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and the Correlation with Their Nongenomic Progestin ActivitiesDepartment of Biology (T.T.), Faculty of Science, National University Corporation Shizuoka University, Shizuoka 422-8529, Japan; and Marine Science Institute (M.T., P.T.), University of Texas at Austin, Port Aransas, Texas 78373
Address all correspondence and requests for reprints to: Dr. Toshinobu Tokumoto, Department of Biology, Faculty of Science, National University Corporation Shizuoka University, Shizuoka 422-8529, Japan. E-mail: sbttoku{at}ipc.shizuoka.ac.jp.
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. Membranes prepared from both ovaries and mPR-transfected cells showed high-affinity, saturable, displaceable, single binding sites specific for the goldfish maturation-inducing steroid, 17,20ß-dihydroxy-4-pregnen-3-one (17,20ß-DHP). DES and DES analogs (dipropionate-DES and hexestrol), which induce OM in goldfish, bound to the receptor and caused concentration-dependent displacement of [3H]-17,20ß-DHP, whereas dimethyl ether-DES had no affinity for the receptor. Scatchard plot analysis of specific 17,20ß-DHP binding in the presence of different amounts of DES showed that DES binding is of the noncompetitive type. The activities of DES and DES analogs to induce meiotic maturation of goldfish oocytes were examined in an in vitro bioassay. Whereas a concentration-dependent induction of OM was observed in response to DES, dipropionate-DES, and hexestrol, dimethyl ether-DES did not show any OM-inducing activity. The close correspondence between binding of DES and its analogs to the mPR protein and their OM-inducing activities suggests a mechanism of endocrine disruption mediated by binding to mPR resulting in its activation, thereby mimicking the nongenomic action of the progestin 17,20ß-DHP. |
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Two progestin MISs with multiple hydroxyl groups on the side chain, 17, 20ß-dihydroxy-4-pregnen-3-one (17,20ß-DHP) and 17, 20ß, 21-trihydroxy-4-pregnen-3-one (20ß-S), have been identified in several fish species. 17,20ß-DHP has been identified as the MIS in amago salmon, Onchorynchus rhodurus (4), whereas 20ß-S is the MIS in Atlantic croaker (5). In addition, the progestin receptors that bind these MISs have been biochemically characterized on the oocyte membranes of spotted sea trout (Cynoscion nebulosus), rainbow trout (O. mykiss), striped bass (Morone saxatilis), yellow tail (Seriola quinqueradiata), and arctic char (Salvelinus alpinus) (6, 7, 8, 9). However, until recently the identities and mechanisms of action of these membrane progestin receptors were unknown.
Recently a novel cDNA, named membrane progestin receptor (mPR)-, was identified in a spotted seatrout ovarian cDNA library that encodes a protein with the characteristics of the membrane progestin receptor mediating the MIS induction of oocyte maturation (OM) in this species (10). The observation that microinjection of antisense oligonucleotides to the homologous zebrafish (Danio rerio) mPR cDNA into zebrafish oocytes was effective in blocking the induction of OM in response to 17,20ß-DHP suggested that mPR is also involved in the induction of OM in this species (10, 11). Subsequently, we described the cloning, sequencing and characteristics of mPR in another well-characterized model of OM, goldfish (Carassius auratus), which is representative of a major family of freshwater teleosts, the Cyprinidae (12). The mPR protein is localized on the plasma membranes of goldfish oocytes and is up-regulated during gonadotropin induction of OM. Microinjection with mPR antisense oligonucleotides also blocked maturation of goldfish oocytes, suggesting the mPR protein is also an intermediary in the MIS induction of OM in goldfish.
The induction of OM in fish by progestins has previously been shown to be a valuable model for investigating interference of nongenomic steroid actions by endocrine-disrupting chemicals (EDCs) (13). For example, we found that treatment of goldfish and zebrafish oocytes with an EDC, diethylstilbestrol (DES), alone induces OM (14). DES is a potent estrogen that was used clinically from the late 1930s to the early 1970s for the prevention of pregnancy complications, but its use was subsequently banned after many adverse effects were reported, including carcinogenicity and teratogenicity (15, 16). Subsequently we demonstrated that tamoxifen (TAM) and its metabolite 4-hydroxytamoxifen (both chemicals are EDCs) also induce OM in zebrafish. Furthermore, a potent inhibitory effect of pentachlorophenol (PCP) was demonstrated on OM induced by 17,20ß-DHP, DES, and TAM (17). The results suggested that these EDCs might interact with the MIS receptor to induce or disrupt OM. The recent identification of the structure of the putative MIS receptor in goldfish as mPR now permits investigations of the molecular mechanism by which EDCs disrupt OM. Therefore, the purpose of the present study was to determine whether DES and its analogs induce OM by binding to mPR (its membrane receptor) in a well-characterized model of OM, the goldfish. Binding of these EDCs to the goldfish ovarian plasma membrane 17,20ß-DHP receptor and membrane fractions prepared from cultured cells transfected with goldfish mPR was examined as well as their abilities to induce meiotic maturation of goldfish oocytes in an in vitro bioassay.
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Ovarian tissue
Goldfish were purchased from a local supplier and maintained in fish holding facilities at the University of Texas Marine Science Institute for at least 2–3 months until the ovaries became full grown. Fish were maintained in 1000-liter recirculating freshwater tanks with external biofilters under low temperature (14–16 C) and photoperiod (14-h light, 10-h dark) conditions. Ovarian tissue containing fully grown oocytes (diameter 1126–1250 µm) was used for both the 17,20ß-DHP membrane receptor competition studies and the in vitro OM bioassays. Ovarian donor fish were humanely killed following procedures approved by the University of Texas Animal Care Committee.
Expression of mPR in bacteria and production of polyclonal antibodies
The entire open reading frame of goldfish mPR was amplified by PCR with primers designed to produce an EcoRI and XhoI site at the 5' and 3'-ends, respectively. PCR fragments were inserted into the pET27b expression vector (Novagen, Madison, WI), which append 33 extra amino acids at the N terminal and penta histidine-tag at the C-terminal end. The recombinant proteins were produced in Escherichia coli BL21 (DE3) and purified by SDS-PAGE (Prep Cell, model 491; Bio-Rad, Richmond, CA). Polyclonal antibodies specific for mPR were raised in guinea pigs against purified recombinant mPR following procedures described previously (18).
Western blot analysis
Ten micrograms of solubilized plasma membrane proteins were resolved in 10% SDS-PAGE gels and transferred to a nitrocellulose membrane for Western blot analysis. The membranes were incubated overnight with blocking solution [5% nonfat milk in TBST buffer: 50 mM Tris/100 mM NaCl/0.1% Tween 20, pH 7.4)]. The membranes were washed with TBST buffer and incubated for 1 h at room temperature with the mPR antibodies diluted 1000-fold in blocking solution. The membranes were washed with TBST buffer and incubated for 1 h at room temperature with horseradish peroxidase conjugated to goat anti-guinea pig antibody (Zymed, San Francisco, CA). The blots were washed three times for 5 min with TBST buffer at the end of the incubation period, treated with enhanced chemiluminescence (Pierce, Rockford, IL), and exposed to x-ray film.
Plasmid constructs
Goldfish mPR cDNA, obtained previously (12), was inserted into a mammalian expression vector containing a cytomegalovirus (CMV) promoter. The coding region of the mPR was amplified by PCR with primers for removal of the stop codon and containing EcoRI and XhoI enzyme cutting sites for insertion into the mammalian expression vector, pBK-CMV (Stratagene, La Jolla, CA). The primers used to amplify mPR cDNA were 5'-CCGAATTCCATGGCGACGGTTGTGATGGAG-3' (forward) and 5'-CCGCTCGAGCTCCTGCTTGTCTTCTAGATAC-3' (reverse). The PCR products were purified by electrophoresis on a 1% agarose gel, extracted with a gel prep kit (QIAGEN, Valencia, CA), ligated with vector, and transformed into chemically competent JM109 E. coli cells following the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Transfected E. coli were spread on kanamycin-coated plates and grown overnight. Resistant colonies were then selected, regrown overnight and the plasmid was purified by a Wizard Plus SV Miniprep DNA purification system (Promega, Madison, WI). Constructs were verified for proper sequence by DNA sequencing using the BigDye terminator mix.
Cell culture and transfection
Human MDA-MB-231 breast carcinoma cells (American Type Culture Collection, Manassas, VA) were stably transfected with the goldfish mPR as described previously for seatrout mPR (10). The cells were cultured and maintained at 37 C with 5% CO2 in DMEM media (Sigma) containing 5% fetal bovine serum (Life Technologies, Inc., Carlsbad, CA) and 100 mg/liter gentamicin (Invitrogen). Media were changed every 2 d, and cells were split among three plates when they became 90% confluent. Purified vector constructs of mPR were then transfected into the cells using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s instructions. Two days after transfection, plasmid-expressing cells were selected using 250 µg/ml G418 (Research Products International, Mt. Prospect, IL). Resistant colonies were then isolated and propagated with 250 µg/ml G418 to produce stably transfected cell lines.
Preparation of membrane fractions
Plasma membrane fractions from ovarian tissues and transfected cells were prepared following procedures described previously (7, 10). From ovarian tissue, fragments (2 g) of ovarian tissue were homogenized in 10 ml ice-cold homogenization assay buffer [HAED: 25 mM HEPES; 10 mM NaCl; 1 mM EDTA; 1 mM dithiothreitol (pH 7.6) at 4 C] using a Teflon-pestle homogenizer followed with a glass-pestle homogenizer. The homogenate was centrifuged at 1000 x g for 7 min at 4 C to remove nuclei. The supernatant was then centrifuged at 20,000 x g for 20 min to pellet the plasma membranes. The crude plasma membrane pellet was resuspended in 10 ml HAED buffer, the membrane suspension was layered on top of 0.5M sucrose in HAED buffer, and the mixture was centrifuged at 9500 x g for 45 min. The material in the layer between the sucrose buffer and HAED buffer was collected and centrifuged at 20,000 x g for 20 min. The resulting partially purified plasma membrane fraction in the pellet was resuspended in HAED buffer and immediately used for the binding assay.
From cultured cells, the cells were washed three times with PBS and scraped into HAED buffer and then sonicated for 15 sec, followed by a 1000 x g centrifugation for 7 min to remove any nuclear and heavy mitochondrial material. The resulting supernatant was further centrifuged at 20,000 x g for 20 min to obtain the plasma membrane fraction.
Membrane binding assays
[1,2,6,7 3H]-17-hydroxyprogesterone (85 Ci/mmol) was purchased from Amersham Biosciences (Piscataway, NJ) and enzymatically converted to radiolabeled 17,20ß-DHP by 3,20ß-hydroxysteroid dehydrogenase (Sigma) as described previously (19). Progestin receptor binding in the membrane fractions was measured following procedures established previously (7). One set of tubes contained 1–10 nM [3H]-17,20ß-DHP alone (total binding); another set also contained cold progestin competitor at a 100-fold greater concentration to measure nonspecific binding. After a 30-min incubation at 4 C with the membrane fractions, the reaction was stopped by filtration [Whatman (Middlesex, UK) GF/B filters, presoaked in HAED buffer containing 2.5% Tween 80]. The filters were washed three times with 5 ml of wash buffer [HEPES, 25 mM; NaCl, 10 mM; EDTA 1 mM (pH 7.4) at 4 C], and bound radioactivity was measured by scintillation counting. The displacement of the radiolabeled 17,20ß-DHP binding by steroids or EDC competitors was expressed as a percentage of the maximum specific binding of the 17,20ß-DHP to the membrane fractions.
Competition studies
The binding of steroids and EDCs to the 17,20ß-DHP membrane receptor was investigated in competitive binding assays. The chemical structures of the EDCs used in this study are shown in Fig. 1
. Full competition curves were generated with 4 nM (ovarian membrane) or 2 nM (culture cell membrane) [3H]-17,20ß-DHP in the presence of steroids or various concentrations of EDCs (0.1 nM to 10 µM). The steroids and EDCs were added to the assay tubes dissolved in ethanol (final concentration < 1%), which did not affect receptor binding. The displacement of [3H]-17,20ß-DHP by the compounds was expressed as a percentage of the specific binding. Several competition curves were run with each compound to confirm the results.
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OM bioassays
The OM-inducing activity of the DES analogs was investigated in in vitro OM bioassays (14). Ovaries were transferred into goldfish Ringer’s solution at 4 C [125 mM NaCl, 2.4 mM KCl, 0.28 mM MgSO4, 0.89 mM MgCl2, 2.4 mM CaCl2, 2 mM HEPES, 5.6 mM glucose, 100 IU/ml penicillin, and 0.2 mg/ml streptomycin (pH 7.5)] before separation of intact follicles for the OM in vitro bioassay. More than 20 fully grown follicle-enclosed goldfish oocytes were incubated for 6 h in each well of a six-well tissue culture plate or in culture dish (40 mm) containing 4 ml goldfish Ringer’s solution. The follicles were exposed with DES or its analogs by addition of each agent (from a 1000-fold stock in ethanol) followed by incubation at room temperature with gentle agitation (40 rpm). To assess the maturation process, germinal vesicles were examined under a binocular microscope (SMZ645; Nikon, Tokyo, Japan) after placing the oocytes in clearing solution (23) or GVBD was assessed by scoring the oocytes that became transparent (17). Percent GVBD was calculated from scoring OM in more than 20 oocytes.
Statistical analysis
All experiments were repeated three times. One-way ANOVA (ANOVA of the data was calculated using the GraphPad Prism program. P < 0.05 was considered statistically significant.
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in plasma membranes from goldfish ovaries and transfected cells against N-terminal and C-terminal peptide fragments of the receptor, in an attempt to obtain a more specific antibody, we produced His-tagged full-length recombinant proteins of goldfish mPR as the antigens. A polyclonal antibody was raised in a guinea pig against these proteins. The antibody specifically cross-reacted with a single band of the recombinant mPR protein produced in the E. coli expression system in a Western blot (Fig. 2A). Western blot analysis of goldfish ovarian membrane proteins using the goldfish mPR antibody showed a single immunoreactive band at approximately 40 kDa (Fig. 2B). The slightly higher molecular mass (45 kDa) of recombinant mPR, compared with the native protein expressed in the goldfish ovary, was due to the added tags that were approximately 5 kDa. To examine the binding activity of the goldfish mPR, the receptor was stably expressed in a human breast cancer cell line lacking the nuclear progesterone receptor, MDA-MB-231 cells (24). The expression of mPR mRNA was verified by RT-PCR of total RNA extracted from cultured cells (Fig. 2C). Expression of the mPR protein was investigated by Western blotting. A distinct band at approximately 47 kDa was present in the membrane proteins extracted from the mPR-transfected cell line, whereas the band was absent in the empty vector-transfected control cells (Fig. 2D). The slightly higher molecular mass (47 kDa) of the mPR protein expressed in the transfected cell line was due to the extension of both of the N and C terminals from the vector sequence that totaled approximately 7 kDa.
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-transfected cells-transfected cells, whereas significantly lower amounts of 17,20ß-DHP binding were detected in empty vector-transfected cell membranes (Fig. 2E
). Saturation analysis showed progestin binding to cell membranes of goldfish ovaries and transfected cells is saturable and of limited capacity (ovary, Bmax = 0.08 nM; mPR, Bmax = 0.05 nM; Fig. 2, F and G). Scatchard analysis showed the presence of a single class of high-affinity binding sites (ovary, Kd = 12.6 nM; mPR, Kd = 3.9 nM, Fig. 2, F and G) in the cell membranes.
Steroid and EDC binding characteristics of ovarian membrane receptor and mPR
Steroid competition studies showed that binding is highly specific for 17,20ß-DHP in membranes prepared from both ovaries and mPR-transfected cells (ovary, Fig. 3, A–C; mPR, Fig. 3, D–F). The relative binding affinities (RBAs) of steroids to the 17,20ß-DHP ovarian membrane receptor and recombinant mPR receptors were similar (Fig. 3 and Table 1). Progesterone (P4) had binding affinities to both receptors with IC50s of 14–30 nM, whereas testosterone and estradiol-17ß caused only similar displacement of [3H]-17,20ß-DHP binding from the receptors at 100-fold higher concentrations, and cortisol showed no displacement at any of the concentrations tested (Fig. 3). The finding that 17,20ß-DHP has high binding affinity for mPR is consistent with our previous results, which suggested mPR is an intermediate in MIS induction of OM. The ability of EDCs, which possess OM-inducing or OM-inhibiting activity in fish, to bind to the ovarian progestin receptor and recombinant mPR membrane protein was examined. DES, an inducer of OM, was a relatively effective competitor of [3H]-17,20ß-DHP binding to both receptors, 1 µM DES displacing almost all of the [3H]-17,20ß-DHP binding to cell membranes expressing mPR (Fig. 3D) and approximately half of the binding to the ovarian 17,20ß-DHP membrane receptor (Fig. 3A). The relative binding affinities of DES to the 17,20ß-DHP ovarian membrane receptor and recombinant mPR were 7 and 33% that of 17,20ß-DHP, respectively. DP-DES and HEX displayed lower binding affinities than DES for both receptors. More than 20-fold higher concentrations of DP-DES and HEX were required to displace 50% of [3H]-17,20ß-DHP binding from the ovarian receptor. Also their affinities for mPR were approximately 10- to 20-fold lower than that of DES (Table 1). In contrast, DM-DES did not displace [3H]-17,20ß-DHP binding to either receptor preparation at any of the concentrations tested (Fig. 3, C and F). TAM had much lower binding affinity than DES for both receptor preparations and caused only 50% displacement of [3H]-17,20ß-DHP binding to mPR at the highest concentration tested (10 µM), which corresponds to its lower potency in inducing OM (17). PCP, a potent inhibitor of 17,20ß-DHP-induced OM, did not compete with 17,20ß-DHP binding to both ovarian and mPR-transfected cell membranes (Fig. 3, B and E).
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). The decrease of Bmax in the presence of increasing DES concentrations suggests that DES binds to the membrane receptor at different site from the 17,20ß-DHP-binding site and that binding is probably of the noncompetitive type. Scatchard plot analysis of inhibition of [3H]-17,20ß-DHP binding to recombinant mPR by DES (Fig. 4B) also suggests that it is probably a noncompetitive inhibitor. The addition of 0, 0.01, 0.1, and 1 µM of DES shifted the apparent Bmax for [3H]-17,20ß-DHP binding from 0.064 to 0.058, 0.046, and 0.01 nM, respectively. In contrast, the natural steroid hormone P4 showed competitive inhibition of [3H]-17,20ß-DHP binding to recombinant mPR in the same Scatchard analysis (Fig. 4C).
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). As described previously, DES-induced GVBD in goldfish oocytes at concentrations of 10 and 2 µM. Although HEX and DP-DES were nearly as effective as DES in inducing GVBD at 10 µM, they were ineffective at the lower concentration of 2 µM. DM-DES did not have any OM-inducing activity, even at the 10 µM concentration.
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. There was a correlation between the relative binding affinities of DES and the DES analogs for mPR and their potencies in mimicking the actions of 17,20ß-DHP on meiotic maturation in the in vitro bioassay. Scatchard analyses suggested that binding of DES to the 17,20ß-DHP membrane receptor is noncompetitive. Taken together, these results suggest that DES and its analogs interact with the progestin membrane receptor, at different site(s) from the steroid binding site, to induce fish oocyte maturation. To our knowledge, the present results provide the first clear evidence of EDCs mimicking progestin actions through binding to a progestin membrane receptor.
DES is the only proven transplacental carcinogen in humans (25). In addition to activating nuclear estrogen receptors (ERs) (26, 27), DES also interacts with the estrogen-related receptors (ERRs), promoting coactivator release from ERRß and inhibiting its transcriptional activity (28) and b binding to ERR
(29). These findings suggest novel pathways for DES action involving other receptors in addition to ERs. Likewise, this study shows that DES interacts with mPR. Our results suggest that estrogen-independent pathways could be involved in mediating some of the developmental effects of DES. Therefore, further characterization of DES actions should lead to a more comprehensive understanding the potential carcinogenic and developmental risks associated with human exposure to a variety of EDCs.
In this study it was demonstrated that both the ovarian plasma membrane receptor in goldfish and goldfish mPR expressed in transfected cells displayed high affinity, limited capacity, displaceable specific binding for [3H]-17,20ß-DHP. The relative binding affinities of various steroids and EDCs to these two receptor preparations were similar, supporting the idea that mPR is a MIS receptor in vivo. This receptor was first identified as a strong candidate for the oocyte membrane progestin receptor mediating OM in spotted seatrout (10). Although the natural MIS, 20ß-S, had low binding affinity for the recombinant seatrout mPR protein produced in E. coli in this initial study, the progestin was a potent activator of signal transduction pathways in MDA-MB-231 breast cancer cells transfected with the receptor, indicating that the recombinant mPR protein produced in this eukaryotic expression system recognizes 20ß-S (10). Recently the ability of recombinant seatrout mPR to bind [3H]-20ß-S was confirmed with this mammalian cell expression system (30).
The same expression system was used to demonstrate specific binding of the MIS, 17,20ß-DHP, to recombinant zebrafish mPR and mPRß proteins (31). They also demonstrated that the mPR proteins are expressed on the cell surface of transfected cells using flow cytometry. In a previous study we described the cloning and identification of goldfish mPR (12). Western blot analysis using a specific goldfish mPR antibody demonstrated the presence of an immunoreactive band of the predicted molecular mass (
40 kDa) in oocyte plasma membranes. Similar to the previous results in spotted seatrout, mPR protein expression in fully grown goldfish oocytes was up-regulated by treatment with gonadotropin and was associated with the development of oocyte maturational competence (i.e. ability to respond to the MIS and undergo OM). Moreover, microinjection of mPR antisense morpholino oligonucleotides into goldfish oocytes blocked both the induction of oocyte maturation in response to 17,20ß-DHP and the up-regulation of mPR protein levels (12). Similarly, oocyte microinjection with antisense oligos to zebrafish mPR had previously been shown to block MIS-induced OM in zebrafish (10). Thus, the results obtained with representatives of two distantly related teleost families, the Sciaenidae and Cyprinidae, support the suggestion that mPR acts as an intermediary in MIS induction of oocyte maturation in teleosts. The present finding that both goldfish oocyte membranes and goldfish mPR expressed in cultured cells display similar high binding affinities for the MIS in this species, 17,20ßDHP, provides further evidence that mPR is a MIS receptor in teleosts.
Interference with MIS induction of oocyte maturation by environmental chemicals has been reported in several fish species and an amphibian (13). Initial evidence for interference with this nongenomic steroid action was obtained with Atlantic croaker oocytes, which showed a dose-dependent inhibition of 20ß-S-induced OM with kepone and o,p'-dichlorodiphenyldichloroethane over the concentration range of 1 nM to 1 µM (32). Interestingly, 5 min exposure to these organochlorine compounds in the presence of 20ß-S was sufficient to block MIS induction of OM, suggesting the actions of these compounds were receptor mediated. Moreover, subsequent washing of the organochlorine-exposed oocytes followed by treatment with 20ß-S completely reversed the interference of OM, further suggesting these inhibitory effects of the compounds are not due to nonspecific toxic mechanisms. Pickford and Morris (33) evaluated the effect of methoxychlor on progesterone-induced maturation of Xenopus laevis. They showed prolonged pretreatment of the progesterone pesticide methoxychlor significantly reduced the rate of maturation. Fort et al. (34) also demonstrated the inhibitory effects of EDCs, such as ethinylestradiol, on Xenopus oocyte maturation. Although methoxychlor did not show any binding affinity for the oocyte membrane MIS receptor, ethinylestradiol was shown to interact with it. In spotted seatrout, kepone and o,p'-DDD were shown to antagonize MIS-induced meiotic maturation by binding to the MIS receptor in a competitive manner (20). Ortho, para derivatives of DDD and dichlorodiphenyltrichloroethane, have also been shown to bind to the ovarian membrane progestin receptor in arctic char (9). We have also shown that DES and its analogs induce OM, and PCP has a potent inhibitory effect on OM in zebrafish (17). It is concluded from these studies that OM in fish and amphibians is sensitive to disturbance by a variety of environmental chemicals and that in vitro OM bioassays are appropriate models for investigating novel mechanisms of endocrine disruption through interference with nongenomic steroid actions.
Overall there was a broad similarity between the relative binding affinities of DES and its analogs for the goldfish ovarian membrane receptor and mPR in the present study (DES > HEX = DP-DES > DM-DES) and their potencies to induce OM (14), which suggests that DES and DES analogs induce oocyte maturation through interaction with the mPR proteins. An interesting finding was that DES and its analogs did not completely inhibit [3H]-17,20ß-DHP binding to the ovarian membrane preparations, whereas they completely inhibited progestin binding to recombinant mPR (Fig. 3, C and F). These results suggest the presence of a second 17,20ß-DHP membrane receptor in goldfish ovaries with a similar Kd to mPR that does not interact with DES. However, possible interactions of DES with one likely candidate for this second 17,20ß-DHP receptor, mPRß, remain to be explored. The saturation and Scatchard analyses indicate that binding of DES to the 17,20ß-DHP ovarian membrane receptor and mPR is noncompetitive. The apparent decrease in the number of 17,20ß-DHP binding sites in the presence of DES could be due to several mechanisms including binding to a second site on the receptor resulting in alteration of the active site (21).
An alternative explanation, that DES denatures the receptor, was not investigated but is unlikely because DES was shown to exert a concentration-dependent agonist effect on OM. DES has been shown to bind to the steroid binding pocket of the nuclear ER to mediate its estrogenic effects (35, 36). Currently the three-dimensional structure of the ligand binding pocket of mPRs is unknown. Information on the structure of the ligand binding pocket of mPRs, including the possibility that mPRs form dimers in the presence of ligands, will be necessary to obtain a better understanding of the residues that account for the specificity of progestin binding as well as those involved with binding DES. Based on our results, our current hypothesis is that DES binds to a second site on the receptor, altering the binding of the natural ligand and at the same time independently causing receptor activation leading to OM. Our limited bioassay data show that the agonist activity of DES is only approximately 1% that of 17,20ß-DHP, whereas the receptor binding affinity of DES is 10–30% that of the natural ligand. An examination of the relative potencies of these two mPR agonists in activating signal transduction pathways involved in OM such as G protein activation should provide insights into the mechanism of DES’s agonistic action. In contrast, a potent inhibitor of fish oocyte maturation, PCP, displayed no binding affinity for the 17,20ß-DHP membrane receptor or the mPR protein. A previous study had shown that no preincubation was required for PCP to exert its inhibitory effect on OM, suggesting an immediate and direct effect of PCP on this process. Therefore, downstream mediators of MIS-induction of OM such as G proteins and adenylyl cyclase should be examined as potential targets of PCP’s toxic action.
The present results provide further evidence that membrane receptor-mediated nongenomic actions of progestins through binding to mPR are particularly sensitive to disruption by EDCs. In addition to the inhibitory effects of EDCs observed previously on progestin receptor membrane function, there is now clear evidence that DES and its analogs can acts as agonists by binding to mPR. Our results emphasize the need for studies to examine more widely the various nongenomic effects of EDCs.
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Acknowledgments |
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,20ß-hydroxysteroid dehydrogenase. We thank Dr. Y. Pang, Dr. A. H. Berg, Dr. S. Rahman, Mrs. J. Dong, M.S., G. Dressing, and Mr. J. Kummer for valuable advice and Mrs. S. Lawson for goldfish maintenance. Part of this study was performed as the National Institute for Basic Biology Cooperative Research Program (5-108 and 6-104 to T.T.). |
Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online April 19, 2007
Abbreviations: Bmax, Binding capacity; CMV, cytomegalovirus; DES, diethylstilbestrol; 17,20ß-DHP, 17, 20ß-dihydroxy-4-pregnen-3-one; DM, dimethyl ether; DP, dipropionate; EDC, endocrine-disrupting chemical; ER, estrogen receptor; ERR, estrogen-related receptor; GVBD, germinal vesicle breakdown; HAED, HEPES, NaCl, EDTA, dithiothreitol; HEX, hexestrol; Kd, dissociation constant; MIS, maturation-inducing steroids; mPR, membrane progestin receptor; OM, oocyte maturation; P4, progesterone; PCP, pentachlorophenol; RBA, relative binding affinity; 20ß-S, 17, 20ß, 21-trihydroxy-4-pregnen-3-one; TAM, tamoxifen.
Received December 18, 2006.
Accepted for publication April 9, 2007.
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,20ß,21-trihydroxy-4-pregnen-3-one as the major ovarian steroid produced by the teleost Micropogonias undulatus during final oocyte maturation. Steroids 47:89–99[CrossRef][Medline],20ß,21-trihydroxy-4-pregnen-3-one on ovarian membranes of striped bass. Biol Reprod 56:266–271[Abstract],20ß, 21-trihydroxy-4-pregnen-3-one in ovaries of spotted seatrout (Cynoscion nebulosus). Gen Comp Endocrinol 78:204–217[CrossRef][Medline],20ß-dihydroxy-4-prognen-3-one to oocyte cortices of rainbow trout (Oncorhynchus mykiss). Fish Physiol Biochem 11:15–24[CrossRef], 20ß-dihydroxy-4-pregnen-3-one in plasma of trout (Salmo gairdneri Richardson): seasonal changes and response to salmon pituitary extract. Gen Comp Endocrinol 46:444–451[CrossRef][Medline]: studies with ERKO and ßERKO mice. Cancer Res 61:6089–6097. Proc Natl Acad Sci USA 98:8880–8884 subtypes and their evolutionary origins. Endocrinology 148:705–718[CrossRef][Medline] and ß in transfected cells. J Endocrinol 190:247–260
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