Soluble Guanylyl Cyclase: MATERIALS AND METHODS
Hormones and Reagents
Highly purified ovine FSH (oFSH-19-SIAFP; 94 X National Institutes of Health [NIH] FSH S1 U/mg; LH activity, 0.025 X NIH LH S1 U/mg) was obtained through the National Hormone and Pituitary Distribution Program (NIDDK, NIH). Androstenedione, eCG, and hCG were purchased from Sigma Chemical Co. (St. Louis, MO), and McCoy 5A medium (modified, serum-free), streptomycin sulfate, penicillin, and L-glutamine were obtained from Life Technologies (Rockville, MD). All other chemicals were reagent grade and obtained from Fisher Scientific (Pittsburgh, PA) unless otherwise specified.
Intact, immature Sprague-Dawley rats (age, 21 days; Harlan Sprague-Dawley, Indianapolis, IN) and young (age, 4 mo) rats of the Long-Evans strain (Charles River Laboratories, Wilmington, MA) were obtained from the commercial suppliers indicated. In addition, neonatal and postnatal females were obtained by mating adult Long-Evans males and females. Animals were maintained under a 16L:8D photoperiod with food and water available ad libitum. Animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All procedures were approved by the Institutional Animal Care and Use Committee of California State University-Los Angeles.
Immature rats received a subcutaneous injection of eCG (10 IU) to stimulate follicular development, which was followed 52 h later by an ovulatory dose of hCG (30 IU). Ovaries were obtained at 0, 24, and 52 h after eCG and at 24 and 72 h after hCG (n = 4 rats/time point). One ovary was fixed in 4% paraformaldehyde and processed for immunohis-tochemical analysis. The remaining ovary was snap-frozen and used for protein extraction and subsequent immunoblot analysis. Similarly, ovaries were collected at approximately 1000 h from rats at Days 5, 10, and 19 of age for immunohistochemical analysis of sGC protein levels (the day of birth was designated Day 1; n = 4 rats/day).
Primary Ovarian Granulosa Cell Culture
To examine the regulation of sGC subunit protein levels in ovarian cells, we used a well-characterized primary granulosa cell culture system as previously described. Ovaries were obtained from immature (age, 25 days) females treated for 5 days with subcutaneous E2 implants to stimulate proliferation of functionally immature granulosa cells. The ovaries were removed aseptically and rinsed in McCoy 5A medium supplemented with streptomycin sulfate (100 U/ml), penicillin (100 U/ml), and L-glutamine (2 mM). Ovarian follicles were then punctured and granulosa cells released with the aid of a 26-guage syringe needle under a stereo-microscope. The cells were harvested, washed, pelleted by centrifugation at low speed, and resuspended in fresh medium. The concentration of viable cells was determined by trypan blue exclusion using a hemocytom-eter. Approximately 1 000 000 viable granulosa cells were cultured per tube in 12- X 75-mm polypropylene, round-bottomed Falcon culture tubes using McCoy 5A medium supplemented with androstenedione (10—7 M) as substrate for E2 synthesis. Cells were cultured with media alone (control) or with FSH at 37°C in a humidified atmosphere of 95% air and 5% CO2 for the specified times, then harvested for immunoblot analysis of sGC subunit protein levels as described below. Each experiment was repeated five times.
Preparation of Protein Homogenates
Protein was obtained from cultured granulosa cells by disrupting the cell membrane with radioimmunoprecipitation (RIPA) lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.5% NP-40, 20% glycerol, 25 mM benzamidine, 0.5 |xg/ml of leupeptin, 0.7 |xg/ml of pepstatin A, 2 |xg/ml of aprotinin, 10 |xg/ml of trypsin inhibitor). The cell lysate was then centrifuged for 20 min at 4°C and 16 000 X g. Similarly, protein was extracted from whole frozen ovaries using RIPA lysis buffer in a Dounce homog-enizer (Wheaton, Millville, NJ). After homogenization, samples were incubated for 30 min on ice and centrifuged for 20 min at 16 000 X g. Total protein concentrations were determined by a modification of the Bradford method (Bio-Rad Protein Assay; Bio-Rad Laboratories, Hercules, CA) using BSA standards prepared in an appropriate amount of RIPA buffer. Microplate absorbance readings were obtained at 595 nm. The remaining supernatant was snap-frozen and stored at — 80°C.
Determination of sGC protein levels was performed by immunoblot analysis. Briefly, the proteins were resolved by 7.5% polyacrylamide-SDS gel electrophoresis under reducing conditions. In each experiment, equal quantities of protein (20 |xg for homogenized ovarian samples and 10 |xg for granulosa cells) were loaded for each sample. Protein was then transferred electrophoretically from the gel onto nitrocellulose membranes. The blots were blocked with 5% milk-TBST (20 mM Tris-buffered saline, 0.05% Tween 20, pH 7.5) at room temperature for 1 h and incubated overnight at 4°C with diluted antisera specific to sGC aj (1:20 000) or Pj (1:5000) subunits (Sigma). These antisera were raised against regions of aj and pj subunits not present in a2 and p2 isoforms and have been shown to immunoprecipitate the a and p type 1, but not type 2, isoforms (unpublished data from commercial supplier). Blots were then washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig) G, followed by washing and detection of immunoreactivity by chemiluminescence detection methods (Pierce Biotechnology, Rockford, IL). Blots were then used to expose radiographic film to visualize immunoreactive signals.
Analysis of Immunoblot Data
The intensity of immunoblot signals was determined by digital image analysis of radiographs using the ImagePC program (Scion Corp., Frederick, MD). To allow statistical comparison of results from different blots, levels were normalized to the value of the eCG Time 0 group in each blot. Differences in protein levels among treatment groups were determined by one- or two-way analysis of variance, followed by the Tukey post-hoc test. A confidence level of P < 0.05 was considered to be statistically significant.
After fixation, ovaries were embedded in paraffin, and sections (thickness, 8 |xm) were cut and mounted on slides. The sections were then processed for immunohistochemical analysis similar to the method used in our previous report. Briefly, sections were deparaffinized with xylene and rehydrated in graded ethanol before being washed with doubledistilled water. To increase epitope exposure, sections were heated for 15 min in sodium citrate buffer (0.01 M, pH 6.0) in a microwave oven. The sections were cooled and washed with 0.01 M PBS (pH 7.2) and then blocked with 5% BSA in TBST for 1 h at room temperature. The sections were incubated overnight at room temperature with polyclonal antibody against either sGC aj and pj subunit (Sigma), respectively, developed in rabbits. After washing three times with PBS, the sections were incubated with a 1:200 dilution of fluorescence-labeled goat anti-rabbit IgG (Alexa Fluor-488; Molecular Probes, Inc., Eugene, OR) for 1 h at room temperature. The sections were observed using an epifluorescence Nikon Inverted Microscope (Nikon, Tokyo, Japan) equipped with a 496-nm excitation-wavelength filter. Specificity of the antibody was examined using normal rabbit serum (NRS) instead of primary antibody. The sections were counterstained with hematoxylin and mounted with coverslips.
Intensity of immunoreactive staining was scored by two independent observers using the following ratings: —, No staining detected; +, weak; + +, moderate; + + +, strong staining. Stages of follicular development were classified as follows: Primordial follicles contained an oocyte surrounded by a single layer of squamous granulosa cells, primary follicles contained an oocyte surrounded by a single layer of cuboidal granulosa cells, preantral follicles contained an oocyte surrounded by multiple layers of granulosa cells but lacking an antrum, antral follicles were those in which the oocyte was contained within multiple layers of granulosa cells in which an antrum was apparent, and preovulatory follicles (diameter >450 |xm) exhibited a large antrum and cumulus oophorus that projected into the antrum.