Hormones and reagents
Purified ovine FSH-20 (oFSH) (lot no.AFP-7028D, 4453 IU/mg, with an FSH activity = 175 times the activity of oFSH-S1) used for culture treatment was a gift from NIDDK, National Hormone Pituitary Program, Bethesda, MD, USA. Recombinant human insulin-like growth factor-I (IGF-I) was from Sigma (St Louis, MO, USA). Recombinant human adiponectin produced in the NSO mammalian cell system was obtained from R&D (Lille, France). Glucose was from VWR (Fontenay sous bois, France).
Rabbit polyclonal antibodies to adiponectin receptor 2 (4–39) and adiponectin receptor 1 (41–65) were from Phoenix Pharmaceuticals Inc. (Belmont, CA, USA). Rabbit polyclonal antibodies to phospho-ERK1/2 (Thr202/Tyr204), and phospho-AMPK alpha Thr172 were purchased from New England Biolabs Inc (Beverly, MA). Rabbit polyclonal antibodies to AMPKα1 were obtained from Upstate Biotechnology Inc (Lake, Placid, NY, USA). Rabbit polyclonal antibodies to ERK2 (C14) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibody to vinculin was obtained from Sigma (St. Louis, MO, USA). Rabbit polyclonal antibodies against p450scc, StAR and 3βHSD were generously provided by Dr. Dale Buchanan Hales (University of Illinois, Chicago, USA) and Dr. Van Luu-The (CHUL Research Center and Laval University, Canada), respectively. Mouse monoclonal antibody against p450 aromatase was purchased from Serotec (Varilhes, France). All antibodies were used at 1/1000 dilution for western blotting.
Animals and experimental procedures
All procedures were approved by the Agricultural Agency and the Scientific Research Agency and conducted in accordance with the guidelines for Care and Use of Agricultural Animals in Agricultural Research and Teaching. Eight week-old female Wistar rats (n = 18) were purchased from Janvier Laboratories (France). They were housed with controlled temperature and photoperiod (10 h darkness, 14 h light, light on from 0600–2000 h). The animals had ad libitum access to food and water. Diabetes was induced in one group of rats (n = 12) by an intraperitoneal injection of 1 ml of 50 mM sodium citrate solution (pH 4.5) containing streptozotocin (STZ, 55 mg/kg body weight). Control female rats (C, n = 6) were injected with 50 mM sodium citrate solution (pH 4.5). One week after injection, plasma glucose levels were checked for each animal and diabetes was confirmed (plasma glucose level >3 g/l). Then, control and STZ-treated rats were killed at diestrus stage. One sample of blood was taken and then tissues (ovaries, leg muscles and liver) were removed and frozen for western blotting.
Isolation and culture of rat granulosa cells
Immature female Wistar rats (21 days) were injected subcutaneously with DES (diethylstilbestrol, 1 mg/day) every day for three days to increase the amount of granulosa cells as previously described [14, 30]. On the third day of DES treatment the animals were killed and the ovaries removed aseptically and transferred to culture medium. Granulosa cells were harvested by puncturing the follicles allowing expulsion of the cells. Cells were recovered by centrifugation, washed with fresh medium and counted in a hemocytometer. The culture medium used was McCoy's 5A supplemented with 20 mmol/L Hepes, penicillin (100 U/ml), streptomycin (100 mg/l), L-glutamine (3 mmol/l), 0.1% BSA, 0.1 μmol/l androstenedione, 5 mg/l transferrin, 20 μg/l selenium and 5% FBS. The cells were first cultured for 48 h with no other treatment and then incubated in DMEM medium without glucose and serum but containing penicillin (100 U/ml), streptomycin (100 mg/l), L-glutamine (3 mmol/l) and 0.1 μmol/l androstenedione with or without test reagents (glucose, FSH, IGF-1) for the appropriate time as indicated in the legend of the figures. All cultures were performed under a water-saturated atmosphere of 95% air/5% CO2 at 37°C.
Lysates of granulosa cells or tissue 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, l'Isle d'Abeau Chesnes, France)]. Lysates were centrifuged at 13,000 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).
Cell extracts were subjected to electrophoresis on 10% (w:v) SDS-polyacrylamide gel under reducing conditions. The proteins were then electrotransferred onto nitrocellulose membranes (Schleicher and Schuell, Ecquevilly, France) for 2 h. The 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 non-specific sites. Then, the membranes were incubated overnight at 4°C with appropriate antibodies (final dilution 1:1,000), in TBS containing 0.1% Tween-20 and 5% NFDMP. They were washed in TBS-0.1% Tween-20, incubated for 2 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (final dilution 1:10,000; Diagnostic Pasteur, Marnes-la-Coquette, France) in TBS, 0.1% Tween-20 5% NFDMP, and washed again in TBS-0.1% Tween-20. The signal was detected by ECL (enhanced chemiluminescence, Amersham Pharmacia Biotech, Orsay, France).
The films were analyzed and signals quantified with the software MacBas V2.52 (Fuji PhotoFilm, USA, Inc.). The results are expressed as the signal intensity in arbitrary units after normalization to the signal for vinculin, used as an internal standard, and each value reported corresponds to the means of three independent experiments.
Thymidine incorporation into granulosa cells
Granulosa cells (2 × 105 viable cells/500 μl) were cultured in 24-well dishes in McCoy's 5A medium containing 10% FBS for 48 h and were then serum starved for 24 h in DMEM medium without glucose: then 1 μCi/μl of [3H] thymidine (Amersham Life Science, Arlington Heights, IL) was added in the presence or absence of various concentrations of glucose and/or FSH (10-8M) and IGF-1 (10-8 M). Cultures were maintained at 37°C under 5% CO2 in air. After 24 h of culture, excess of thymidine was removed by washing twice with phosphate-buffered saline (PBS), and the samples were fixed with cold trichloroacetic acid 50% for 15 min and lysed by addition of 0.5 N NaOH. The radioactivity was determined by addition of scintillation fluid (Packard Bioscience) and counting in a β-photomultiplier.
Progesterone and oestradiol radioimmunoassay
The concentration of progesterone (P) and oestradiol (E2) in the culture medium of granulosa cells was measured after 48 h of culture by a radioimmunoassay protocol as previously described [31–33] and adapted to measure steroids in cell culture media. The limit of detection of P was 12 pg/tube (60 pg/well) and the intra- and interassay coefficients of variation were less than 10% and 11%, respectively. The limit of detection of E2 was 1.5 pg/tube (7.5 pg/well) and the intra-and interassay coefficients of variation were less than 7% and 9%, respectively. Results are expressed as the amount of steroids secreted per 48 h per 100 μg of protein, and values reported are means ± SE of three cultures of granulosa cells. For each culture, about 30 to 40 rats were used and each condition (FSH, IGF-1, glucose alone or combined with IGF-1 or FSH) was analyzed four times independently.
After extraction of steroids from serum, the concentration of progesterone was measured by the RIA method as previously described . The oestradiol concentration in the serum was measured with a RIA KIT (DIASORIN, Antony, France).
Glucose, adiponectin, resistin and insulin plasma levels
Plasma glucose was assayed by the glucose oxidase method (Glucose Beckman Analyser 2, Beckman, Palo Alto, CA). Plasma adiponectin and resistin were assayed with a rat adiponectin ELISA kit (Phoenix Pharmaceuticals, Inc, Belmont, CA, USA) and a rat resistin ELISA kit (BioVendor laboratory Medicine, Inc, Heidelberg, Germany), respectively. Serum insulin levels were determined using a rat insulin ELISA kit (Mercodia AB, Uppsala, Sweden).
All experimental data are presented as the mean ± SE. One t-test (for comparison of two means, Control and STZ) or one-way analysis of variance (ANOVA) (for comparison of various means) were used to test differences; if ANOVA revealed significant effects, the means were compared by Newman's test, with P < 0.05 considered significant.