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BOR - Papers in Press, published online ahead of print April 26, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.047407
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BIOLOGY OF REPRODUCTION 75, 217–225 (2006)
DOI: 10.1095/biolreprod.105.047407
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Distinct Regulation by Steroids of Messenger RNAs for FSHR and CYP19A1 in Bovine Granulosa Cells

Wenxiang Luo , and Milo C. Wiltbank 1 

Endocrinology-Reproductive Physiology Program and Department of Dairy Science, University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT

Steroidalregulation of gene expression in follicular cells is not completely defined. Granulosa cells from 5 mm bovine follicles were cultured and treated and steady-state mRNA levels determined forFSHR(follicle-stimulating hormone receptor) andCYP19A1(aromatase). Cells were treated for 5 days with (0.1–300 ng/ml) 17beta-estradiol (E2), testosterone (T), or 5alpha-dihydrotestosterone (DHT).FSHRmRNA was increased by T and DHT but not E2. In contrast,CYP19A1mRNA was induced by all doses of E2 but only high doses of T and DHT. Similarly, varying treatment duration (1–5 days) showed thatFSHRwas increased by T and DHT andCYP19A1mRNA increased by E2 and T at all times. Synergism between steroid hormones and FSH or forskolin was also evaluated. FSH or E2 did not alterFSHRmRNA and did not enhance DHT stimulation ofFSHRmRNA. In contrast, DHT alone had no effect onCYP19A1mRNA but synergized with FSH plus E2 to increaseCYP19A1mRNA, probably due to induction ofFSHRby DHT. Effects of E2 and T onCYP19A1were blocked by ICI 182,780, indicating mediation by estrogen receptors. However, the specific androgen receptor antagonist bicalutamide did not block E2 or T effects onCYP19A1but did block T and DHT stimulation ofFSHR. Thus,FSHRis specifically regulated through androgen receptor, whereasCYP19A1is regulated by multiple pathways, including estrogen receptors and cAMP/protein kinase A induced by FSHR activation in granulosa cells. These inter- and intracellular regulatory mechanisms may be critical for normal follicle growth and dominant follicle selection.

androgen receptor, estradiol, estradiol receptor, follicle-stimulating hormone receptor, granulosa cells

INTRODUCTION

Ovarian follicular development is regulated by both systemically and locally produced regulators [1, 2]. The systemic roles of follicular steroids, particularly estrogens and gonadotropins, in regulating follicular development are well known [3, 4]. In addition, intrafollicular steroidal and nonsteroidal factors are known to mediate the trophic effects of gonadotropins and directly regulate some aspects of follicular development [2, 3, 5]. The important role of intrafollicular estrogen in regulating follicular development in rodents was highlighted by early studies showing that diethylstilbestrol treatment increased ovarian weight and number of follicles in superovulated rodents [6]. Nevertheless, key questions have not yet been resolved concerning the role of intrafollicular estrogen and androgen in regulating follicle growth in monovular species such as the human and cow [7].

The enzyme CYP19A1 is present in granulosa cells of healthy growing antral follicle, and this enzyme converts androgens to estrogens. In hypophysectomized rats, treatments with estrogen stimulated proliferation of granulosa cells in small preantral follicles [6]. Estrogen also facilitated differentiation of granulosa cells by increasing LHCGR (known as luteinizing hormone receptor) expression [8], increasing the number of gap junctions between granulosa cells [9], and inhibiting granulosa cell apoptosis [10]. Estrogens act via ESR1 (known as estrogen receptor {alpha}) and ESR2 (known as estrogen receptor ß), which act as transcription factors to regulate expression of specific genes and ultimately proteins and thereby alter cellular physiology in numerous tissues, including ovarian cells. Although ESR1 has been reported in granulosa cells of some species [11], there is prominent expression of ESR2 in granulosa cells of all species that have been examined [11]. In recent years, mice with ESR1 knockout (ERKO), ESR2 knockout, double ESR knockout ({alpha}ß ERKO), and CYP19A1 knockout (AromKO) have provided opportunities to further define the roles of estrogen in folliculogenesis. Knockout of either ESR1 or ESR2 altered but did not completely prevent follicle growth. However, double {alpha}ß ERKO mice showed dramatically impaired follicular growth with some ovarian Sertoli-like seminiferous tubules indicating a partial reversal of the female phenotype in the ovaries [3, 12, 13]. In AromKO mice, follicular development was initially arrested shortly before ovulation, but in 1-year-old AromKO mice, secondary and antral follicles were absent [13, 14]. In contrast, inhibition of follicular estrogen production in primates has not provided definitive evidence for a role for intrafollicular estrogens in follicular development [7]. In addition, the precise gene expression pathways that are regulated by estrogens in granulosa cells of monovular species have not yet been defined.

Classically, androgens have been implicated as atretogenic factors [15]. However, recent investigations demonstrate a stimulatory role for androgen in follicular development. Testosterone (T) and dihydrotestosterone (DHT) stimulated growth of cultured mouse follicles [16]. The number of follicles as well as thecal and granulosa cell proliferation increased in rhesus monkeys treated with T and DHT [17]. Androgens induced transcription of the mRNAs for IGF1 (know as insulin-like growth factor-I [IGF-I]) and IGF1R (known as IGF-I receptor) in ovarian follicles up to the antral stage in rhesus monkeys [18]. A recent study showed that knockout of the AR (known as androgen receptor) increased apoptosis of granulosa cells in preovulatory follicles and decreased the rate of ovulation and the number of corpora lutea generated [19]. Nevertheless, one recent study found that treatment of rhesus monkeys with DHT reduced ovarian estrogen production and prevented the increase in ovarian weight induced by FSH and LH treatments [20]. Thus, much, but not all, of the recent evidence points to a stimulatory role of androgens in follicular development; however, direct mediators of this stimulatory effect have not yet been elucidated. In addition, androgen could act by directly activating AR in granulosa cells and/or could be converted to estrogens in granulosa cells and subsequently signal through ESR1 and/or ESR2. In this study we designed experiments to investigate whether 17ß-estradiol, testosterone, and dihydrotestosterone act alone or interact with FSH in regulating granulosa cell expression of two gene transcripts that have been shown to be critical for follicular development, FSHR [21] and CYP19A1 [13, 14].

MATERIALS AND METHODS

Culture of Bovine Granulosa Cells

Bovine ovaries from the slaughterhouse were stored on ice and quickly rinsed with 70% ethanol before dissecting follicles that were ~5 mm in diameter (as estimated by eye). No attempt was made to determine if 5-mm follicles were growing, stable, or atretic prior to dissection of granulosa cells. In cattle, emergence of a new follicular wave occurs with growth of a number of 3–4-mm follicles (average of 24 follicles during each wave) until selection of a single dominant follicle occurs at ~8.5 mm in diameter [22]. Thus, most 5-mm follicles would be expected to be after emergence of a new follicular wave, during FSH-dependent growth, but prior to selection of a single dominant follicle. To harvest the granulosa cells, follicles were bisected in medium 199 (M199; Sigma, St. Louis, MO) and granulosa cells scraped from the internal surface of the follicular wall with a sterile rubber policeman. Cells were washed three times using low-speed centrifugation (1500 x g) followed by resuspension in M199, and the number and viability of granulosa cells were determined by trypan blue exclusion. In all experiments 2 x 105 cells/well were seeded into 24-well cell culture plates in M199 supplemented with 10% fetal bovine serum (FBS; not charcoal stripped). Granulosa cells were allowed to attach overnight and then washed and cultured in fresh M199 supplemented with 1% FBS and 10 ng insulin/ml plus any experimental hormonal treatments. Since the pH indicator dye, phenol red, has been reported to have weak estrogenic activity [23], granulosa cells were cultured in M199 with and without phenol red for comparative purposes in some of the experiments with estrogen treatments. Each treatment was done in triplicate wells in each experimental replicate, and all experiments were replicated on at least four occasions.

Isolation of mRNA

Messenger RNA was isolated from the cultured granulosa cells using Magnetight oligo (dT) magnetic beads (Novagen, Madison, WI). After treatments, the cells were lysed with 100 µl (per well) lysis buffer (4 M guanidine isothiocyanate, 100 mM Tris [pH 8.0], 1% DTT, and 0.5% N-lauroylsarcosine) and neutralized with 200 µl binding buffer (100 mM Tris [pH 8.0], 400 mM NaCl, and 20 mM EDTA). Samples were centrifuged at 16 000 x g for 5 min at 4°C to pellet cellular debris. The supernatant of each sample was transferred to a tube containing 50 µl of oligo (dT) beads and allowed to hybridize for 10 min. Beads were then captured on a magnetic stand and washed three times with 300 µl wash buffer (10 mM Tris [pH 8.0], 150 mM NaCl, and 1 mM EDTA). The mRNA was eluted by heating 10 µl of elution buffer (2 mM EDTA) to 65°C for 5 min and the isolated mRNA was stored at –70°C.

Reverse Transcription and Real-Time PCR

Reverse transcription (RT) was performed in a 20-µl volume of RT master mix that contained 1x RT reaction buffer, 5 µM random hexamer primers, 200 µM dNTP, 40 U M-MLV reverse transcriptase (Promega, Madison, WI), and 2 µl of mRNA sample [24]. The RT reaction was carried out at 37°C for 1.5 h followed by heating to 95°C for 10 min in a programmable thermocycler (MJ research, Watertown, MA).

Steady-state concentrations of investigated mRNAs were quantified by real-time PCR using a GeneAmp 5700 Sequence Detection System (PE Biosystems, Foster City, CA) with PCR products detected with SYBR Green I (Molecular Probes, Eugene, OR). Primers for amplification were designed using Primer Express (PE Biosystems). Each PCR reaction mix (25 µl) contained 1x PCR Buffer (Promega) with 1:20 000 dilution of SYBR Green I, 1.5 mM MgCl2, 200 µM dNTP, 250 nM forward primer, 250 nM reverse primer, 2 µl RT products, and 1.25 U Taq polymerase (Promega) [25]. Thermal cycling conditions were 95°C for 30 sec; followed by 40 cycles at 95°C for 30 sec, 57°C for 30 sec, and 72°C for 30 sec; and finally 72°C for 10 min. Melting curve analyses and agarose gel electrophoresis were performed after real-time PCR reactions to monitor PCR product purity. Primers for bovine FSHR were forward 5'-AGCCCCTTGTCACAACTCTATGTC-3' and reverse 5'-GTTCCTCACCGTGAGGTAGATGT-3'. Primers for bovine CYP19A1 were forward 5'-GTGTCCGAAGTTGTGCCTATT-3' and reverse 5'-GGAACCTGCAGTG-GGAAATGA-3'.

The threshold cycle (CT) numbers were determined for the amplified cDNA for each investigated mRNA and for the housekeeping gene, RPLPO (known as acidic ribosomal phosphoprotein [PO]), in each unknown sample during real-time PCR [26]. The relative quantification of gene expression across treatments was evaluated using the {Delta}{Delta}CT method [27]. The {Delta}CT is calculated as the difference between the CT of the investigated gene and the CT of PO in each sample. The {Delta}{Delta} CT of each investigated gene is calculated as the difference between the {Delta}CT in each treated sample and the {Delta}CT in each control sample. The fold change in relative mRNA concentrations for treated versus control samples was calculated using the formula 2–{Delta}{Delta}CT. The effects of treatments on the two mRNAs are shown graphically as fold change compared to mRNA concentration in control (untreated) wells and expressed as percentage change from control (2-fold increase = 200% of control) in the text.

Experimental Design

A total of six different cell culture experiments were designed to determine the intrafollicular roles of estrogen and androgen in regulating mRNAs for FSHR and CYP19A1 in cultured bovine granulosa cells. Estrogen actions were evaluated by treatment with 17ß-estradiol. Androgen actions were evaluated using T and the nonaromatizable androgen DHT. Testosterone was anticipated to act directly through AR and/or indirectly through the ESR pathway after being converted to estrogen by the actions of CYP19A1. A dose response for these compounds was performed in experiment 1. Subsequent experiments utilized the 30-ng/ml concentration based on the results of experiment 1, and 30 ng/ml approximates normal concentrations of E2 and T found in bovine follicular fluid [28]. The effect of duration of treatments was evaluated in experiment 2. In experiment 3, synergistic effects of FSH, E2, and DHT on expression of FSHR and CYP19A1 mRNAs were studied. Cells were treated with all combinations of FSH (0.3 ng/ml), E2 (30 ng/ml), and DHT (30 ng/ml) and each hormone alone for 3 days. The FSH dose of 0.3 ng/ml was chosen on the basis of recent studies showing this to be the peak circulating FSH concentration in cattle using a purified FSH preparation (Dr. A. F. Parlow, National Hormone and Peptide Program, Harbor-UCLA Medical Center) for a standard curve [29]. Experiment 4 also examined synergism between E2 (30 ng/ml) and DHT (30 ng/ml) combined with forskolin as a direct pharmacological stimulator of ADCY (known as adenylate cyclase) in regulating FSHR and CYP19A1. The design of this experiment was identical to experiment 3 except that forskolin was used in place of FSH in order to directly stimulate the cAMP-dependent protein kinase A (PKA) pathway. The final experiments determined the specificity of regulatory pathways using a specific estrogen receptor antagonist (ICI 182,780 [ICI]; Tocris, Ellisville, MO), androgen receptor antagonist (bicalutamide, a gift from AstraZeneca, Macclesfield, Cheshire, UK), or inhibitor of PKA (H89 [30]; Calbiochem, La Jolla, CA).

Statistical Analyses

Comparisons of mRNA concentrations between control and each treatment were performed using Student t-test. Paired t-test was used to compare mRNA levels between the group treated with hormone alone and each group treated with hormone plus antagonist. Unpaired t-test was used in comparing results from cultures in M199 with and without phenol red. A one-way ANOVA was used to analyze the differences among hormone-treated groups, followed by a Fisher least significant difference test. Each treatment was done in triplicate wells in each experimental replicate, and all experiments were replicated on at least four occasions. Data were averaged for all wells of a replicate, and statistical analyses were done using replicate as the experimental unit. Graphical data are plotted as the mean fold change in mRNA levels over control ± SEM and P < 0.05 was considered to be statistically significant.

RESULTS

The first experiment focused on the doses of E2, T, and DHT required to regulate expression of mRNA for FSHR and CYP19A1. The mRNA encoding for the FSHR was increased by both T and DHT at concentrations greater than 3 and 1 ng/ml, respectively, but was not significantly increased with any concentration of E2 tested (Fig. 1A). Further analyses with one-way ANOVA showed a dose response in the stimulatory effects of DHT and T on FSHR expression at concentrations greater than 3 ng/ml (Fig. 1A), and the peak response of FSHR expression occurred between 30 and 100 ng/ml of androgens.


Figure 1
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  		FIG. 1. Dose responses for 17ß-estradiol (E2), testosterone (T), and 5{alpha}-dihydro-testosterone (DHT) on the steady-state concentrations of mRNAs encoding FSHR (A) and CYP19A1 (B). Cell culture duration was 5 days, and values shown are mean ± SEM for four experiments. * P < 0.05 compared to control gene expression

In contrast to FSHR, the mRNA for CYP19A1 was increased by all concentrations of E2 that were evaluated (Fig. 1B; 0.1–300 ng/ml). However, T increased CYP19A1 mRNA only at concentrations greater than 3 ng/ml, and DHT was effective only at concentrations greater than at 10 ng/ml (Fig. 1B). Within the treatment groups, there were no differences in CYP19A1 mRNA concentrations among E2 treatment groups, but the fold increase in expression peaked at 30 ng/ml (Fig. 1B). Only the higher concentrations of T increased CYP19A1 expression, and this expression also peaked at 30 ng/ml (Fig. 1B). The stimulatory effect of DHT on CYP19A1 was greatest with 30 ng/ml DHT but was still significantly elevated with 10 ng/ml DHT (Fig. 1B).

The second experiment utilized the dose of steroids that was found to be maximally effective in experiment 1 (30 ng/ml) to evaluate the amount of time required for these steroidal effects on FSHR and CYP19A1 mRNA. There was an increase in FSHR mRNA in response to T and DHT at all the times that were tested, with maximal increases observed after 5 days (492% and 442%, respectively) (Fig. 2A). Only on the first day of culture did E2 slightly stimulate FSHR expression (150%; Fig. 2A).


Figure 2
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  		FIG. 2. Time course for expression of mRNAs for FSHR (A) and CYP19A1 (B) after treatments with 17ß-estradiol (E2), testosterone (T), and 5{alpha}-dihydrotestosterone (DHT) (all at 30 ng/ml). Values are mean ± SEM for four experiments. * P < 0.05 in comparison with control

In contrast to its effects on the expression of the FSHR mRNA, E2 increased CYP19A1 expression on all days tested. The magnitude of the effect did not differ between days ranging from 434% to 621% above control levels (Fig. 2B). Similar to the effects produced by E2, T also increased CYP19A1 expression at all days that were tested, and this expression level ranged from 350% to 711% above control (Fig. 2B). In contrast, DHT did not increase CYP19A1 mRNA on day 1 or day 3 but had a moderate stimulatory effect on day 5 (336%) (Fig. 2B). Thus, FSHR mRNA appeared to be primarily if not exclusively regulated by androgens, whereas CYP19A1 mRNA was regulated by estrogen or an androgen-like testosterone that could be aromatized to estrogen.

These previous experiments were done by treating with only a single steroid, and therefore our next experiment focused on how combinations of estrogen and androgen may interact with FSH to regulate gene expression. These previous experiments were also done in media containing phenol red to monitor pH; however, phenol red has been previously shown to have some estrogenic activity [23]. Thus, the third experiment had a technical objective (test the effect of phenol red in the culture media) and an important biological objective: to test the synergistic effects of E2, DHT (as a nonaromatizable androgen receptor agonist), and FSH on mRNAs for FSHR and CYP19A1. Removal of phenol red from M199 had slight quantitative changes (discussed later), but biological results are similar (for simplicity, listed values are for media with phenol red). Treatment with FSH, E2, or FSH + E2 did not change the level of FSHR mRNA. In contrast, any treatment that involved DHT, that is, DHT (alone), FSH + DHT, FSH + E2 + DHT, and E2 + DHT, showed an increase in FSHR mRNA ranging from 328% to 430% above control (P < 0.05) (Fig. 3A). There were no significant differences between the different treatments containing DHT on FSHR mRNA concentrations, but each of the DHT treatments was significantly different from any of the treatments that did not contain DHT (Fig. 3A).


Figure 3
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  		FIG. 3. Effects of combining 17ß-estradiol (E2) and 5{alpha}-dihydrotestosterone (DHT) with FSH on expression of mRNAs for FSHR (A) and CYP19A1 (B). Cells were cultured for 3 days in medium 199 with or without phenol red. Values are mean ± SEM for four experiments. * P < 0.05 in comparison to control in same medium; # P < 0.05 in comparison between cells cultured with and without phenol red

The mRNA for CYP19A1 (Fig. 3B) was increased by either E2 (519%) or FSH (166%) and was greatly increased when FSH and E2 were combined (1022%). At the concentration tested, DHT alone did not elevate CYP19A1 mRNA and did not significantly enhance FSH-stimulated CYP19A1 mRNA (Fig. 3B). However, DHT synergized with FSH and E2 to increase mRNA for CYP19A1 to 2373% above control (Fig. 3B). Comparison of the treatment groups showed that the greatest concentration of CYP19A1 was with the combination of FSH + E2 + DHT. The concentration of CYP19A1 mRNA was greater with FSH + E2 than for FSH, DHT, or FSH + DHT (Fig. 3B).

Use of ANOVA indicated a trend that when the DHT experiments did not have phenol red (black bars, Fig. 3A), the FSHR mRNA levels were higher when E2 was present than when it was not, that is, FSH + E2 + DHT and E2 + DHT greater than FSH + DHT or only DHT. In addition, FSH did produce a slightly greater increase in CYP19A1 expression in cells that were cultured in M199 without phenol red (Fig. 3B) compared to when it was present.

Based on the results of the third experiment, it was clear that there was synergism between FSH, estrogen, and androgen in regulation of CYP19A1 mRNA. Therefore, the next experiment (experiment 4) used forskolin in place of FSH to evaluate if bypassing the FSHR would still allow these synergistic effects to be manifest. Similar to experiment 3, treatments containing FSK, E2, and FSK + E2 did not increase FSHR mRNA, but any treatment containing DHT (i.e., DHT, FSK + DHT, FSK + E2 + DHT, and E2 + DHT) increased FSHR mRNA (Fig. 4A). Also similar to the results in experiment 3, CYP19A1 mRNA was increased by FSK (161%) and E2 (569%) alone, and E2 further increased FSK-stimulated CYP19A1 mRNA (1448%) (Fig. 4B). However, in contrast to experiment 3, DHT did not synergize with FSK and E2 to increase CYP19A1 mRNA, suggesting that this effect of DHT may be mediated by the increase in FSHR induced by DHT (Fig. 4B). Analysis within treatment groups showed that the concentrations of CYP19A1 in groups treated with FSK + E2, FSK + E2 + DHT, and E2 + DHT were not different (Fig. 4B).


Figure 4
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  		FIG. 4. Effects of combining 17ß-estradiol (E2) and 5{alpha}-dihydrotestosterone (DHT) with forskolin (FSK) on expression of mRNAs for FSHR (A) and CYP19A1 (B). Cells were cultured for 3 days in medium 199 with or without phenol red. F, FSK. Values are mean ± SEM for four experiments. * P < 0.05 in comparison with control cells cultured in same medium; # P < 0.05 in comparison between cells cultured with and without phenol red

Similar to experiment 3, the results from each treatment were not substantially different for cells cultured in M199 with or without phenol red. There was a slight increase in CYP19A1 mRNA for FSK + DHT-treated cells cultured in M199 without phenol red (Fig. 4B). In addition, E2 had slight effects in M199 without phenol red on FSHR (FSK + E2 slightly greater than control; FSK + E2 + DHT and E2 + DHT slightly greater than FSK + DHT and DHT).

To determine if steroidal and FSH actions were mediated by ESR and/or PKA, specific antagonists of ESR (ICI) or PKA (H89) were utilized in experiment 5. ICI was chosen on the basis of high specificity as an antiestrogen, extremely high affinity for ESR1 and ESR2 (100-fold greater than tamoxifen), and lack of agonistic activity (unlike tamoxifen, toremifene, droloxifene, idoxifene, and raloxifene). As expected, treatment with E2 increased CYP19A1 mRNA (838%) but not FSHR mRNA. Treatment with 1 µM ICI inhibited E2-induced CYP19A1 mRNA (70%) but did not alter basal concentration of CYP19A1 mRNA (Fig. 5A). ICI at doses greater than 1 µM inhibited both E2-induced and basal CYP19A1 (Fig. 5A). In addition, ICI slightly inhibited FSHR mRNA at 3 µM (basal only) and 10 µM (basal and with E2) (Fig. 5A). The second and third part of experiment 5 utilized much lower doses (nM) of ICI that were found to be effective during 1 day of treatment, and in addition these doses did not inhibit basal expression of mRNA for FSHR or CYP19A1. The effects of T on CYP19A1 were completely inhibited by ICI at 3 and 10 nM, whereas these doses produced no inhibition of the T-induced increase in FSHR (Fig. 5B). This result is consistent with the effects of T on CYP19A1 being mediated by conversion to estrogen and subsequent interaction with ESR. In Figure 5C are shown the results when both ICI and H89 were utilized to determine the involvement of ESR and PKA in the effects of FSH and T on FSHR and CYP19A1. Treatment with FSH + T increased both FSHR (200%) and CYP19A1 (520%) mRNAs. Treatment with H89 (5 µM) and ICI (1, 3, and 10 nM) inhibited the increase in CYP19A1 induced by FSH + T but not the basal concentration of this mRNA (Fig. 5C). The increase in FSHR mRNA induced by FSH + T was not changed by treatment with ICI (1, 3, and 10 nM) but was inhibited by H89 (Fig. 5C).


Figure 5
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  		FIG. 5. Effects on steady-state mRNAs concentrations for FSHR and CYP19A1 using the ESR (known as estrogen receptor) antagonist ICI 182,780 (ICI) at three different doses (In A with 1, 3, or 10 µM for 5 days; in B and C with 1, 3, or 10 nM for 1 day) and protein kinase A inhibitor (H89 at 5 µM for 1 day). E2, 17ß-Estradiol; F, FSH; T, testosterone. * P < 0.05 in comparison to control group for the same mRNA. # P < 0.05 in comparison to E2-induced (A), T-induced (B), or FSH + T-induced (C) concentrations of mRNA

The final experiment focused on evaluating the role of AR in steroidal regulation of FSHR and CYP19A1. In preliminary experiments, we utilized flutamide at a variety of different doses. At high doses (50 µM or more), flutamide was still not an effective inhibitor of DHT effects on FSHR mRNA but appeared to be toxic to cells as indicated by cellular morphology and decreases in basal concentrations of mRNA. Therefore, we obtained another AR antagonist, bicalutamide, from Zeneca Pharmaceutical Company. This compound has been shown to be a more effective antiandrogen with 4-fold greater affinity for the rat AR than hydroxyflutamide [31]. FSHR mRNA was increased by T (347%) or DHT (275%) but not by E2 (Fig. 6A). The CYP19A1 mRNA was increased by E2 (468%) or T (474%) treatment but not by DHT (Fig. 6B). Treatment with 10 µM bicalutamide significantly inhibited the T- and DHT-induced increase in FSHR mRNA but not the increases in CYP19A1 induced by E2 or T (Fig. 6, A and B). Bicalutamide did not alter basal expression of either FSHR or CYP19A1 (Fig. 6, A and B).


Figure 6
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  		FIG. 6. Effects on expression of mRNAs for FSHR (A) and CYP19A1 (B) using the AR (known as androgen receptor) antagonist bicalutamide (10 µM) during 3-day cultures. * P < 0.05 in comparison to control group for the same mRNA. # P < 0.05 in comparison between the treatment with steroid alone and the treatment with steroid plus bicalutamide. Bica, Bicalutamide; E2, 17ß-estradiol; DHT, 5{alpha}-dihydrotestosterone; T, testosterone

DISCUSSION

The intrafollicular roles of steroids in regulating follicular development have been postulated and directly tested in a number of previous studies. Nevertheless, this is the first study to provide evidence for a direct role of AR in stimulating FSHR expression in granulosa cells. Surprisingly, FSH, cAMP/protein kinase A, and estrogen-mediated pathways were not found to be involved in regulating expression of FSHR mRNA in bovine granulosa cells. The specific androgen-mediated regulation of FSHR provided a distinct contrast to the intriguing results on regulation of CYP19A1 in bovine granulosa cells. There was little direct effect of androgen on CYP19A1 but very dramatic and synergistic effects of FSH, acting through the cAMP/protein kinase A pathway, and E2, acting through ESR, on CYP19A1 mRNA in granulosa cells. There also appeared to be indirect, AR-mediated effects on CYP19A1, probably due to the androgen-stimulated increase in FSHR.

Based on these results, a physiological model (Fig. 7) is proposed that is an important extension of the two-cell/two-gonadotropin model of follicular development and previous models on the intrafollicular actions of androgens [32]. Androgen production by the thecal cells is stimulated by the action of LH. This theca-derived androgen acts as an intercellular regulator of granulosa cell function by directly stimulating FSHR expression. Androgen could also indirectly regulate granulosa cell function by being converted to estrogen that would act through ESR (probably ESR2) in concert with FSH-stimulated pathways to induce expression of CYP19A1. Clearly, the cAMP/PKA pathway is a critical part of the FSH-stimulated pathways; however, other pathways, such as p38 mitogen-activated protein kinase (MAPK), AKT1 (known as protein kinase B), FOXO1A, and direct interaction of APPL1 with the FSHR, have all been found to be involved in FSH signaling pathways [33, 34]. The increase in CYP19A1 could act as a type of positive feedback system to increase estrogen production and thus increase intrafollicular as well as systemic estrogen action.


Figure 7
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  		FIG. 7. Model depicting role of androgen in elevating FSHR gene expression in granulosa cells and in synergistically enhancing CYP19A1 expression in the presence of FSH and 17ß-estradiol (E2). Dotted lines indicate that stimulation may be mediated through indirect mechanisms and not directly by ER or AR. FSK, Forskolin; AR, androgen receptor; ER, ESR (known as estrogen receptor); AC, ADCY (known as adenylate cyclase); cAMP, cyclic adenosine monophosphate; PKA, protein kinase A; CREB-1, cAMP-responsive element binding protein 1; ICI, ICI 182,780; Bica, bicalutamide

An important part of this model is the novel finding of a direct action of androgen, acting through the AR, on expression of FSHR in granulosa cells. A number of studies using the mouse model have suggested that androgens can increase granulosa cell apoptosis [15] and have atretogenic effects on follicular development [10]. Nevertheless, knockout of AR reduced follicular development, resulting in fewer corpora lutea [19]. In addition, AR knockout mice had reduced FSHR mRNA in follicles after 2 days of stimulation with eCG [19]. These results are consistent with AR having a role in FSHR expression in granulosa cells, although direct effects were not convincingly demonstrated. Other evidence consistent with androgen regulation of FSHR is found in the results of a study utilizing in vivo treatment of monkeys with T [35]. Treatment with T increased FSHR mRNA in follicles of all sizes; however, it was not determined whether T was acting directly through the AR or by an action on the ESR after conversion of T to E2. Our results, combined with these previous studies, provide strong evidence that activated AR causes increased FSHR mRNA concentrations in granulosa cells, and this may be critical for normal follicular development. Nevertheless, future studies will be required to determine if AR directly acts on the FSHR promoter or through other transcription factors or mRNA stabilization mechanisms to increase FSHR mRNA.

These direct effects of androgens on granulosa cell function may have key functions in follicular development. In cultured follicles of mice, T and DHT initiated and increased responses of type 4 follicles to FSH, which resulted in further enhancing the androgen-induced increase in follicular diameter [36]. Murray et al. also demonstrated that DHT promoted growth of murine follicles at concentrations of FSH that are marginal for stimulating follicular development [16]. Granulosa cells isolated from small follicles from the marmoset monkey had greatly increased steroidogenic responses to FSH treatment when combined with androgen treatment, although steroidogenesis in granulosa cells from larger follicles was found to be inhibited by androgen [37]. Thus, normal follicular development at physiological FSH concentrations may depend on androgen-stimulated increases in FSHR. Nevertheless, there may also be negative effects of androgens on follicular development as evidenced by the previously mentioned study in granulosa cells from large follicles in the marmoset [37], previous studies in mouse [15], and a recent study in GnRH antagonist-treated rhesus monkeys [20]. In the study by Zeleznik et al. [20], DHT treatment was found to decrease estrogen secretion and the normal augmentation in ovarian weight that occurred in response to 15 days of i.v. infusion of FSH and LH.

Studies on the regulation of FSHR mRNA in granulosa cells by signal transduction pathways other than androgen have produced somewhat varied results. For example, we found no effect of E2, FSH, or pharmacological activation of cAMP/PKA by forskolin on concentrations of FSHR mRNA in granulosa cells. Consistent with our results, FSHB (known as FSH beta subunit) knockout female mice had decreased levels of mRNA for CYP19A1, INHA (known as inhibin alpha), and INHBA and B (known as inhibin or activin beta A and B) but no change in FSHR mRNA [38]. These mice had a well developed thecal layer, high androgen production, and high thecal mRNA for CYP17A1 (known as cytochrome P450 17{alpha}-hydroxylase) and LHCGR [38]. These data are consistent with our model that FSHR expression is not dependent on FSH and E2 but may primarily depend on thecal cell-derived androgen action. The lack of an effect of ICI on androgen-mediated stimulation of FSHR is also consistent with a lack of regulation of FSHR expression by ESR. However, removal of phenol red, which has been reported to have estrogenic effects in monkey granulosa cells [23], did appear to allow a slight but significant effect of E2 on FSHR mRNA, suggesting that there may be a role for basal levels of estrogen in FSHR expression. In addition, estrogen priming of hypophysectomized immature rats is well known to increase ovarian FSH binding in rats; however, this effect appears to be due primarily to estrogen-induced proliferation of granulosa cells with a fixed number of FSHR per cell [39]. Our lack of effect of FSH on FSHR mRNA is consistent with the lack of change in FSHR mRNA after knockout of FSHB production [38] but varied from other studies reporting stimulation by FSH and cAMP of FSHR expression in rat [40] and porcine [41] granulosa cells. In addition, we had a slight but significant inhibition of FSHR expression by H89 in granulosa cells stimulated with FSH plus testosterone. This may indicate basal activity of PKA may modulate transcriptional regulation of FSHR expression. Indeed the FSHR promoter is known to contain a NR5A1 (known as SF-1) binding site and an E-box [42], which can bind transcription factors that can be regulated by PKA, such as NR5A1, NR5A2 (known as LRH-1), USF1 (known as upstream stimulatory factor-1), and USF2. However, H89 targets the ATP binding site of PKA, and while it clearly inhibits PKA with high affinity, it also has very high affinity for a number of other kinases, including P70S6K [30], involved in signal transduction pathways for other key regulatory hormones, including IGF1. Previous studies have shown regulation of FSHR mRNA by activin, nerve growth factor, and IGF1 [43, 44]. Thus, the mechanism for H89 action on FSHR mRNA expression is not clear.

In contrast to the relatively specific regulation of FSHR by androgen, CYP19A1 mRNA was regulated by multiple pathways in a synergistic manner. Consistent with previous results, we found that FSH acting through the cAMP/PKA pathway had a rapid stimulatory effect on CYP19A1. In addition, the stimulatory effect of FSH plus testosterone on CYP19A1 mRNA was completely inhibited by ICI, indicating the critical role of ESR in regulation of CYP19A1 mRNA. Clearly, the effects of testosterone were not directly mediated by androgen as evidenced by the lack of similar effects of DHT on CYP19A1 and lack of inhibition of testosterone action by the specific AR antagonist, bicalutamide. This contrasts with the clear inhibitory effect of the same dose of bicalutamide on FSHR stimulation by testosterone or DHT. The inhibitory effect of H89 on stimulation of CYP19A1 by FSH plus testosterone may also indicate a critical role of cAMP in regulating CYP19A1 in granulosa cells or again may be due to effects of H89 on other kinases.

The clear synergism between FSH or forskolin with estradiol or testosterone on CYP19A1 expression is consistent with multiple pathways being required for optimal CYP19A1 expression. Nevertheless, previous studies using hypophysectomized, immature rats (either with or without diethylstilbestrol treatment) failed to find a stimulatory effect of estradiol on CYP19A1 activity in granulosa cells [45, 46]. However, it is well known that the physiological state of cells can dramatically influence the response of CYP19A1 to stimulation [45]. In primate granulosa cells, pretreatment with estradiol increased the CYP19A1 activity induced by FSH [23], consistent with our results with bovine granulosa cells. Although the mechanisms involved in estrogen stimulation of CYP19A1 mRNA are undefined, cAMP-stimulated CYP19A1 transcription is regulated by an interaction in the promoter region of CYP19A1 between a cAMP responsive element (CRE) and an NR5A1 site [47, 48]. The dramatic stimulation of CYP19A1 by estrogen or testosterone appears to be directly mediated by ESR, but it is unclear if this effect is mediated via cross talk with the cAMP-signaling cascade triggered by FSH, direct ESR effects on transcription, and/or effects of ESR on CYP19A1 mRNA degradation. An intriguing possibility is that E2 may stimulate expression of prostaglandin-endoperoxide synthase 2 and production of prostaglandins (PG), particularly PGE2, that may stimulate expression of CYP19A1. This mechanism appears to be important in regulation of CYP19A1 expression in breast cancer cells [49] and endometriosis [50]. In granulosa cells this positive feedback loop between estradiol and prostaglandin also seems plausible based on previous findings of PGE2 stimulation of CYP19A1 activity in granulosa cells [51]. However, other mechanisms may also be important because previous studies have found that FSH-responsive ADCY (also known as adenylate cyclase) was increased by treatment with estradiol alone [52] and that estrogen increased CREB (known as cAMP responsive element binding protein) phosphorylation to promote CRE-dependent gene transcription [53–55]. Further studies are needed to dissect the molecular events leading to the synergism between FSH/cAMP and ESR in regulating CYP19A1.

There was also a synergistic effect of DHT on FSH plus estrogen stimulation of CYP19A1 expression. This effect was lost when FSHR was bypassed by direct stimulation of ADCY by forskolin. Thus, it seems likely that the androgen-mediated synergism with FSH and estrogen are due to increased expression of FSHR. Nevertheless, the combination of forskolin + E2 only attained the levels of CYP19A1 expression reached during FSH + E2 treatment and not the extremely high levels reached during the synergistic actions of FSH + E2 + DHT. This suggests that the synergistic actions of DHT may lie in other pathways besides the FSH-stimulated cAMP pathway, such as other FSH-stimulated signal transduction pathways or DHT-induced pathways that are independent of FSHR. There were also some direct effects of DHT on CYP19A1 that were detectable only after prolonged exposure to androgen (5 days) at fairly high doses (≥10 ng/ml).

The expression of AR and ESR in granulosa cells is consistent with the steroidal effects that we observed. Granulosa cells are likely to be regulated by androgen from very early developmental stages because AR is initially expressed in granulosa cells of bovine follicles with only 1–1.5 layers of cuboidal granulosa cells, and this expression increases during the early stages of antral follicle development [56]. In primates, expression of AR protein [57] and mRNA [58] was found primarily in granulosa cells of healthy follicles, was negatively correlated to granulosa cell apoptosis, and was decreased in the preovulatory follicle and corpus luteum. These results were consistent with a protective or stimulatory role of AR in granulosa cell development, which could be explained by the direct action of androgen on FSHR expression. Expression of ESR2 mRNA is also very pronounced in granulosa cells of antral bovine follicles at various developmental stages [59]. In addition, bovine granulosa cells express ESR1, but the expression level is much lower than found in thecal cells [60]. These two ESR isoforms could act independently, form homodimers, or form heterodimers in order to mediate E2 actions in granulosa cells [3].

In conclusion, androgen from thecal cells appears to regulate granulosa cell function by directly inducing expression of the FSHR and by synergizing with FSH and estrogen to induce CYP19A1 (Fig. 7). Thus, there is an intriguing and complex interplay between direct actions of thecal androgens on gonadotropin receptor expression, production of estrogen from thecal androgen, and synergistic interaction of these steroids with other hormones and intracellular effector systems within the granulosa cells. These inter- and intracellular regulatory mechanisms are likely to be critical for normal follicle growth and dominant follicle selection and may be important in pathological follicular problems.

FOOTNOTES

1 Correspondence: Milo C. Wiltbank, 1675 Observatory Dr., Madison, WI 53706. FAX: 608 262 9412; e-mail:Wiltbank{at}wisc.edu Back

Received: 16 September 2005.

First decision: 13 October 2005.

Accepted: 20 April 2006.

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