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BOR - Papers in Press, published online ahead of print July 9, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.015479
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BIOLOGY OF REPRODUCTION 69, 1642–1650 (2003)
DOI: 10.1095/biolreprod.103.015479
© 2003 by the Society for the Study of Reproduction, Inc.

Mechanisms of Hormone Action

Androgens Inhibit Estradiol-17ß Synthesis in Atlantic Croaker (Micropogonias undulatus) Ovaries by a Nongenomic Mechanism Initiated at the Cell Surface1

Alyssa M. Braun2,4, and Peter Thomas3

Marine Science Institute,3 University of Texas at Austin, Port Aransas, Texas 78373 Department of Biological Sciences,4 University of Nevada, Las Vegas, Las Vegas, Nevada 89154-4004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The presence of androgen receptors in the ovaries of several vertebrate species, including Atlantic croaker, suggests that androgens may have important roles in ovarian function. In the current study the effects of androgens on ovarian steroidogenesis in Atlantic croaker were investigated. Addition of 17ß-hydroxy-5{alpha}-androstan-3-one (DHT), 11-ketotestosterone (11-KT), or Mibolerone to ovarian incubations caused dose-dependent decreases in gonadotropin-stimulated in vitro estradiol production, which was not reversed by cotreatment with the antiandrogens, cyproterone acetate or 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene. Androgen treatment also caused significant decreases in estradiol production in the presence of 17-hydroxyprogesterone, which suggests that the site of androgen action is downstream of this steroid in the steroidogenic pathway. The mechanism of androgen action on ovarian steroidogenesis was also investigated. Coincubation with actinomycin D did not reverse the inhibitory effect of the androgens, which suggests that the mechanism of androgen action is nongenomic. An androgen conjugated to bovine serum albumin (DHT-BSA), which does not enter the cell, also caused inhibition of estradiol production in vitro, indicating that the androgen is acting at the cell surface. In addition, time course experiments revealed that the androgen action is rapid; 5-min exposure to DHT was sufficient to cause a significant reduction in estradiol production. Finally, preliminary evidence was obtained for the existence of a high-affinity, low-capacity androgen binding site in croaker ovarian plasma membranes. These studies suggest that androgens can down-regulate estrogen production in croaker ovaries via a rapid, cell surface-mediated, nongenomic mechanism.

androgen receptor, estradiol, ovary, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In addition to the well-known functions of androgens in males, androgens and their receptors have been identified in female reproductive tissues, indicating that they also influence reproduction in females (reviewed by [1]). Studies showing that androgens can modulate ovarian steroidogenesis in several vertebrate species have provided additional evidence for an important physiological role for these steroids in female reproduction [2–8]. For example, androgens have been shown to inhibit gonadotropin-stimulated progesterone secretion from granulosa cells of primates and the domestic hen [5–7, 9]. Additionally, there is evidence for androgen modulation of ovarian steroidogenesis at multiple sites along the steroidogenic pathway. Specifically, aromatase activity in large follicles isolated from primate granulosa cells is inhibited after androgen treatment [9], whereas cytochrome P450 side chain cleavage (P450scc) enzyme activity is inhibited in the domestic hen [7, 8]. Androgens not only competitively inhibit P450scc activity in hen granulosa cells but also decrease protein levels of this enzyme, indicating that androgens can regulate steroid production through multiple mechanisms [8].

Several lines of evidence suggest that androgens can regulate certain steroidogenic enzymes in males via the classic, genomic mechanism of steroid action mediated by intracellular steroid binding to nuclear androgen receptors. Studies in mouse Leydig cells implicate nuclear androgen receptors in the androgen-dependent inhibition of de novo synthesis of the cytochrome P450 17{alpha}-hydroxylase/C17-20 lyase (P45017{alpha}) protein [10] via repression of Cyp17, the gene encoding this enzyme [11]. Nuclear androgen receptors have also been shown to mediate androgen regulation of cytochrome P450 aromatase (P450arom) activity in the brain of adult male rats [12], an action that involves regulation of P450arom mRNA [13].

In addition to the classical nuclear mechanism of steroid action, evidence has gradually accumulated that androgens and other steroid hormones can act via rapid, nongenomic mechanisms initiated at the cell surface (reviewed by [14]). For example, in Atlantic croaker, estradiol-17ß has been shown to exert a cell surface-mediated, nongenomic action on testicular androgen production [15]. In contrast, little information is available on possible androgen regulation of steroidogenesis via this mechanism. Androgens have been shown to cause rapid changes in Ca2+ levels in the steroidogenic cells of the human ovary through a nongenomic mechanism [16]. Moreover, alteration of intracellular Ca2+ is one suggested mechanism of autoregulation of androgen production in rat thecal interstitial cells [17]. However, to the best of our knowledge, direct experimental evidence that androgens influence steroidogenesis by a cell surface-mediated, nongenomic mechanism is currently lacking.

Recently a nuclear androgen receptor has been biochemically characterized in the ovary of Atlantic croaker [18, 19], but its functional role remains unclear. Therefore, the purposes of the current study were to determine the effects of androgens on ovarian steroidogenesis in this species and to investigate the possible involvement of genomic and nongenomic mechanisms in mediating the androgen effects. The results show that gonadotropin-stimulated estradiol (E2) secretion is inhibited by androgens acting via a rapid, cell surface-mediated, nongenomic mechanism. Consequently, a preliminary search for the presence of high-affinity androgen binding sites on the plasma membrane of croaker ovaries was also conducted.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

All unlabeled steroids were purchased from Steraloids (Newport, RI) or from Sigma (St. Louis, MO). The synthetic androgen Mibolerone was a gift from Upjohn Laboratories. The conjugated androgen 5{alpha}-androstan-17ß-ol-3-one hemisuccinate:BSA (DHT-BSA) (molar ratio = 30 DHT:1 BSA) was purchased from Steraloids. Cyproterone acetate, Dulbecco modified Eagle Medium without phenol-red (DMEM, nutrient mixture F-12 HAM), actinomycin D, streptomycin sulfate, penicillin G, and human chorionic gonadotropin (hCG) were purchased from Sigma. 1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) was purchased from Chem Service (West Chester, PA). [2,4,6,7-3H]-Estradiol-17ß ([3H]-estradiol) (specific activity = 87 Ci/mmol) and [1,2,6,7-3H]-testosterone (3H-T) (specific activity = 95 Ci/mmol) were purchased from New England Nuclear (Boston, MA). All radioactivity was counted in a liquid scintillation counter (LS 6000SC; Beckman Instruments, Fullerton, CA). The scintillation cocktail was composed of reagent-grade toluene (4 L), 7,5-diphenyl-oxazole (PPO, Sigma) (16 g), and 1,4-bis[5-phenyl-2-oxazolyl]-benzene (POPOP, Sigma) (0.4 g).

Animals and Tissue Collection

Atlantic croaker undergoing gonadal recrudescence were captured by gill net or otter trawl during the period of August–October (1996–2001) from Redfish Bay near Port Aransas, Texas. Fish were acclimated to laboratory conditions for at least 3 wk prior to experimentation and maintained in circular, recirculating tanks under constant photoperoid (11L:13D) and temperature (22–25°C). Fish were fed a diet of commercial pellets daily. Female fish were visually inspected for signs of gonadal maturity. Typically six females with fully grown ovaries containing large vitellogenic oocytes were humanely killed by rapid decapitation following procedures approved by the University of Texas at Austin Institutional Animal Care and Use Committee according to NIH guidelines, and gonadal tissue was removed and immediately placed in ice-cold DMEM. The DMEM was supplemented with streptomycin (100 mg/L), penicillin (60 mg/L), and sodium bicarbonate (1.2 g/L) and adjusted to pH 7.4. The stage of ovarian and follicular development was determined by measuring the diameters of the largest follicles under a microscope. The gonadosomatic index (GSI) was determined from the equation GSI = (gonad weight/total weight) x 100 and used as an index of gonadal growth. The average GSI of the fish chosen for in vitro incubations was 13.54% ± 0.45% (mean ± standard error). Each treatment was replicated six times with tissue from six different donors. Occasionally, fewer than six donor fish at the same reproductive stage were available for an in vitro experiment and tissues from fewer donors were used. However, in all cases experiments were repeated until observations were obtained for each treatment from at least six donors.

General In Vitro Incubation Procedures

Immediately after tissue collection, 100-mg fragments of ovarian follicles per well were placed in 24-well culture plates (Costar) containing 1 ml DMEM and incubated for 1–3 h prior to hormone treatment. At the end of this preincubation period, the media were replaced with 1 ml fresh DMEM. Treatment chemicals were either dissolved in ethanol (final concentration of vehicle <=1%) prior to addition to the media or directly dissolved in DMEM. Treatment duration varied depending on the specific experiment, and except where noted, tissue fragments were washed two to five times with 1 ml media after the treatment period and incubated for a further 9 h in DMEM alone. After this posttreatment incubation, media were removed and frozen at -20°C for later analysis of estradiol-17ß (E2) production by radioimmunoassay (RIA). Estradiol RIAs were conducted on 100 µl unextracted media after heat treatment at 70°C for 1 h as described previously [20].

Effect of Androgens and Antiandrogens on In Vitro Estradiol Production

The effects of a range of concentrations of three androgens, 17ß-hydroxy-5{alpha}-androstan-3-one (DHT) (10–10 000 ng/ml = 34 nM–34 µM), 11-ketotestosterone (11-KT) (1 nM–10 µM = 0.3–3000 ng/ml), and Mibolerone (33 nM–33 µM = 10–10 000 ng/ml) and the synthetic antiandrogen cyproterone acetate (CA) (0.12–120 µM = 0.05–50 µg/ml) on hCG-stimulated (1 or 5 IU/ml) ovarian estradiol production were investigated. Tissues were treated with DHT for 6 h, whereas the treatment period was extended to 12 h for the other three compounds prior to washing and posttreatment incubations. The effects of various combinations of androgens and antiandrogens were also investigated. Tissues were exposed to cyproterone acetate (1.2 µM = 0.5 µg/ml), a suspected xenobiotic antiandrogen; p,p-DDE (625 µM), or vehicle control for 2 h in the presence of 5 IU hCG/ml prior to the addition of DHT (3.4 µM = 1 µg/ml) for a further 12 h. In another study, tissues were exposed to increasing concentrations of Mibolerone ± 12 µM cyproterone acetate for 12 h prior to washing and the posttreatment incubation.

Steroid Specificity

Ovarian fragments were exposed to various teleost C21 steroids (corticosteroids and progestins) in order to test whether the effects on estrogen production were specific to androgens. Tissues were incubated for 12 h in the presence of 2.5 IU hCG/ml and two concentrations of cortisol (F), 17{alpha},20ß,21-trihydroxy-4-pregnen-3-one (20ß-S), 17{alpha},20ß-dihydroxy-4-pregnen-3-one (17,20ß-P), or Mibolerone (Mib). After the treatment period, tissues were washed and incubated for a further 9 h in DMEM.

Effects of Androgen Treatment on In Vitro Ovarian Estradiol Production in the Presence of Steroid Precursors

In order to investigate the site of androgen action along the steroidogenic pathway, ovarian fragments were coincubated with androgen precursors. Tissue was incubated for 2 h in DMEM prior to the addition of 3.4 µM DHT and 300 nM of one of two steroid precursors, progesterone (P4), or 17-hydroxyprogesterone (17-OH-P4). Controls contained precursor but no androgen. Immediately after the 9-h treatment incubation, media were removed and frozen for E2 analysis.

Effects of Actinomycin D on Ovarian Estradiol Production in Response to Androgen Treatment

In vitro steroid production was stimulated for 3 h in the presence of 5 IU hCG/ml; tissue fragments were subsequently washed with 1 ml DMEM and treated with DHT (3.4 µM) ± 10 µg/ml actinomycin D (Act D) for 9 h. Treatment media were removed for analysis of E2.

Effects of Treatment with an Androgen Conjugate on Ovarian Estradiol Production

Ovarian tissues were treated in vitro with DHT (1 µM), DHT-BSA (75 µM based on DHT), BSA (1 µM), or vehicle control in the presence of 1 IU hCG/ml for 3 h prior to washing and posttreatment incubation.

Time Course of Androgen Effects

Ovarian tissue fragments were incubated in vitro for various time periods ranging from 5 min to 12 h in DHT (3.4 µM) in the presence of 5 IU hCG/ml. At the end of the treatment period, tissue fragments were washed five times prior to the 9-h posttreatment incubation in DMEM alone.

Androgen Binding to a Plasma Membrane Preparation

Ovarian tissue was removed from croaker after they had been killed humanely as described previously. Tissue was immediately placed in ice-cold assay buffer (HAED) (25 mM HEPES, 10 mM NaCl, 1 mM dithioerythritol, 1 mM EDTA, pH 7.6). A 2-g fragment of ovarian tissue was initially homogenized in 15 ml HAED using a Polytron Tekmar Tissumizer on high setting for 5 sec prior to three passes with a glass Tenbroeck tissue grinder (Wheaton Science Products, Millville, NJ). The resulting homogenate was centrifuged at 1000 x g for 7 min. The pellet was discarded, and the supernatant was centrifuged at 20 000 x g for 20 min. The resulting supernatant was discarded, and the pellet was resuspended in 10 ml HAED and further separated over a sucrose pad [21]. The pellet suspension was layered over 1.2 M sucrose prepared in HAED (1 vol pellet resuspension:1 vol sucrose buffer) and centrifuged at 6900 x g for 45 min. After centrifugation, the middle membrane layer was removed and diluted to the appropriate protein concentration for the radioreceptor assay.

The membrane preparation was incubated in the presence of increasing concentrations of 3H-testosterone (3H-T) (0.1–20 nM) for 30 min at 4°C (reaction vol = 500 µl) to estimate total ligand binding. Nonspecific binding was assessed in a parallel set of tubes containing 1000-fold excess unlabeled testosterone. Bound steroid was separated from free steroid by filtration over presoaked Whatman glass-fiber filters, pore size 1 µm, at 4°C. Each filter was immediately washed with 12.5 ml wash buffer (HAED without dithioerythritol). Bound radioactivity on each filter was counted in a liquid scintillation counter. Kd and Bmax were estimated from nonlinear regression of the saturation curve (GraphPad Prism version 3.02 for Windows, GraphPad Software, San Diego, CA; www.graphpad.com). Data were linearly transformed according to the method of Scatchard [22].

Statistics

All incubation E2 levels are reported as means ± SEM. Data were analyzed by Student t-test (GraphPad Prism) or, where multiple treatments were being compared, ANOVA with Student-Newman-Keuls multiple comparison procedures (SNK) (SigmaStat, SPSS, Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Androgens and Antiandrogens on In Vitro Estradiol Production

Androgen treatment consistently caused a decrease in gonadotropin-stimulated estradiol production in a concentration-dependent manner. Treatment with 34 nM of the nonaromatizable androgen DHT caused a significant decrease in hCG-stimulated E2 production (13.6 ± 2.8 pg/mg tissue vs. 16.3 ± 3.5 for controls, P <= 0.05, n = 6; Fig. 1a). Similar significant concentration-dependent decreases in E2 production after DHT treatment were observed in three additional experiments (results not shown). The major fish androgen 11-KT also caused significant decreases in E2 production within the physiological range of steroid concentrations in two separate experiments (P < 0.02). For example, treatment with 1 nM 11-KT decreased E2 production from 12.8 ± 0.6 pg/mg tissue to 8.0 ± 0.5 pg/mg tissue (P <= 0.01, n = 6; Fig. 1b). The minimum effective concentrations of DHT and 11-KT causing inhibition of estradiol production varied considerably among repeated dose-response experiments, ranging from 1 nM to 34 µM androgen. Treatment with Mib, a synthetic androgen, also caused a concentration-dependent decrease in E2 production. Estradiol production decreased from 17.2 ± 2.1 pg/mg tissue in controls to 10.4 ± 1.6 pg/mg tissue as a result of exposure to 33 nM Mib (P < 0.01, n = 6; Fig. 1c). The experiment was repeated several times with similar results in all cases.



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  		FIG. 1. Effects of androgen treatments on gonadotropin-stimulated estradiol production by croaker ovarian tissue fragments. Ovarian fragments were incubated in various concentrations of androgen in the presence of hCG, washed, and incubated in control DMEM for a further 9 h. Tissue was exposed to increasing concentrations of (a) DHT in the presence of 5 IU hCG/ml for 6 h, (b) 11-KT in the presence of 1 IU hCG/ml for 12 h, or (c) Mibolerone in the presence of 5 IU hCG/ml for 12 h. Each point is the mean ± SEM from six replicate incubations. Representative results for DHT, T, and Mibolerone treatments are shown and are obtained with ovarian tissue from three, one, and six donors, respectively. Asterisks denote means significantly different from controls as computed by a one-tailed, paired Student t-test, and squares denote means significantly different from controls as computed by a one-tailed, two-sample (unpaired) Student t-test

The synthetic antiandrogen cyproterone acetate slightly stimulated gonadotropin-stimulated E2 production at the low dose of 0.12 µM (22.4 ± 2.4 pg/mg tissue vs. 17.6 ± 2.5 pg/mg tissue for controls; Fig. 2a); however, this effect was not significant. In contrast, at the highest dose, 120 µM, cyproterone acetate decreased estradiol production to approximately 20% of control levels (3.6 ± 0.6 pg/mg tissue, n = 6). In experiments designed to test the ability of antiandrogens to block the effects of DHT, tissues were exposed to both an antiandrogen, cyproterone acetate or p,p'-DDE, and androgen concurrently. DHT treatment significantly reduced E2 production compared to controls (25.7 ± 3.6 pg/mg tissue vs. 47.2 ± 7.6 pg/mg tissue, respectively, P < 0.05), whereas concurrent treatment of DHT with cyproterone acetate (at a noninhibitory concentration, 1.2 µM) or p,p'-DDE (625 µM = 200 µg/ml) did not significantly alter E2 production compared to that of DHT treatment alone (n = 6; Fig. 2b). In order to determine whether the inhibitory effect of cyproterone acetate at higher doses was additive to the effects of androgen exposure, tissues were treated with increasing concentrations of Mib in the presence or absence of 12 µM cyproterone acetate (Fig 2c). Cotreatment with cyproterone acetate at all concentrations of Mib significantly decreased estradiol production compared to either treatment alone (n = 6).



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  		FIG. 2. Effects of antiandrogen treatment on gonadotropin-stimulated estradiol production by croaker ovarian tissue fragments in vitro. Each point is the mean ± SEM from six replicate incubations with ovarian tissue from six different donors. a) Ovarian fragments were incubated in increasing concentrations of cyproterone acetate (CA) as described in Materials and Methods. Asterisk denotes mean significantly different from control (no CA present) in a two-tailed, paired Student t-test. b) Ovarian fragments were preincubated in cyproterone acetate or p,p'-DDE for 2 h prior to the addition of DHT or vehicle control as described in Materials and Methods. Asterisks denote means significantly different from control computed by ANOVA with SNK multiple comparisons test. c) Tissue fragments were incubated in Mibolerone (Mib) with and without CA as described in Materials and Methods. Significant effects of the Mib treatments alone, (-) CA, on mean E2 production compared to their respective control group are denoted by squares; significant effects of the combined Mib and CA treatments, (+) CA, on mean E2 production as compared to their respective control group (control [+] CA) are denoted by stars; and significant effects of coincubation with CA on the mean estrogen production in response to each Mib concentration are denoted by asterisks. Significance was determined by two-way ANOVA with SNK multiple comparisons procedure

Steroid Specificity

Mibolerone significantly inhibited estradiol production at a concentration of 33 nM (18.8 ± 4.9 pg/mg tissue) as compared to control levels (22.7 ± 4.8 pg/mg tissue, P < 0.05; Fig. 3). Of all the C21 steroids tested, cortisol, 20ß-S, and 17,20ß-P, only the natural maturation-inducing steroid (MIS), 20ß-S, at a concentration of 167 nM caused a significant reduction in E2 production (14.8 ± 2.4 pg/mg tissue, P < 0.05, n = 6).



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  		FIG. 3. Effects of various teleost C21 steroids on hCG-stimulated E2 production. Tissue fragments were exposed to steroids (see text for abbreviations) at concentrations 33 or 167 nM for 12 h prior to washing and postincubation. Each point is the mean ± SEM from six replicate incubations with ovarian tissue from six different donors. Asterisks denote significantly different E2 production as compared to control as calculated by a two-tailed, paired Student t-test

Effects of Androgen Treatment on In Vitro Ovarian Estradiol Production in the Presence of Steroid Precursors

Estradiol production in the presence of either progesterone or 17-OH-progesterone precursor was significantly decreased by treatment with DHT (P < 0.05, n = 6; Fig. 4).



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  		FIG. 4. Effects of androgen treatment on in vitro ovarian estradiol production in the presence of steroid precursors. Tissue was incubated for 2 h in DMEM alone prior to the addition of either progesterone (P4) or 17-OH-progesterone (17-OH-P4) ± DHT and incubated for an additional 9 h. Treatment media were assayed for E2 production. Each point is the mean ± SEM from six replicate incubations with ovarian tissue from three different donors (two replicates/fish). Asterisks denote means significantly different from precursor alone as determined by a one-tailed, paired Student t-test

Effects of Actinomycin D on Ovarian Estradiol Production in Response to Androgen Treatment

Treatment with DHT caused a 50% reduction in in vitro estradiol production compared to controls, which was not reversed by coincubation with actinomycin D at a concentration (10 µg/ml) known to block transcription in the incubation system (P < 0.05, n = 6; Fig. 5). Actinomycin D treatment alone did not alter E2 production as compared to controls. In an additional experiment, tissues were washed after the same treatments and allowed to incubate for an additional 9 h in control DMEM. In this experiment, similar to the previous results, coincubation with androgen and actinomycin D (3.95 ± 0.89 pg/mg tissue) did not reverse the inhibitory effects of DHT alone (4.81 ± 0.56 pg/mg tissue), although actinomycin D alone (8.98 ± 1.316 pg/mg tissue) did slightly inhibit E2 production compared to control (11.98 ± 0.69 pg/mg tissue; P <= 0.05, n = 6; data not shown).



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  		FIG. 5. Effects of actinomycin D (Act D) on the androgen-induced inhibition of estradiol production. Ovarian fragments were incubated in the presence of 5 IU hCG/ml, washed, and exposed to Act D (10 µg/ml), DHT (3.4 µM) , or Act D + DHT for 9 h. Treatment media were assayed directly for E2 production. Each point is the mean ± SEM from six replicate incubations with ovarian tissue from three donors (two replicates/fish). Asterisks denote means significantly different from control as calculated by ANOVA with the SNK multiple comparisons test

Effects of Treatment with an Androgen Conjugate on In Vitro Ovarian Estradiol Production

The androgen conjugate DHT-BSA, which cannot diffuse across the plasma membrane into the cell, caused a significant decrease in gonadotropin-stimulated E2 production (P < 0.001, n = 6; Fig. 6). The greater inhibition of steroidogenesis observed with DHT-BSA treatment compared to DHT could be the result of the higher concentration of DHT in the conjugate treatment. BSA (1 µM) alone had no effect on E2 production. In three additional experiments, 70–100 µM DHT-BSA (based on steroid concentration) significantly reduced E2 production as compared to controls (P <= 0.05 in all three experiments, n = 6; data not shown).



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  		FIG. 6. Effects of an androgen conjugate on gonadotropin-stimulated estradiol production. Ovarian fragments were incubated in DHT (1 µM), DHT-BSA (75 µM steroid), or BSA 1 µM in the presence of 1 IU hCG/ml for 3 h, washed, and incubated for 9 h in control DMEM. Each point is the mean ± SEM from six replicate incubations. Representative results are shown with ovarian tissue obtained from a single donor. Asterisks denote means significantly different from control as calculated by ANOVA with the SNK multiple comparisons test

Time Course of Androgen Effects

Ovarian fragments were incubated in 5 IU hCG/ml ± 3.4 µM DHT for various time periods, washed, and subsequently incubated for a further 9 h in DMEM alone. The 9-h posttreatment incubation was necessary in order for estradiol accumulation to reach levels detectable by RIA. Estradiol production increased with duration of exposure to hCG in both control and DHT-treated tissue fragments (Fig. 7). Significant differences in estradiol production between control and DHT-exposed tissues were observed after 5 min of androgen treatment (control = 2.0 ± 0.3 pg/mg tissue, DHT = 1.7 ± 0.2 pg/mg tissue, P < 0.05, n = 6). In a different time course experiment, tissues were preincubated in 1 IU hCG/ml for 3 h to up-regulate steroidogenesis prior to the addition of the androgen and subsequently incubated for various periods with 1 µM DHT or media alone (controls), washed, and then incubated a further 9 h in media alone. In this experiment, there was a trend of decreased E2 production after a 5-min treatment with androgen, but the effect was not significant until 30 min of androgen exposure (data not shown).



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  		FIG. 7. Time course of androgen inhibition of gonadotropin-stimulated estradiol production. Ovarian fragments were incubated in 3.4 µM DHT in the presence of 5 IU hCG/ml for 5 min to 12 h of exposure. After the treatments, tissues were washed and incubated a further 9 h in control DMEM. Each point is the mean ± SEM from six replicate incubations with ovarian tissue from six different donors. Asterisks denote significant differences in estradiol production between DHT treatment and control (Con) at each time point as calculated by a one-tailed, paired Student t-test.

Androgen Binding to a Plasma Membrane Preparation

In order to investigate whether an androgen binding moiety could be detected in a plasma membrane fraction of croaker ovarian tissue, a radioreceptor assay was performed using 3H-T as the labeled ligand. Total binding and nonspecific binding measurements are the average of two replicates from one fish. Initial binding studies showed the presence of a high-affinity (Kd = 5.69 nM), saturable (Bmax = 2.68 pmol/mg protein) androgen binding site in a plasma membrane preparation (R2 = 0.98 of the nonlinear regression of the specific binding curve; Fig. 8a). Linear Scatchard analysis indicates one-site binding (R2 = 0.81; Fig. 8b).



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  		FIG. 8. a) Saturation curve and (b) Scatchard analysis of androgen binding to a croaker ovarian plasma membrane tissue preparation. Nonspecific binding (NSB), total binding (TB), and specific binding (SB) were calculated from a radioreceptor assay using 3H-T as the labeled ligand


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the current study show that androgens, within a physiological range of concentrations, inhibit in vitro production of estradiol by Atlantic croaker ovarian tissue. This androgenic inhibition is rapid and exhibited in incubations with the androgen conjugate DHT-BSA, which does not diffuse into the cell, and in coincubations of androgen with the transcription blocker actinomycin D. To our knowledge this is the first direct evidence of a nongenomic, rapid, and specific action of androgens on in vitro ovarian steroidogenesis initiated at the cell surface. Interestingly, preliminary binding studies show the presence of an androgen binding moiety in the plasma membrane fraction of mature croaker ovarian tissue, which could be mediating this androgen action. Previous studies in other vertebrates have shown that androgens can regulate steroidogenesis through genomic mechanisms [10–13]. The present findings suggest that androgen modulation of steroidogenesis in ovarian tissues involves rapid, nongenomic mechanisms in addition to slower genomic processes, resulting in integrated, sequential cellular responses to the hormonal stimulus.

Whereas steroid hormones including androgens have been shown to act through nongenomic mechanisms to alter the release of protein hormones such as prolactin [23–25], insulin [26], and follicle-stimulating hormone [27]; little is known about the nongenomic effects of steroids on steroidogenesis. Interestingly, androgens rapidly alter Ca2+ signaling in human steroidogenic cells [16] and rat Sertoli cells [28, 29], and alterations in Ca2+ levels influence brain aromatase activity [30]. Furthermore, a nongenomic androgen action on Ca2+ signaling was suggested as one mechanism of androgen autoregulation in rat steroidogenic cells [17]. The preliminary identification of an androgen membrane receptor in the current study provides a plausible mechanistic explanation of how androgens can rapidly alter calcium levels and steroid production in steroidogenic tissues. However, the possibility that the nongenomic actions of androgens to alter steroidogenesis may involve a calcium-dependent signaling pathway remains to be investigated in the croaker model.

The fact that the transcription blocker actinomycin D did not reverse the inhibitory effects of androgens on ovarian estradiol production indicates that the androgen action is nongenomic. Nongenomic steroid actions have been classified into two main categories, direct and indirect actions, and both of these categories have been further subdivided into nonspecific and specific effects [31]. Steroid effects on membrane fluidity, generally at doses >=10 µM, have been cited as examples of direct, nonspecific, nongenomic steroid actions (reviewed by [31]). Although androgens may be acting through this mechanism at the highest doses tested in the current study (34 µM), the androgenic inhibition of gonadotropin-stimulated E2 production occurs within a physiological range of concentrations (nM), suggesting that this effect of androgens is not a nonspecific effect on the plasma membrane but rather should be classified as a direct, specific action mediated by a receptor-type molecule.

The finding that cortisol and 17,20ß-P have no significant effects on in vitro ovarian estradiol production at physiological concentrations further indicates that the androgen action is a specific effect. Interestingly, the MIS of this species, 20ß-S, decreases estradiol production, which is similar to the 20ß-S-induced inhibition of gonadal steroidogenesis observed previously in croaker testes [15]. A 20ß-S plasma membrane receptor has been characterized in the ovaries and testes of a closely related sciaenid species, the spotted seatrout (Cynoscion nebulosus) [32, 33], and identified on the membrane of croaker ovaries (unpublished results) and sperm [34], so it is possible that the MIS inhibits gonadal steroidogenesis through this 20ß-S receptor. The MIS membrane receptor demonstrates low affinity for androgens [32], which suggests that the nongenomic actions of androgens on croaker ovarian steroidogenesis are unlikely to be mediated by this receptor.

Several androgens, including DHT, have been shown to act intracellularly to competitively inhibit aromatase activity [35], which could be classified as a direct, specific, nongenomic, nonreceptor-mediated effect. Doolan et al. [36] have also demonstrated a specific, nongenomic, nonreceptor-mediated steroid action on protein kinase C activity [36]. However, in the current study, DHT-BSA, which is unable to enter the cell, causes an inhibition of E2 production similar to unconjugated DHT. These results indicate that androgens can decrease E2 production through a different nongenomic mechanism other than competitive enzyme inhibition and further suggest that this mechanism is mediated through a binding site located on the cell surface. Several other studies have used androgen conjugates to demonstrate cell surface-mediated effects of androgens, including the stimulatory effects of androgens on prolactin secretion by rat lactotrophs [25], the increase in intracellular Ca2+ levels in mouse T cells caused by exposure to testosterone [37, 38], androgen-regulated alterations of second messengers in rat osteoblasts [39], and androgen-induced increases in cytosolic Ca2+ in rat Sertoli cells [28, 29].

Falkenstein et al. [31] further subclassified direct, specific, nongenomic steroid actions into those mediated by steroid nuclear receptors and those involving other, nonclassical steroid receptors. Similar to the methods of the current experiments, several studies have used nuclear receptor antagonists (antiandrogens) as tools to determine which type of receptor mediates the nongenomic actions of androgens. For example, the antiandrogen hydroxyflutamide inhibits the androgen-induced, rapid increase in cytosolic Ca2+ in rat Sertoli cells, suggesting that the nuclear receptor acting at the cell surface may have a role in this nongenomic androgen action [29]. However, the results of the current study show that the antiandrogens cyproterone acetate and p,p'-DDE do not affect the androgenic inhibition of in vitro gonadotropin-stimulated estradiol production despite the previous identification of a nuclear androgen receptor (AR2) in the croaker ovary [18]. These results, along with other studies showing that antiandrogens do not inhibit rapid, nongenomic androgen actions [16, 37–40], further support the existence of nonclassical androgen receptors. However, it should be noted that although cyproterone acetate and p,p'-DDE are weak competitors for AR2 [19], the ability of these synthetic compounds to act as antiandrogens has not been established in croaker.

In addition to the experiments with antiandrogens, other results from the current study indicate that this nongenomic androgen action is mediated through an unconventional receptor. Previous characterization of AR2 shows that the half-time of androgen association is 44 min [18], whereas the current results show that significant inhibition of estradiol production occurs after only 5 min of androgen exposure prior to treatment removal and a 9-h postincubation in plain media. Since only a small percentage of androgen would bind to AR2 in 5 min, it is unlikely that the AR2 protein mediates these effects.

Since the results suggested the existence of a plasma membrane receptor for androgens in the croaker ovary, preliminary binding experiments were performed on an isolated membrane fraction. Those results indicate the presence of a high-affinity, saturable androgen binding moiety that is a possible mediator of the current nongenomic androgen action in the ovary. Other studies have identified membrane androgen binding sites in synaptosomes in the rat [41, 42], the olfactory tissue from brown and rainbow trout [43], rat testis [44], the liver and prostate cells from male rats [45], and mouse splenic T cells and a macrophage cell line [38, 46]. Further experiments are required to characterize fully the biochemical nature of this membrane androgen binding site in the croaker ovary.

The observation that androgens inhibit estradiol production in croaker ovaries in the presence of progesterone or 17-OH-progesterone suggests that at least one site of androgen action is distal to the formation of 17-OH-progesterone along the steroidogenic pathway to E2 synthesis. Possible targets of androgen regulation include the amount and/or activity of C17-20 lyase or aromatase. It has been shown that androgen treatment inhibits synthesis of P45017{alpha} in cultured mouse Leydig cells [47] and aromatase activity in large follicles isolated from primate granulosa cells [9], although androgen inhibition of aromatase was not observed in other tissues [2, 12]. In addition to defining possible enzyme targets of androgen action on croaker ovarian steroidogenesis, the experiment with the steroid precursors suggests that the effect of androgens on croaker ovarian estrogen production is present not only in gonadotropin-stimulated tissues but also when enzyme substrate is increased. Similarly, Lee and Bahr [7] showed that androgens regulate steroidogenesis in the absence of gonadotropin when precursors are added.

The physiological significance of the nongenomic androgen action identified in croaker is uncertain but may be one of the mechanisms by which steroid hormones autoregulate steroidogenesis during the reproductive cycle to control gametogenesis. Androgens have also been shown to act through nuclear receptors to alter steroidogenesis [10–13, 47]. Taken together, these results indicate the possibility of an integrated mechanism of androgen regulation of steroidogenesis involving both genomic and nongenomic mechanisms as suggested in a previous study of androgen-regulated steroidogenesis in female rat ovarian cells [17].

In conclusion, this study shows that treatment with physiological concentrations of androgens causes inhibition of ovarian E2 synthesis in response to gonadotropin stimulation. Moreover, this androgen action is specific, relatively rapid, nongenomic, and mediated at the cell surface, which are characteristics of nonclassical steroid actions mediated by steroid membrane receptors. Although the precise physiological significance of androgenic inhibition of E2 synthesis is not understood, this novel autocrine mechanism for regulating the ovarian production of estrogens with their immediate androgen precursors represents an additional level of control of steroid hormone synthesis that is likely to be of physiological importance during the reproductive cycle of female Atlantic croaker.


   ACKNOWLEDGMENTS
 
The authors wish to thank Upjohn Laboratories for the donation of Mibolerone. We also thank A. Katrina Loomis and Abby Benninghoff for assistance with the tissue incubations and Susan Lawson for animal care.


   FOOTNOTES
 
1 This work was supported by an E.J. Lund Fellowship to A.B. from the University of Texas Marine Science Institute and by Environmental Protection Agency STAR Grants R826126 and R82902401 to P.T. Back

2 Correspondence: Alyssa M. Braun, Department of Biological Sciences, University of Nevada, Las Vegas, 4505 Maryland Parkway, Las Vegas, NV 89154-4004. alyssa.braun{at}ccmail.nevada.edu Back

Received: 13 January 2003.

First decision: 4 February 2003.

Accepted: 1 July 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Biochemical Characterization of a Membrane Androgen Receptor in the Ovary of the Atlantic Croaker (Micropogonias undulatus)
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