All experimental procedures were approved by the Chancellor's Animal Research Committee at the University of California at Los Angeles.
Primary cell cultures
Primary hypothalamic astrocyte cultures pooled from two to six animals were obtained from 50-day-old adult Long-Evans rats (Charles River, Wilmington, MA, USA) and from 60-day-old adult mice (C57/Bl6 wild type and estrogen receptor-alpha (ERα) knockout (Jackson Laboratory, Bar Harbor, ME, USA) and C57BL/6J FCG mice (gift from Dr. Arthur Arnold, UCLA, Los Angeles, CA, USA)). FCG mice were obtained by breeding XX female mice with XY-
Sry male mice, which possess a Y chromosome with the Sry gene deleted and a functional Sry transgene inserted onto an autosome. The presence of the Sry gene leads to differentiation of the indeterminate gonads into testes, and its absence results in formation of ovaries [19, 20]. This cross generates four genotypes: XY-
Sry gonadal males (XYM), XY- gonadal females (XYF), XXSry gonadal males (XXM) and XX gonadal females (XXF) .
The hypothalamus was dissected with the following boundaries: rostral extent of the optic chiasm, rostral extent of the mammillary bodies, lateral edges of the tuber cinereum and the top of the third ventricle. Hypothalamic tissue was dissociated with 2.5% trypsin solution (Invitrogen, Eugene, OR, USA) and a fire polished glass Pasteur pipette. Cultures were grown in Dulbecco's modified Eagle's medium (DMEM)/F12 (Mediatech, Manassas, VA, USA) with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and 1% penicillin (10,000 IU/ml)-streptomycin (10,000 μg/ml) solution (PS) (Mediatech) at 37°C in 5% CO2. Astrocyte cultures were grown to confluency and purified from other glial cells [14–16] using a technique modified from McCarthy and de Vellis . Sry expression in the astrocytes of FCG mice was analyzed and confirmed by reverse transcription (RT)-PCR.
Before the experiments, the DMEM/F12 medium with 10% FBS and 1% PS was removed, and primary astrocyte cultures were washed with Hanks' balanced salt solution (HBSS) (Mediatech), dissociated with a 2.5% trypsin solution and resuspended in DMEM/F12 medium with 10% FBS. Astrocytes were centrifuged for 3 minutes at 80 g, then the supernatant was removed and the pellet containing astrocytes resuspended. Astrocytes were counted, plated and incubated in DMEM/F12 medium with 10% FBS and 1% PS at 37°C in 5% CO2 for 24 to 48 hours before Ca2+ imaging and progesterone radioimmunoassay (RIA). For biotinylation, astrocytes were counted, plated and grown in flasks for 2 weeks before experimentation. Cultures were routinely checked for purity using immunocytochemistry for glial fibrillary acidic protein (Chemicon, Temecula, CA, USA) with Hoechst 3342 nuclear stain (Sigma-Aldrich, St. Louis, MO, USA). Cultures were determined to be > 95% pure astrocytes, as previously reported [14–16],
Intracellular Ca2+ measurements
Astrocytes (5000) were plated onto 15 mm glass coverslips coated with 0.1 mg/ml poly-D lysine (Sigma-Aldrich) in 12-well culture plates and grown in DMEM/F12 medium with 10% FBS and 1% PS at 37°C in 5% CO2 for 24 to 48 hours. The astrocytes were then starved of steroid for 18 hours by incubating in DMEM/F12 medium with 5% charcoal-stripped FBS at 37°C in 5% CO2. Before imaging, astrocytes were incubated for 45 minutes with HBSS and 4.5 μmol/l of the calcium indicator Fluo-4 AM (Invitrogen) dissolved in dimethyl sulfoxide (DMSO) and methanol. Glass coverslips were placed into a 50 mm chamber insert (Warner Instruments, Hamden, CT, USA) fixed into a 60 × 15 mm cell culture dish (Corning Inc., Corning, NY, USA) and placed into a quick exchange platform (QE-2; Warner Instruments) for imaging under a laser confocal microscope (Axioplan2-LSM 510 Meta; Zeiss, Thornwood, New York, NY, USA). Astrocytes were gravity perfused with HBSS, and media were removed by vacuum suction. Cyclodextrin encapsulated 17β-estradiol (1 nmol/l) (Sigma-Aldrich) was prepared in HBSS and used to induce [Ca2+]i release. Controls were stimulated with HBSS only. For Fluo-4 AM imaging, a water immersion objective (IR-Achroplan 40X/0.80; Zeiss, Jena, Germany) was used with 488 nm laser excitation and emission monitored through a low-pass filter with a cutoff at 505 nm. The increase in Ca2+ fluorescence (relative fluorescence units (RFU)) was calculated for each astrocyte as the difference between baseline fluorescence and peak response to drug stimulation.
Approximately 500,000 astrocytes were plated into six-well culture plates and grown for 24 hours. Astrocytes were then starved of steroid in DMEM/F12 medium with 5% charcoal-stripped FBS for 18 hours before treatment with cyclodextrin encapsulated 17β-estradiol (1 or 100 nmol/l dissolved in HBSS) (Sigma-Aldrich) or HBSS with no steroids for 60 minutes at 37°C. After 1 hour of drug treatment, the supernatant for each well was collected and frozen at -20°C for up to 1 week before the RIA.
For the progesterone assay, samples were thawed, mixed with diethyl ether (Fisher Scientific, Fair Lawn, NJ, USA) and then mixed by vortex for 2 minutes. To freeze the aqueous layer, samples were placed into a methanol and dry ice bath. The upper ether layer was decanted into a separate tube and the ether was allowed to evaporate overnight. The extract was reconstituted in isooctane (Mallinkrodt Baker, Phillipsburg, NJ, USA) and a diatomaceous earth column (Celite column™; Celite Corp., Lompoc, CA, USA) with ethylene glycol as the stationary phase was used to isolate the progesterone. Progesterone was then eluted off the column using 4 ml of isooctane. Standards and samples (100 μl) were incubated with rabbit polyclonal antibody against progesterone (Sigma-Aldrich) for 30 minutes at 37°C. Tritium radiolabeled progesterone (2000 counts/minute/ml) was added and incubated for an additional 60 minutes at 37°C. Standards and samples were cooled to 4°C, and a 0.05% charcoal dextran solution (Sigma-Aldrich) added to remove all unbound progesterone. The mixture was centrifuged at 1500 × g for 15 minutes at 4°C. The supernatant was then collected for chromatographic detection of progesterone. All samples were run in duplicate, and sample progesterone concentrations determined by extrapolation from a curve determined from the progesterone standards.
Primary astrocyte cultures were starved of steroids in DMEM/F12 medium with 5% charcoal-stripped FBS 12 hours before treatment with vehicle or 1 nmol/l of 17β-estradiol (Sigma-Aldrich) for 30 minutes at 37°C. Cells in each flask were washed three times with ice cold phosphate buffered saline (PBS) and incubated with freshly prepared biotin (0.5 mg/ml) (EZ-Link Sulfo-NHS-LC-Biotin; Pierce Biotechnology Inc., Rockford, IL, USA) in PBS for 30 minutes at 4°C with gentle agitation. Excess biotin reagent was quenched by rinsing the cells three times with ice cold glycine buffer (50 mmol/l glycine in PBS). Cells were scraped into 10 ml of PBS solution, transferred into a 50 ml conical tube and centrifuged at 500 × g for 3 minutes. The pellet was washed twice with ice-cold PBS and resuspended in 200 ml radioimmunoprecipitation assay (RIPA) lysis buffer containing the protease inhibitors 1 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l EDTA, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 1 μg/ml aprotinin and 1 mmol/l sodium orthovanadate (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). The cells were homogenized by passing them through a 25-gauge needle, and the cell extract was centrifuged at 10,000 × g for 2 minutes at 4°C. The protein concentration of the supernatant was determined using the Bradford Assay (Bio-Rad, Hercules, CA, USA). Samples with equal protein concentration were added to a washed immobilized gel (NeutrAvidin™Gel; Pierce Biotechnology Inc.) for 2 hours at room temperature and then centrifuged at 1,000 × g for 1 minute. The beads were washed four times with 1 ml of RIPA buffer (Santa Cruz Biotechnology) containing the protease inhibitors previously mentioned. The bound proteins were eluted with SDS-PAGE sample buffer supplemented with 50 mmol/l dithiothreitol (DTT) for 1 hour at 37°C.
Samples were separated in a 10% Tris-HCl gel (Ready Gel; Bio-Rad) and transferred to polyvinylidene fluoride membranes (GE Healthcare, Piscataway, NJ, USA). ERα was detected with primary rabbit polyclonal antibody (1:1000) (C1355; Upstate Biotechnology, Inc., Lake Placid, NY, USA). β-actin was used as a loading control and detected by rabbit polyclonal antibody (1:5000) (Abcam, Cambridge, MA, USA). A secondary donkey anti-rabbit IgG (H+L) antibody (1:5000) (Jackson ImmunoResearch, West Grove, PA, USA) and an anti-biotin horseradish peroxidase-linked antibody (1:1000) (Cell Signaling Technology, Danvers, MA, USA) were then used (1.5 hour incubation). To estimate the molecular weight, samples were run alongside a biotinylated protein ladder (Cell Signaling Technology). Immunoreactive bands were visualized using an enhanced chemoluminesence (ECL) kit and ECL hyperfilm (GE Healthcare). Routine exposures varied from 0.5 to 2 minutes. The optical density of each immunoreactive band was determined. For each sample, immunoreactive ERα was normalized with β-actin to obtain the percentage of ERα protein to β-actin protein ratio (% relative ratio).
Total RNA was extracted from several primary cultures of astrocytes from Long-Evans post-pubertal rats using Trizol reagent (Invitrogen), following the manufacturer's recommended protocol. To prevent DNA contamination, RNA was treated with DNase I (Ambion, Austin, TX, USA) at 37°C for 30 minutes followed by inactivation with DNase inactivation reagent (Ambion). Total RNA quality and concentration were assessed using a spectrophotometer (NanoDrop 1000; Thermo Scientific, Wilmington, DE, USA). RT was then performed using 1 μg of total RNA to synthesize single-stranded cDNA in a 20 μl reaction with a reverse transcriptase (SuperScript III; Invitrogen) and a combination of random hexamers and oligo (dT)20 primers, following the manufacturer's protocol. Briefly, RT was performed at 50°C for 50 minutes, then the reaction terminated at 85°C for 5 minutes and the RNA destroyed with 1 μl of RNase H at 37°C for 20 minutes. cDNAs were subjected to PCR with primers specific to rat Sry (Fwd: 5-GCAGCGTGAAGTTGCCTCAAC-3 and Rev: 5-TGCAGCTCTAGCCCAGTCCTG-3) in an RT-PCR system (Mx3000p Real-Time PCR System; Stratagene, Santa Clara, CA, USA). PCR conditions used for amplification were as follows: initial denaturation at 94°C for 10 minutes, 35 cycles of denaturation at 94°C for 45 seconds, annealing at 60°C for 45 seconds and elongation at 72°C for 1 minute, with a final extension at 72°C for 7 minutes. Amplified products were separated by electrophoresis in a 2% agarose gel with ethidium bromide and visualized with ultraviolet light. Gel images were captured digitally to confirm product size and the absence of non-specific products. Negative controls (no cDNA) were included in every PCR run.
Data are presented as means ± standard error (SEM) in RFU, pg/ml or % relative ratio. Statistical comparisons were made using one-way analysis of variance (ANOVA) with Student-Newman-Keuls post hoc test when comparing means across at least three independent groups. For the FCG mice, mean comparisons and contrasts under the ANOVA model were made using the Tukey-Fisher least significant difference (LSD) criterion. Statistical calculations were carried out using SAS (version 9.2; SAS Institute, Cary, NC, USA) and GraphPad Prism (version 5; GraphPad Software, La Jolla, CA, USA) software programs. P < 0.05 was considered significant.