by incubating the slurry for 1 h at 55 °C in 100 µl 2xSDS sample buffer. Final samples were spun down, extracted from the resin and then analyzed by western blotting as described previously.

Flow cytometry

Expression of the receptors was further verified by flow cytometry using a fluorescein-5-isothiocyanate (FITC)-conjugated V5 antibody (Invitrogen) or His (Cell Signaling) and mPR primary antibody with a FITC-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA). Approximately 1x10⁶ cells were collected by scraping into 2 ml PBS and then spun down at 1350 r.p.m. for all the washing steps. The cells were decanted, permeabilized for 15 min on ice with 90% methanol for staining with V5 and His antibodies, or left on ice for mPR antibody staining. Following two washes in PBS, the harvested cells were washed with blocking solution consisting of 0.5% BSA in PBS. The cells were then incubated with unlabeled primary antibodies (His or mPR, 1:200 dilution in blocking solution) or FITC-conjugated V5 antibodies for 30 min at 4 °C in the dark. Those cells that reacted with the unlabeled primary antibodies were further incubated with FITC-conjugated anti-rabbit antibodies (1:500 dilution) following two washes with blocking solution. These samples were resuspended in 100 µl PBS for analysis on the flow cytometer following two washes in blocking solution. Analysis of 10 000 events was performed on a FACScan flow cytometer using CELLQest software (BD Biosciences, Palo Alto, CA, USA). For expression analysis, a gate (a minimum fluorescence intensity) was set above 95% of the untransfected control cell population. The percentage of recombinant cells expressing fluorescence intensity above the gate was reported after subtracting 5% from the total.

Receptor-binding assays

[1,2,6,7 ³H]17-Hydroxyprogesterone (85 Ci/mmol) was purchased from New England Nuclear (Boston, MA, USA) and enzymatically converted to 4-pregnen-17,20ß-diol-3-one (17,20ß-DHP) as described previously (Scott et al. 1982). Plasma membrane fractions of MDA-MB-231 cells were obtained following established procedures with few modifications (Patiño and Thomas 1990b, Zhu et al. 2003a). The cells were washed with assay buffer and then sonicated for 15 s, followed by a 1000 g centrifugation for 7 min to remove any nuclear and heavy mitochondrial material. The resulting supernatant was centrifuged at 20 000 g for 20 min to obtain the plasma membrane fraction. Progestin receptor binding in the membrane fractions was characterized as described previously (Patiño & Thomas 1990b). One set of tubes contained radiolabeled 17,20ß-DHP alone (total binding), another set also contained cold progestin competitor at a 1000-fold greater concentration to measure nonspecific binding. For competition assays, a third set of tubes contained radiolabeled steroids and various concentrations of the steroid competitors ranging from 100 pM–10 µM (dissolved in 1–5 µl ethanol (EtOH)), which do not affect ligand binding in the receptor assay. After a 30-min incubation at 4 °C with the membrane fractions, the reaction was stopped by filtration (Whatman GF/B filters, presoaked in assay buffer; Fisher Scientific, Pittsburgh, PA, USA). The filters were washed twice with 25 ml assay buffer and the bound radioactivity was measured by scintillation counting. The displacement of the radiolabeled 17,20ß-DHP binding by the steroid competitors was expressed as a percentage of the maximum specific binding of the 17,20ß-DHP for the mPRs.

Immunofluorescence staining of mPR in zebrafish oocytes

Oocytes were collected from adult zebrafish, separated by size, fixed in Bouin’s fixative (70% v/v Picric acid, 23% v/v of 40% aqueous formaldehyde, and 4.7% v/v glacial acetic acid) for 24 h at 4 °C and stored in 100% methanol at –20 °C until processed for immunohistochemistry. Oocytes were then dehydrated for 5 min with 75, 90, 100% EtOH, 100% xylene, and then mounted in paraffin wax cassettes. Paraffin sections were cut at 6 µm and placed on Superfrost glass slides (Fisher Scientific). The oocyte sections were then deparaffinized by 5-min washes with 100% xylene twice, 100, 95, 70% EtOH, and twice with PBS.

Blocking was carried out at room temperature for 1 h in PBS containing 2% BSA and 1% goat normal serum. The slides were then incubated overnight at 4 °C with mPR antibody (1:400 dilution) in PBS containing 2% BSA, washed three times with PBS and then incubated for 2 h at room temperature with FITC-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch; 1:500 dilution) in PBS with 2% BSA and 1% goat normal serum. The slides were then washed three times with PBS, treated with 70% EtOH for 5 min, equilibrated with PBS, and then mounted using an antifade reagent (1% 1,4-diazabicyclo[2,2,2]octane; Sigma), in 90% glyceraldehyde, phosphate buffered) and observed under a fluorescence microscope (Olympus BX-40 fluorescence microscope).

MAPK activation and ligand screening

MAPK activation was measured by both western blotting and flow cytometry using a phospho-specific p44/p42 antibody (1:2000 dilution for western-blots and 1:200 dilution for flow cytometry; Cell Signaling). Cells were split at least 48 h prior to testing to allow mPR protein expression to recover and then serum starved for 72 h to reduce basal MAPK activity. Activation was measured after 5-min exposure to various steroids (Steraloids, Newport, RI, USA) added directly to the media. Steroids were dissolved in EtOH, so an equivalent amount of EtOH was added in the control group. Epidermal growth factor was used as a positive control of activation. Cells were then either immediately lysed in 1xSDS sample buffer for western analysis or fixed with 2% paraformaldehyde dissolved in PBS for 10 min for flow cytometry analysis, both using the same phospho-specific p44/p42 antibody. Following western blotting with phospho-specific antibody, the membrane blots were stripped for 30 min at 50 °C in stripping buffer (2% SDS, 62.5 mM Tris (pH 6.8), 100 mM ß-mercaptoethanol) and washed five times in TBST. Then, the membrane blots were reblotted with total MAPK antibody in order to confirm equal protein loading. For MAPK flow cytometry analysis, the following modifications were made to the flow cytometry methods described previously. The cells were washed two more times with PBS before scraping from the plate. Then, the cells were permeabilized and incubated with the phospho-specific MAPK primary antibody (1:250), and a FITC-conjugated goat anti-rabbit secondary antibody (1:500 dilution; Jackson ImmunoResearch).

cAMP measurements

The levels of cAMP in transfected cells were measured using a nonradioactive enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA). Cells were prepared by splitting at least 48 h prior to testing to allow mPR protein expression to recover and then serum starved for 18 h to reduce the background levels of steroids in the media. For pertussis toxin pretreatment, cells were incubated overnight with 200 ng/ml pertussis toxin added to the serum-free media. For cholera toxin treatment, cells were incubated with 1 µg/ml cholera toxin 2 h before treatment. Cells were then stimulated with 1 µM forskolin for 15 min followed by steroid treatment for 15 min. Samples were then placed on ice, the media removed, 66% EtOH added, and then samples were immediately frozen at –80 °C. Samples were thawed and cAMP was measured in aliquots of supernatant according to the manufacturer’s instructions. Data were analyzed by a spreadsheet program provided by Cayman Chemical, which calculated cAMP content in picomoles per milliliter. For each sample, the amount of protein was measured by the Bradford assay and then standardized to express the final value as picomoles cAMP per microgram protein.

Statistical analysis

All experiments were repeated at least three times. Western-blots were analyzed for fluorescent intensity by Alpha Innotech analysis software and expressed as the mean-fold change (MFC) in band intensity±S.E.M. Data were analyzed using the GraphPad Prism Program (San Diego, CA, USA) to calculate a one-way ANOVA. A Newman–Keuls multiple comparison test was used to determine specific differences between means when determined as significant by ANOVA. A P value <0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Western-blot analysis of zebrafish ovarian membrane proteins using the seatrout mPR antibody showed a single immunoreactive band at ~40 kDa (Fig. 1). The membrane localization of the receptor in zebrafish oocytes was further confirmed by immunohistochemistry using the same mPR antibody (Fig. 2), in agreement with previous results showing membrane localization of the mPR in spotted seatrout oocytes.

View larger version (25K): [in this window] [in a new window]   Figure 1 Western-blot analysis of zebrafish ovary and transfected membrane progestin receptor (mPR and mPRß) cells using antibody against seatrout mPR. A unique band at 40 kDa was seen for the ovarian membrane sample corresponding to the mPR protein and bands at 45 kDa corresponding to the recombinant mPR and mPRß receptors from 50 µg total proteins (A) extracted from zebrafish ovarian membrane or MDA-MB-231 cell membranes respectively, and plasma membrane proteins (B) following biotin-surface labeling and immunoprecipitation of cell-surface proteins.

View larger version (54K): [in this window] [in a new window]   Figure 2 Immunofluorescent localization of mPR in zebrafish oocytes. (A) An oocyte reacted with mPR antibody under normal light. (B) Positive fluorescence reaction (GFP) was observed on the ovarian membrane of the same oocyte under GFP fluorescent light. Insert, mPR was observed on the oocyte membrane at a high magnification. (C) An oocyte reacted with secondary antibody only under normal light. (D) No GFP was observed in the same oocyte under GFP fluorescent light. Bar=100 µm.

View larger version (23K): [in this window] [in a new window]   Figure 3 Flow cytometry analysis of mPR and mPRß in stably transfected cells using His or mPR primary antibody with FITC-conjugated secondary antibody or FITC-conjugated V5 antibody. Cells were permeabilized for His and V5 staining of C-terminal receptor tag and nonpermeabilized for staining with the N-terminal mPR antibody. Results, expressed as percentage of mPR- and mPRß-transfected cells, show greater fluorescent intensity than a gated control population. Horizontal bar in the figure represents a gate set above 95% of the untransfected control population. The number in the figure indicates the percentage of recombinant cells under the gate after 5% were subtracted.

View larger version (11K): [in this window] [in a new window]   Figure 4 Specific [3H]4-pregnen-17,20ß-diol-3-one (17,20ß-DHP) binding assessed by a single point radioreceptor assay (total binding tubes: 4 nM [3H]17,20ß-DHP, nonspecific-binding tubes: +4 µM 17,20ß-DHP) to the plasma membranes of nontransfected MBA-MD-231 cells (231), zebrafish mPR-transfected (tr-mPR) or zebrafish mPRß-transfected (tr-mPRß) cells. *P<0.05 compared with controls. Values are means±S.E.M. from three independent assays with different batches of cells.

View larger version (13K): [in this window] [in a new window]   Figure 5 Saturation analyses and Scatchard plots of specific [3H]4-pregnen-17,20ß-diol-3-one (17,20ß-DHP) binding to the plasma membranes of mPR- and mPRß-transfected cells. Representative plots are shown. Nearly identical results were obtained in three independent assays with different batches of cells.

View larger version (15K): [in this window] [in a new window]   Figure 6 Competition curves of steroid binding expressed as a percentage of maximum specific [3H]4-pregnen-17,20ß-diol-3-one (17,20ß-DHP) binding to plasma membranes prepared from mPR- and mPRß-transfected cells ([3H]17,20ß-DHP: 4 nM). , 17,20ß-DHP; , progesterone; , 20ß-S; , testosterone; , estradiol-17ß; , cortisol. Values are means from three independent assays with different batches of cells.

View larger version (39K): [in this window] [in a new window]   Figure 7 MAPK pathwayactivation in mPR- and mPRß-transfected cells after 5 min stimulation with 1 µM of various steroids – progesterone (P4), 4-pregnen-17,20ß diol-3-one (17,20ß-DHP), 20ß-S, 4-pregnen-20ß-ol-3-one (20HP), 4-pregnen-17-ol-3,20-dione (17HP), cortisol (Cort), 11-desoxycortisol (11DC), testosterone (Test), and estradiol-17ß (E2). Representative western-blots for active phosphorylated p-ERK and total ERK (top). Mean-fold change (MFC) in active p-ERK over untreated samples, expressed as the mean±S.E.M. forat least four separate experiments (bottom). P4 as well as the fish maturation-inducing progestins, 17,20ß-DHP and 20ß-S, show significant MAPK activation over control treatments. 17,20ß-DHP showed the most significant activation of the steroids tested. Epidermal growth factor (EGF) was used as a positive control of MAPK activation and the vehicle for steroid delivery, EtOH, was used as a negative control. Bars (mean±S.E.M.) with different letters are significantly different from one another (P<0.05). UN, untreated control.

View larger version (28K): [in this window] [in a new window]   Figure 8 MAPK activation in mPR- and mPRß-transfected cells after 5 min stimulation with various steroids at various concentrations (1 nM–1 µM) – progesterone (P4), 4-pregnen-17,20ß-diol-3-one (17,20ß-DHP), 4-pregnen-17,20ß,21-triol-3-one (20ß-S), and estradiol-17ß (E2). MFC in active p-ERK over untreated samples, expressed as an average±S.E.M. for at least four separate experiments (*P<0.05). Un, untreated control.

View larger version (35K): [in this window] [in a new window]   Figure 9 MAPK activation in mPR- and mPRß-transfected cells after 5 min stimulation with 1 µM progestins (solid line) compared with untreated mPR-transfected cells (solid filled line) and negative control of secondary antibody only (dotted line) as detected by flow cytometry. The mPR-transfected cells showed a 1.64±0.06 MFI for progesterone stimulation (A) and a 1.79±0.08 MFI for 17,20ß-DHP stimulation (B). The mPRß-transfected cells showed a 1.41± 0.04 MFI for progesterone stimulation (C) and a 1.61±0.06 change for 17,20ß-DHP stimulation (D). Activation was measured for 10 000 cells (MFI; median fluorescent intensity of treated/median fluorescent intensity of untreated, mean MFI±S.E.M., n=3).

View larger version (23K): [in this window] [in a new window]   Figure 10 cAMP levels in mPR- and mPRß-transfected cells after 15 min stimulation with 1 µM various steroids – progesterone (P4), 4-pregnene-17,20ß diol-3-one (17,20ß-DHP), 4-pregnen-17,20ß,21-triol-3-one (20ß-S), 4-pregnen-20ß-ol-3-one (20HP), 4-pregnen-17-ol-3,20-dione (17HP), cortisol (Cort), 11-desoxycortisol (11DC), testosterone (Test), and estrogen (E2). Samples were pretreated with 1 µM forskolin (Fskln) for 15 min prior to the addition of steroid (mean±S.E.M., n=6, *P<0.05). Un, untreated control.

View larger version (21K): [in this window] [in a new window]   Figure 11 cAMP inhibition by various doses of 17,20ß-DHP for 15 min in mPR- and mPRß-transfected cells. Samples were pretreated with 1 µM forskolin (Fskln) for 15 min prior to addition of steroid (mean±S.E.M., n=4, *P<0.05). Un, untreated control.

View larger version (17K): [in this window] [in a new window]   Figure 12 cAMP inhibition of mPR- and mPRß-transfected cells at various time points after addition 1 µM 17,20ß-DHP. Samples were pretreated with 1 µM forskolin (Fskln) for 15 min prior to addition of steroid (mean±S.E.M., n=3, *P<0.05). Un, untreated control.

View larger version (15K): [in this window] [in a new window]   Figure 13 Effects of pertussis toxin on cAMP levels in mPR- and mPRß-transfected cells after 15 min stimulation with 1 µM 17,20ß-DHP. Samples were pretreated as indicated with 1 µM forskolin (Fskln) for 15 min and 200 ng/ml active pertussis toxin (PTX) or inactive pertussis toxin (inPTX) for 12 h prior to the addition of steroid (mean±S.E.M., n=3, *P<0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Cell-surface localization, specific mPR-binding activity, and potential nongenomic-signaling pathways initiated by progestins acting through zebrafish mPR and mPRß were identified by stably transfecting the nonprogestin responsive and nuclear progestin receptor-deficient MDA-MB-231 cell line with the receptors. Clear evidence was obtained for expression and cell-surface localization of the recombinant receptors by three independent methods, western blotting, flow cytometry, and biotin-surface labeling. Moreover, expression of both zebrafish mPR proteins was associated with marked increases in specific membrane-bound 17,20ß-DHP. These binding moieties have characteristics typical of progestin membrane receptors. High affinity, saturable, displaceable, single-binding sites specific for 17,20ß-DHP, and other progestins were identified in plasma membrane preparations from both mPR- and mPRß-transfected cells. In addition, a comprehensive series of experiments showed rapid activation of signaling pathways via mPR and mPRß after progestin treatment, indicating that the two mPR proteins can function as receptors in the transfected cells to transduce progestin signaling. Progesterone, together with the known MISs in fish, 17,20ß-DHP and 20ß-S, significantly increased MAPK activity in receptor-transfected cells within 5 min. The zebrafish MIS, 17,20ß-DHP, caused activation of MAPK for both receptors at lower concentrations (1–10 nM) compared with P4 (10–100 nM) and 20ß-S (1 µM). Furthermore, cAMP levels in both receptor-transfected cell lines were significantly decreased in a concentration- and time-dependent manner in response to physiologically relevant concentrations of 17,20ß-DHP. Although circulating levels of 17,20ß-DHP have not yet been measured in zebrafish due to their small size, MIS concentrations (1–10 nM) that were effective in significantly decreasing cAMP levels and activating MAPK in this experiment are within the range found in the plasma of a variety of fish species during oocyte maturation and ovulation (Kobayashi et al. 1987, Truscott et al. 1992, King et al. 1994). The finding that the MIS, 17,20ß-DHP, is the preferred ligand for the zebrafish mPRs is important for establishing their ovarian functions in the presence of a variety of similar progestin derivatives in the ovary. The observation that both the untransfected MDA-MB-231 cells and receptor-transfected cells showed significant increases in MAPK in response to testosterone, and a significant decrease in cAMP levels in response to estrogen, raises the possibility that membrane receptors for these steroids are expressed in this cell line. However, no rapid signaling in response to progestins or corticosteroids was observed (see Supplementary data) in nontransfected cells. Taken together, the results provide clear evidence that both zebrafish mPR and mPRß can mediate rapid nongenomic actions of progestins and that this can occur independent of the nuclear progestin receptor since it is not present in this cell model (Horwitz et al. 1978).

Previous results showing co-localization of mPR and mPRß in the brain and reproductive tissues of zebrafish, and inhibition of oocyte maturation in this species by microinjection of antisense oligos to mPR and/or mPRß microinjection, support the suggestion that the receptors share common functions (Zhu et al. 2003a, Thomas et al. 2004, Kazeto et al. 2005). Zebrafish mPR and mPRß may have a similar involvement in oocyte maturation to that proposed for the seatrout mPR and goldfish mPR as intermediaries in MIS induction of this process. The seatrout mPR is localized in the oocyte plasma membrane, its hormonal regulation and pattern of expression are consistent with its involvement in oocyte maturation, and the signal transduction pathway initiated through the receptor is the same as that identified during MIS induction of oocyte maturation in seatrout and other fish species (Nagahama 1997, Thomas et al. 2002, Zhu et al. 2003a, Pace & Thomas 2005). In the zebrafish, mPR and mPRß have been co-localized primarily in the ovaries, the receptors have been shown to be upregulated prior to oocyte maturation and antisense microinjections for both receptors have been shown to block MIS-induced oocyte maturation (Zhu et al. 2003a, Thomas et al. 2004, Kazeto et al. 2005, Kohli et al. 2005). In the present study, the zebrafish MIS shows the greatest activation of MAPK and inhibition of cAMP, signaling that is consistent with previous studies of fish oocyte maturation, through both zebrafish mPR and mPRß. The mPRs have also been localized primarily on the plasma membrane in zebrafish oocytes, in agreement with the predicted localization of the MIS receptor.

The zebrafish is an excellent model for further examination of the actions of mPR and mPRß in oocyte maturation and early development, because it spawns daily, responds to a known MIS, develops rapidly, and their embryos survive externally. However, additional studies are required to demonstrate direct roles for each of the receptors in mediating MIS induction of ooctye maturation in vivo. Detailed information is also needed on the progestin-mediated signaling pathways involved in induction of oocyte maturation in this species, on the pattern of daily changes in receptor expression and functionality, and possible interactions between mPR and mPRß. A better understanding of the functions mediated by the mPRs in zebrafish has the potential to significantly advance our understanding of progestin-mediated nongenomic signaling in vertebrate reproductive tissues. For example, mPR and mPRß have been localized in mammalian ovaries, where they may have similar roles in progestin signaling (Zhu et al. 2003b, Cai & Stucco 2005).

	Acknowledgements

 
This work was supported in part by the National Science Foundation Grant IBN-0315349 to Y Z and the EPA STAR Grant No R-82902401 to P T. We want to thank Prakash Peddi for the assistance with immunohistochemistry. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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Received in final form 24 April 2006
Accepted 9 May 2006
Made available online as an Accepted Preprint 29 May 2006

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