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Biology of Reproduction 66, 190-198 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Putative Activation of the Peroxisome Proliferator-Activated Receptor {gamma} Impairs Androgen and Enhances Progesterone Biosynthesis in Primary Cultures of Porcine Theca Cells1

Pamela D. Schoppeea, James C. Garmeya, and Johannes D. Veldhuis2,a

a Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Medical School, Charlottesville, Virginia 22908


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ovarian theca cells are the predominant source of gonadotropin-stimulated androgen biosynthesis in vivo. Troglitazone (TG), a synthetic agonist of the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) and a thiazolidinedione used to treat insulin resistance, decreases serum androgen concentrations in women with hyperthecosis and/or polycystic ovary syndrome. Using reverse transcription-polymerase chain reaction (RT-PCR), we demonstrated the presence of PPAR{gamma} mRNA in the porcine ovary. Since activation of ovarian PPAR{gamma} may alter hormone-stimulated steroidogenesis in vitro, we cultured porcine theca cells for 48 h in the presence of two different PPAR{gamma} ligands, TG and 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2). Putative TG-mediated activation of PPAR{gamma} resulted in a 53%–69% decrease in LH- and/or insulin-stimulated androstenedione and testosterone accumulation. Although TG reduced 3-isobutylmethylxanthine-enhanced LH-stimulated cAMP accumulation by 74%–78%, it did not alter basal cAMP concentrations. Exposure to 8Br-cAMP did not overcome the TG-induced inhibition of androgen accumulation. In contrast, TG administration amplified basal and hormone-stimulated progesterone accumulation, particularly in the presence of insulin, without altering levels of 17{alpha}-hydroxyprogesterone. The putative natural PPAR{gamma} ligand, 15d-PGJ2, inhibited androgen biosynthesis and stimulated progesterone production. RT-PCR-based amplification of cytochrome P450 cholesterol side-chain cleavage (CYP11A) and cytochrome P450 17{alpha}-hydroxylase/C-17,20-lyase (CYP17) transcripts indicated that TG moderately enhanced expression of these genes. However, TG did not affect CYP17 protein expression. We conclude that putative ligand-mediated activation of PPAR{gamma} decreases LH- and/or insulin-driven theca cell androgen production by impairing the ability of CYP17 to synthesize androstenedione from available progestins. The corresponding augmentation of progesterone production could suggest that PPAR{gamma} activation induces theca cell differentiation toward a progestin-synthesizing phenotype.

gene regulation, mechanisms of hormone action, ovary, steroid hormones, theca cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polycystic ovary syndrome (PCOS), which is characterized by amenorrhea or oligomenorrhea, hirsutism, acne, and variable subfertility, is the most common endocrine disorder of premenopausal women [1]. These clinical symptoms and signs are associated with ovarian hyperandrogenism, which is marked biochemically by elevated secretion of androstenedione, testosterone, and/or dihydrotestosterone and a reciprocal decrease in sex steroid-binding globulin concentrations [1]. The most common corresponding ovarian phenotype is the presence of multiple nonestrogenic developing follicles (<=6 mm) and the failure of emergence of a dominant estrogenic follicle [1, 2]. Many patients with anovulatory hyperandrogenism exhibit hyperinsulinism and measurable insulin resistance in tissues such as muscle, liver, and fat [3]. In vitro studies of theca cells from women with PCOS show that both LH and insulin can potently enhance androgen biosynthesis [4]. Several lines of evidence further suggest that LH and insulin jointly contribute to dysregulation of cytochrome P450 17{alpha}-hydroxylase/C-17,20-lyase (CYP17), the rate-limiting enzyme in androgen biosynthesis [5–7].

CYP17 encodes a single enzyme that catalyzes both 17{alpha}-hydroxylase and C17,20-lyase activities. The first reaction is constitutive in the presence of adequate NADPH:cytochrome P450 (c) oxidoreductase (P450OR) [8], whereas the second is dependent on posttranslational protein modification and the presence of P450OR and cytochrome b5 [8–10]. Luteinizing hormone hypersecretion in women with PCOS may contribute to enhanced androgen biosynthesis through cAMP-mediated phosphorylation and subsequent stimulation of CYP17 lyase activity and possibly also via phosphorylation of the insulin receptor [6, 9].

Thiazolidinedione compounds, including ciglitazone, pioglitazone, and troglitazone (TG), effectively reduce insulin resistance in non-insulin-dependent diabetes mellitus. These novel agents activate the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) [11], a transcription factor that stimulates expression of glucose transporters, GLUT1 and GLUT4, and reduces expression of the ob (leptin), tumor necrosis factor {alpha}, and hepatic glucokinase genes [12]. One member of this class of insulin-sensitizing drugs, TG, has been used to treat women with PCOS. Patients treated with 400 mg/day TG orally for 3 mo exhibit a fall in serum androstenedione, total and biologically available testosterone, dehydroepiandrosterone, 17{alpha}-hydroxyprogesterone, estradiol-17ß, estrone, and LH concentrations. Sex steroid-binding globulin levels, insulin sensitivity, and glucose use rise concomitantly [13–15]. The rates of ovulation and conception are also improved [13, 15, 16].

Recent reports indicate that both TG and 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2), a putative endogenous ligand for PPAR{gamma} [17], inhibit progesterone synthesis in cultured porcine and human granulosa cells [18–20] and inhibit cytochrome P450 aromatase activity in cultured human granulosa and breast adipose stromal cells [21, 22]. We hypothesized that TG may decrease circulating androgen concentrations in women with PCOS by inhibiting theca cell steroidogenesis. The population of growing follicles in prepubertal porcine ovaries are a good model for testing this hypothesis because they are nonestrogenic, they are ultimately destined for atresia, and their theca cells constitutively synthesize androgens even after the follicle itself has become atretic. Accordingly, the current study investigated the presence of PPAR{gamma} transcripts in the pig ovary, the impact of TG and 15d-PGJ2 on LH- and insulin-stimulated androgen biosynthesis, and the expression of cytochrome P450 cholesterol side-chain cleavage (CYP11A) and CYP17 in primary cultures of porcine theca cells.


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

Ovine LH (NIDDK-oLH-26) was obtained from the National Hormone and Pituitary Program, National Institutes of Health (Bethesda, MD); porcine insulin, 8Br-cAMP, collagenase type IV, DNase I, 3-isobutyl-1-methylxanthine (IBMX), and androstenedione were purchased from Sigma Chemical Co. (St. Louis, MO); Dulbecco modified Eagle medium:Ham F-12 medium, and penicillin/streptomycin were obtained from Life Technologies (Grand Island, NY); and dimethyl sulfoxide (DMSO) was obtained from Mallinckrodt Baker Inc. (Paris, KY). Troglitazone was donated by Dr. Toshihiko Hashimoto of Sanykyo Research Laboratories Ltd. (Tokyo, Japan). The 15d-PGJ2 was purchased as a solution in methyl acetate from Cayman Chemical (Ann Arbor, MI); the methyl acetate was evaporated under a stream of nitrogen and replaced with DMSO. Luteinizing hormone and insulin were reconstituted in sterile distilled water containing 0.1% BSA fraction V (Sigma Chemical Co.) or 0.01 N HCl, respectively; 8Br-cAMP was reconstituted in sterile distilled water. Troglitazone and IBMX were dissolved in DMSO. Androstenedione was dissolved in 100% ethanol and then diluted in culture media containing 0.1% BSA.

Theca Cell Preparation

Ovaries from prepubertal pigs (ovaries had follicles measuring <6 mm in diameter and lacked evidence of ovulation) were collected at a local abattoir, placed in cold saline, and transported to the laboratory within 4 h. The combined theca and membrana granulosa layers from follicles measuring 2–5 mm were separated from the ovary with forceps, placed in serum-free culture media, and washed three times (mixed vigorously with a glass rod for 3 min each time) to separate granulosa cells from the basement membrane. The membranes were then separated from the remaining loose cells by passing fresh medium through a filter containing the follicle walls and a nylon membrane (177 µm pores). The cellular complexes were then placed in medium containing 1 mg/ml collagenase and 10 µg/ml DNase to digest the extracellular matrix [23]. After the tissue was digested to a uniform consistency, the cells were filtered again to remove undigested tissue. The remaining cells were pelleted at 1000 rpm for 5 min. The pellet was washed in warm media (37°C) twice, resuspended in 2 ml media, and semipurified over a Percoll centrifugation gradient, as described by Magoffin and Erickson [24]. The Percoll layer containing theca cells was removed, pelleted, washed thrice, and then counted in a hemocytometer.

Culture Conditions

Theca cells were cultured in serum-free Dulbecco modified Eagle medium:Ham F-12 medium (1:1) containing L-glutamine, sodium bicarbonate (2.438 g/L), pyridoxine hydrochloride, and added antibiotics (99 U penicillin and 99 µg streptomycin per milliliter of medium). Theca cells were plated in 96-well (50 µl/well containing 105 cells) or 24-well (200 µl/well containing 106 cells; Western blot experiments) tissue culture plates (Falcon Multiwell, Becton Dickinson, Lincoln Park, NJ) containing the treatment medium (200 or 800 µl, respectively), as specified. The cells were incubated at 37°C in a humidified 5% CO2, 95% air environment for 48 h.

Treatments

Theca cells were cultured in serum-free medium without hormones (control) or in medium containing LH and/or insulin (100 ng/ml each). These hormone concentrations induce maximal theca cell androstenedione synthesis under the present culture conditions [25]. Follicle-stimulating hormone (100 ng/ml) had no effect on either progesterone or androstenedione levels (unpublished results). Theca cells were also cultured with TG, 15d-PGJ2, or vehicle (DMSO). To evaluate any effects of DMSO, control cells were exposed to medium alone or medium containing DMSO (0.6%) in 10 independent cultures. DMSO had no effect (P > 0.60) on theca cell progesterone or androstenedione synthesis (data not shown). Thus, thereafter, all control cells were cultured with DMSO.

Troglitazone was used as the primary PPAR{gamma} ligand in these studies. Initially, a dose-response experiment was conducted with 0.8–8 µg/ml (1.8–18.1 µM) to determine concentrations of TG affecting theca cell steroidogenesis without impairing cell viability. To test the effect of TG (2.4 µg/ml) on cAMP accumulation, theca cells were cultured in the presence or absence of the phosphodiesterase inhibitor IBMX (200 µM) for 48 h. To bypass LH receptor-stimulated adenylate cyclase, we also assessed the effect of TG (2.4 µg/ml) on steroidogenesis stimulated by 8Br-cAMP (800 µM) ± insulin. To confirm that the effects of TG on androgen and progesterone synthesis were due to PPAR{gamma} receptor activation, theca cells were cultured with 0.08–0.8 µg/ml 15d-PGJ2 (0.25–2.5 µM). These doses were chosen based on the reported effects of 15d-PGJ2 in cultured bovine luteal cells [26].

The effects of putative PPAR{gamma} receptor activation on CYP11A and CYP17 mRNA accumulation were evaluated semiquantitatively via homologous reverse transcription-polymerase chain reaction (RT-PCR). Experiments were conducted with paired 96-well culture plates containing relevant effectors. Cells in one culture plate were used to assess steroidogenesis and DNA content, and cells in the second plate were used for the collection of total RNA.

The effect of PPAR{gamma} receptor activation on immunoreactive CYP17 protein content was assessed by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Total cellular protein was collected and pooled from six culture wells per treatment after adding 200 µl of Dulbecco phosphate-buffered saline (DPBS) and scraping the wells. The cells were pelleted via centrifugation at 2000 rpm in a 1.5-ml tube. After the DPBS was removed, the cells were immediately frozen at -20°C for 15 min, then 150–200 µl of hot Laemmli buffer (125 mM Tris, 0.1% SDS, 10% glycerol, and 0.2% ß-mercaptoethanol at 65°C, pH 6.8) was added to each tube. Protein concentrations were determined with the NanoOrange fluorescent dye assay.

Radioimmunoassays

Steroid accumulation was assessed by measuring medium steroid concentrations in samples stored at -20°C. Solid-phase radioimmunoassays (RIAs) were used to measure levels of progesterone and androstenedione (ICN Pharmaceuticals, Inc., Costa Mesa, CA) as well as levels of 17{alpha}-hydroxyprogesterone and testosterone (Diagnostic Systems Laboratories, Inc., Webster, TX). Accumulation of medium cAMP was determined with an automated Gammaflow assay after acidification with 0.1 N HCl [27].

NanoOrange Fluorescent Assay for Protein

A sensitive protein assay using NanoOrange fluorescent dye [28] was validated according to the manufacturer's protocol (Molecular Probes Inc., Eugene, OR). Two microliters of sample protein was diluted 1:10 in DPBS, then 2 µl was further diluted in 500 µl of assay dye diluent, heated to 95°C for 10 min, and cooled to room temperature for 20 min. The sample was then pipetted into 96-well tissue culture plates in duplicate (200 µl/well). Fluorescence was detected with a FluorImager 595 Optical Scanner (version 5.01, Molecular Dynamics, Sunnyvale, CA) and quantitated via densitometry by ImageQuant analysis (Molecular Dynamics).

PicoGreen Fluorescent Assay for DNA

A sensitive double-stranded DNA assay using PicoGreen fluorescent dye was validated according to the manufacturer's protocol (Molecular Probes Inc.). To quantitate total cellular DNA, the culture medium was replaced with 150 µl sterile, deionized water, and cultures were incubated at room temperature for 15–30 min and then frozen at -80°C until assayed [29]. After the cultures were thawed, 150 µl of 2x TE buffer (1x TE buffer: 20 mM Tris-HCl, 1 mM EDTA, pH 7.5) was added to each well and mixed. An aliquot of the cellular lysate (30–50 µl) was brought up to 100 µl in 1x TE buffer and assayed in duplicate in a separate 96-well tissue culture plate [30]. PicoGreen dye (100 µl diluted 1:200 in TE buffer) was added, and the incubation was continued for 2–5 min [31]. Fluorescence was detected with a FluorImager 595 Optical Scanner and quantitated via densitometry by ImageQuant analysis.

The 18S, CYP17, and CYP11A PCR products were also quantitated using the PicoGreen fluorescent dye method. An aliquot of each reaction was first diluted 1:10 in TE buffer to ensure that the DNA concentration would fall within the linear range of the standard curve. A negative control, consisting of representative cDNA from each culture replicate (n = 5) that was PCR-amplified without primers for the respective cycle number, was included in the PicoGreen assay. The fluorescence detected in these samples was treated as background and subtracted from the fluorescent value of the cDNA unknowns.

Reverse Transcription-Polymerase Chain Reaction

Total RNA was extracted from whole tissue or from cultured theca cells with TriReagent (Molecular Research Center, Inc., Cincinnati OH) according to the manufacturer's procedure. The optical density at 260 nm was used to quantitate total RNA. Synthesis of cDNA from 2.5 µg total RNA per sample was performed in individual 50-µl reactions containing 2.5 mM MgCl2; 50 mM KCl; 10 mM Tris-HCl (pH 8.3); 1 mM each of dTTP, dATP, dCTP, and dGTP (Roche Diagnostics, Indianapolis, IN); 50 U of RNase inhibitor (Perkin Elmer, Boston, MA); and 50 U of murine leukemia virus reverse transcriptase (Perkin Elmer). Reverse transcription of polyadenylated mRNA and 18S rRNA (used to correct for differences in RT of total RNA) was performed in one reaction per sample with 2.5 µM of oligo(dT)15 (Roche Diagnostics) and 200 nM of reverse primer specific for 18S (Table 1). AmpliWax PCR beads (Perkin Elmer) were used to form the moisture barrier. The reaction was performed at 42°C for 15 min and then terminated at 99°C for 5 min before being cooled to 4°C. The resultant cDNA was used in three subsequent PCR reactions per sample.


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  		TABLE 1. Primer pairs used for PCR amplification of porcine CYP11A, CYP17, and PPAR{gamma} mRNA and 18S rRNA

PCR amplification of CYP11A, CYP17, and 18S cDNA was performed in separate 100 µl reactions containing 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 200 µM of each dNTP, 1 µM of each primer (Table 1), 2.5 U of AmpliTaq Gold DNA polymerase (Perkin Elmer), and 5 µl of RT cDNA product with AmpliWax PCR beads as a moisture barrier. With a thermocycler (Thermolyne Amplitron I; Barnsted/Thermolyne, Dubuque, IA), all PCR reactions were heated to 95°C for 12 min to activate the DNA polymerase. The CYP11A and CYP17 cDNAs were amplified for 28 cycles with denaturation at 94°C for 60 sec and annealing/extension at 62°C for 2 min. After a final extension step at 62°C for 10 min, the reaction was rapidly cooled to 4°C [25]. The program used to amplify 18S cDNA consisted of 13 cycles of denaturation at 95°C for 35 sec, annealing at 62°C for 30 sec, and extension at 67°C for 90 sec. After a final extension step at 67°C for 10 min, the reaction was rapidly cooled to 4°C. Initially, PCR amplification with primers and no cDNA, cDNA without primers, and cDNA plus the forward or reverse primer only were tested as negative controls. The presence of specific PCR products was confirmed by visualization in a 1.25% agarose gel stained with ethidium bromide. The DNA was extracted from the agarose with the Wizard PCR DNA purification system (Promega, Madison, WI) and sequenced via an ABI Prism automated sequencer, model 377 (Biomolecular Research Facility, University of Virginia, Charlottesville, VA). Thereafter, PCR amplifications of representative cDNA (a pool generated from samples within each experiment) with or without primers were used as the positive and negative controls, respectively. The PCR products (double-stranded DNA) were quantified using the PicoGreen fluorescent dye assay described above.

RT-PCR was also performed on total RNA collected from whole ovary or pig testis and from semipurified theca and granulosa cells collected from preovulatory follicles to confirm the presence of PPAR{gamma} in the porcine ovary. Preovulatory follicles were chosen for theca and granulosa cell collection because treatment of theca cells with 100 ng/ml of LH for 48 h in vitro may induce biochemical changes analogous to those occurring during an LH surge in vivo. The primers used for PCR amplification are listed in Table 1. Synthesis of cDNA from 2.5 µg total RNA was performed as described previously with the following modifications. First, the RNA was heated to 65°C to relax the secondary structure, before the reaction was performed at 42°C for 45 min. Second, PPAR{gamma} cDNA (5 µl) was amplified by heating the reactions to 95°C for 8 min, amplification for 40 cycles with denaturation at 94°C for 60 sec, annealing at 62°C for 30 sec, extension at 72°C for 90 sec, and a final extension step at 72°C for 10 min. The presence of specific PCR products was confirmed by purification and sequencing.

Western Blot Analysis

Equal aliquots of total theca cell protein (40 µg) were separated by SDS-PAGE using 12.5% bisacrylamide gels and electrophoretically transferred to nitrocellulose (Hybond-ECL, Amersham Pharmacia Biotech, Inc., Piscataway, NJ) [32]. Membranes were blocked with Tris-buffered saline containing 0.1% Tween-20 and 5% skim milk (TBS-TM), washed, and incubated for 1 h at room temperature in TBS-TM containing primary antibody diluted 1:3000 (rabbit polyclonal antibody generated against CYP17 purified from porcine testis [33, 34]). The blots were washed again and incubated at room temperature for 1 h in TBS-TM containing secondary antibody diluted 1:10 000 (donkey anti-rabbit IgG, Amersham Life Science). Visualization of CYP17 was accomplished with the ECL chemiluminescence kit (Amersham Pharmacia Biotech, Inc.). The images were scanned with a Personal Densitometer SI Scanner (Molecular Dynamics) and quantitated via densitometry using ImageQuant analysis.

Statistics

Experiments were replicated at least four times each (except for those shown in Table 2) with separate batches of 50–100 ovaries. Theca cells were plated in triplicate cultures per treatment, and the media were pooled for steroid analysis (described previously). The DNA content in individual wells and the mean DNA per treatment were determined. Steroid accumulation was normalized to DNA content. Means represent data from four independent experiments unless otherwise noted. The data were log transformed for analysis via the general linear model procedure in SAS [35]. Means were separated post hoc using the Duncan multiple range test. The basic model tested the significance of hormones (control, LH, insulin, and LH plus insulin), PPAR{gamma} ligand (2–5 doses), and the hormones x PPAR{gamma} ligand interaction.


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  		TABLE 2. Testosterone accumulation in porcine theca cell cultures supplemented with androstenedione.a


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Presence of PPAR{gamma} mRNA in the Porcine Ovary

Figure 1A demonstrates that PPAR{gamma} mRNA is expressed in the porcine ovary. The PPAR{gamma}2 transcript was evident in theca and granulosa cells, but not in testis. The signal was stronger for the band containing a sequence common to both PPAR{gamma}1 and PPAR{gamma}2, suggesting that both isoforms are present. Both bands were isolated, purified, and sequenced. The resulting sequences (Fig. 1B) were homologous with published porcine PPAR{gamma} sequences [36].



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  		FIG. 1. Expression of specific PPAR{gamma} mRNAs in porcine ovarian tissue (A). RT-PCR-based amplification of PPAR{gamma} mRNA was performed as described in the Materials and Methods using total RNA from the whole ovary or testis and theca or granulosa cells (Gran) collected from preovulatory follicles. Forward primers (B) were specific to PPAR{gamma}2 (single underlined letters) or common to both PPAR{gamma}1 and PPAR{gamma}2 (double underlined letters). A single reverse primer (double underlined letters) was used. The two PCR products were synthesized in separate tubes. The first lane corresponds to PPAR{gamma}2 (256 bp) and the second to the common region of PPAR{gamma}1 and PPAR{gamma}2 (164 bp)

Dose-Dependent Effect of TG and 15d-PGJ2 on DNA Content and Steroid Accumulation

The data in Figure 2 represent the overall DNA content or steroid levels detected in cultures stimulated without hormones or with LH (100 ng/ml), insulin (100 ng/ml), or LH plus insulin for each dose of the respective PPAR{gamma} ligands. As an indirect assessment of theca cell viability, we determined theca cell DNA content after 48 h of culture using the PicoGreen fluorescent dye assay. Only the maximum concentration of TG or 15d-PGJ2 decreased the culture DNA content relative to control (P < 0.05; Fig. 2A). Exposure to >=2.4 µg/ml TG increased progesterone and decreased androstenedione accumulation relative to control (P <= 0.005; Fig. 2, B and C, respectively). However, at 8 µg/ml, TG decreased progesterone synthesis (P < 0.05). Based on these data, theca cells were exposed to 2.4 µg/ml TG (5.4 µM) in subsequent experiments. Administration of 15d-PGJ2 also increased progesterone accumulation (0.8 µg/ml; P < 0.05, Fig. 2B) and decreased androstenedione accumulation (at 0.4–0.8 µg/ml; P < 0.005, Fig. 2C).



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  		FIG. 2. Dose-dependent effects of troglitazone or 15-deoxy-{Delta}12,14-prostaglandin J2 on A) porcine theca cell DNA concentrations and on B) progesterone and C) androstenedione accumulation in serum-free cultures. The data represent the overall DNA content or steroid levels detected in cultures stimulated for 48 h. In B and C, the left Y-axis represents steroid accumulation in cultures exposed to troglitazone, and the right Y-axis represents steroid accumulation in cultures exposed to 15-deoxy-{Delta}12,14-prostaglandin J2. The data are presented as the mean ± SEM of 4 independent experiments for the respective PPAR{gamma} ligands. PPAR{gamma} activation increased progesterone and decreased androstenedione synthesis (P < 0.001). abcWithin each PPAR{gamma} ligand group, bars with unshared superscripts differ at P < 0.05

Effect of Hormone Stimulation and PPAR{gamma} Activation on Steroidogenesis

Figure 3 illustrates the individual effects of LH, insulin, or LH plus insulin on progesterone and androstenedione concentrations in the presence or absence of TG (2.4 µg/ml). Luteinizing hormone and insulin alone each stimulated (P < 0.0002) accumulation of progesterone (Fig. 3A, 3- to 4-fold); and androstenedione (Fig. 3B, 2- to 3-fold) relative to control. Luteinizing hormone plus insulin increased both progesterone (8-fold) and androstenedione (4-fold) production, but the effect was synergistic only for progesterone. Troglitazone enhanced progesterone synthesis (P < 0.001), particularly in the presence of insulin (11-fold) and LH plus insulin (27-fold relative to control; hormone x TG interaction, P < 0.02). In preliminary experiments, the effect of TG on theca cell pregnenolone accumulation was similar to that on progesterone (data not shown). In contrast, TG inhibited (P < 0.001) LH- and/or insulin-stimulated androstenedione accumulation by 53%–63% and basal androstenedione accumulation by 46% (P <= 0.01). When 15d-PGJ2 was used as the PPAR{gamma} ligand, the individual effects of LH ± insulin on progesterone and androstenedione levels were not different from those observed after exposure to TG (data not shown).



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  		FIG. 3. Impact of troglitazone (TG, 2.4 µg/ml) on A) progesterone, B) androstenedione, and C) 17{alpha}-hydroxyprogesterone accumulation in serum-free cultures of porcine theca cells incubated without hormones (control) or with LH (100 ng/ml), insulin (INS, 100 ng/ml), or LH plus insulin for 48 h. Data are presented as the mean ± SEM of 13 (A, B) or 7 (C) independent experiments. Hormonal stimulation increased the accumulation of all 3 steroids relative to control (P < 0.001) but had no affect on accumulation of 17{alpha}-hydroxyprogesterone (P > 0.65). Addition of TG enhanced progesterone and inhibited androstenedione production (P < 0.001). abcWithin TG concentration, means with unshared superscripts differ at P < 0.05

We evaluated media concentrations of 17{alpha}-hydroxyprogesterone (and cellular CYP17 mRNA accumulation; described subsequently) to determine whether TG inhibited androstenedione synthesis by altering its enzymatic activity or CYP17 gene expression. Figure 3C illustrates that TG had no effect on 17{alpha}-hydroxyprogesterone production (P > 0.27). However, LH or insulin alone stimulated 17{alpha}-hydroxyprogesterone accumulation by 2-fold (P < 0.0002) and together exerted a synergistic effect (6-fold).

The individual effects of TG (2.4 µg/ml) and 15d-PGJ2 (0.4 and 0.8 µg/ml) on testosterone accumulation are illustrated in Figure 4. Luteinizing hormone and LH plus insulin increased testosterone output (P < 0.001), albeit the latter effect was not synergistic. Troglitazone and 15d-PGJ2 each inhibited (P < 0.001) hormone-stimulated and basal testosterone production by 62%–69% and 32%, respectively (P <= 0.01). The TG-induced decrease in testosterone accumulation did not solely reflect a lack of available androstenedione substrate, inasmuch as TG still decreased testosterone synthesis by 36%–60% when theca cell cultures were supplemented with androstenedione (100 ng/ml; Table 2).



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  		FIG. 4. Effects of putative PPAR{gamma} activation with troglitazone (TG, 2.4 µg/ml; n = 8 experiments) or 15-deoxy-{Delta}12,14-prostaglandin J2 (n = 4) on testosterone accumulation in serum-free cultures of porcine theca cells incubated without hormones (control) or with LH (100 ng/ml), insulin (INS, 100 ng/ml), or LH plus insulin for 48 h. Data are presented as the mean ± SEM. Hormonal stimulation enhanced testosterone accumulation relative to control (P < 0.001). Both TG and 15d-PGJ2 impaired testosterone production (P < 0.001). abc Within each PPAR{gamma} ligand concentration, means with unshared superscripts differ at P < 0.05

Effect of PPAR{gamma} Activation on cAMP Accumulation

The effects of TG on cAMP accumulation are illustrated in Figure 5. In the absence of TG and IBMX, only exposure to LH plus insulin elevated cAMP accumulation relative to control (Fig. 5A, P < 0.04). IBMX enhanced basal cAMP levels (Fig. 5B, P < 0.0002) and mediated a 13-fold increase in LH- or LH plus insulin-stimulated cAMP accumulation (P < 0.001). Insulin alone had no effect. An effect of TG on cAMP accumulation was only observed in the presence of IBMX; LH- or LH plus insulin-stimulated cAMP accumulation was decreased by 74%–78% (P < 0.02). However, these cAMP concentrations did not approach the basal levels detected in the absence of IBMX. Troglitazone exerted no effect on cAMP accumulation in insulin-treated or control cells cultured with IBMX (Fig. 5B; hormone x TG interaction, P < 0.03).



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  		FIG. 5. Relative impact of troglitazone (TG, 2.4 µg/ml) on cAMP accumulation in cultures of porcine theca cells exposed to control solvent (control), LH (100 ng/ml), insulin (100 ng/ml), or both LH and insulin without (A) or with (B) IBMX (200 µM) for 48 h. Data are presented as the mean ± SEM of 4 independent experiments. In the absence of IBMX (A), LH plus insulin increased cAMP accumulation (P < 0.05; hormone x TG, P > 0.85). Cotreatment with IBMX and with LH ± insulin (B) enhanced this effect (P < 0.001). Insulin alone was inactive. An effect of TG on cAMP levels was observed only in the presence of IBMX. Accumulation of cAMP stimulated by LH and LH plus insulin decreased (P < 0.02), whereas no effect was exerted in insulin-treated or control cultures (hormone x TG interaction, P < 0.03). abWithin TG concentration, means with unshared superscripts differ at P < 0.05

Impact of PPAR{gamma} Activation on 8Br-cAMP-Stimulated Steroidogenesis

Theca cells were exposed to 8Br-cAMP ± insulin or LH ± insulin to determine whether the TG-induced inhibition of androgen accumulation could be overcome by direct stimulation of protein kinase A (PKA). The effects of LH or 8Br-cAMP did not differ (P > 0.55; data not shown), and exposure to 8Br-cAMP did not overcome the effects of TG (Table 3).


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  		TABLE 3. Effect of TG on theca cell androgen synthesis stimulated by 8Br-cAMP without or with insulin

Influence of PPAR{gamma} Activation on CYP11A and CYP17 Gene Expression

The results of semiquantitative RT-PCR amplification of CYP11A and CYP17 mRNA (expressed as a ratio to 18S rRNA) are summarized in Figure 6. Luteinizing hormone plus insulin increased CYP11A transcript levels by 1.5-fold (Fig. 6A, P < 0.001) and CYP17 mRNA by 2.5-fold (Fig. 6B, P < 0.001). Treatment with 8Br-cAMP ± insulin achieved the same effects. Insulin and 8Br-cAMP alone increased accumulation of CYP11A but not CYP17 mRNA. No effect of LH alone was detected at the end of 48 h of culture [25]. Troglitazone moderately increased overall accumulation of both CYP11A (cells cultured with TG at 0 µg/ml or 2.4 µg/ml: 5.4 ± 0.2 vs. 6.8 ± 0.3, respectively) and CYP17 (cells cultured with TG at 0 µg/ml or 2.4 µg/ml: 3.1 ± 0.3 vs. 4.1 ± 0.4, respectively) mRNA (P < 0.002) in basal and hormone-stimulated cultures.



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  		FIG. 6. Concentration of theca cell mRNAs encoding CYP11A (cytochrome P450 cholesterol side-chain cleavage, A) or CYP17 (cytochrome P450 17{alpha}-hydroxylase/C17,20-lyase, B) mRNA relative to 18S rRNA after stimulation with LH (100 ng/ml) or 8Br-cAMP (800 µM), insulin (INS, 100 ng/ml), LH + INS, 8Br-cAMP + INS, or no hormones (control) for 48 h. Data are presented as the mean ± SEM of 5 independent experiments. Insulin augmented LH- or 8Br-cAMP-stimulated CYP17 and CYP11A mRNA levels, but the effect was synergistic only for CYP17. Exposure to TG moderately increased gene expression regardless of the type (or lack of) hormone stimuli (P < 0.002). abWithin TG concentration, means with different superscripts differ at P < 0.05

Effect of TG on Immunoreactive CYP17 Protein

A representative Western blot of theca cell immunoreactive CYP17 is illustrated in Figure 7. In porcine testis, CYP17 protein migrates as three bands with molecular weights of 52, 46, and 45 kDa [34]. Although a 45- to 46-kDa band was occasionally detected in theca extracts, only the 52-kDa band of CYP17 was consistently evident. Total cellular protein from lung tissue and uncultured granulosa cells (collected via aspiration from follicles measuring 2–5 mm) was used as a negative control. As expected, a very faint band corresponding to CYP17 was visible in granulosa cell lysate because these cells were not purified over a Percoll gradient. Surprisingly, a band of similar low intensity was detected consistently in lung tissue lysate. No change in the amount of immunoreactive CYP17 protein was detected after 48 h of culture in the presence or absence of hormonal stimulation. Likewise, TG had no effect on the amount of 52-kDa CYP17 protein present in theca cells after 48 h of culture (P > 0.65).



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  		FIG. 7. A) Western blot of immunoreactive CYP17 in theca cells cultured for 48 h without hormones (C, control) or with LH (L, 100 ng/ml), insulin (I, 100 ng/ml), or LH plus insulin (LI) along with control solvent (DMSO, 0.6%) or troglitazone (TG, 2.4 µg/ml). Positive controls were porcine testis (Ts) and uncultured theca cells (Tc), and negative controls were lung (Lg) and uncultured granulosa cells (Gc). Two CYP17 bands (52 and 45–46 kDa) were detected in the testis and in uncultured theca cells. However, the 45- to 46-kDa band was not consistently visible in theca cells. B) The bar graph depicts the mean density (arbitrary units) of the 52-kDa band from 4 independent experiments. TG did not affect the amount of immunoreactive CYP17 (P > 0.65). Note that the Y-axis has been truncated to the mean background value (0.3) for all 4 blots


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present studies establish the presence of PPAR{gamma} gene transcripts in the porcine ovary and demonstrate for the first time that putative in vitro activation of PPAR{gamma} with structurally distinct synthetic (TG) or natural (15d-PGJ2) ligands inhibits LH- and/or insulin-stimulated theca cell androgen production. Evidence of an inhibitory action of these PPAR{gamma} ligands on theca cell androstenedione and testosterone production is consistent with the in vivo ability of similar serum concentrations of TG to reduce circulating androgens in women with PCOS [13–15]. In vivo, this PPAR{gamma} agonist might also modulate other biochemical reactions that could indirectly influence ovarian function. The current in vitro model documents that putative PPAR{gamma} activation can inhibit theca cell output of androstenedione via direct theca cell actions that are independent of any repression of CYP17 mRNA.

Ligand-mediated activation of PPAR{gamma} may modify theca cell steroidogenesis by inhibiting one or more distal effects of PKA on the catalytic activity of CYP17. Given that 17{alpha}-hydroxyprogesterone accumulation was not altered, we speculate that TG and 15d-PGJ2 may selectively inhibit lyase activity by impeding the PKA-dependent phosphorylation of CYP17. Although we did not directly determine the CYP17 state of phosphorylation here, this postulate is consistent with the failure of exogenous cAMP to antagonize TG-induced inhibition of androgen biosynthesis. It is conceivable that TG and 15d-PGJ2 impair the catalytic activity of PKA as initially suggested for ciglitazone in rat adipocytes [37]. This conjecture is less likely, given that TG enhanced the PKA-mediated stimulatory actions of LH on progestin accumulation.

Alternatively, the lack of change in 17{alpha}-hydroxyprogesterone accumulation despite increased accumulation of progesterone and pregnenolone substrate may indicate that PPAR{gamma} activation impairs the 17{alpha}-hydroxylase activity as well as the lyase activity of CYP17. An inhibitory effect of TG on the 17{alpha}-hydroxylase activity of CYP17 was recently demonstrated using a humanized yeast system and high concentrations of TG [38]. A reduction in the interaction between P450OR and CYP17 or a suppression of P450OR activity or cytosolic NADPH availability could compromise CYP17 hydroxylase and lyase activities. However, in some other systems, PPAR activation enhances P450OR mRNA accumulation [39, 40]. A limiting availability of reducing equivalents is plausible, because PPAR{gamma} activation blocked hormone-stimulated testosterone accumulation by more than 60% even in the presence of excessive androstenedione substrate. Since PPAR{gamma} activation plays a role in the regulation of the cell cycle [41–43] and the pentose phosphate pathway is the primary source of cytosolic NADPH [44], it is plausible that activation of PPAR{gamma} influences the net availability of NADPH through this pathway. Accordingly, the present findings provide a framework for testing several potential new mechanisms of PPAR{gamma} actions on theca cell steroidogenesis.

The PPAR{gamma} activator-induced shift from predominantly androgen to progestin biosynthesis in theca cells is analogous to the in vivo steroidogenic transition following an LH surge [45]. Ciglitazone and 15d-PGJ2 stimulate progesterone synthesis and inhibit cellular proliferation of LH-responsive (small) bovine luteal cells that express PPAR{gamma} [26]. Given the apparent similarity of PPAR{gamma} ligand effects on follicular cell steroidogenesis across species (unpublished results and [18, 19]), one could speculate that sustained activation of the PPAR{gamma} pathway influences the steroidogenic cytodifferentiation of theca cells toward a luteal phenotype. PPAR{gamma} regulation of cytodifferentiation has been inferred for numerous other cell types [17, 46–48]. Since PPAR{gamma} is only expressed in porcine theca cells isolated from preovulatory follicles exposed to an LH surge [49], LH itself may modulate activity of this regulatory pathway. The ability of PKA stimulants to influence PPAR activity in the mouse liver has recently been reported [50].

The present data indicate that activators of PPAR{gamma} enhance insulin-stimulated progesterone synthesis in theca cells. Werman et al. [51] reported that insulin stimulates ligand-independent transactivation by both PPAR{gamma} isoforms. In addition, thiazolidinediones can activate PPAR{gamma} independently of its phosphorylation state [52, 53]. Whether one or both of these interactions mediate the present effects of TG and 15d-PGJ2 or explicate the clinical efficacy of TG in reducing androgen production in women with PCOS is not known. However, the present in vitro model of PPAR{gamma} activation provides a means to study such signaling mechanisms in further detail.


   ACKNOWLEDGMENTS
 
Appreciation is expressed to Dr. G. Zhang for his design of the primers used for PCR of CYP11A and CYP17, to Valerie Long in the Ligand Assay and Analysis Core Laboratory in the NIH Center for Research in Reproduction for performing the RIAs, and to Dr. A. Payne for her donation of the CYP17 antibody.


   FOOTNOTES
 
First decision: 2 May 2001.

1 This research was supported in part by an NIH Training Grant 5T32 DK0764609 in Reproductive Neuroendocrinology (to P.D.S.), by NIH grants 16393 and HD 16806 (to J.D.V.), and by NICHD/NIH through cooperative agreement (U54 HD28934) as part of the Specialized Cooperative Centers Program in Reproduction Research. Back

2 Correspondence: Johannes D. Veldhuis, Endocrine Division, Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, VA 22908. FAX: 434 982 1911; jdv{at}virginia.edu Back

Accepted: August 28, 2001.

Received: April 9, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ehrmann DA, Barnes RB, Rosenfield RL. Polycystic ovary syndrome as a form of functional ovarian hyperandrogenism due to dysregulation of androgen secretion. Endocr Rev 1995; 16:322-353[Abstract/Free Full Text]
  2. Young RL, Goldzieher JW. Clinical manifestations of polycystic ovarian disease. Endocrinol Metab Clin North Am 1988; 17:621-635
  3. Dunaif A. Insulin resistance and the polycystic ovary syndrome: mechanism and implications for pathogenesis. Endocr Rev 1997; 18::774-800[Abstract/Free Full Text]
  4. Franks S, Gilling-Smith C, Watson H, Willis D. Insulin action in the normal and polycystic ovary. Endocrinol Metab Clin North Am 1999;; 28:361-378[CrossRef][Medline]
  5. Rosenfield RL, Barnes RB, Cara JF. Dysregulation of cytochrome P450c17{alpha} as the cause of polycystic ovarian syndrome. Fertil Steril 1990; 53:785-791[Medline]
  6. Qin K-N, Rosenfield RL. Role of cytochrome P450c17 in polycystic ovary syndrome. Mol Cell Endocrinol 1998; 145:111-121[CrossRef][Medline]
  7. Strauss III JF. Molecular mysteries of polycystic ovary syndrome. Mol Endocrinol 1999; 13:800-805[Free Full Text]
  8. Miller WL, Auchus RJ, Geller DH. The regulation of 17,20 lyase activity. Steroids 1997; 62:133-142[CrossRef][Medline]
  9. Zhang L-H, Rodriguez H, Ohno S, Miller WL. Serine phosphorylation of human P450c17 increases 17,20-lyase activity: implications for adrenarche and the polycystic ovary syndrome. Proc Natl Acad Sci U S A 1995; 92:10619-10623[Abstract/Free Full Text]
  10. Auchus RJ, Lee TC, Miller WL. Cytochrome b5 augments the 17,20-lyase activity of human P450c17 without direct electron transfer. J Biol Chem 1998; 273:3158-3165[Abstract/Free Full Text]
  11. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}). J Biol Chem 1995; 270:12953-12956[Abstract/Free Full Text]
  12. Henry RR. Thiazolidinediones. Endocrinol Metab Clin North Am 1997; 26:553-573[CrossRef][Medline]
  13. Dunaif A, Scott D, Finegood D, Quintana B, Whitcomb R. The insulin-sensitizing agent troglitazone improves metabolic and reproductive abnormalities in the polycystic ovary syndrome. J Clin Endocrinol Metab 1996; 81:3299-3306[Abstract]
  14. Ehrmann DA, Schneider DJ, Sobel BE, Cavaghan MK, Imperial J, Rosenfield RL, Polonsky KS. Troglitazone improves defects in insulin action, insulin secretion, ovarian steroidogenesis, and fibrinolysis in women with polycystic ovary syndrome. J Clin Endocrinol Metab 1997; 82:2108-2116[Abstract/Free Full Text]
  15. Hasegawa I, Murakawa H, Suzuki M, Yamamoto Y, Kurabayashi T, Tanaka K. Effect of troglitazone on endocrine and ovulatory performance in women with insulin-resistance-related polycystic ovary syndrome. Fertil Steril 1999; 71:323-327[CrossRef][Medline]
  16. Mitwally MFM, Kuscu NK, Yalcinkaya TM. High ovulatory rates with use of troglitazone in clomiphene-resistant women with polycystic ovary syndrome. Hum Reprod 1999; 14:2700-2703[Abstract/Free Full Text]
  17. Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC, Lehmann JM. A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor {gamma} and promotes adipocyte differentiation. Cell 1995; 83:813-819[CrossRef][Medline]
  18. Gasic S, Bodenburg Y, Nagamani M, Green A, Urban RJ. Troglitazone inhibits progesterone production in porcine granulosa cells. Endocrinology 1998; 139:4962-4966[Abstract/Free Full Text]
  19. Willis DS, White J, Brosens J, Franks S. Effect of 15-deoxy-delta (12,14)-prostaglandin J2 (PGJ2) A peroxisome proliferator activating receptor {gamma} (PPAR{gamma}) ligand on human ovarian steroidogenesis. In: Program of the 81st annual meeting of the Endocrine Society; 1999; San Diego, CA. Abstract P3-247
  20. Gasic S, Nagamani M, Green A, Urban RJ. Troglitazone is a competitive inhibitor of 3ß-hydroxysteroid dehydrogenase enzyme in the ovary. Am J Obstet Gynecol 2001; 184:575-579[CrossRef][Medline]
  21. Rubin GL, Kalus AM, Simpson ER. PPAR{gamma} ligands inhibit CYP19 gene expression in breast adipose tissue: potential therapy for breast cancer. In: Program of the 81st annual meeting of the Endocrine Society; 1999; San Diego, CA. Abstract OR28-6
  22. Mu, YM Yanase T, Nishi Y, Waseda N, Tanaka A, Takayanagi R, Nawata H. Insulin sensitizer, troglitazone, directly inhibits aromatase activity in human ovarian granulosa cells. Biochem Biophys Res Commun 2000; 217:710-713
  23. May JV, Bidge AJ, Gotcher ED, Gangrade BK. The regulation of porcine theca cell proliferation in vitro: synergistic actions of epidermal growth factor and platelet-derived growth factor. Endocrinology 1992; 131:689-697[Abstract/Free Full Text]
  24. Magoffin DA, Erickson GF. Purification of ovarian theca-interstitial cells by density gradient centrifugation. Endocrinology 1988; 122::2345-2347[Abstract/Free Full Text]
  25. Zhang G, Garmey JC, Veldhuis JD. Interactive stimulation by luteinizing hormone and insulin of the steroidogenic acute regulatory (StAR) protein and 17{alpha}-hydroxylase/17,20-lyase (CYP17) genes in porcine theca cells. Endocrinology 2000; 141:2735-2742[Abstract/Free Full Text]
  26. Löhrke J, Viergutz T, Shahi SK, Pöhland R, Wollenhaupt K, Goldammer T, Walzel H, Kantiz W. Detection and functional characterization of the transcription factor peroxisome proliferator-activated receptor {gamma} in lutein cells. J Endocrinol 1998; 159:429-439[Abstract]
  27. Brooker G, Terasaki WL, Price MG. Gammaflow: a completely automated radio-immunoassay system. Science 1976; 194:270-276[Free Full Text]
  28. Harvey MD, Bablekis V, Skinner CD. Utilization of the non-covalent fluorescent dye, NanoOrange, as a potential clinical diagnostic tool. Nanomolar human serum albumin quantitation. J Chromatogr B Biomed Appl 2001; 754:345-356[CrossRef]
  29. Rago R, Mitchen J, Wilding G. DNA fluorometric assay in 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water. Anal Chem 1990; 191:31-34
  30. Singer VL, Jones LJ, Yue ST, Haugland RP. Characterization of PicoGreen reagent and development of a fluorescent-based solution assay for double-strand DNA quantitation. Anal Biochem 1997; 249::228-238[CrossRef][Medline]
  31. Ahn SJ, Costa J, Emanuel JR. PicoGreen quantitation of DNA: effective evaluation of samples pre- or post-PCR. Nucleic Acids Res 1996;; 24:2623-2625[Free Full Text]
  32. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Short Protocols in Molecular Biology: A Compendium of Methods From Current Protocols in Molecular Biology. 4th ed. New York: John Wiley & Sons, Inc.; 1999
  33. Hales DB, Sha L, Payne AH. Testosterone inhibits cAMP-induced de novo synthesis of Leydig cell cytochrome P-45017{alpha} by an androgen receptor-mediated mechanism. J Biol Chem 1987; 262:11200-11206[Abstract/Free Full Text]
  34. Kuwada M, Takizawa N, Sone Y. The relation between two molecular species of P-450 in adult testis and 17{alpha}-hydroxylase and 17,20-lyase activities. Biochem Biophys Res Commun 1996; 218:524-529[CrossRef][Medline]
  35. Cody RP, Smith JK. Applied Statistics and the SAS Programming Language. 3rd ed. Englewood Cliffs, NJ: Prentice Hall Inc.; 1991
  36. Houseknecht KL, Bidwell CA, Portocarrero CP, Spurlock ME. Expression and cDNA cloning of porcine peroxisome proliferator receptor gamma (PPAR{gamma}). Gene 1998; 225:89-96[CrossRef][Medline]
  37. Kirsch DM, Bachmann W, Häring HU. Ciglitazone reverses cAMP-induced post-insulin receptor resistance in rat adipocytes in vitro. FEBS Lett 1984; 176:49-54[CrossRef][Medline]
  38. Arlt W, Auchus RJ, Miller WL. Thiazolidinediones but not metformin directly inhibit the steroidogenic enzymes p450c17 and 3ß-hydroxysteroid dehydrogenase. J Biol Chem 2001; 276:16767-16771[Abstract/Free Full Text]
  39. Prough RA, Johnson Webb S, Wu H-Q, Lapenson DP, Waxman DJ. Induction of microsomal and peroxisomal enzymes by dehydroepiandrosterone and its reduced metabolite in rats. Cancer Res 1994; 54::2878-2886[Abstract/Free Full Text]
  40. Waxman DJ. Role of metabolism in the activation of dehydroepiandrosterone as a peroxisome proliferator. J Endocrinol 1996; 150::S129-S147[CrossRef][Medline]
  41. Altiok S, Xu M, Spiegelman BM. PPAR{gamma} induces cell cycle withdrawal: inhibition of E2F/DP DNA-binding activity via down-regulation of PP2A. Genes Dev 1997; 11:1987-1998[Abstract/Free Full Text]
  42. Shao D, Lazar MA. Peroxisome proliferator activated receptor {gamma}, CCAAT/enhancer-binding protein {alpha} and cell cycle status regulate the commitment to adipocyte differentiation. J Biol Chem 1997; 272::21473-21478[Abstract/Free Full Text]
  43. Butler R, Mitchell SH, Tindall DJ, Young CY. Nonapoptotic cell death associated with S-phase arrest of prostate cancer cells via the peroxisome proliferator-activated receptor gamma ligand, 15-deoxy-{Delta}12,14-prostaglandin J2. Cell Growth Differ 2000; 11:49-61[Abstract/Free Full Text]
  44. Mathews CK, van Holde KE. Biochemistry. Redwood City, CA: The Benjamin/Cummings Publishing Company, Inc.; 1990
  45. Baird DT, Swanston IA, McNeilly AS. Relationship between LH, FSH and prolactin concentrations and the secretion of androgens and estrogens by the preovulatory follicle in the ewe. Biol Reprod 1981; 24::1013-1025[Abstract/Free Full Text]
  46. Mueller E, Sarraf P, Tontonz P, Evans RM, Martin KJ, Zhang M, Fletcher C, Singer S, Spiegelman BM. Terminal differentiation of human breast cancer through PPAR{gamma}. J Mol Cell 1998; 1:465-470
  47. Sarraf P, Mueller E, Jones D, King FJ, DeAngelo DJ, Partridge JB, Holden SA, Chen LO, Singer S, Fletcher C, Spiegelman BM. Differentiation and reversal of malignant changes in colon cancer through PPAR{gamma}. Nat Med 1998; 4:1046-1052[CrossRef][Medline]
  48. Johnson TE, Vogel R, Rutledge SJ, Rodan G, Schmidt A. Thiazolidinedione effects on glucocorticoid receptor-mediated gene transcription and differentiation in osteoblastic cells. Endocrinology 1999; 140::3245-3254[Abstract/Free Full Text]
  49. Schoppee PD, Guthrie DH, Veldhuis JD. Expression of peroxisome proliferator activated receptor {gamma} is associated with specific stages of follicular cell differentiation in porcine ovaries. Biol Reprod 2000; 62:: 195 (abstract)
  50. Lazennec G, Canaple L, Saugy D, Wahli W. Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators. Mol Endocrinol 2000; 14:1962-1975[Abstract/Free Full Text]
  51. Werman A, Hollenberg A, Solanes G, Bjorbaek C, Vidal-Puig AJ, Flier JS. Ligand-independent activation domain in the N-terminus of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}): differential activity of PPAR{gamma}1 and -2 isoforms and influence of insulin. J Biol Chem 1997; 32:20230-20235
  52. Hu E, Kim JB, Sarraf P, Spiegelman BM. Inhibition of adipogenesis through MAP kinase-mediated phosphorylation of PPAR{gamma}. Science 1996; 274:2100-2103[Abstract/Free Full Text]
  53. Camp HS, Tafuri SR. Regulation of peroxisome proliferator-activated receptor {gamma} activity by mitogen-activated protein kinase. J Biol Chem 1997; 272:10811-10816[Abstract/Free Full Text]



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