Gonadotropin-Releasing Hormone Antagonist: MATERIALS AND METHODS
Hormones and Reagents
The GnRH-a leuprolide acetate (Lupron), was a donation from Abbott Laboratories (Buenos Aires, Argentina). The original ampoule (2.8 mg/5 ml) was dissolved in saline solution. SYNTEX S.A (Buenos Aires) generously provided eCG (Novormon). HEPES, SDS, Antide [W-Ac-D-Nal1, D-pCl-Phe2, D-Pal3, Ser4, Nic-Lys5, D-Nic-Lys6, Leu7, Ipr-Lys8, Pro9, D-Ala10NH2; Nal-Lys antagonist] was purchased from Sigma Chemical Co. (St. Louis, MO). Dulbecco modified Eagle medium (DMEM, 4.5 g glucose/L), Ham F-12 nutrient mixture (F12), fungizone (250 |xg/ml), and gentamicine (10 mg/ml), were from Gibco Laboratories (Grand Island, NY). Polyclonal primary antibodies for BAX (N-20), cytochrome C (H-104), FAS (FL-335), and FASL (Q-20) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit antibody against bovine cytochrome P450scc was a generous gift from Dr. Anita H. Payne (Stanford, CA). Anti-rabbit secondary antibody conjugated with horseradish peroxidase was purchased from Sigma.
In Vivo GnRH Analog Treatment and Superovulation
General care and housing of rats was carried out at the Instituto de Biologia y Medicina Experimental in Buenos Aires. Prepubertal rats were from our own colony. Female Sprague-Dawley rats, 23-25 days old, were allowed food and water ad libitum, kept at a room temperature of 21-23°C, on a 12L:12D cycle. GnRH analogs were diluted in saline to the desired concentrations (GnRH-a, 0.5 ^g/rat; GnRH-ant, 5 ^g/rat) and injected in 0.1 ml of vehicle. Animals were injected s.c. with 0.1 ml eCG (25 IU/rat, control group) to induce multiple follicular growth. They were injected at Time 0 with LA, Ant, or both, and then at 12-h intervals during 48 h. LA, Ant, or both were given at the same dose. Control animals were injected with vehicle only. The last LA and Ant injections were administrated 3 h before rats were killed by cervical dislocation. The ovaries were removed and cleaned of adhering tissue in culture medium for subsequent assays. All animals were treated and cared for in accordance with standard international animal care protocols (e.g., Canadian Council of Animal Care, Guide to the Care and Use of Experimental Animals). The experimental protocols were approved by the Animal Experimentation Committee of the IBYME.
Ovarian Morphology and Apoptosis
To evaluate changes in general structure, representative ovaries from the control and treated groups were immediately fixed in 4% neutral buffered formalin for 12 h and then embedded in paraffin. Three-micrometer step sections were mounted at 50-^m intervals onto microscope slides to prevent counting the same follicle twice, according to the method described by Woodruff et al.. Slides were stained with hematoxylin-eosin to count the number of follicles and apoptotic cells per ovary section under a light microscope. The apoptotic cells were counted in 400X microscopic fields of preantral follicles (PFs), early antral follicles (EAFs), and preovulary follicles (POFs). Atresia was defined as the presence of more than 10 pyknotic nuclei per follicle; in the smallest follicles, the criteria for atresia was a degenerate oocyte, precocious antrum formation, or both. The number of preantral, antral, and atretic follicles was determined in ovarian sections obtained from animals (n = 8) after 48 h of vehicle injection or GnRH-a, GnRH-ant, or both. To study ovarian morphology or the number of apoptotic cells, five randomly selected fields were analyzed from each ovarian section (6 sections/ovary, 6-8 ovaries). Apoptotic cells were recognized in hematoxylin-eosin stained tissue sections on the basis of morphological criteria following the procedure described by Goyeneche et al.. The apoptotic index was calculated as the number of death cells from 100 cells per follicle in EAFs and POFs. In PFs, 70 cells were counted per follicle. Apoptotic index is expressed in percentages.
Follicle Isolation and Incubation
Healthy preovulatory follicles (>400 |xm in diameter) from 12 ovaries were dissected microscopically using fine needles. The culture was initiated within 1 h of ovary removal. For DNA, four follicles per glass vial were incubated during 24 h under serum-free conditions at 37°C in 500 |xl of DMEM:F12 (1:1), containing 10 mM HEPES, supplemented with fungizone (250 |xg/ml) and gentamicine (10 mg/ml). Follicles were gassed with 95% O2-5% CO2 at the start of culture. This model has the advantage of maintaining the integrity of the follicle. In addition, the incubation in serum-free conditions during 24 h allows it to exhibit the typical apoptotic DNA ladder: presence of internucleosomal fragments of 180 base-pair multiples.
DNA Isolation and Fragmentation Analysis
Cellular DNA was extracted from follicles incubated for 24 h under serum-free conditions in 500 |xl of DMEM supplemented with streptomycin and gentamicine in the absence of hormones as previously described. Briefly, the follicles from each culture were homogenized in a buffer containing 100 mM NaCl, 4 mM EDTA, 50 mM Tris-HCl, 0.5% SDS pH 8, and proteinase K (100 |xg/ml) at 55°C for 4 h to facilitate membrane and protein disruption. After incubation, samples were cooled for 30 min on ice in 1 M potassium acetate and 50% chloroform to initiate protein precipitation, and centrifuged at 9000 X g for 8 min at 4°C. Su-pernatants were then precipitated for 30 min in 2.5 volumes of ethanol at -70°C and centrifuged for 20 min at 5000 X g at 4°C. Finally, samples were extracted in 70% ethanol and resuspended in water. DNA content was measured by reading the absorbance at 260 nm, and incubated for 1 h with RNase (10 |xg/ml) at 37°C. DNA samples (4 |xg) were electropho-retically separated on 1.9% agarose gels containing ethidium bromide (0.4 |xg/ml) in Tris-borate-EDTA buffer. Within each agarose gel, equal amounts of DNA were loaded into each well. To enhance sensitivity, gels were further stained with ethidium bromide for 15 min. DNA was visualized with a UV (302 nm) transilluminator, and photographed with a Polaroid camera system. Densitometric analysis of low-molecular-weight (<15 kilobase) DNA was performed with an Image Scanner (Genius) using the software program Image Quant (Molecular Dynamics). Quantitative results obtained by densitometric analysis of the low-molecular-weight DNA fragments represent the mean ± SEM of three or four independent gel runs.
Preparation of Mitochondria and Cytosolic Fractions
Fresh (t0), healthy preovulatory follicles were resuspended in ice-cold separation buffer (200 mM mannitol, 50 mM sucrose, 10 mM KCl, 1 mM EDTA, 10 mM Hepes-KOH pH 7.4, 1 mM PMSF, 10 ^g/ml leupeptin, 10 |xg/ml aprotinin), and homogenized with an Ultra-Turrax (IKA Werk, Breisgau) homogenizer. Samples were centrifuged in Eppendorf tubes (at 900 X g for 5 min at 4°C) to remove nuclei, followed by centrifugation at 10 000 X g (25 min at 4°C) to obtain a membrane pellet enriched in mitochondria. The supernatant was collected and used as the cytosolic fraction. The mitochondrial pellet was resuspended in 20 |xl of PBS, 0.2% Triton X-100. The resuspended mitochondrial fraction and the cytosolic fraction were either used immediately or stored at —70°C. Mitochondrial preparation efficiency (mitochondrial fraction/[cytosol + mitochondrial fractions] = 0.80) was estimated by the presence of P450scc enzyme known to be specific for the mitochondrial membrane. Protein concentration was measured by the Bradford assay.
Proteins were resolved on 15% SDS-PAGE and transferred to a nitrocellulose membrane. The blot was preincubated in blocking buffer (5% nonfat dry milk, 0.05% Tween-20 in 20 mM TBS pH 8.0) for 1 h at room temperature and incubated with appropriate primary antibodies (FAS, FASL, BAX, cytochrome C, P450scc) in blocking buffer for 1 h at room temperature. Then, it was incubated with anti-rabbit secondary antibody conjugated with horseradish peroxidase and finally detected by chemilu-minescence and autoradiography using x-ray film. Negative controls were obtained in the absence of the primary antibody.
Quantification for Western Blot Assay
The loading protein ranged from 20 to 40 |xg for both antibodies. In each experiment, equal amounts of protein were loaded for all samples, and all groups in one experiment were loaded on the same gel. For quantification, a screening was performed on blots with x-ray film using different times of exposure to optimize the signal. The levels of protein were compared in extracts from the different groups, and analyzed by densito-metric studies. Optical density data are expressed as arbitrary units ± SEM (n = 3). Data are expressed as mean (cytosol or mitochondria fraction)/ [cytosol + mitochondrial fractions]) for both proteins analyzed (BAX and cytochrome C).
The proper loading was evaluated by staining the membranes with Ponceau-S. As an internal control, the density of each protein was normalized to the density of a band that was observed in the protein transference pattern in all Ponceau-S stained membranes. This band was selected because it was unchanged under the different treatments (data not shown).
Data are expressed as the mean ± SEM of triplicate incubations of at least three experiments using six animals per group. Representative gels are shown in the figures. Statistical analyses were performed using oneway analysis of variance followed by either the Tukey or Newman-Keuls Multiple test. Values of P < 0.05 were considered significant.