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Mechanisms of Hormone Action |
University of Texas at Austin, Marine Science Institute, Port Aransas, Texas 78373
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-dihydrotestosterone, T, and 11-ketotestosterone had relative binding affinities (RBAs) of 193%, 100%, and 13%, respectively, whereas none of the C18 or C21 steroids tested bound with high affinity except for progesterone (RBA = 191%). This androgen-binding moiety with high affinity for progesterone is unlikely to mediate the physiological actions of progestins in croaker, because it has low binding affinity for fish progestin hormones. Androgen-binding sites were also detected in membrane fractions of the brain, liver, kidney, and drumming muscle, whereas little or no binding was detected in the trunk muscle, heart, gills, or intestine. Receptor levels increased 10-fold during ovarian recrudescence, reaching maximum levels in fully mature ovaries, which suggests a likely physiological role for this receptor during the reproductive cycle of female croaker. It is concluded that the androgen-binding moiety identified in the plasma membrane fraction of Atlantic croaker ovarian tissue fulfils all the criteria for its designation as a steroid receptor.
androgen receptor, ovary, steroid hormone receptors, steroid hormones, testosterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Overall, rapid actions of androgens and membrane androgen receptors (mARs) have received less attention than those of most other classes of steroids. Nevertheless, several rapid, nongenomic effects of androgens have been described. Intracellular free Ca2+ levels are rapidly regulated by androgen treatment in a variety of tissues and cell-culture systems, including both male and female reproductive tissues [6–9], skeletal muscle [10], osteoblasts [11, 12], and T cells and macrophages [13–15]. Other signaling pathways rapidly activated by androgens include mitogen-activated protein kinases (MAPKs) and protein kinase C [16, 17]. Although these studies have provided indirect evidence for the existence of membrane-associated androgen-binding sites, in many cases the receptors mediating these rapid androgen actions have not been characterized.
Androgen-binding sites have been partially characterized in the membranes of mammalian endothelial cells [18], synaptosomes in the rat [19, 20], olfactory tissues from brown and rainbow trout [21], and testis [22], liver, and prostate from male rats [23]. Through the use of confocal laser-scanning microscopy and flow cytometry, putative testosterone (T) receptors were also identified on the plasma membranes of T cells and macrophages [14, 15]. However, these androgen-binding sites have not been extensively investigated. Therefore, comprehensive information is currently lacking on the binding characteristics of androgen membrane receptors.
Previously, we described a rapid, nongenomic action of androgens on ovarian steroidogenesis in the Atlantic croaker (Micropogonias undulatus) [24]. Evidence was obtained that androgens inhibit in vitro gonadotropin-stimulated production of estradiol by croaker ovaries through a mechanism initiated at the cell surface. Moreover, preliminary binding experiments showed the existence of an androgen-binding site in an ovarian membrane preparation. Previously, a nuclear androgen receptor was identified in croaker ovaries, and its steroid-binding characteristics were described in detail [25–27]. The purpose of the current study was to investigate fully the binding characteristics of the ovarian membrane binding moiety to determine whether it fulfills all the criteria for its designation as a steroid receptor and whether its steroid-binding characteristics are similar to those of the nuclear androgen receptor in this tissue.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The radioactive tracer, [1,2,6,7-3H]T (specific activity, 95 Ci/mmol), and the synthetic androgen, 17ß-hydroxy-17-methyl-4,9,11-estratiene-3-one (R1881), were purchased from New England Nuclear (Boston, MA). Other unlabeled steroids were purchased from Steraloids (Newport, RI) or Sigma (St. Louis, MO). The synthetic androgen, mibolerone, was a gift from Upjohn Laboratories (Kalamazoo, MI). The antiandrogens, cyproterone acetate and flutamide, were purchased from Sigma. Vinclozolin and its metabolites (M1 and M2) were a gift from L. Earl Gray (U.S. Environmental Protection Agency, Research Triangle Park, NC). All para,para- and ortho,para-congeners of dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene (DDE), and dichlorodiphenyldichloroethane (DDD) were obtained from Supelco (St. Louis, MO). The steroids, antiandrogens, and xenobiotics were dissolved and stored in 95% ethanol at –20°C. The diagnostic kit for the measurement of 5'-nucleotidase activity, Dulbecco modified Eagle medium (DMEM; nutrient mixture, F-12 HAM), and all other chemicals used to prepare the buffers were of reagent grade and purchased from Sigma. The scintillation cocktail was prepared by dissolving 16 g of 7,5-diphenyl-oxazole (Sigma) and 0.4 g of 1,4-bis[5-phenyl-2-oxazolyl]-benzene (Sigma) in 4 L of reagent-grade toluene.
Animals and Tissue Collection
Atlantic croaker were captured by gill net or otter trawl from the bays near Port Aransas, TX, during late summer and early fall (1998–2001), which is the period of gonadal recrudescence. Fish were maintained in circular, recirculating tanks under constant photoperiod and temperature (22–25°C) and fed a diet of commercial pellets daily. Before tissue collection, fish were acclimated to laboratory conditions for at least 3 wk. The gonadosomatic index (GSI), calculated by the equation GSI (%) = (gonad weight/somatic weight) x 100, was used to determine the stage of gonadal maturity. The average GSI for females in the present study except for the seasonal experiment, was 14.26% ± 0.74%. The average male GSI was 6.56% ± 1.22%.
Fish were humanely killed by rapid decapitation following procedures approved by the University of Texas Animal Care and Use Committee according to NIH guidelines. Tissues were immediately removed and placed in ice-cold buffer or frozen at –80°C for later analysis. Androgen binding remained constant in frozen tissues for at least 1 yr.
Tissue Preparation
Two-gram aliquots of individual or pooled ovaries were homogenized in 15 ml of HED buffer (25 mM Hepes, 10 mM NaCl, 1 mM dithioerythritol, and 1 mM EDTA, pH 7.6) at 4°C using a Polytron Tekmar Tissumizer (Tekmar, Cincinnati, OH) on a high setting for 5 sec followed by three to five passes with a glass Tenbroek tissue grinder (Wheaton Science Products, Millville, NJ). All the remaining preparation steps were conducted at 4°C. The homogenate was centrifuged at 1000 x g for 7 min to pellet the nuclear material, and the resulting supernatant was centrifuged at 20 000 x g for 20 min to pellet the membrane fraction. The supernatant from this spin was considered to be the crude cytosolic fraction. To further purify the membrane fraction, the membrane pellet was resuspended in 10 ml of HED, layered over an equal volume of 1.2 M sucrose dissolved in HED, and centrifuged at 6900 x g for 45 min based on the method of Peck and Kelner [28]. After this spin, the middle membrane layer was removed with a Pasteur pipette, diluted (1:2) with HED, and centrifuged at 20 000 x g for 20 min. The pellet of this final spin was resuspended in 5 ml of HED and either used immediately in a binding assay or frozen at –80°C for later analysis. Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA).
Radioreceptor-Binding Assays
All steps of the binding assays were conducted at 4°C. The membrane preparations were diluted to 0.15–0.5 mg protein/ml in HED immediately before all binding assays. Total binding saturation curves were generated by incubating 250 µl of membrane preparation and 250 µl of [3H]T, dissolved in HED for final reaction concentrations ranging from 0.3 to 25 nM, for 40 min. Nonspecific binding was determined from a parallel set of reaction tubes, which also contained 1000-fold excess unlabeled T. The same procedure was used in one-point binding assays except that the membrane preparations were incubated with only one concentration of [3H]T. After the 40-min incubation, the binding reactions were terminated by rapidly filtering 400 µl of the reaction over a presoaked Whatman, glass-fiber filter (pore size, 1 µm) to separate bound steroid from free steroid. The filter was immediately washed twice with 12.5 ml of wash buffer (HED without dithioerythritol) and placed in a scintillation vial. Radioactivity was counted in a liquid scintillation counter (LSC 6000SC; Beckman Instruments, Fullerton, CA). Specific [3H]T binding was calculated as the difference between total and nonspecific binding, and in saturation experiments, specific binding was transformed according to the method of Scatchard [29]. The number of receptors (Bmax) and the affinity constant (Kd) were determined from the nonlinear regression of the specific [3H]T binding curve.
Verification of the Plasma Membrane Preparation
To verify that the androgen binding was in the membrane fraction of the ovarian tissue, 5'-nucleotidase activity was measured as a marker of the plasma membrane according to the protocol provided with the kit by Sigma (procedure no. 265-UV). In addition, each fraction of the ovarian homogenate was diluted to 0.2–0.4 mg protein/ml, and binding was determined subsequently by one-point assays.
Association and Dissociation Rates
The rates of association and dissociation of androgen binding to ovarian membrane preparations were determined at 4°C. For association assays, membrane preparations of individual ovaries or a pool of three ovaries were incubated with 8 or 10 nM [3H]T for various time periods ranging from 1 min to 23 h. A parallel set of reaction tubes contained 1000-fold excess unlabeled T to determine nonspecific binding. Dissociation rates were determined by incubating membrane preparations with 10 nM [3H]T (total binding) or with 10 nM [3H]T plus 10 µM unlabeled T (nonspecific binding) for 40 min before the addition of 1000-fold excess unlabeled T. Reactions were stopped by filtration at various time periods ranging from 1 min to 2 h after addition of the unlabeled T.
Competition Studies
Steroid competitors were dissolved in ethanol, added to the reaction tubes for final reaction concentrations of 0.1 nM to 10 µM, and evaporated under nitrogen. Next, 250 µl of membrane preparation diluted to 0.15–0.5 mg protein/ml in HED and 250 µl of [3H]T (final reaction concentration, 10 nM) were added to the reaction tubes and incubated at 4°C for 40 min. Maximum specific [3H]T binding was defined as the difference between total binding at 10 nM [3H]T (no competitor present) and nonspecific binding (in the presence of 1000-fold excess unlabeled T). Antiandrogens and xenobiotics were tested in competition assays using the same method; however, the range of competitor concentrations was 100 nM to 1 mM. The DDT congeners were tested in the presence of two different concentrations of [3H]T (5 and 10 nM). The concentration of competitor needed to displace 50% of maximum specific [3H]T binding (EC50) was calculated from the one-site competitive binding curve. In this analysis, the top and the bottom of the curve were defined as 100% and 0%, respectively, and held constant. The relative binding affinity (RBA) was calculated from the ratio of the EC50 of T to the EC50 of the competitor and expressed as a percentage (T = 100%).
Tissue Specificity
Various tissue types were collected from female croaker, and the membrane fraction of each tissue was prepared as described previously. Androgen binding in the membrane fractions of the ovary, liver, brain, drumming muscle, kidney, trunk muscle, intestine, gills, and heart was detected with semiquantitative, one-point assays. Full saturation curves were performed on membrane fractions from brain and testicular tissues.
Biological Relevance: Effects of Stage of Ovarian Development and Hormonal Treatments on Receptor Concentrations
Samples of croaker ovaries were collected throughout the reproductive cycle, and membrane fractions were prepared as described previously. The GSI for each fish was calculated and used as a measure of the reproductive stage of the ovary. Androgen binding in these seasonal samples was detected by one-point assays. In addition, the effects of in vitro steroid treatment on receptor concentrations in mature ovaries were investigated. Aliquots (
1 g) of fresh, mature, ovarian tissue were incubated in 50 ml of DMEM containing 500 nM concentrations of various steroids for 11 h at room temperature. After the incubation, ovarian fragments were removed from the steroid treatment and frozen at –80°C for later analysis. Membrane fractions were prepared as described previously and analyzed for androgen binding by one-point assays.
Statistical Analysis
Values are reported as the mean ± SEM. All linear and nonlinear regression analyses were generated by GraphPad Prism for Windows (Version 3.02; GraphPad Software, San Diego, CA; http://www.graphpad.com). Data were transformed when necessary to conform to assumptions of normality and homoscedasticity, then analyzed by one-way ANOVA and Tukey tests for multiple comparisons (Sigma Stat; SPSS, Chicago, IL).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nonlinear regression of the specific [3H]T binding curves (Fig. 1) indicates the presence of a high-affinity (Kd = 15.3 ± 2.7 nM), low-capacity (Bmax = 2.8 ± 0.3 pmol/ mg protein or 9.5 ± 1.6 pmol/g tissue), androgen-binding site in the membrane fraction of ovarian tissue (n = 5 preparations, R2 
0.92). Linear Scatchard transformations indicate a one-site model of androgen binding (Fig. 1, inset).
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To verify that the androgen binding was located in the membrane preparation, 5'-nucleotidase activity was measured in all subcellular fractions. Enzyme activity was approximately 10-fold higher in the membrane fraction than in the crude nuclear and cytosolic fractions (Fig. 2a). In addition, one-point assays of all fractions indicate that androgen binding was primarily located in the membrane fraction, with values in the cytosolic and nuclear fractions less than 10% of the binding in the membrane fraction (Fig. 2b). Binding was also significantly reduced in boiled membrane fractions (P = 0.01).
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Association and Dissociation Rates
The results of the association and dissociation experiments indicated that androgen binding in the membrane fraction was relatively rapid. The half-time of association was 3.7 ± 1.7 min, with maximum binding achieved after 30 min (n = 2) (Fig. 3a). Androgen binding remained constant for at least 23 h. Dissociation was also rapid, with a half-time of 4.7 ± 0.2 min and complete dissociation after 40 min (n = 2) (Fig. 3b). The half-time values were calculated by nonlinear regression analysis of the specific [3H]T binding of association or dissociation (R2 0.82).
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Competition Studies
Curves used to calculate RBA values (Table 1) accurately reflected the data; coefficients of determination (R2) were greater than or equal to 0.87 except for one assay with estradiol, which had an R2 of 0.52 (n = 2–5). In steroid specificity experiments that tested the binding affinity of steroid hormones found in fish, only T and 11-ketotestosterone substantially displaced 10 nM [3H]T within physiological concentrations (Fig. 4a). The binding affinity of estradiol was two orders of magnitude lower than that of T. Further testing of androgens indicated that in addition to T and 11-ketotestosterone, dihydrotestosterone (DHT) competed for binding, demonstrating approximately twice the affinity of T (Fig. 4b). No other androgens tested effectively competed for binding within this range of concentrations. All the progestogens tested had RBA values 
1.1% except for progesterone (RBA = 191%) (Fig. 4c). The parallel binding curves of T and progesterone suggest that binding is competitive. The antiandrogens flutamide and cyproterone acetate had RBA values of 0.13% and 0.01%, respectively (Fig. 5a). Of the xenobiotics tested, vinclozolin and its metabolites (M1 and M2) displaced [3H]T (Fig. 5a), whereas the highest concentrations of the DDT congeners tested did not compete for binding (Fig. 5b).
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Tissue Specificity
Substantial androgen binding was detected in membrane fractions of the ovary, brain, drumming muscle, and kidney from female croaker with the semiquantitative, one-point assay, whereas little binding was detected in the trunk muscle, intestine, gills, and heart (Fig. 6). Results of the one-point binding assay with liver tissue indicated very high levels of androgen binding (645 dpm/µg protein). However, the membrane layer from the sucrose pad procedure was not clearly defined in the preparations of the liver; therefore, the accuracy of the binding in this tissue is uncertain.
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Saturation analysis of pooled brain tissue of both males and females indicates that androgen binding in the membrane fraction of the brain is of significantly higher affinity (Kd = 1.55 ± 0.51 nM) and lower capacity (Bmax = 0.60 ± 0.003 pmol/mg protein or 3.12 ± 0.63 pmol/g tissue) compared to the binding in the ovary (n = 3, R2 0.80 for the nonlinear regression of the specific [3H]T binding curves, P < 0.01) (Fig. 7a). Scatchard analysis indicates a one-site model of binding (Fig. 7a, inset). In contrast, the affinity of binding in the testes was lower than that in the ovary (Kd = 27.94 ± 2.01 nM, R2 0.98 for the nonlinear regression of the specific [3H]T binding curves, P < 0.05, n = 2). Androgen-binding capacity in the membrane fraction of testicular tissue was significantly lower (Bmax = 0.36 ± 0.03 pmol/mg protein or 1.56 pmol/g tissue) as compared to the ovary (P < 0.01) (Fig. 7b).
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Biological Relevance: Effects of Stage of Ovarian Development and Hormonal Treatments on Receptor Concentrations
The amount of androgen binding in croaker ovarian membranes was dependent on the reproductive stage of the fish (Fig. 8a). One-point assays indicate that binding was 10-fold greater in fully mature ovaries than in regressed tissue. Androgen-binding concentrations in ovarian fragments were not altered after an 11-h treatment with steroid hormones in vitro (Fig. 8b). One-way ANOVA detected no significant differences among the different treatment groups (P > 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To our knowledge, only a few reports describe partial biochemical characterizations of androgen-binding sites associated with cell membranes. The brown trout (Salmo trutta) olfactory mAR [21] demonstrates higher binding affinity (Kd = 0.5–1.9 nM) and lower capacity (Bmax = 30–60 fmol/mg protein) than the croaker ovarian mAR (Kd = 15.3 nM, Bmax = 2.8 pmol/mg protein). In contrast, the androgen-binding site identified in the membrane fraction of rat brain tissue [20] demonstrated an affinity of the same order of magnitude (Kd = 31.8 nM) as the croaker ovarian mAR, although it had a considerably higher binding capacity (202 pmol/mg protein). Thus, there appear to be marked species and/or tissue differences in the binding affinities and capacities of the few membrane androgen-binding moieties investigated so far, although the physiological significance of these differences remains unclear.
The rapid association and dissociation of membrane androgen binding in the croaker ovary is similar to that of other membrane androgen-binding sites, which show complete association after 16 min in brain tissue [20] and after 45 min in fish olfactory tissue [21]. Additionally, both the membrane maturation-inducing steroid (MIS) receptor in spotted seatrout (Cynoscion nebulosus) and the membrane estrogen receptor in Atlantic croaker associate rapidly, reaching equilibrium within 5 and 30 min, respectively [30, 31]. In contrast, complete androgen association to the nuclear androgen receptor in the croaker ovary is only observed after an 8-h incubation [25]. The rapid rate of association of ligands to steroid membrane receptors would theoretically enable steroids acting at the cell surface to initiate cellular responses within a few minutes and, therefore, is consistent with the rapid changes in the intracellular levels of second-messenger molecules frequently reported within minutes of steroid application.
The steroid specificity of the croaker ovarian mAR is unusual in several respects. To our knowledge, the high affinity of progesterone for this receptor has not been observed in other studies of androgen binding to membrane preparations, in which progesterone failed to compete for androgen binding [19, 20, 23]. Furthermore, the common synthetic androgen, R1881, does not bind to the croaker ovarian mAR, which is similar to the lack of R1881 competition for androgen binding in membranes prepared from of rat liver and prostate cells [23]. Interestingly, a nuclear androgen receptor has been described in the shark testis that binds progesterone, but not R1881, with high affinity [32]. Moreover, it was suggested that this shark androgen receptor is related to a common ancestral nuclear-receptor molecule. The possible phylogenetic significance of the high binding affinity of progesterone to the croaker ovarian mAR cannot be determined in the absence of information regarding the structures of steroid membrane receptors. However, from a physiological standpoint, this binding is unlikely to be significant, because progesterone is not present at physiologically relevant concentrations in fish. The mAR appears to mediate only the actions of androgens, because the progestins 17,20ß,21-trihydroxy-4-pregnen-3-one and 17,20ß-dihydroxy-4-pregnen-3-one, the maturation-inducing steroids in fish, do not bind to this receptor. The lack of binding of two other progestins, 17-hydroxy-4-pregnene-3,20-dione and 20ß-hydroxy-4-pregnen-3-one, to the receptor further indicates it is not a progestin receptor and has a distinctly different steroid specificity from that of progestin membrane receptors.
Possible binding of antiandrogens to other mARs can only be inferred indirectly from studies of their effects on rapid androgen actions. For example, cyproterone acetate and flutamide do not reverse the rapid effects of androgens on intracellular calcium in macrophages [11, 15], suggesting that these compounds do not act as androgen-receptor antagonists in this system. However, both flutamide and cyproterone acetate displace T from the croaker ovarian mAR at high concentrations, indicating that these compounds, acting as either agonists or antagonists, could potentially alter androgen signaling via this receptor. Interestingly, flutamide has been shown to mimic the nongenomic actions of androgens on MAPKs in human breast cancer cells [17].
Androgen binding to plasma membrane fractions was tissue specific in the Atlantic croaker. In addition to the ovary, significant binding was detected in other tissues, including the brain, where rapid, nongenomic effects of androgens have been demonstrated previously in other vertebrates [33–36]. Studies concerning nongenomic steroid actions in the brain have shown that steroids are capable of modulating the actions of gamma-aminobutyric acid (GABA) by noncompetitively binding to its receptor (for review, see [37]). Evidence suggests that the androgen, 5-androstane-3,17ß-diol, inhibits sexual behavior via this mechanism in rats [38]. In croaker, both GABA and androgens decrease LH secretion in fully mature females [39, 40]. However, to our knowledge, competition studies to determine whether androgens and GABA bind to the same receptor in croaker brain have not been conducted. Therefore, it is not known whether androgens could potentially influence LH secretion through binding to the GABA-receptor complex in this species. Interestingly, T binding in the croaker brain was of a higher affinity and lower capacity compared to that of the ovarian mAR, which suggests these two binding sites may represent two distinct receptors or the same receptor in two different conformations. Additionally, preliminary research indicates that in contrast to the ovarian mAR, T binds to the membrane site in the brain with higher affinity than DHT (unpublished results), which is similar to the higher binding affinity of T to the nuclear receptor identified in the croaker brain [25]. However, a complete characterization of the androgen-binding site in the brain will be necessary to confirm the existence of two distinct croaker mARs.
The presence of specific androgen-binding sites in croaker testicular membranes suggests the testis is also a site of nongenomic androgen actions initiated at the cell surface, although the concentrations of androgen-binding sites in the testis were significantly lower than those in the ovaries and brain. The binding capacity of the membrane binding site in croaker testis was 360 fmol/mg protein, which is similar to the levels of membrane androgen binding observed in the rat testes (Bmax = 328 fmol/mg protein) [22]. However, the rat testicular binding site had a higher binding affinity (Kd = 1.6 nM) than that of the croaker testicular binding site (Kd = 27.94 nM). Although the cellular localization and physiological role of the croaker testicular membrane androgen-binding site is yet unknown, androgens have been shown to rapidly alter Ca2+ signaling in rat Sertoli cells [6, 7].
One-point assays revealed that androgen binding was also present in membrane fractions of croaker kidneys and drumming (sonic) muscle, although the saturation points and binding affinities of these binding sites were not determined. It is interesting to note that a role of androgens has been established in both these tissues in certain species of fish. Androgen treatment increases the sonic muscle mass in male weakfish (C. regalis), a species closely related to Atlantic croaker, mimicking the changes observed in this species during the reproductive season [41]. Androgens also cause hypertrophy of the stickleback kidney, which is associated with production of the nest-building glue protein, spiggin [42, 43]. To our knowledge, the type of receptor mediating these androgen actions has not yet been determined. Therefore, the involvement of nuclear androgen receptors and/or mARs remains a possibility in these tissues, although an mAR has yet to be identified in the stickleback kidney.
Because steroid membrane receptors have not been as thoroughly characterized as their nuclear counterparts, relatively little information is available regarding their binding affinities for xenobiotic chemicals. Certain metabolites of the pesticide DDT were found to bind to a membrane estrogen receptor in the testis of the Atlantic croaker [31], and o,p'-DDE was found to bind to the membrane progestogen receptor on croaker sperm [44]. In addition, o,p'-DDD was found to bind to the membrane MIS receptor in croaker ovaries and to disrupt the process of final oocyte maturation (for review, see [45]). However, the DDT metabolites, including p,p-DDE, a xenobiotic antiandrogen [46, 47] that binds to the nuclear androgen receptor in the croaker ovary [26], fail to displace T binding from the ovarian mAR at high concentrations. Other xenobiotics that compete for binding to nuclear androgen receptors in mammals and Atlantic croaker ovaries include the metabolites of the fungicide vinclozolin, M1 and M2 [26, 48]. In contrast to the results with the DDT metabolites, M1 and M2 were capable of binding to the croaker ovarian mAR, which suggests that these xenobiotics have the potential to disrupt androgen signaling through this receptor.
Currently, much research is focused on the molecular nature of the membrane steroid receptors. Unlike the nuclear receptors, current evidence suggests that the proteins mediating the rapid, nongenomic effects of steroid hormones cannot be classified into a single superfamily of shared molecular characteristics. In some tissues, the membrane-binding sites appear to be structurally related to the nuclear receptors, as found in some studies of membrane estrogen binding [49, 50]. However, the binding results from the current study provide indirect evidence that the structures of the binding pockets of the nuclear androgen receptors and mARs in the croaker ovary differ. Competition studies show that the synthetic androgens, mibolerone and R1881, do not bind to the mAR, whereas these androgens compete for binding to the ovarian nuclear androgen receptor, AR2, with RBA values of 42% and 62%, respectively [25, 27]. The mAR demonstrates little affinity for 4-androstene-3,11,17-trione, whereas this steroid displays a biphasic binding curve in AR2 competition assays and binds with high affinity (RBA = 2000%; [27]). Furthermore, the high affinity of the mAR for progesterone is not a characteristic of the nuclear androgen receptor [25]. In addition, the mAR has a lower affinity and a higher capacity than the nuclear androgen receptor (Kd = 0.62 nM, Bmax = 0.38 pmol/g tissue [25]). Although these differences in binding characteristics suggest that the croaker ovarian mAR is a novel receptor, as in the case of the T receptors in the plasma membranes of T cells [14], without specific antibodies directed against these proteins or the protein or nucleic acid sequences the possibility remains that the androgen receptor on the membrane is a modification of the nuclear receptor. The finding that the croaker ovarian mAR is colocalized with the nuclear receptor described by Sperry and Thomas [25] in croaker ovarian, testicular, and brain tissues supports this possibility.
In addition to modified nuclear receptors, membrane steroid-binding sites can be novel proteins and are often G protein-coupled receptors (GPCRs) [4]. A membrane progestin receptor with the characteristics of a GPCR has been discovered in the ovary of spotted seatrout, a species closely related to the croaker [51, 52]. However, preliminary studies indicate that the croaker ovarian mAR is not coupled to a G protein (unpublished results). Alternatively, other proteins could potentially mediate the rapid, nongenomic actions of steroids initiated at the cell surface. Androgens can increase the growth of prostate cancer cells through a nongenomic mechanism mediated by the steroid hormone-binding globulin (SHBG) [53]. Whereas an SHBG has been identified in the plasma of the spotted seatrout [54], this fish SHBG demonstrated a higher affinity for androgens (Kd = 4.89 nM), a substantially higher affinity for estradiol (Kd = 3.13 nM), and a lower affinity for progesterone [54] compared to the mAR currently identified in the ovary of the Atlantic croaker. Because evidence suggests that the steroid-binding characteristics of the SHBG do not change once it has bound to its receptor on the plasma membrane [55], the mAR described in the ovary of the Atlantic croaker is unlikely to be the SHBG/receptor complex.
A rapid, nongenomic action of androgens initiated at the cell surface to down-regulate ovarian estradiol production in croaker has been described previously [24]. It is possible that the mAR currently described is responsible for mediating this nongenomic effect of androgens. The current study supports a role for the mAR in the reproductive cycle of female Atlantic croaker. Moreover, a positive relationship appears to exist between plasma sex-steroid levels and mAR concentrations during the reproductive cycle. The highest concentrations of mAR were detected in fully mature ovaries, which produce large amounts of androgens, whereas only a small amount of binding was present in regressing ovaries, which synthesize lower amounts of steroids. These results suggest that the nongenomic actions of androgens mediated by the mAR are of greatest importance at the end of ovarian and oocyte growth. Therefore, the mAR may have an important role in the changes of ovarian estradiol production observed at the end of this phase of the reproductive cycle. However, mibolerone, which is an inhibitor of estradiol production in the in vitro system [24], does not bind to the mAR, suggesting that this synthetic steroid is not acting via the mAR. In this case, mibolerone may be acting through the nuclear receptor, because only DHT was clearly shown to act at the cell surface through a rapid, nongenomic mechanism to inhibit in vitro estradiol production [24]. Therefore, it is possible that androgens decrease estradiol synthesis through binding to both membrane and nuclear receptors in the croaker ovary. The finding that the membrane receptor has a lower affinity and higher capacity than the nuclear receptor [25] raises the possibility that these two receptors are activated under different androgen concentrations and that the rapid effects of androgens in the croaker ovary are not always turned on but, rather, are initiated only at times of higher androgen concentrations.
In conclusion, an androgen-binding site has been characterized in a membrane fraction of Atlantic croaker ovaries. This moiety demonstrates high-affinity binding that is both reversible and of finite capacity. Androgen binding in the membrane is both steroid and tissue specific and likely of biological relevance in the reproductive cycle of female croaker. Thus, it fulfills all the binding requirements and should be designated as a steroid receptor. This is one of the first reports providing conclusive evidence for the existence of mARs in vertebrates.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence and current address: Alyssa M. Braun, University of Nevada, Las Vegas, Department of Biological Sciences, 4505 Maryland Pkwy, Las Vegas, NV 89154-4004. FAX: 702 895 2396; alyssa.braun{at}ccmail.nevada.edu
Received: 21 November 2003.
First decision: 13 December 2003.
Accepted: 24 February 2004.
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  Y. Pang, J. Dong, and P. Thomas Estrogen Signaling Characteristics of Atlantic Croaker G Protein-Coupled Receptor 30 (GPR30) and Evidence It Is Involved in Maintenance of Oocyte Meiotic Arrest Endocrinology, July 1, 2008; 149(7): 3410 - 3426. [Abstract] [Full Text] [PDF]
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Y. Katsu, R. Ichikawa, T. Ikeuchi, S. Kohno, L. J. Guillette Jr., and T. Iguchi Molecular Cloning and Characterization of Estrogen, Androgen, and Progesterone Nuclear Receptors from a Freshwater Turtle (Pseudemys nelsoni) Endocrinology, January 1, 2008; 149(1): 161 - 173. [Abstract] [Full Text] [PDF]
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E. Filardo, J. Quinn, Y. Pang, C. Graeber, S. Shaw, J. Dong, and P. Thomas Activation of the Novel Estrogen Receptor G Protein-Coupled Receptor 30 (GPR30) at the Plasma Membrane Endocrinology, July 1, 2007; 148(7): 3236 - 3245. [Abstract] [Full Text] [PDF]
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J. W. Gatson and M. Singh Activation of a Membrane-Associated Androgen Receptor Promotes Cell Death in Primary Cortical Astrocytes Endocrinology, May 1, 2007; 148(5): 2458 - 2464. [Abstract] [Full Text] [PDF]
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 Y. Takeshita, R. Fujinaga, C. Zhao, A. Yanai, and K. Shinoda Huntingtin-associated protein 1 (HAP1) interacts with androgen receptor (AR) and suppresses SBMA-mutant-AR-induced apoptosis Hum. Mol. Genet., August 1, 2006; 15(15): 2298 - 2312. [Abstract] [Full Text] [PDF]
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J. W. Gatson, P. Kaur, and M. Singh Dihydrotestosterone Differentially Modulates the Mitogen-Activated Protein Kinase and the Phosphoinositide 3-Kinase/Akt Pathways through the Nuclear and Novel Membrane Androgen Receptor in C6 Cells Endocrinology, April 1, 2006; 147(4): 2028 - 2034. [Abstract] [Full Text] [PDF]
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 R Mindnich, F Haller, F Halbach, G Moeller, M H. de Angelis, and J Adamski Androgen metabolism via 17{beta}-hydroxysteroid dehydrogenase type 3 in mammalian and non-mammalian vertebrates: comparison of the human and the zebrafish enzyme J. Mol. Endocrinol., October 1, 2005; 35(2): 305 - 316. [Abstract] [Full Text] [PDF]
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 J. M. Kaufman and A. Vermeulen The Decline of Androgen Levels in Elderly Men and Its Clinical and Therapeutic Implications Endocr. Rev., October 1, 2005; 26(6): 833 - 876. [Abstract] [Full Text] [PDF]
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P. Thomas, Y. Pang, E. J. Filardo, and J. Dong Identity of an Estrogen Membrane Receptor Coupled to a G Protein in Human Breast Cancer Cells Endocrinology, February 1, 2005; 146(2): 624 - 632. [Abstract] [Full Text] [PDF]
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