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BOR - Papers in Press, published online ahead of print July 30, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.017244
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BIOLOGY OF REPRODUCTION 69, 1665–1674 (2003)
DOI: 10.1095/biolreprod.103.017244
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Expression and Functional Role of Peroxisome Proliferator-Activated Receptor-{gamma} in Ovarian Folliculogenesis in the Sheep1

Pascal Froment3, Stéphane Fabre3, Joëlle Dupont3, Claudine Pisselet3, Didier Chesneau3, Bart Staels4, and Philippe Monget2,3

Physiologie de la Reproduction et des Comportements,3 UMR 6073 INRA-CNRS-Université F. Rabelais de Tours, 37380 Nouzilly, France Département d'Athérosclerose,4 U.545 INSERM, Institut Pasteur de Lille, and Université de Lille II, 59006 Lille, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxisome proliferator-activated receptor (PPAR{gamma}) is a nuclear receptor that is activated by fatty acids and derivatives and the antidiabetic glitazones, which plays a role in the control of lipid and glucose homeostasis. In the present work, we tested the hypothesis that PPAR{gamma} plays a role in reproductive tissues by studying its expression and function in the hypothalamo-pituitary-ovary axis in the sheep. PPAR{gamma} 1 and PPAR{gamma} 2 proteins and mRNAs were detected in whole ovine pituitary and ovary but not in hypothalamic extracts. In situ hybridization on ovarian section localized PPAR{gamma} mRNA in the granulosa layer of follicles. Interestingly, PPAR{gamma} expression was higher in small antral (1–3 mm diameter) than in preovulatory follicles (>5 mm diameter) (P < 0.001) and was not correlated with healthy status. To assess the biological activity of ovarian PPAR{gamma}, ovine granulosa cells were transfected with a reporter construct driven by PPAR{gamma}-responsive elements. Addition of rosiglitazone, a PPAR{gamma} ligand, stimulated reporter gene expression, showing that endogenous PPAR{gamma} is functional in ovine granulosa cells in vitro. Moreover, rosiglitazone inhibited granulosa cell proliferation (P < 0.05) and increased the secretion of progesterone in vitro (P < 0.05). This stimulation effect was stronger in granulosa cells from small than from large follicles. In contrast, rosiglitazone had no effect on LH, FSH, prolactin and growth hormone secretion by ovine pituitary cells in vitro. Overall, these data suggest that PPAR{gamma} ligands might stimulate follicular differentiation in vivo likely through a direct action on granulosa cells rather than by modulating pituitary hormone secretion.

granulosa cells, ovary, pituitary, pituitary hormones, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanisms controlling the interaction between energy balance and reproduction are the subject of intensive investigations. For example, negative energy balance caused by inadequate nutrient supply or excessive consumption is able to affect the fertility of female mammals. In particular, excessive loss of body weight during lactation in cattle and swine accentuates the decrease in energy balance and is able to extend the interval from parturition or weaning to estrus, respectively (for review, [1, 2]). In contrast, in the sheep, short periods of improved nutrition before and during mating are able to increase the proportion of ewes bearing twins. In this species as well, a supplement of lupin grain (a legume high in digestible energy and protein), supplemental fat, or intravenous glucose infusion increases the number of growing ovarian follicles (for review, [1, 2]).

It has been suggested that polyunsaturated fatty acids (PUFAs) play a role in the regulation of reproduction by influencing energy homeostasis [3], these effects being possibly mediated by lipid- or glucose-sensing mechanisms. One of these sensors, peroxisome proliferator-activated receptor gamma (PPAR{gamma}), is a fatty acid receptor that belongs to the PPAR family. The PPAR family is composed of three members: PPAR{alpha}, PPARß ({delta}), and PPAR{gamma}, each being encoded by a specific gene (for review, [4, 5]). PPARs bind to specific responsive elements (PPREs) as a heterodimer with the retinoid X receptor (RXR). PPAR{gamma} ligands are fatty acids and derivatives such as 15deoxy-,12, 14-prostaglandin J2 (15d-PGJ2) [6] as well as the synthetic glitazones (rosiglitazone, troglitazone, and pioglitazone) [7]. Glitazones are insulin sensitizers acting via PPAR{gamma} to increase sensitivity of cells to the action of insulin. In particular, PPAR{gamma} has been shown to stimulate lipid metabolism as well as glucose and lipid transport in adipose tissue (for review, [8]). The glitazones are a recently developed class of drugs used to treat hyperglycemia associated with type 2 diabetes and, more generally, disorders associated with insulin resistance. Interestingly, insulin-sensitizing agents such as troglitazone [9] were also shown to ameliorate the ovulatory function of polycystic ovarian syndrome patients [10, 11]. Moreover, a recent study has shown that PPAR{gamma} is expressed in rodent granulosa cells and that PPAR{gamma} agonists modulate their steroidogenic capability [12, 13].

To assess a role of PPAR{gamma} in the regulation of reproductive axis in female, we decided to perform an overall study of the expression and the role of a PPAR{gamma} agonist in the different compartments of the hypothalamo-pituitary-ovarian axis in the ewe.

In particular, we have studied the expression and the functionality of endogenous PPAR{gamma} in granulosa and pituitary cells and the action of a PPAR{gamma} agonist on proliferation and steroidogenesis.

In particular, we have studied 1) the expression of PPAR{gamma} in the hypothalamo-pituitary-ovary axis by reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and immunoblot; 2) the functionality of endogenous PPAR{gamma} in granulosa and pituitary cells by transfection; and 3) the action of PPAR{gamma} agonist on proliferation and steroidogenesis of two different ovine granulosa cell populations in vitro from small (1–3 mm) and large (>5 mm) antral follicles. In addition, we tested the ability of PPAR{gamma} ligand to modulate the secretion of pituitary hormones, which control folliculogenesis, in particular FSH and LH, by ovine pituitary cells in vitro.


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

Fluorogestone acetate sponges used to synchronize estrous cycles and hCG used for injections to animals were obtained from Intervet (Angers, France). Porcine FSH (pFSH) from pituitary extract (pFSH activity = 1.15 times activity of NIH pFSH-P1) used for injections to animals was obtained from Dr. Y. Combarnous (Nouzilly, France). Purified ovine FSH-20 (oFSH) (lot no. AFP-7028D, 4453 IU/mg, FSH activity = 175 times activity of oFSH-S1) used for culture treatment was a gift from NIDDK, National Hormone Pituitary Program (Bethesda, MD). Recombinant human insulin-like growth factor-I (IGF-I) was a gift from Dr. P. Swift (Ciba-Geigy, Saint Aubin, Switzerland). Rosiglitazone and LG1069 were obtained from A. Bril (GSK, Rennes, France).

Animals

All procedures were approved by the Agricultural Agency and the Scientific Research Agency (approval number A 37801) and conducted in accordance with the guidelines for Care and Use of Agricultural Animals in Agricultural Research and Teaching. Sixty adult cyclic Romanov ewes were treated with intravaginal sponges impregnated with progestogen (fluorogestone acetate, 40 mg) for 15 days to mimic a luteal phase. Cells for culture were collected from animals in the luteal phase of the estrous cycle (10 days after sponge removal, plus intramuscular injections of 6 IU and 5 IU pFSH 24 h and 12 h prior to slaughter, respectively). The ovaries from five ewes were embedded in Tissue Tek (Miles Laboratories, Elkhart, IN), immediately frozen in liquid nitrogen, and stored at -80°C until in situ hybridization.

RNA Analysis

Total RNA was extracted from whole tissue (hypothalamus, pituitary, and white adipose tissue) or cultured granulosa cells using RNAble reagent according to the manufacturer's procedure (Eurobio, Les Ulis, France). RNA was quantified by measuring the absorbance at 260 nm. Samples were stored at -80°C until use.

Reverse transcription was performed for 1 h at 42°C in a total volume of 25 µl with 2 µg total RNA per sample, 1.5 mM deoxynucleotide triphosphate (dNTP), reverse transcriptase buffer 5x, 20 U of RNase inhibitor, 2 µM lower primer and 200 U of M-MLV reverse transcriptase. The resulting cDNA was subjected to PCR. PCR amplification of PPAR{gamma} cDNA was performed in 50 µl reaction containing 1 U of AmpliTaq polymerase, 2 mM MgCl2, 0.2 mM dNTP, 1 µM each primer, AmpliTaq polymerase buffer 10x, and 5 µl of RT cDNA product.

Amplification of PPAR{gamma} cDNA (472-bp fragment) for 30 cycles (denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min) by using upper primer: 5' CCA CCA ACT TTG GGA TCA G 3' and lower primer 5' TTC TGA AAC CGA CAG TAC TGA C 3'. PPAR{gamma}2 cDNA (301 bp) and the common region of PPAR{gamma} isoforms (164 bp) were amplified for 30 cycles (denaturation at 94°C for 1 min, annealing at 55°C for 1 min with PPAR{gamma}2 primers, at 60°C for 1 min with the primers of the common region of PPAR{gamma} and extension at 72°C for 1 min) by using PPAR{gamma}2 upper primer: 5' ATG GGT GAA ACT CTG GGA GAT 3' or the common region upper primer: 5' CTC CGT GGA CCT TTC TAT GAT 3' and the lower primer 5' CTT GGA GCT TCA GGT CAT ACT 3'.

The cDNA amplification was performed on a geneAmp PCR system 9700 (Perkin-Elmer, Norwalk, CT). After a final extension step at 72°C for 7 min, the reaction was rapidly cooled to 4°C. After migration in a 2% agarose gel stained with ethidium bromide, the DNA was extracted from the agarose using the gel extraction kit QIAEX II (Qiagen, Hilden, Germany) and sequenced in both directions using Dye terminator kit on ABI Prism automated sequencer, model 377 (Biomolecular Research Facility, University of Virginia, Charlottesville, VA). PCR amplifications with RNA were performed in parallel as negative controls. RT-PCR consumables were purchased from Sigma (l'Isle d'Abeau Chesnes, France), except Moloney murine leukemia virus reverse transcriptase and RNase inhibitor (RNasin) from Promega (Madison, WI).

The pGEMT-PPAR{gamma} antisense and sense constructs used for in situ hybridization were generated by inserting the fragment of PPAR{gamma} cDNA (472 bp) into pGEM-T vector (Promega) and selecting a clone with the appropriate antisense or sense orientation.

In Situ Hybridization

Frozen ovaries were serially sectioned at a thickness of 10 µm with a cryostat to perform in situ hybridization experiments using 35S-labeled ovine PPAR{gamma} cRNA as probe as previously described [14]. For prehybridization, sections were incubated for 2 h at 50°C in the following prehybridization buffer: 50% formamide (v/v) (Merck, Nogent-sur-Marne, France), 0.6 M NaCl, 10 mM Tris, 1 mM EDTA, 1% SDS (Serva Biowhittaker, Fonternay-ous-Bois, France), 10 mM dithiothreitol (Boehringer Mannheim, Meylan, France), 250 µg/ml tRNA (Sigma), 2% Denhardt reagent (Eurogentec, Angers, France), and 100 µg/ml salmon sperm DNA. For hybridization, 35S-labeled antisense and sense cRNA probe were diluted in the prehybridization buffer with 10% Dextran Sulfate (w/v) and without salmon sperm DNA, denatured at 80°C for 3 min and applied on sections (200 000 cpm/50 µl) at 50°C overnight in a sealed humidified container. Nonspecifically bound RNA transcripts were removed by washing sections in Tris buffer (10 mM Tris, 0.5 M NaCl, pH 8) containing 20 µg/ml RNase A (Boehringer Mannheim) during 1 h at 37°C. Sections were then washed in the following: 1) Tris buffer without RNase A for 30 min at 37°C; 2) 50% formamide, 0.1% ß-mercaptoethanol, and single-strength SSC (single strength SCC = 150 mM sodium chloride, 15 mM sodium citrate, pH 7) for 30 min at 50°C; 3) 50% formamide, 0.1% ß-mercaptoethanol, and 0.1-single-strength SSC for 30 min at 37°C. Then sections were dehydrated and air dried, and slides were dipped in Kodak NTB2 emulsion (Integra Bioscience, Cergy-Pontoise, France) and exposed at 4°C for 6–12 wk in a desiccated dark box. Slides were developed and counterstained with hematoxylin. Specificity of hybridization was assessed by comparing signal obtained with the cRNA antisense probe and the corresponding cRNA sense probe.

Histological determination of follicular size and degree of atresia was performed on adjacent sections stained with Feulgen. As previously described [14], follicles were judged normal or atretic using classical histological criteria (normal: frequent mitosis, no pyknosis in granulosa cells; atretic: no mitosis, frequent pyknotic bodies in granulosa cells).

Microscopic Analysis of Autoradiography

Quantitative autoradiographic analysis of [35S]-PPAR{gamma} expression was performed using a microscope-linked PC-based image analyzer (SAMBA 2005, Alcatel TITN, Meylan, France). Each section was analyzed with an objective 40x. Quantification of labeling was performed by measuring the area occupied by silver grains present in a constant area (250 µm2). This quantification has been performed on granulosa cells from 34 small antral follicles (1–3 mm diameter) and 23 large follicles (>5 mm diameter). Labeling was estimated from 10 measurements on each follicle. Specific binding was obtained by subtracting the values of labeling associated with nonspecific binding from the total binding values [14].

Isolation and Culture of Granulosa Cells

Culture of granulosa cells and reagents was described previously [15]. Briefly, ovaries were immersed for 15 min in isotonic solution containing Fungizone (Gibco BRL, Cergy-Pontoise, France) and antibiotics (penicillin and streptomycin) immediately after slaughter. Then ovaries were placed in B2 medium and follicles larger than 1 mm diameter were dissected within 1 h after slaughtering. B2 medium was prepared according to Menezo [16]. Each follicle was then slit open in B2 medium and granulosa cells were removed by gently scraping the interior surface of the follicle with a platinum loop.

Granulosa cell suspensions were pooled according to the size of follicles (small follicles [SF] antral: 1–3 mm, and large follicles [LF] antral: >=5 mm). Cell suspensions were centrifuged at 300 x g for 7 min. For Western blotting experiments, the pellets were stored at -80°C until experiments. For culture experiments, pellets were resuspended in culture medium (McCoys 5a medium [Sigma] containing bicarbonate supplemented with 20 mmol/L Hepes, 100 kIU/L penicillin, 0.1 g/L streptomycin, 3 mmol/L L-glutamine, 0.1% BSA [w/v], 100 µg/L insulin, 10 µg/L IGF-I, 0.1 µmol/L androstenedione, 5 mg/L transferrin, 20 µg/L selenium).

Granulosa cells were then cultured in serum-free conditions, according to the method described by Campbell et al. [17]. Briefly, cells were counted and the viability (ranged between 60% and 80%) was estimated by trypan blue exclusion. Cultures were performed in 96-well plates or in chamber slides. Cells were seeded at 105 viable cells/well and cultured at 37°C in a humidified atmosphere with 5% CO2 in serum-free culture medium containing either no exogenous factors, or 5 ng oFSH/ml in combination with increasing doses of rosiglitazone (1 µM or 10 µM) or 1 µM LG1069 or control solvent (dimethylsulfoxide [DMSO] 0.01%).

Each combination of treatment was tested in triplicate in at least three independent cultures. Cultures of granulosa cells from small and large follicles were performed for 96 h. Culture media were partially changed at 48 h, by replacing 180 µl of the total (250 µl) with prewarmed medium. The spent medium between 48 and 96 h was stored at -20°C prior to progesterone assay.

Determination of Thymidine Labeling Index

The effects of rosiglitazone on proliferation were studied in granulosa cells from SFs and LFs by measuring the thymidine labeling index (percentage of labeled cells) after 48 h of culture in vitro. Briefly, cells were washed with B2 medium without thymine and then incubated with [3H]-thymidine (0.25 µCi/ml) at 37°C for 2 h (specific activity 6.7 Ci/nmol, Dupont, De Nemours, Les Ulis, France). After two washes with B2 medium (with thymine), cells were fixed in 3% glutaraldehyde for 1.5 h at room temperature. Cells were stained with Feulgen (Schiff reagent, Merck, Schuchardt, Germany), dipped in NTB2 emulsion, air dried, and exposed for autoradiography for 6 days at 4°C. The thymidine labeling index was estimated by counting the number of labeled and unlabeled cells in 20 different microscopic fields (objective 100x). Measures were made on 500–1000 cells per slide, this number being sufficient to estimate proliferation, as previously described [18].

Progesterone Radioimmunoassay

The concentration of progesterone (P4) in the culture medium of granulosa cells from SFs and LFs was measured after 96 h of culture by a radioimmunoassay protocol previously described [19] and adapted to measure steroids in cell culture media. The limit of detection of P4 was 12 pg/tube (60 pg/well) and the intra- and interassay coefficients of variation were less than 10% and 11%, respectively. Results were expressed as the amount of steroids secreted between 48 and 96 h of culture per 5 x 104 cells recovered at the end of the culture period.

Pituitary Cell Cultures

Primary cultures of ovine anterior pituitary cells were prepared by collagenase/deoxyribonuclease dispersion. Pituitary glands from Romanov ewes were minced and cells were dispersed in Hams F-12 medium (Gibco, Cergy Pontoise, France) buffered with Hepes (2.5 mg/ml) and containing 0.4 mg/ml collagenase type A (Boehringer Mannheim), 0.025 mg/ml deoxyribonuclease (Sigma), 100 U/ml penicillin, 100 µg/ml streptomycin, and fetal bovine serum (FBS) (5%) as previously described [20, 21]. Cells were resuspended in Dulbeccos modified Eagle medium without phenol red (DMEM, Sigma) containing 5% FBS, 5 µg/ml transferrin, 100 µM ascorbic acid, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Cells were seeded in 48-well culture plates at 4 x 105 viable cells/well and cultured at 37°C in a humidified atmosphere with 5% CO2. Culture media were totally changed at 48 h, and cells were incubated in culture medium containing either no exogenous factors or 10 nM GnRH in combination with 1 µM or 10 µM rosiglitazone and/or 1 µM LG1069 or control solvent (DMSO 0.01% final) during 24 or 72 h. Each combination of treatment was tested in quadruplicate in at least three independent cultures. The spent medium during the last 24 or 72 h was stored at -20°C prior to LH, FSH, prolactin, and growth hormone (GH) assays.

Pituitary Hormones Immunoassays

Pituitary cell-conditioned media were assayed for oLH and oFSH concentrations by using a specific two-site ELISA sandwich (enzyme-linked immunoassays, EIA) that employed two anti-LH or anti-FSH monoclonal antibodies, kindly provided by Dr. J.F. Roser (University of California, Davis, CA) [22] and Dr. K.M. Henderson (Wallaceville Animal Research Centre, Upper Hutt, New Zealand) [23], respectively. Ovine LH (batch 1083, INRA, Nouzilly, France) and oFSH (NIH RP2, kindly provided by Dr. A.F. Parlow, Harbor-UCLA Medical Center, Torrance, CA) were used as assay standards. The first antibody was directed against the ß-subunit of the molecule, whereas the second biotinylated monoclonal antibody (Serotec MCA 1026) [24] was directed against the {alpha}-subunit. The detection limits of LH and FSH ELISAs were 0.1 ng/ml (2 pg/tube) and 0.4 ng/ml (20 pg/ tube), respectively. The intraassay variables were 6.7% for oLH and 5.1% for oFSH. Results were expressed as nanograms per milliliter as appropriate according to the particular standard preparation analyzed. These LH and FSH EIA systems exhibited 0.01% and 0.07% cross-reactivity with highly purified oFSH and oLH, respectively.

Ovine prolactin and ovine GH were measured in pituitary cell-conditioned media using the radioimmunoassay protocol previously described [25, 26]. The limit of detection of prolactin and GH RIA was 0.3 ng/ml and 0.62 ng/ml, respectively. The intraassay coefficient of variation was 4.5% for prolactin and 5.8% for GH. GH was measured as previously described with minor modifications [26]. Volumes were adjusted with a solution of 0.03 M NaH2PO4, 3.72 g/L of EDTA, 500 µl/L Tween 20, 200 mg/L of protamine sulphate and 200 mg/L of azide, (pH 7.4) for a final volume of 550 µl. After 48 h of incubation, samples were incubated with sheep serum raised against rabbit IgG (6 µl/tube), polyethylene glycol (PEG 6000, 0.06 g/tube) in PBS (2 ml/tube) overnight at 4°C. Then samples were subjected to centrifugation, and radioactivity was counted in the pellet.

Transient Transfection of Granulosa and Pituitary Cells

After 72 h of culture in McCoy or DMEM, respectively, containing 3% fetal ovine serum (FOS), granulosa (at 5 x 104 viable cells/well in 48-well culture plates) and pituitary cells (at 2 x 105 viable cells/well in 48-well culture plates) were transfected with 250 ng/well of a vector containing PPREs driving the firefly luciferase gene (PPRE-Luc) [27] and 25 ng of renilla luciferase plasmid to normalize transfection efficiency. Transfections were carried out using the DAC-30 DNA transfection reagent (Eurogentec S.A, Seraing, Belgium) according to the manufacturer's instructions. Transfection efficiency of granulosa cells was approximately 15% (unpublished data). The transfection medium was removed after 3 h and cells were incubated in fresh McCoy medium without FOS and IGF-I for granulosa cells and DMEM medium without FOS for pituitary cells in the presence of various reagents for 60 h (see Results). Then cells were lysed according to the manufacturer's instructions (luciferase assay, Promega, Charbonnières, France), and both firefly and renilla luciferase activities were quantified on a luminometer (TD-20/20, Turner Designs, Sunnyvale, CA). Each combination of treatment was tested in quadruplicate in at least four independent cultures.

Western Immunoblotting

Cell lysates of granulosa cells, anterior pituitary, or hypothalamus were prepared on ice with an Ultraturax homogenizer in lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Igepal) containing various protease inhibitors (2 mM PMSF, 10 mg/ml leupeptin, 10 mg/ml aprotinin) and phosphatase inhibitors (100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate [Sigma]). Lysates were centrifuged at 15 000 x g for 20 min at 4°C, and the protein concentration in the supernatants was determined using a colorimetric assay (kit BC assay, Uptima Interchim, Montluçon, France). Extracts from Cos-7 cells transiently transfected with a vector expressing hPPAR{gamma}2 were used as positive controls [28].

Cell extracts were submitted to electrophoresis on 12% (w:v) SDS-polyacrylamide gel under reducing conditions. The proteins were then electrotransferred onto nitrocellulose membranes (Schleicher and Schuell, Ecquevilly, France) for 2 h. Membranes were incubated for 1 h at room temperature with Tris-buffered saline (TBS, 2 mM Tris-HCl, pH 8.0, 15 mM NaCl, pH 7.6), containing 5% nonfat dry milk powder (NFDMP) and 0.1% Tween-20 to saturate nonspecific sites. Thereafter, membranes were incubated overnight at 4°C with anti-CYP11A (final dilution 1:2000), anti-3ß-hydroxysteroid dehydrogenase (3ß-HSD) (final dilution 1:500), antiactin (final dilution 1:1000; Sigma) or anti-PPAR{gamma} (final dilution 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies in TBS containing 0.1% Tween-20 and 5% NFDMP. Rabbit polyclonal antibody raised against bovine CYP11A and against human placental 3ß-HSD were kindly provided by Dr. D.B. Hales (University of Illinois, Chicago) and Dr. V. Luu-The (Centre de Recherche en Endocrinologie Moléculaire, Québec, PQ, Canada), respectively. After washing in TBS-Tween-20 0.1%, nitrocellulose membranes were incubated for 2 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit, anti-mouse IgG (final dilution 1:10 000; Diagnostic Pasteur, Marnes-la-Coquette, France) in TBS-0.1% Tween-20 NFDMP 5%. After washing in TBS-Tween-20 0.1%, the signal was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Orsay, France).

The films were analyzed and signals quantified with the software ImageMaster version 4.10 (Amersham Pharmacia Biotech). The results are expressed as the intensity signal in arbitrary units after normalization allowed by the presence of actin as an internal standard and correspond to the average of five or three independent experiments (n = 5 granulosa cells from SFs; n = 3 granulosa cells from LFs).

Statistical Analysis

All experimental data are presented as the mean ± SEM. The effects of rosiglitazone on granulosa cell steroidogenesis, pituitary cell secretion, and PPAR{gamma} activity were analyzed using two-way ANOVA to appreciate the effect of rosiglitazone as well as the culture experiment effect. Post hoc comparisons were performed by Newman-Keuls and Scheffe tests. The effects of rosiglitazone on thymidine labeling index, pyknotic index, and quantification were analyzed by paired t-test. The comparison of the fold induction of progesterone secretion by rosiglitazone was compared by a one-way ANOVA or in the case of heterogeneity of variance, the Mann and Whitney U test was used to compare means between two groups. P > 0.05 was considered nonsignificant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RT-PCR analysis performed on RNA from ovine anterior pituitary gland and granulosa cells resulted in the amplification of two cDNA corresponding to a fragment of the common region of ovine PPAR{gamma} gene (472 base pair [bp]) and to a specific region of PPAR{gamma}2 isoform (301 bp) (Fig. 1, a and b). A total of 590 bp PPAR{gamma} cDNA were sequenced in ovine granulosa cells. This cDNA presented 98.3% and 90.3% identity with bovine and porcine sequences, respectively [29, 30] (Fig. 1c). By immunoblotting on protein extracts, two bands of PPAR{gamma} were detected in granulosa cells and pituitary cell extracts, showing that both isoforms of PPAR{gamma} (PPAR{gamma}1 and PPAR{gamma}2) were expressed in these samples (Fig. 1d). Neither mRNA (even after 40 cycles of PCR) nor PPAR{gamma} protein were detected in ovine hypothalamus (Fig. 1, a and d).



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  		FIG. 1. Detection of PPAR{gamma} protein and transcripts in the hypothalamo-pituitary-ovary axis of ewe. (a, b) RT-PCR analysis of PPAR{gamma} mRNA. Total RNAs extracted from ovine white adipose tissue (AT), hypothalamus (HT), pituitary (PT), and granulosa cells (GC) were submitted to RT-PCR as described in Materials and Methods by using primers designed to amplify (a) a fragment of PPAR{gamma} (472 bp with the PPAR{gamma} primers) or (b) 164 bp of the common region of PPAR{gamma} isoforms and 301 bp corresponding to PPAR{gamma}2 (with the forward primers PPAR{gamma}1 and PPAR{gamma}2, respectively, and a single reverse primer PPAR{gamma}2). CT, PCR amplifications with RNA as negative control. c) Alignment of PPAR{gamma} N-terminal ovine sequence with the bovine and porcine sequence [29, 30]. d) Detection of PPAR{gamma} proteins by immunoblotting. Protein extracts (30 µg) were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with a specific monoclonal antibody raised against PPAR{gamma} isoforms (PPAR{gamma}1 and PPAR{gamma}2) and actin. Lane 1, Cos-7 cells; lane 2, Cos-7 cells overexpressing hPPAR{gamma}-2; lane 3, ovine hypothalamus; lane 4, whole ovine pituitary; lane 5, ovine granulosa cells. The arrows indicate the two PPAR{gamma} isoforms, the upper band corresponding to PPAR{gamma}2 (lane 2 vs. lane 1).

By in situ hybridization on ovarian sections, PPAR{gamma} mRNA was found to be highly expressed in granulosa cells of antral follicles (Fig. 2a). The cumulus oophorus was labeled as intensively as mural granulosa cells. The intensity of labeling was 3-fold higher in small (1–3 mm diameter, SF) in comparison with large (>5 mm diameter, LF) antral follicles (P < 0.001), suggesting that PPAR{gamma} expression decreases during terminal follicular growth (Fig. 2b). A weak labeling was observed in corpus luteum and to a lesser extent, in theca of some follicles. Healthy and atretic follicles expressed similar levels of PPAR{gamma} mRNA.



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  		FIG. 2. Localization of PPAR{gamma} mRNA in the ewe ovary by in situ hybridization. a) Localization of PPAR{gamma} in small antral healthy follicles (1–2, 5–6, 7–8), large healthy follicles (9–10), large atretic follicles (11–12), and corpus luteum (13–14) by in situ hybridization. Bright-field (1, 5, 7, 9, 11, and 13) and dark-field photomicrographs (2, 6, 8, 10, 12, and 14) of follicles and corpus luteum hybridized with ovine [35S]-PPAR{gamma} cRNA antisense probe. Bright-field (1 and 3) and dark-field photomicrographs (2 and 4) of the same follicle hybridized with antisense and sense [35S]-PPAR{gamma} cRNA probe, respectively. Note that PPAR{gamma} expression is observed in granulosa cells of healthy and atretic follicles as well as in cumulus oophorus. A, Antrum; C, cumulus oophorus; CL, corpus luteum; G, granulosa cells; T, thecal cells. Scale bar, 100 µm. b) Quantification of labeling of [35S]-PPAR{gamma} mRNA localized in SF and LF. ***P < 0.001

Addition of rosiglitazone dose dependently increased luciferase activity in granulosa cells from SF and LF (Fig. 3a). The action of 1 µM rosiglitazone on luciferase expression was further enhanced by addition of 1 µM RXR ligand (LG1069) (P < 0.001) (Fig. 3b), indicating that endogenous RXR{alpha}/PPAR{gamma} heterodimers are functional in ovine granulosa cells in vitro. Moreover, transient transfection experiments showed that neither FSH nor IGF-I were able to alter basal (Fig. 3c, P > 0.44) or rosiglitazone-induced activity of endogenous PPAR{gamma} from ovine granulosa cells in vitro (Fig. 3c, P > 0.43).



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  		FIG. 3. Endogenous PPAR{gamma} activation by rosiglitazone in ovine granulosa cells. Granulosa cells from SFs and LFs were transfected with the PPRE-Luc plasmid as described in Materials and Methods. a) After transfections, granulosa cells from SFs and LFs were stimulated with 1 µM or 10 µM of rosiglitazone for 60 h (n = 5–7 independent experiments). b) Granulosa cells from SFs were stimulated with 1 µM rosiglitazone alone or in presence of 1 µM LG1069 for 60 h (n = 4 independent experiments). c) Granulosa cells from SFs were incubated in the presence or absence of 5 ng/ml FSH, 10 ng/ml IGF-I with or without stimulation by 10 µM rosiglitazone for 60 h (n = 5–7 independent experiments). Luciferase activities of both Firefly and Renilla were measured in cell lysates and values were expressed in percentage ± SEM of control, the activity of the unstimulated control being taken as 100%. *P < 0.05, **P < 0.01, ***P < 0.001, significant differences from control conditions. In each experiment, measures were obtained as the mean of four replicates

In the presence and in the absence of IGF-I, rosiglitazone induced a 2-fold decrease in the [3H]-thymidine incorporation by granulosa cells from SF (P = 0.026; Fig. 4). In the absence of FSH, rosiglitazone dose dependently increased progesterone secretion by ovine SF and LF granulosa cells (Fig. 5a). In the presence of FSH, the effect of rosiglitazone on progesterone secretion was attenuated (SF: 5.05 ± 0.89 vs. 1.69 ± 0.25-fold induction, n = 5, basal vs. FSH-treated P = 0.008; LF: 3.07 ± 0.39 vs. 1.30 ± 0.12-fold induction, n = 5, basal vs. FSH-treated P = 0.008; Fig. 5b). The action of rosiglitazone on progesterone secretion was enhanced by addition of LG1069 (1 µM; Fig. 5c).



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  		FIG. 4. Effects of rosiglitazone on the proliferation rate of ovine granulosa cells. Effects of 10 µM rosiglitazone on proliferation of ovine granulosa cells from SFs cultured in absence or presence of IGF-I (10 ng/ml) during 48 h as described in Materials and Methods. Results are expressed as a percentage of control, i.e., basal conditions without rosiglitazone and IGF-I of thymidine-labeled cells. *P < 0.05, significant differences from control conditions. $P < 0.05, significant differences from IGF-1 treated vs. basal conditions. Experimental data are expressed as the mean ± SEM of five independent experiments



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  		FIG. 5. Effects of rosiglitazone on progesterone secretion by ovine granulosa cells. a, b) Granulosa cells from SFs and LFs were cultured during 96 h in serum-free medium with 10 ng/ml IGF-1 in absence (a) or presence (b) of 5 ng/ml FSH and increasing concentrations of rosiglitazone (1 µM and 10 µM). c) Granulosa cells from SFs were cultured during 96 h in serum-free medium in presence of 1 µM rosiglitazone alone or in presence of 1 µM LG1069. The amount of P4 secreted by 5 x 104 cells during the last 48 h was measured at 96 h of culture. *P < 0.05, **P < 0.01, ***P < 0.001, significant differences from control conditions. Experimental data are expressed as the mean ± SEM of four to five independent experiments. In each experiment, measures were obtained as the mean of three replicates

The stimulation of progesterone secretion was not explained by an effect on the expression of two key enzymes of steroidogenesis. Indeed, immunoblot analysis failed to detect any effect of rosiglitazone on CYP11A and 3ß-HSD protein levels in extracts from SF and LF granulosa cells in vitro (P > 0.05) (Fig. 6, a and b).



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  		FIG. 6. Effects of rosiglitazone on the expression of CYP11A and 3ßHSD in ovine granulosa cells. a) Proteins extracts from granulosa cells cultured for 96 h in presence or absence of 10 µM rosiglitazone were subjected to SDS-PAGE electrophoresis as described in Materials and Methods. The membrane was incubated with antibodies raised against CYP11A and 3ß-HSD. All SF and LF protein extracts contained equal amounts of proteins, as confirmed by reprobing membrane with an anti-actin antibody. Results are representative of at least three independent experiments. b) Quantification of the expression of CYP11A and 3ßHSD (fold induction of protein level)

Rosiglitazone (10 µM) was able to stimulate the expression of luciferase gene (1.9 ± 0.46-fold induction, n = 7, P = 0.01), indicating the presence of functional PPAR{gamma} in ovine pituitary cells (Fig. 7a). Rosiglitazone did not affect LH and FSH secretion by ovine pituitary cells in vitro, whatever the duration (24 or 72 h) of induction and irrespective of the presence of GnRH (10 nM; Fig. 7, b and c). However, in the presence of GnRH, a slight increase in LH secretion under 10 µM rosiglitazone was observed at 24 h (P < 0.05). Rosiglitazone also had no effect on prolactin or GH secretion by ovine pituitary cells in vitro (Fig. 7d). Interestingly, a decrease in GH secretion was observed when LG1069 was added (P = 0.003).



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  		FIG. 7. Effects of rosiglitazone on LH, FSH, prolactin, GH secretion by ovine pituitary cells. a) Ovine pituitary cells were transfected with the PPRE-Luc plasmid as described in Materials and Methods. Cells were stimulated with 10 µM rosiglitazone for 60 h (n = 7 independent experiments). b, c) Ovine pituitary cells were stimulated with 1 µM or 10 µM rosiglitazone alone or in presence of 1 µM LG1069 and/or 10-8 M GnRH for 24 or 72 h. The amount of LH (b), FSH (c), prolactin, and GH (d) secreted during the last 24 or 72 h of culture was measured. Results are expressed as a percentage of control, i.e., basal conditions without rosiglitazone and GnRH. *P < 0.05, **P < 0.01, ***P < 0.001, significant differences from control conditions. Experimental data are expressed as the mean ± SEM of three independent experiments (24 h) and four independent experiments (72 h). In all experiments, measures were obtained as the mean of four replicates


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present data show that PPAR{gamma} is expressed in granulosa cells from antral follicles, in cumulus oophorus as well as in the corpus luteum in the sheep ovary. Expression of PPAR{gamma} mRNA was shown to decrease with follicular size and was not associated with the healthy status of the follicle. These results are in concordance with a previous study in the rat ovary showing PPAR{gamma} expression in granulosa cells [12]. In the present work, we have detected by immunoblotting and by RT-PCR the presence of both PPAR{gamma}1 and PPAR{gamma}2 isoforms in ovine granulosa and pituitary cell extracts, as previously observed in bovine lutein cells [29] and porcine theca cells [30]. Furthermore, our results obtained by in vitro transfection of granulosa cells showed that endogenous ovarian PPAR{gamma} is functional and responds to rosiglitazone treatment.

One of the known functions of PPAR{gamma} is to modulate cell proliferation and differentiation. We have shown that PPAR{gamma} activation inhibits proliferation of granulosa cells from ovine small follicle, which are known to proliferate in vivo as well as in vitro. The mechanism that underlies this inhibition is unknown. However, in human hepatoma cells and in nonprecursor fibroblast cell lines, troglitazone has been shown to increase the expression of cyclin-dependent kinase inhibitors of cell proliferation, such as p21, p27, and p18(INK4c) [31–34], suggesting that PPAR{gamma} ligands could inhibit cell proliferation by preventing the G1/S progression. Further investigation is needed to determine the mechanisms by which rosiglitazone regulates cell proliferation in ovine granulosa cells.

The consequences of PPAR{gamma} activation on steroidogenesis of ovine granulosa cells was further analyzed in vitro. The present data showed that rosiglitazone treatment increases progesterone secretion, these effects being enhanced by addition of LG1069 (1 µM). Of note, preliminary experiments showed a slight (1.6-fold) but reproducible increase in estradiol secretion, another marker of the follicle differentiation, in presence of both ligands (data not shown). Our results are in concordance with recent studies. Indeed, PG2, a natural ligand of PPAR{gamma}, was also shown to induce a 14-fold increase in progesterone production and a 30% increase in estradiol production by rat granulosa cells in vitro [12]. Similarly PPAR{gamma} ligands are also able to increase the progesterone secretion by bovine lutein cells [35] and porcine theca cells [30] in vitro. However, other studies reported a troglitazone-induced inhibition of the progesterone secretion by porcine and human granulosa cells in vitro [36, 37]. The discrepancies between these different reports could be explained by species difference, and/or the state of differentiation of granulosa cells (nonluteinized granulosa cells vs granulosa-lutein cells). They also can be due to differences between the in vitro models. Of note, in our hands, the inhibitory and stimulatory effects of PPAR{gamma} ligands on cell proliferation and progesterone secretion, respectively, were very reproducible and observed cell culture conditions (cell density, duration of cell culture). Finally, we cannot exclude a PPAR{gamma}-independent action of troglitazone in human and porcine granulosa cells culture, as suggested by a recent study [38].

In our experimental conditions, rosiglitazone did not induce the expression of CYP11A (P450scc) nor 3ß-HSD. This suggests that rosiglitazone may stimulate progesterone secretion by increasing activity rather than expression of steroidogenic enzyme and/or speeding up cholesterol transport. Troglitazone was also shown to alter 3ß-HSD activity in porcine granulosa cells without affecting its mRNA expression [37].

In addition, in the present work, the magnitude of effect of rosiglitazone on progesterone secretion tended to be lower in LF, compared with SF granulosa cells, and was clearly lower in FSH-stimulated compared with unstimulated cells. Similar results were obtained by Schoppee et al. [30] in porcine theca cells stimulated with LH. One could hypothesize that FSH may have exerted a maximal effect on progesterone secretion, rosiglitazone not being able to have any additional effect. We can also not exclude a modulation of PPAR{gamma} activity by the protein kinase A pathway, as recently shown in the mouse liver [39]. However, our results from the transfection experiments indicate that neither IGF-I nor FSH alter basal or rosiglitazone-stimulated endogenous PPAR{gamma} activity. Alternatively, PPAR{gamma} may modulate the activity and/or the binding of transcription factors on the promoter of steroidogenic enzyme genes. For example, the ability of the PPAR{gamma}/RXR{alpha} heterodimer to modulate the binding of Sp1 to GC-box to the promoter of interleukin 1-beta gene has recently been reported [40].

Overall, our results in ovine granulosa cells, as well as studies on rodents, suggest that PPAR{gamma} ligands could favor follicular maturation and corpus luteum functionality. Interestingly, conditional inactivation of PPAR{gamma} in the ovary of mice leads to a reduction of the number of implanted embryos associated with a slight decrease in the progesterone levels in serum [41]. This suggests a role for PPAR{gamma} in the final maturation of oocytes during terminal follicular growth and/or in the ability of newly formed corpus luteum to secrete sufficient concentrations of progesterone to supply implantation. Of note, in cattle, the concentration of PPAR{gamma} is higher in the corpus luteum of pregnant heifers in comparison with nonpregnant heifers [42]. In this species, PPAR{gamma} expression decreases during luteal phase progress [35, 42]. In several cell types, PPAR{gamma} activators modulate the expression of cyclooxygenase-2 [43], plasminogen activator [44], or vascular endothelial growth factor [45], genes known to be involved in follicular and luteal development as well as in oocyte maturation. Further experiments are required to study if PPAR{gamma} action could act on oocyte maturation or angiogenesis by modulating expression of these genes in the ovary.

In the second part of this study, we have shown by RT-PCR and immunoblotting that PPAR{gamma} is expressed in the pituitary but not in the hypothalamus in sheep. A previous study also failed to detect expression of PPAR{gamma} in the brain of adult rat as well [46]. Moreover, transfection experiments showed a small but significant effect of rosiglitazone on endogenous pituitary PPAR{gamma}, indicating that PPAR{gamma} is functional and could play a physiological role in this tissue. However, our studies performed on ovine pituitary cells in vitro revealed no effect of rosiglitazone on basal- or GnRH-stimulated LH and FSH secretion. Similarly, rosiglitazone had no effect on basal or GnRH-stimulated LH secretion by the mouse gonadotrope-derived LßT2 cell line (data not shown). This suggests that in the pituitary, PPAR{gamma} is not expressed in FSH/LH cells and/or exerts no role on gonadotropin secretion. However, the involvement of gonadotropins in the local actions mediated by PPAR{gamma} may not be completely ruled out. For example, we cannot exclude an action of steroids on the expression or the actions of PPAR{gamma} [47]. We also failed to detect any effects of rosiglitazone on prolactin or GH secretion by ovine pituitary cells in vitro.

In vivo, several studies have reported a role of nutrients such as fatty acids on reproductive performances. For example, in the sheep, a short-term nutritional supplementation [2] is able to increase ovulation rate and the size of preovulatory follicles [48, 49, 2] without any effect on circulating gonadotropins concentrations. Moreover, granulosa cells collected from follicles of fat-supplemented cows were shown to exhibit an increase in progesterone secretion in vitro [50], suggesting an ovarian effect of fatty acids on steroidogenesis. Overall, these results suggest that part of the effects of nutrients on reproduction could be, at least partly, mediated by the activation of PPAR{gamma} by its natural ligands in granulosa cells.

In conclusion, the present data suggest that PPAR{gamma} action is able to inhibit granulosa cell proliferation and to stimulate follicular differentiation by a direct action on the ovary rather than by acting at the hypothalamo-pituitary level. Further investigations are needed to identify the molecular mechanisms underlying the action of PPAR{gamma} and to determine whether it could mediate some effects of nutritional factors on ovarian functions in vivo.


   ACKNOWLEDGMENTS
 
We thank Alice Pierre and Sabine Mazerbourg for technical assistance. We are grateful to Francis Paulmier, Francis Dupont, and their technical staff for animal management. We thank the staff of the Laboratoire de Dosages Hormonaux (INRA, Nouzilly, France), Joël Fontaine for ELISAs, and Dr. Kaïs Al-Gubory for GH RIA reagents. We acknowledge Michèle Peloille for sequencing, and Danielle Monniaux and Giulia Chinetti for helpful discussion. We wish to thank Dr. Dale Buchanan Hales, Dr. Van Luu-The, Dr. Janet F. Roser, and Dr. Keith Henderson for generously providing the CYP11A, 3ß-HSD, ß-oLH, and ß-oFSH antibodies, respectively, and Dr Albert Parlow for oFSH RP2.


   FOOTNOTES
 
1 This work was supported by Institut National de la Recherche Agronomique. P.F. was supported by a fellowship from Institut National de la Recherche Agronomique and Région Centre. Back

2 Correspondence: Philippe Monget, Physiologie de la Reproduction et des Comportements, UMR 6073 INRA-CNRS-Université F. Rabelais de Tours, 37380 Nouzilly, France. FAX: 33 2 47 42 77 43; monget{at}tours.inra.fr Back

Received: 24 March 2003.

First decision: 20 April 2003.

Accepted: 14 May 2003.


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