Cross-Talk between Fas/Fas Ligand System and Nitric Oxide in the Pathway Subserving Granulosa Cell Apoptosis: A Possible Regulatory Mechanis
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内分泌学杂志 2005年第2期
Department of Obstetrics and Gynecology, Faculty of Medicine, The University of Tokyo, Tokyo 113-8655, Japan
Address all correspondence and requests for reprints to: Dr. Tetsu Yano, Associate Professor, Department of Obstetrics and Gynecology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: tetu-tky@umin.ac.jp.
Abstract
Recent studies have shown the involvement of Fas/Fas ligand (FasL) system and nitric oxide (NO) in ovarian follicle atresia. Here we asked whether Fas/Fas ligand system interacts with NO using rat granulosa cell culture. Soluble recombinant Fas ligand (rFasL), at 100 ng/ml, significantly decreased cell viability, as measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, in the presence of 200 U/ml interferon-, whereas the concurrent addition of a caspase inhibitor, Z-VAD-FMK, at 20 μM, significantly inhibited rFasL-induced cytotoxicity. Hoechst 33342 staining and flow cytometric analysis confirmed the induction of apoptosis in granulosa cells by 100 ng/ml rFasL in the presence of interferon-, which was blocked by the concomitant addition of an NO donor, S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated that rFasL significantly up-regulated caspase-3, -8, and -9 activities in granulosa cells, which were attenuated by concurrent treatment with S-nitroso-N-acetylpenicillamine. Real-time quantitative RT-PCR revealed a significant decrease in inducible NO synthase mRNA levels in rFasL-induced apoptotic granulosa cells. In conclusion, we demonstrated the involvement of Fas/FasL system in inducing apoptosis through activation of a caspase-mediated cascade in rat granulosa cells, which is coupled with a decrease in inducible NO synthase expression. We further showed that NO inhibited Fas/FasL system-induced apoptosis by suppressing activation of the caspases, pointing to a cross-talk between Fas/FasL system-induced apoptosis pathway and NO-mediated antiapoptotic pathway in ovarian follicle atresia.
Introduction
MORE THAN 99.9% of ovarian follicles undergo the degenerative change known as atresia at varying stages of follicular development (1). Over the past decade, a substantial set of studies (2, 3, 4) suggested that apoptosis, a physiological form of cell death, is an underlying mechanism of ovarian follicle atresia. Although many researchers have attempted to determine mechanisms that regulate apoptotic cell death during follicular atresia, the precise molecular system that controls the initiation and progression of apoptosis in granulosa cells remains to be clarified.
Fas (APO-1/CD95) is a transmembranous glycoprotein that belongs to the TNF receptor superfamily and mediates apoptosis in a variety of lymphoid and tumor cells after the binding of Fas ligand (FasL) (5, 6). Fas was detected in human granulosa/luteal cells isolated from patients at the time of ovum retrieval for in vitro fertilization and an antihuman Fas monoclonal antibody-induced apoptosis in these cells by binding to Fas on them (7). We also reported that the interaction of Fas on granulosa cells and FasL on and/or from oocytes, by triggering apoptosis in granulosa cells, may function as a part of complex process of ovarian follicle atresia (8). Other studies (9, 10, 11, 12) demonstrated the presence of both Fas and FasL in granulosa cells and the dynamic changes in their expression during follicular development and atresia, proposing the autocrine/paracrine system of apoptosis within granulosa cells themselves. Binding of the adaptor protein, Fas-associated death domain protein (termed FADD), to the cytoplasmic death domain of Fas activates the caspase cascade, resulting in apoptotic cell death (13, 14). Recently several studies (15, 16, 17, 18, 19, 20) have shown important roles of caspase-3, -8, and -9 in executing granulosa cell apoptosis.
Endogenously generated nitric oxide (NO) synthesized from L-arginine by NO synthase is a mediator of a variety of physiological and pathological phenomena (21, 22). Current studies have shown that NO manifests both pro- and antiapoptotic properties and that endogenous NO synthesis or exposure to appropriate amounts of NO inhibits apoptosis in diverse cell types (23). In the ovary, NO has been considered to be involved in the regulation of several physiological functions such as steroidogenesis, ovulation, and folliculogenesis (24, 25, 26). Worth emphasizing is a report that demonstrated inhibition of follicular apoptosis by the addition of NO generator in cultured preovulatory follicles (24). Our own recent studies (27, 28, 29) also revealed that inducible NO synthase (iNOS) is predominantly localized in granulosa cells of healthy immature follicles in the rat ovary and suggested a role of NO as a cytostatic factor in granulosa cells.
Ovarian follicular development and atresia are delicately regulated by the complex cross-talk of cell death and cell survival signals (2, 3, 4). However, little is known about the interaction between Fas/FasL system-induced apoptosis pathway and NO-mediated antiapoptotic pathway in the ovarian follicle. In the present study, we attempted to examine the impact of exogenous NO on Fas/FasL system-induced apoptosis pathway and determine the change in iNOS expression levels in response to the stimulation of Fas/FasL system in cultured rat granulosa cells.
Materials and Methods
Chemicals
Soluble recombinant Fas ligand (rFasL) was purchased from Upstate Biotechnology (Lake Placid, NY). All other chemicals, unless otherwise mentioned, were obtained from Sigma Chemical Co. (St. Louis, MO).
Preparation and culture of granulosa cells
Guidelines for the care and use of laboratory animals as adopted and promulgated by the University of Tokyo were followed. Twenty-five-day-old immature female Wistar rats were purchased from Takasugi Experimental Animal Inc. (Saitama, Japan) and housed in a temperature-controlled room with a 12-h light, 12-h dark schedule. Pelleted food and water were provided ad libitum. Rats were injected ip with 10 IU pregnant mare serum gonadotropin (Teikokuzoki, Tokyo, Japan) in 0.2 ml saline and killed 48 h later by cervical dislocation. Removed ovaries were immediately cleaned of surrounding connective tissues, and granulosa cells were collected from ovarian follicles as previously described (8). Granulosa cells were suspended in RPMI 1640 medium (Invitrogen Corp., Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B, unless otherwise stated, and cultured in a humidified atmosphere of 5% CO2 in 95% air at 37 C. It is known that pretreatment or cotreatment with interferon (IFN)- increases Fas expression on granulosa cells (7, 8, 30). Therefore, granulosa cells were cultured in media containing 200 U/ml IFN (30) for Fas-mediated apoptosis to be potentiated. After 24 h pretreatment with IFN, cells were incubated with rFasL or S-nitroso-N-acetyl-DL-penicillamine (SNAP), an NO donor, in the presence of 200 U/ml IFN during the whole period of treatment. Cultured granulosa cells treated with IFN alone were considered as control. Twenty-six animals were used in each preparation of all the following experiments.
Cell viability assay
Cell viability was examined by using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay kit (CellTiter 96 Aqueous One solution cell proliferation assay; Promega Corp., Madison, WI) according to the manufacturer’s instructions. Briefly, granulosa cells were seeded into 96-well plates (Becton Dickinson and Co., Franklin Lakes, NJ) at a density of 5 x 104 cells/well in 200 μl of the culture medium. After 24 h, the medium was replaced with fresh medium containing 5% FBS and 200 U/ml recombinant rat IFN (Genzyme/Techne, Minneapolis, MN). After an additional 24 h, granulosa cells were exposed to 100 ng/ml rFasL (31), and cell culture was continued for a further 24, 48, or 72 h. Some cells were pretreated with Z-VAD-FMK (Promega), a caspase inhibitor (32), at 20 μM for 30 min before addition of rFasL. Finally, the medium was replaced with 100 μl of fresh medium containing 20 μl of MTS solution and incubated for an additional 2 h. Mitochondrial dehydrogenase enzymes of viable cells converted MTS tetrazolium into a colored formazan product. The OD of samples was read at 492 nm in the DigiScan microplate reader (ASYS Hitech GmbH, Eugendorf, Austria).
Hoechst 33342 staining
Hoechst staining was performed to confirm the apoptotic profile as a result of morphological change in the nucleus in which Hoechst 33342 binds specifically to the A-T base region in DNA and emits fluorescence. Granulosa cells were seeded into 16-well chamber slides (Nalge Nunc, Naperville, IL) at a density of 2 x 104 cells/well in 200 μl of the culture medium. After 24 h, the medium was replaced with fresh medium containing 5% FBS and 200 U/ml recombinant rat IFN. After an additional 24 h, 100 ng/ml rFasL and 0.5 mM SNAP, an NO donor, were added to indicated wells. After 72 h incubation, granulosa cells were rinsed in PBS (pH 7.4) and fixed with 4% paraformaldehyde in PBS at room temperature for 30 min. Then cells were rinsed in PBS twice and stained with Hoechst 33342 (10 μg/ml in PBS) for 3 min. The specimens were mounted with Vectashield medium (Vector Laboratories Inc., Burlingame, CA), and photographs were taken at x200 magnification under a fluorescent microscope (Olympus, Tokyo, Japan). The proportion of cells with nuclear fragmentation was calculated by counting the number of stained cells per more than 200 cells. Three different individuals made these observations three times each.
Flow cytometry
Granulosa cells were seeded into a 10-cm culture dish (Iwaki, Tokyo, Japan) at a density of 2 x 106 cells/dish in 10 ml of the culture medium. After 24 h, the medium was replaced with fresh medium containing 5% FBS and 200 U/ml recombinant rat IFN. After an additional 24 h, granulosa cells were treated with 100 ng/ml rFasL and SNAP at concentrations of 0.05, 0.2, and 0.5 mM (29), and cell culture was continued for a further 72 h. Then the cells were harvested by trypsin (0.05%)/EDTA (0.02%), washed twice with ice-cold PBS (pH 7.4), and fixed with 70% ethanol at –20 C overnight. After washing twice with ice-cold PBS, the cells were incubated in 0.25 mg/ml ribonuclease solution (QIAGEN GmbH, Hilden, Germany) for 30 min at 37 C and stained with 50 μg/ml propidium iodide for 30 min on ice, followed by filtration through a 40-μm nylon mesh (Becton Dickinson) to remove cell clumps. A total of 20,000 stained cells per treatment were analyzed in the EPICS XL flow cytometry (Beckman Coulter, Inc., Fullerton, CA). Sub-G1 phase represents low-molecular-weight DNA derived from apoptotic cells.
Western blotting
Granulosa cells were seeded into a 10-cm culture dish at a density of 2 x 106 cells/dish in 10 ml of the culture medium. After 24 h, the medium was replaced with fresh medium containing 5% FBS and 200 U/ml recombinant rat IFN. After an additional 24 h, the medium was replaced with fresh medium containing 100 ng/ml rFasL with or without 0.5 mM SNAP. After a further 72 h, granulosa cells were harvested with trypsin (0.05%)/EDTA (0.02%) and scraped into lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, and 0.5% sodium deoxycholate for 60 min on ice. Insoluble material was removed by centrifugation at 12,000 x g for 20 min at 4 C. The supernatants were recovered, and the protein concentrations were measured using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Equivalent amounts of lysate protein (100 μg) were subjected to 15% SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes (Millipore Corp., Billerica, MA) by using the Bio-Rad semidry electrophoretic transfer cell. After blocking nonspecific binding sites by incubation for 1 h with Tris-buffered saline [25 mM Tris and 150 mM NaCl (pH 7.6)] containing 5% nonfat milk and 0.1% Tween 20, the filters were blotted with the rabbit polyclonal antibody to cleaved caspase-3 and -9 (Cell Signaling Technology, Inc., Beverly, MA), caspase-8 (H-134), and Fas (M-20) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 C. Reactive proteins were detected with the horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences, Little Chalfont, UK) for 60 min at room temperature and developed with ECL Plus Western blotting detection reagents (Amersham Biosciences). The membranes were stripped with the buffer containing 100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate and 62.5 mM Tris-HCl (pH 6.7) and then reprobed with the polyclonal antibody of actin (Santa Cruz Biotechnology) to confirm equivalent protein loading. Densitometry was performed on Fas and caspase cleavage product bands (P17 and P19 for caspase-3, P18 for caspase-8, and P17 for caspase-9) by the fluorescence scanning system STORM (Molecular Dynamics, Sunnyvale, CA). Values were normalized to actin levels on the corresponding reprobed filters and then expressed as a percentage of the control value.
Reverse transcription (RT) and real-time PCR
Granulosa cells were seeded into a 10-cm culture dish at a density of 2 x 106 cells/dish in 10 ml of the culture medium. After 24 h, the medium was replaced with fresh medium containing 5% FBS and 200 U/ml recombinant rat IFN. After an additional 24 h, granulosa cells were treated with 100 ng/ml rFasL, and cell culture was continued for a further 72 h. RNA was isolated from cultured granulosa cells by the acid guanidinium-phenol-chloroform method using ISOGEN (Nippongene, Toyama, Japan). First-strand cDNA was synthesized in a reaction volume of 15 μl containing 5 μg total RNA and 0.2 μg of random hexamer primers by using the first-strand cDNA synthesis kit (Amersham Biosciences) according to the manufacturer’s instructions. After the RT reaction, cDNA was amplified to determine iNOS expression using the following primer pair: iNOS, 5'-CATGGTGAACACGTTCTTGG-3' (sense) and 5'-GTGGTGACAAGCACATTTGG-3' (antisense). Expression of iNOS mRNA was normalized to RNA loading for each sample using glycerol-3-phosphate dehydrogenase (G3PDH) mRNA as an internal standard. The primers of G3PDH were used as follows: G3PDH, 5'-ACCACAGTCCATGCCATCAC-3' (sense) and 5'-TCCACCACCCTGTTGCTGTA-3' (antisense).
Real-time PCR was performed using the LightCycler (Roche Applied Science, Mannheim, Germany) in 20 μl including 1.6 mM MgCl2, 2 μl LightCycler-FastStart reaction mix SYBR Green 1 (Roche Applied Science), 0.25 μM of each primer, and 50 ng cDNA from RT reactions as template. After an initial denaturation at 95 C for 10 min, the amplification program for iNOS and G3PDH consisted of 40 cycles of denaturation for 15 sec at 95 C, annealing for 10 sec at 62 C, and extension for 18 sec at 72 C. Finally, the temperature was raised gradually (0.2 C/sec) from the annealing temperature to 95 C for the melting curve analysis.
The samples from cultured granulosa cells treated with or without rFasL were run in triplicate and analyzed as follows. The concentrations of the samples were extrapolated from the standard curve by LightCycler software. Exogenous cDNA standards for iNOS and G3PDH were produced by inserting PCR products, which were generated using sample primers noted above and granulosa cell cDNA as templates, into the pCR2.1 vector using the TOPO TA cloning kit (Invitrogen). The concentration of each standard was determined by measuring the OD260, and the copy number was calculated. Relative expression levels of iNOS were calculated by subtracting the signal threshold cycle of the internal standard (G3PDH) from the threshold cycle of iNOS. The PCR products (444 bp size) were characterized by using a DNA sequencer (ABI PRISM 310 genetic analyzer; PerkinElmer Applied Biosystems).
Statistical analysis
Data represent the mean ± SEM from at least three independent experiments using separate groups of rats (26 animals/group). Statistical analyses were carried out by Mann-Whitney U test for paired comparison and one-way ANOVA with post hoc test for multiple comparisons by using StatView software (SAS Institute Inc., Cary, NC). Repeated-measures ANOVA was applied for analysis of time course study of the effect of rFasL on granulosa cell viability. P < 0.05 was considered statistically significant.
Results
Effect of rFasL on cultured granulosa cell viability
The effect of rFasL on granulosa cell viability was examined by MTS assay. As shown in Fig. 1A, treatment of granulosa cells with 100 ng/ml rFasL in the presence of 200 U/ml IFN decreased granulosa cell viability in a time-dependent manner. At 48 and 72 h, the cell viability was significantly reduced to 86.7 ± 2.3 and 77.6 ± 1.7% of baseline (0 h). Control incubation at 24, 48, and 72 h did not result in significant loss of viability. To investigate whether cell death induced by rFasL is associated with the activation of caspase cascade, Z-VAD-FMK (20 μM), a caspase inhibitor, was added 30 min before rFasL administration, and the combined treatment was continued for 72 h. The rFasL-induced granulosa cell death was inhibited by concurrent treatment with Z-VAD-FMK (98.4 ± 5.2% of the control) (Fig. 1B).
FIG. 1. Effects of rFasL and Z-VAD-FMK, a caspase inhibitor, on cultured granulosa cell viability estimated by MTS assay. A, Relative viability was calculated based on percentage absorbance of the samples (rFasL-treated) and untreated control at 24, 48, and 72 h with respect to that of the 0 h cultures (baseline) in media with 5% FBS in the presence of 200 U/ml IFN. Results are shown as the mean percentage of the untreated control (IFN alone) at 0 h ± SEM of eight wells of five independent experiments using separate groups of rats. *, P < 0.05 vs. 0 h. B, Z-VAD-FMK (20 μm) inhibited rFasL-induced granulosa cell apoptosis at 72 h of the treatment. Results are shown as the mean percentage of the untreated control (IFN alone) at 72 h ± SEM. *, P < 0.05 vs. control and rFasL + Z-VAD-FMK.
Effect of SNAP on rFasL-induced apoptosis in cultured granulosa cells
The effects of rFasL and SNAP, an NO donor, on the incidence of apoptotic cells were determined by Hoechst 33342 nuclear staining and flow cytometry in cultured granulosa cells. The apoptotic cells exhibiting shrunken nuclei, chromatin condensation, and nuclear fragmentation were recognized by Hoechst 33342 nuclear staining. The in vitro induction of granulosa cell apoptosis by 100 ng/ml rFasL was markedly inhibited by the addition of 0.5 mM SNAP (Fig. 2, A–C). The exact frequency of cells with nuclear fragmentation by Hoechst 33342 staining is shown in Fig. 2D. The percentage of dead cells was significantly increased after 72 h of incubation with rFasL (18.2 ± 0.7%; P < 0.05 vs. control), compared with that in the control (6.5 ± 0.8%), and this increment was suppressed by concurrent supplement with SNAP (6.2 ± 0.7%; P < 0.05 vs. rFasL alone). Figure 3A is a representative result of three independent experiments showing the proportion of sub-G1 phase, an apoptotic cell fraction. As shown in Fig. 3B, flow cytometric analysis revealed that the proportion of sub-G1 phase was significantly increased by the addition of rFasL (19.6 ± 1.4%; P < 0.05 vs. control), compared with that for the control (8.1 ± 0.6%) at 72 h of treatment. The increased proportion of sub-G1 phase by rFasL was dose-dependently suppressed by SNAP at 0.05, 0.2, and 0.5 mM to 17.1 ± 1.5, 10.3 ± 0.5, and 8.0 ± 0.5%, respectively.
FIG. 2. Hoechst 33342 staining of 100 ng/ml rFasL-treated granulosa cells with or without SNAP, an NO donor, in the presence of 200 U/ml IFN (magnification, x200). Twenty-six rats were killed for preparation of granulosa cells; a part of granulosa cells were cultured in 16-well chamber slide for Hoechst 33342 staining. A, Control (IFN alone). B, Treatment with rFasL increased the rate of cells with nuclear fragmentation at 72 h. C, Addition of SNAP (0.5 mM) suppressed rFasL-induced apoptosis. D, The frequency of cells with nuclear fragmentation by Hoechst 33342 staining was calculated by counting the number of stained cells per more than 200 cells. Results are shown as the mean ± SEM of three independent experiments using separate groups of rats. *, P < 0.05 vs. control and rFasL + SNAP.
FIG. 3. DNA histograms of 100 ng/ml rFasL-treated granulosa cells with or without SNAP at concentrations of 0.05, 0.2, and 0.5 mM in the presence of 200 U/ml IFN. At 72 h of the treatment, cells were stained with propidium iodine (PI) and analyzed by flow cytometry. Sub-G1 indicates an apoptotic cell fraction. A, The result of flow cytometric analysis is representative of three independent experiments. B, Effects of rFasL and SNAP on the proportion of sub-G1 phase. Results are shown as the mean ± SEM of three independent experiments using separate groups of rats. *, P < 0.05 vs. control (IFN alone).
Effect of SNAP on Fas expression in cultured granulosa cells
As shown in Fig. 4, Western blot analysis revealed that the expression level of Fas was significantly increased to 188.5 ± 9.5% of the control after treatment with 100 ng/ml rFasL for 72 h (P < 0.05 vs. control). This increment was not significantly changed by concurrent addition of 0.5 mM SNAP.
FIG. 4. Western blot analysis of Fas expression in 100 ng/ml rFasL-treated granulosa cells with or without SNAP (0.5 mM), an NO donor, in the presence of 200 U/ml IFN. At 72 h of the treatment, cell protein lysates were analyzed by immunoblotting with anti-Fas antibody. A, The result is representative of three independent experiments. B, Results show quantitative analysis of Fas protein level and are expressed as the mean percentage of the untreated control (IFN alone) ± SEM of three independent experiments using separate groups of rats. *, P < 0.05 vs. control.
Effect of SNAP on rFasL-induced caspase activation in cultured granulosa cells
Cleaved caspase protein levels were evaluated by Western blot analysis. Figure 5, A–C (left) illustrates representative results, exhibiting the 19- and 17-kDa cleavage fragments of activated caspase-3, the 18-kDa cleavage fragments of activated caspase-8, and 17-kDa cleavage fragments of activated caspase-9, respectively. As shown in Fig. 5, A–C (right), the expression levels of cleaved caspase-3, -8, and-9 were significantly up-regulated to 201.7 ± 6.0, 158.8 ± 12.1, and 141.3 ± 15.7% of the control, respectively, after treatment with 100 ng/ml rFasL for 72 h, and these increments were attenuated by concurrent addition of 0.5 mM SNAP (120.7 ± 5.2, 112.3 ± 2.0, and 105.8 ± 2.9%, respectively; P < 0.05 vs. rFasL alone).
FIG. 5. Western blot analysis of protein levels of cleaved caspase-3, -8, and -9 in 100 ng/ml rFasL-treated granulosa cells with or without SNAP (0.5 mM), an NO donor, in the presence of 200 U/ml IFN. At 72 h of the treatment, cell protein lysates were analyzed by immunoblotting with anticleaved caspase-3, anti-caspase-8, and anti-cleaved caspase-9 antibody, which recognizes active cleavage fragments of 19 and 17 kDa (A), 18 kDa (B), and 17 kDa (C), respectively. Results on the left are representative films exhibiting bands for active forms of caspases with corresponding actin bands. Results on the right show quantitative analysis of caspase-3, -8, and -9 cleavage into active forms. Results are shown as the mean percentage of the untreated control (IFN alone) ± SEM of three independent experiments using separate groups of rats. *, P < 0.05 vs. control and rFasL + SNAP.
Effect of rFasL on iNOS expression in cultured granulosa cells
The effect of rFasL on iNOS mRNA levels was investigated by real-time quantitative RT-PCR in cultured granulosa cells. After 72 h culture of granulosa cells with 100 ng/ml rFasL, iNOS mRNA levels were significantly decreased to 61.1 ± 6.1% of the control (P < 0.05).
Discussion
It has been documented that Fas/FasL system is one of the critical mediators of apoptosis in ovarian follicle atresia (3, 4, 7, 8, 9, 10, 11, 12). In the present study, using cultures of rat granulosa cells, we demonstrated that Fas/FasL-mediated apoptosis is accompanied by a remarkable increase in caspase-3, -8, and -9 activities and a concomitant decrease in iNOS expression and that exogenous NO inhibits Fas/FasL-mediated apoptosis in association with an attenuation of caspase cascade activation.
The process of apoptosis was characterized by nuclear shrinkage, plasma membrane blebbing, chromatin condensation, and internucleosomal DNA fragmentation. Concomitant with these changes is the activation of a cascade of intracellular proteolytic events catalyzed by a family of cysteine aspartate proteases known as caspases (33, 34, 35). Caspases consisting of at least 14 isoforms are activated in response to multiple apoptotic stimuli and propagate death signals by cleaving a number of cellular protein substrates. These substrates include inactive procaspase zymogens that are cleaved into active proteases leading ultimately to apoptosis. Activated caspases specifically cleave the target substrates on the carboxyl side of aspartate residues. Caspase-8, -9, and -10 are initiator caspases that can transduce apoptotic signals by directly activating the downstream effector caspase-3, -6, and -7. Two pathways have been described for Fas-induced apoptosis (14). First, the assembly of a membrane-bound death-inducing signaling complex (DISC) is initiated by Fas-FasL interaction and subsequent receptor trimerization (14). Then the adaptor protein FADD binds via its own death domain to the death domain in Fas. Interaction of the death domain of Fas with that of FADD recruits procaspase-8 to complete the DISC, which triggers autoprocessing of the enzyme. Active caspase-8 can both directly activate downstream caspases such as caspase-3 (type I) and induce mitochondrial apoptotic signaling, including mitochondrial cytochrome c release (type II). In type II pathway, cytosolic cytochrome c induces formation of the apoptosome by stimulating oligomerization of the adapter protein (Apaf-1), apoptotic protease-activating factor 1 and, consequently, recruitment of the initiator caspase-9 (36). As a result, activated caspase-9 can then directly cleave and activate caspase-3. In the immune system, binding of FasL to Fas causes apoptosis by activating the caspase cascade, resulting in the cleavage and activation of caspase-3 (13, 14). Previously we demonstrated that activation of the Fas/FasL system was capable of initiating apoptosis in cultured rat granulosa cells (8). The present study showed the prevention of Fas/FasL-mediated granulosa cell death by a caspase inhibitor, Z-VAD-FMK, and a positive correlation between the enzyme activity of caspases and the occurrence of apoptosis in granulosa cells.
These findings are in accordance with other observations that caspase-3 mRNA and its protein were detected in rat granulosa cells during atresia and down-regulated by gonadotropin-promoted follicular survival (15, 16). The notion that active caspase-3 is a key functional participant in granulosa cell death program was reinforced by the study demonstrating the presence of aberrant atretic follicles in caspase-3-null mouse ovaries (18).
NO can prevent or induce apoptosis, depending on its concentration, cell type, and the oxidative milieu (23, 37). Increasing evidence has demonstrated NO as a potential regulator of follicular development (24, 25, 26, 27, 28, 29). It was reported that administration of SNAP, an NO donor, directly inhibited spontaneously occurring apoptosis in cultured rat granulosa cells, suggesting that NO acts as a follicle survival factor (24, 28). Recently we showed that iNOS mRNA levels in rat granulosa cells were decreased by administration of GnRH agonist, an atretogenic agent for ovarian follicles or epidermal growth factor, a mitogenic and antiapoptotic factor, and that treatment with SNAP attenuates both GnRH agonist-induced apoptosis and epidermal growth factor-induced DNA synthesis in granulosa cells (29). In the present study, iNOS mRNA levels were decreased in granulosa cells exposed to proapoptotic stimulation by rFasL, whereas treatment with SNAP suppressed Fas/FasL-mediated apoptosis in a dose-dependent manner. The present data strengthen our hypothesis that endogenous NO synthesized by iNOS in granulosa cells acts as a cytostatic factor in ovarian follicles, with a loss of NO being associated with the conversion into a different developmental status in whichever direction (i.e. progression into differentiation or demise via apoptotic process) (27, 28, 29).
To further address the role of NO in the regulation of ovarian follicle atresia, the impact of NO on rFasL-induced death pathway was examined. Our present study revealed that NO inhibits Fas/FasL system-mediated apoptosis, at least in part, by directly interfering with caspase-8, leading to the down-regulation of caspase-9 and -3 activities. However, we cannot exclude the possibility that NO directly inhibits the activation of caspase-9 and/or -3. Similar findings were reported using other cell types, in which NO inhibits apoptosis by inactivating several members of the caspase family through S-nitrosylation of a cysteine thiol at the catalytic site of caspase (37, 38, 39, 40, 41, 42, 43, 44, 45, 46). On the other hand, it was reported that the Fas/FasL system activates caspase-3 not only by inducing the cleavage of the caspase zymogen to its active subunits but also by stimulating the denitrosylation of its active-site thiol (47). Thus, caspase S-nitrosylation/denitrosylation appears to regulate Fas/FasL-mediated death pathway. Recent studies also revealed various NO-mediated inhibitory mechanisms of follicular apoptosis (24, 48, 49). It has recently been shown that NO might inhibit Fas-mediated apoptosis through a decreased expression of Fas on granulosa cells (49). However, in our additional study, Western blot analysis failed to detect the inhibitory effect of SNAP on Fas expression at the same concentration as SNAP inhibited Fas-mediated apoptosis. The most likely explanation for this discrepancy may be the difference of experimental designs. It might be possible that stimulation of cGMP production (24) and modulation of apoptosis- and antiapoptosis-related gene expression such as induction of heat shock protein 70 (48), suppression of Bax expression (48), and stimulation of Bcl-2 expression (49) ultimately prevent activation of the effector caspase-3 in granulosa cells (23, 41, 50).
In conclusion, using rat granulosa cell culture system, we have demonstrated that there is a cross-talk between Fas/FasL system-induced apoptosis pathway and NO-mediated antiapoptotic pathway in the ovarian follicle. The shift in a balance between cell death and cell survival signaling pathways in granulosa cells might determine the fate of ovarian follicles. Further studies on the precise molecular mechanisms underlying the alterations of this balance would provide clues to devise diagnostic and therapeutic strategies for various pathologic conditions of the ovary.
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Address all correspondence and requests for reprints to: Dr. Tetsu Yano, Associate Professor, Department of Obstetrics and Gynecology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: tetu-tky@umin.ac.jp.
Abstract
Recent studies have shown the involvement of Fas/Fas ligand (FasL) system and nitric oxide (NO) in ovarian follicle atresia. Here we asked whether Fas/Fas ligand system interacts with NO using rat granulosa cell culture. Soluble recombinant Fas ligand (rFasL), at 100 ng/ml, significantly decreased cell viability, as measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, in the presence of 200 U/ml interferon-, whereas the concurrent addition of a caspase inhibitor, Z-VAD-FMK, at 20 μM, significantly inhibited rFasL-induced cytotoxicity. Hoechst 33342 staining and flow cytometric analysis confirmed the induction of apoptosis in granulosa cells by 100 ng/ml rFasL in the presence of interferon-, which was blocked by the concomitant addition of an NO donor, S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated that rFasL significantly up-regulated caspase-3, -8, and -9 activities in granulosa cells, which were attenuated by concurrent treatment with S-nitroso-N-acetylpenicillamine. Real-time quantitative RT-PCR revealed a significant decrease in inducible NO synthase mRNA levels in rFasL-induced apoptotic granulosa cells. In conclusion, we demonstrated the involvement of Fas/FasL system in inducing apoptosis through activation of a caspase-mediated cascade in rat granulosa cells, which is coupled with a decrease in inducible NO synthase expression. We further showed that NO inhibited Fas/FasL system-induced apoptosis by suppressing activation of the caspases, pointing to a cross-talk between Fas/FasL system-induced apoptosis pathway and NO-mediated antiapoptotic pathway in ovarian follicle atresia.
Introduction
MORE THAN 99.9% of ovarian follicles undergo the degenerative change known as atresia at varying stages of follicular development (1). Over the past decade, a substantial set of studies (2, 3, 4) suggested that apoptosis, a physiological form of cell death, is an underlying mechanism of ovarian follicle atresia. Although many researchers have attempted to determine mechanisms that regulate apoptotic cell death during follicular atresia, the precise molecular system that controls the initiation and progression of apoptosis in granulosa cells remains to be clarified.
Fas (APO-1/CD95) is a transmembranous glycoprotein that belongs to the TNF receptor superfamily and mediates apoptosis in a variety of lymphoid and tumor cells after the binding of Fas ligand (FasL) (5, 6). Fas was detected in human granulosa/luteal cells isolated from patients at the time of ovum retrieval for in vitro fertilization and an antihuman Fas monoclonal antibody-induced apoptosis in these cells by binding to Fas on them (7). We also reported that the interaction of Fas on granulosa cells and FasL on and/or from oocytes, by triggering apoptosis in granulosa cells, may function as a part of complex process of ovarian follicle atresia (8). Other studies (9, 10, 11, 12) demonstrated the presence of both Fas and FasL in granulosa cells and the dynamic changes in their expression during follicular development and atresia, proposing the autocrine/paracrine system of apoptosis within granulosa cells themselves. Binding of the adaptor protein, Fas-associated death domain protein (termed FADD), to the cytoplasmic death domain of Fas activates the caspase cascade, resulting in apoptotic cell death (13, 14). Recently several studies (15, 16, 17, 18, 19, 20) have shown important roles of caspase-3, -8, and -9 in executing granulosa cell apoptosis.
Endogenously generated nitric oxide (NO) synthesized from L-arginine by NO synthase is a mediator of a variety of physiological and pathological phenomena (21, 22). Current studies have shown that NO manifests both pro- and antiapoptotic properties and that endogenous NO synthesis or exposure to appropriate amounts of NO inhibits apoptosis in diverse cell types (23). In the ovary, NO has been considered to be involved in the regulation of several physiological functions such as steroidogenesis, ovulation, and folliculogenesis (24, 25, 26). Worth emphasizing is a report that demonstrated inhibition of follicular apoptosis by the addition of NO generator in cultured preovulatory follicles (24). Our own recent studies (27, 28, 29) also revealed that inducible NO synthase (iNOS) is predominantly localized in granulosa cells of healthy immature follicles in the rat ovary and suggested a role of NO as a cytostatic factor in granulosa cells.
Ovarian follicular development and atresia are delicately regulated by the complex cross-talk of cell death and cell survival signals (2, 3, 4). However, little is known about the interaction between Fas/FasL system-induced apoptosis pathway and NO-mediated antiapoptotic pathway in the ovarian follicle. In the present study, we attempted to examine the impact of exogenous NO on Fas/FasL system-induced apoptosis pathway and determine the change in iNOS expression levels in response to the stimulation of Fas/FasL system in cultured rat granulosa cells.
Materials and Methods
Chemicals
Soluble recombinant Fas ligand (rFasL) was purchased from Upstate Biotechnology (Lake Placid, NY). All other chemicals, unless otherwise mentioned, were obtained from Sigma Chemical Co. (St. Louis, MO).
Preparation and culture of granulosa cells
Guidelines for the care and use of laboratory animals as adopted and promulgated by the University of Tokyo were followed. Twenty-five-day-old immature female Wistar rats were purchased from Takasugi Experimental Animal Inc. (Saitama, Japan) and housed in a temperature-controlled room with a 12-h light, 12-h dark schedule. Pelleted food and water were provided ad libitum. Rats were injected ip with 10 IU pregnant mare serum gonadotropin (Teikokuzoki, Tokyo, Japan) in 0.2 ml saline and killed 48 h later by cervical dislocation. Removed ovaries were immediately cleaned of surrounding connective tissues, and granulosa cells were collected from ovarian follicles as previously described (8). Granulosa cells were suspended in RPMI 1640 medium (Invitrogen Corp., Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B, unless otherwise stated, and cultured in a humidified atmosphere of 5% CO2 in 95% air at 37 C. It is known that pretreatment or cotreatment with interferon (IFN)- increases Fas expression on granulosa cells (7, 8, 30). Therefore, granulosa cells were cultured in media containing 200 U/ml IFN (30) for Fas-mediated apoptosis to be potentiated. After 24 h pretreatment with IFN, cells were incubated with rFasL or S-nitroso-N-acetyl-DL-penicillamine (SNAP), an NO donor, in the presence of 200 U/ml IFN during the whole period of treatment. Cultured granulosa cells treated with IFN alone were considered as control. Twenty-six animals were used in each preparation of all the following experiments.
Cell viability assay
Cell viability was examined by using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay kit (CellTiter 96 Aqueous One solution cell proliferation assay; Promega Corp., Madison, WI) according to the manufacturer’s instructions. Briefly, granulosa cells were seeded into 96-well plates (Becton Dickinson and Co., Franklin Lakes, NJ) at a density of 5 x 104 cells/well in 200 μl of the culture medium. After 24 h, the medium was replaced with fresh medium containing 5% FBS and 200 U/ml recombinant rat IFN (Genzyme/Techne, Minneapolis, MN). After an additional 24 h, granulosa cells were exposed to 100 ng/ml rFasL (31), and cell culture was continued for a further 24, 48, or 72 h. Some cells were pretreated with Z-VAD-FMK (Promega), a caspase inhibitor (32), at 20 μM for 30 min before addition of rFasL. Finally, the medium was replaced with 100 μl of fresh medium containing 20 μl of MTS solution and incubated for an additional 2 h. Mitochondrial dehydrogenase enzymes of viable cells converted MTS tetrazolium into a colored formazan product. The OD of samples was read at 492 nm in the DigiScan microplate reader (ASYS Hitech GmbH, Eugendorf, Austria).
Hoechst 33342 staining
Hoechst staining was performed to confirm the apoptotic profile as a result of morphological change in the nucleus in which Hoechst 33342 binds specifically to the A-T base region in DNA and emits fluorescence. Granulosa cells were seeded into 16-well chamber slides (Nalge Nunc, Naperville, IL) at a density of 2 x 104 cells/well in 200 μl of the culture medium. After 24 h, the medium was replaced with fresh medium containing 5% FBS and 200 U/ml recombinant rat IFN. After an additional 24 h, 100 ng/ml rFasL and 0.5 mM SNAP, an NO donor, were added to indicated wells. After 72 h incubation, granulosa cells were rinsed in PBS (pH 7.4) and fixed with 4% paraformaldehyde in PBS at room temperature for 30 min. Then cells were rinsed in PBS twice and stained with Hoechst 33342 (10 μg/ml in PBS) for 3 min. The specimens were mounted with Vectashield medium (Vector Laboratories Inc., Burlingame, CA), and photographs were taken at x200 magnification under a fluorescent microscope (Olympus, Tokyo, Japan). The proportion of cells with nuclear fragmentation was calculated by counting the number of stained cells per more than 200 cells. Three different individuals made these observations three times each.
Flow cytometry
Granulosa cells were seeded into a 10-cm culture dish (Iwaki, Tokyo, Japan) at a density of 2 x 106 cells/dish in 10 ml of the culture medium. After 24 h, the medium was replaced with fresh medium containing 5% FBS and 200 U/ml recombinant rat IFN. After an additional 24 h, granulosa cells were treated with 100 ng/ml rFasL and SNAP at concentrations of 0.05, 0.2, and 0.5 mM (29), and cell culture was continued for a further 72 h. Then the cells were harvested by trypsin (0.05%)/EDTA (0.02%), washed twice with ice-cold PBS (pH 7.4), and fixed with 70% ethanol at –20 C overnight. After washing twice with ice-cold PBS, the cells were incubated in 0.25 mg/ml ribonuclease solution (QIAGEN GmbH, Hilden, Germany) for 30 min at 37 C and stained with 50 μg/ml propidium iodide for 30 min on ice, followed by filtration through a 40-μm nylon mesh (Becton Dickinson) to remove cell clumps. A total of 20,000 stained cells per treatment were analyzed in the EPICS XL flow cytometry (Beckman Coulter, Inc., Fullerton, CA). Sub-G1 phase represents low-molecular-weight DNA derived from apoptotic cells.
Western blotting
Granulosa cells were seeded into a 10-cm culture dish at a density of 2 x 106 cells/dish in 10 ml of the culture medium. After 24 h, the medium was replaced with fresh medium containing 5% FBS and 200 U/ml recombinant rat IFN. After an additional 24 h, the medium was replaced with fresh medium containing 100 ng/ml rFasL with or without 0.5 mM SNAP. After a further 72 h, granulosa cells were harvested with trypsin (0.05%)/EDTA (0.02%) and scraped into lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, and 0.5% sodium deoxycholate for 60 min on ice. Insoluble material was removed by centrifugation at 12,000 x g for 20 min at 4 C. The supernatants were recovered, and the protein concentrations were measured using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Equivalent amounts of lysate protein (100 μg) were subjected to 15% SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes (Millipore Corp., Billerica, MA) by using the Bio-Rad semidry electrophoretic transfer cell. After blocking nonspecific binding sites by incubation for 1 h with Tris-buffered saline [25 mM Tris and 150 mM NaCl (pH 7.6)] containing 5% nonfat milk and 0.1% Tween 20, the filters were blotted with the rabbit polyclonal antibody to cleaved caspase-3 and -9 (Cell Signaling Technology, Inc., Beverly, MA), caspase-8 (H-134), and Fas (M-20) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 C. Reactive proteins were detected with the horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences, Little Chalfont, UK) for 60 min at room temperature and developed with ECL Plus Western blotting detection reagents (Amersham Biosciences). The membranes were stripped with the buffer containing 100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate and 62.5 mM Tris-HCl (pH 6.7) and then reprobed with the polyclonal antibody of actin (Santa Cruz Biotechnology) to confirm equivalent protein loading. Densitometry was performed on Fas and caspase cleavage product bands (P17 and P19 for caspase-3, P18 for caspase-8, and P17 for caspase-9) by the fluorescence scanning system STORM (Molecular Dynamics, Sunnyvale, CA). Values were normalized to actin levels on the corresponding reprobed filters and then expressed as a percentage of the control value.
Reverse transcription (RT) and real-time PCR
Granulosa cells were seeded into a 10-cm culture dish at a density of 2 x 106 cells/dish in 10 ml of the culture medium. After 24 h, the medium was replaced with fresh medium containing 5% FBS and 200 U/ml recombinant rat IFN. After an additional 24 h, granulosa cells were treated with 100 ng/ml rFasL, and cell culture was continued for a further 72 h. RNA was isolated from cultured granulosa cells by the acid guanidinium-phenol-chloroform method using ISOGEN (Nippongene, Toyama, Japan). First-strand cDNA was synthesized in a reaction volume of 15 μl containing 5 μg total RNA and 0.2 μg of random hexamer primers by using the first-strand cDNA synthesis kit (Amersham Biosciences) according to the manufacturer’s instructions. After the RT reaction, cDNA was amplified to determine iNOS expression using the following primer pair: iNOS, 5'-CATGGTGAACACGTTCTTGG-3' (sense) and 5'-GTGGTGACAAGCACATTTGG-3' (antisense). Expression of iNOS mRNA was normalized to RNA loading for each sample using glycerol-3-phosphate dehydrogenase (G3PDH) mRNA as an internal standard. The primers of G3PDH were used as follows: G3PDH, 5'-ACCACAGTCCATGCCATCAC-3' (sense) and 5'-TCCACCACCCTGTTGCTGTA-3' (antisense).
Real-time PCR was performed using the LightCycler (Roche Applied Science, Mannheim, Germany) in 20 μl including 1.6 mM MgCl2, 2 μl LightCycler-FastStart reaction mix SYBR Green 1 (Roche Applied Science), 0.25 μM of each primer, and 50 ng cDNA from RT reactions as template. After an initial denaturation at 95 C for 10 min, the amplification program for iNOS and G3PDH consisted of 40 cycles of denaturation for 15 sec at 95 C, annealing for 10 sec at 62 C, and extension for 18 sec at 72 C. Finally, the temperature was raised gradually (0.2 C/sec) from the annealing temperature to 95 C for the melting curve analysis.
The samples from cultured granulosa cells treated with or without rFasL were run in triplicate and analyzed as follows. The concentrations of the samples were extrapolated from the standard curve by LightCycler software. Exogenous cDNA standards for iNOS and G3PDH were produced by inserting PCR products, which were generated using sample primers noted above and granulosa cell cDNA as templates, into the pCR2.1 vector using the TOPO TA cloning kit (Invitrogen). The concentration of each standard was determined by measuring the OD260, and the copy number was calculated. Relative expression levels of iNOS were calculated by subtracting the signal threshold cycle of the internal standard (G3PDH) from the threshold cycle of iNOS. The PCR products (444 bp size) were characterized by using a DNA sequencer (ABI PRISM 310 genetic analyzer; PerkinElmer Applied Biosystems).
Statistical analysis
Data represent the mean ± SEM from at least three independent experiments using separate groups of rats (26 animals/group). Statistical analyses were carried out by Mann-Whitney U test for paired comparison and one-way ANOVA with post hoc test for multiple comparisons by using StatView software (SAS Institute Inc., Cary, NC). Repeated-measures ANOVA was applied for analysis of time course study of the effect of rFasL on granulosa cell viability. P < 0.05 was considered statistically significant.
Results
Effect of rFasL on cultured granulosa cell viability
The effect of rFasL on granulosa cell viability was examined by MTS assay. As shown in Fig. 1A, treatment of granulosa cells with 100 ng/ml rFasL in the presence of 200 U/ml IFN decreased granulosa cell viability in a time-dependent manner. At 48 and 72 h, the cell viability was significantly reduced to 86.7 ± 2.3 and 77.6 ± 1.7% of baseline (0 h). Control incubation at 24, 48, and 72 h did not result in significant loss of viability. To investigate whether cell death induced by rFasL is associated with the activation of caspase cascade, Z-VAD-FMK (20 μM), a caspase inhibitor, was added 30 min before rFasL administration, and the combined treatment was continued for 72 h. The rFasL-induced granulosa cell death was inhibited by concurrent treatment with Z-VAD-FMK (98.4 ± 5.2% of the control) (Fig. 1B).
FIG. 1. Effects of rFasL and Z-VAD-FMK, a caspase inhibitor, on cultured granulosa cell viability estimated by MTS assay. A, Relative viability was calculated based on percentage absorbance of the samples (rFasL-treated) and untreated control at 24, 48, and 72 h with respect to that of the 0 h cultures (baseline) in media with 5% FBS in the presence of 200 U/ml IFN. Results are shown as the mean percentage of the untreated control (IFN alone) at 0 h ± SEM of eight wells of five independent experiments using separate groups of rats. *, P < 0.05 vs. 0 h. B, Z-VAD-FMK (20 μm) inhibited rFasL-induced granulosa cell apoptosis at 72 h of the treatment. Results are shown as the mean percentage of the untreated control (IFN alone) at 72 h ± SEM. *, P < 0.05 vs. control and rFasL + Z-VAD-FMK.
Effect of SNAP on rFasL-induced apoptosis in cultured granulosa cells
The effects of rFasL and SNAP, an NO donor, on the incidence of apoptotic cells were determined by Hoechst 33342 nuclear staining and flow cytometry in cultured granulosa cells. The apoptotic cells exhibiting shrunken nuclei, chromatin condensation, and nuclear fragmentation were recognized by Hoechst 33342 nuclear staining. The in vitro induction of granulosa cell apoptosis by 100 ng/ml rFasL was markedly inhibited by the addition of 0.5 mM SNAP (Fig. 2, A–C). The exact frequency of cells with nuclear fragmentation by Hoechst 33342 staining is shown in Fig. 2D. The percentage of dead cells was significantly increased after 72 h of incubation with rFasL (18.2 ± 0.7%; P < 0.05 vs. control), compared with that in the control (6.5 ± 0.8%), and this increment was suppressed by concurrent supplement with SNAP (6.2 ± 0.7%; P < 0.05 vs. rFasL alone). Figure 3A is a representative result of three independent experiments showing the proportion of sub-G1 phase, an apoptotic cell fraction. As shown in Fig. 3B, flow cytometric analysis revealed that the proportion of sub-G1 phase was significantly increased by the addition of rFasL (19.6 ± 1.4%; P < 0.05 vs. control), compared with that for the control (8.1 ± 0.6%) at 72 h of treatment. The increased proportion of sub-G1 phase by rFasL was dose-dependently suppressed by SNAP at 0.05, 0.2, and 0.5 mM to 17.1 ± 1.5, 10.3 ± 0.5, and 8.0 ± 0.5%, respectively.
FIG. 2. Hoechst 33342 staining of 100 ng/ml rFasL-treated granulosa cells with or without SNAP, an NO donor, in the presence of 200 U/ml IFN (magnification, x200). Twenty-six rats were killed for preparation of granulosa cells; a part of granulosa cells were cultured in 16-well chamber slide for Hoechst 33342 staining. A, Control (IFN alone). B, Treatment with rFasL increased the rate of cells with nuclear fragmentation at 72 h. C, Addition of SNAP (0.5 mM) suppressed rFasL-induced apoptosis. D, The frequency of cells with nuclear fragmentation by Hoechst 33342 staining was calculated by counting the number of stained cells per more than 200 cells. Results are shown as the mean ± SEM of three independent experiments using separate groups of rats. *, P < 0.05 vs. control and rFasL + SNAP.
FIG. 3. DNA histograms of 100 ng/ml rFasL-treated granulosa cells with or without SNAP at concentrations of 0.05, 0.2, and 0.5 mM in the presence of 200 U/ml IFN. At 72 h of the treatment, cells were stained with propidium iodine (PI) and analyzed by flow cytometry. Sub-G1 indicates an apoptotic cell fraction. A, The result of flow cytometric analysis is representative of three independent experiments. B, Effects of rFasL and SNAP on the proportion of sub-G1 phase. Results are shown as the mean ± SEM of three independent experiments using separate groups of rats. *, P < 0.05 vs. control (IFN alone).
Effect of SNAP on Fas expression in cultured granulosa cells
As shown in Fig. 4, Western blot analysis revealed that the expression level of Fas was significantly increased to 188.5 ± 9.5% of the control after treatment with 100 ng/ml rFasL for 72 h (P < 0.05 vs. control). This increment was not significantly changed by concurrent addition of 0.5 mM SNAP.
FIG. 4. Western blot analysis of Fas expression in 100 ng/ml rFasL-treated granulosa cells with or without SNAP (0.5 mM), an NO donor, in the presence of 200 U/ml IFN. At 72 h of the treatment, cell protein lysates were analyzed by immunoblotting with anti-Fas antibody. A, The result is representative of three independent experiments. B, Results show quantitative analysis of Fas protein level and are expressed as the mean percentage of the untreated control (IFN alone) ± SEM of three independent experiments using separate groups of rats. *, P < 0.05 vs. control.
Effect of SNAP on rFasL-induced caspase activation in cultured granulosa cells
Cleaved caspase protein levels were evaluated by Western blot analysis. Figure 5, A–C (left) illustrates representative results, exhibiting the 19- and 17-kDa cleavage fragments of activated caspase-3, the 18-kDa cleavage fragments of activated caspase-8, and 17-kDa cleavage fragments of activated caspase-9, respectively. As shown in Fig. 5, A–C (right), the expression levels of cleaved caspase-3, -8, and-9 were significantly up-regulated to 201.7 ± 6.0, 158.8 ± 12.1, and 141.3 ± 15.7% of the control, respectively, after treatment with 100 ng/ml rFasL for 72 h, and these increments were attenuated by concurrent addition of 0.5 mM SNAP (120.7 ± 5.2, 112.3 ± 2.0, and 105.8 ± 2.9%, respectively; P < 0.05 vs. rFasL alone).
FIG. 5. Western blot analysis of protein levels of cleaved caspase-3, -8, and -9 in 100 ng/ml rFasL-treated granulosa cells with or without SNAP (0.5 mM), an NO donor, in the presence of 200 U/ml IFN. At 72 h of the treatment, cell protein lysates were analyzed by immunoblotting with anticleaved caspase-3, anti-caspase-8, and anti-cleaved caspase-9 antibody, which recognizes active cleavage fragments of 19 and 17 kDa (A), 18 kDa (B), and 17 kDa (C), respectively. Results on the left are representative films exhibiting bands for active forms of caspases with corresponding actin bands. Results on the right show quantitative analysis of caspase-3, -8, and -9 cleavage into active forms. Results are shown as the mean percentage of the untreated control (IFN alone) ± SEM of three independent experiments using separate groups of rats. *, P < 0.05 vs. control and rFasL + SNAP.
Effect of rFasL on iNOS expression in cultured granulosa cells
The effect of rFasL on iNOS mRNA levels was investigated by real-time quantitative RT-PCR in cultured granulosa cells. After 72 h culture of granulosa cells with 100 ng/ml rFasL, iNOS mRNA levels were significantly decreased to 61.1 ± 6.1% of the control (P < 0.05).
Discussion
It has been documented that Fas/FasL system is one of the critical mediators of apoptosis in ovarian follicle atresia (3, 4, 7, 8, 9, 10, 11, 12). In the present study, using cultures of rat granulosa cells, we demonstrated that Fas/FasL-mediated apoptosis is accompanied by a remarkable increase in caspase-3, -8, and -9 activities and a concomitant decrease in iNOS expression and that exogenous NO inhibits Fas/FasL-mediated apoptosis in association with an attenuation of caspase cascade activation.
The process of apoptosis was characterized by nuclear shrinkage, plasma membrane blebbing, chromatin condensation, and internucleosomal DNA fragmentation. Concomitant with these changes is the activation of a cascade of intracellular proteolytic events catalyzed by a family of cysteine aspartate proteases known as caspases (33, 34, 35). Caspases consisting of at least 14 isoforms are activated in response to multiple apoptotic stimuli and propagate death signals by cleaving a number of cellular protein substrates. These substrates include inactive procaspase zymogens that are cleaved into active proteases leading ultimately to apoptosis. Activated caspases specifically cleave the target substrates on the carboxyl side of aspartate residues. Caspase-8, -9, and -10 are initiator caspases that can transduce apoptotic signals by directly activating the downstream effector caspase-3, -6, and -7. Two pathways have been described for Fas-induced apoptosis (14). First, the assembly of a membrane-bound death-inducing signaling complex (DISC) is initiated by Fas-FasL interaction and subsequent receptor trimerization (14). Then the adaptor protein FADD binds via its own death domain to the death domain in Fas. Interaction of the death domain of Fas with that of FADD recruits procaspase-8 to complete the DISC, which triggers autoprocessing of the enzyme. Active caspase-8 can both directly activate downstream caspases such as caspase-3 (type I) and induce mitochondrial apoptotic signaling, including mitochondrial cytochrome c release (type II). In type II pathway, cytosolic cytochrome c induces formation of the apoptosome by stimulating oligomerization of the adapter protein (Apaf-1), apoptotic protease-activating factor 1 and, consequently, recruitment of the initiator caspase-9 (36). As a result, activated caspase-9 can then directly cleave and activate caspase-3. In the immune system, binding of FasL to Fas causes apoptosis by activating the caspase cascade, resulting in the cleavage and activation of caspase-3 (13, 14). Previously we demonstrated that activation of the Fas/FasL system was capable of initiating apoptosis in cultured rat granulosa cells (8). The present study showed the prevention of Fas/FasL-mediated granulosa cell death by a caspase inhibitor, Z-VAD-FMK, and a positive correlation between the enzyme activity of caspases and the occurrence of apoptosis in granulosa cells.
These findings are in accordance with other observations that caspase-3 mRNA and its protein were detected in rat granulosa cells during atresia and down-regulated by gonadotropin-promoted follicular survival (15, 16). The notion that active caspase-3 is a key functional participant in granulosa cell death program was reinforced by the study demonstrating the presence of aberrant atretic follicles in caspase-3-null mouse ovaries (18).
NO can prevent or induce apoptosis, depending on its concentration, cell type, and the oxidative milieu (23, 37). Increasing evidence has demonstrated NO as a potential regulator of follicular development (24, 25, 26, 27, 28, 29). It was reported that administration of SNAP, an NO donor, directly inhibited spontaneously occurring apoptosis in cultured rat granulosa cells, suggesting that NO acts as a follicle survival factor (24, 28). Recently we showed that iNOS mRNA levels in rat granulosa cells were decreased by administration of GnRH agonist, an atretogenic agent for ovarian follicles or epidermal growth factor, a mitogenic and antiapoptotic factor, and that treatment with SNAP attenuates both GnRH agonist-induced apoptosis and epidermal growth factor-induced DNA synthesis in granulosa cells (29). In the present study, iNOS mRNA levels were decreased in granulosa cells exposed to proapoptotic stimulation by rFasL, whereas treatment with SNAP suppressed Fas/FasL-mediated apoptosis in a dose-dependent manner. The present data strengthen our hypothesis that endogenous NO synthesized by iNOS in granulosa cells acts as a cytostatic factor in ovarian follicles, with a loss of NO being associated with the conversion into a different developmental status in whichever direction (i.e. progression into differentiation or demise via apoptotic process) (27, 28, 29).
To further address the role of NO in the regulation of ovarian follicle atresia, the impact of NO on rFasL-induced death pathway was examined. Our present study revealed that NO inhibits Fas/FasL system-mediated apoptosis, at least in part, by directly interfering with caspase-8, leading to the down-regulation of caspase-9 and -3 activities. However, we cannot exclude the possibility that NO directly inhibits the activation of caspase-9 and/or -3. Similar findings were reported using other cell types, in which NO inhibits apoptosis by inactivating several members of the caspase family through S-nitrosylation of a cysteine thiol at the catalytic site of caspase (37, 38, 39, 40, 41, 42, 43, 44, 45, 46). On the other hand, it was reported that the Fas/FasL system activates caspase-3 not only by inducing the cleavage of the caspase zymogen to its active subunits but also by stimulating the denitrosylation of its active-site thiol (47). Thus, caspase S-nitrosylation/denitrosylation appears to regulate Fas/FasL-mediated death pathway. Recent studies also revealed various NO-mediated inhibitory mechanisms of follicular apoptosis (24, 48, 49). It has recently been shown that NO might inhibit Fas-mediated apoptosis through a decreased expression of Fas on granulosa cells (49). However, in our additional study, Western blot analysis failed to detect the inhibitory effect of SNAP on Fas expression at the same concentration as SNAP inhibited Fas-mediated apoptosis. The most likely explanation for this discrepancy may be the difference of experimental designs. It might be possible that stimulation of cGMP production (24) and modulation of apoptosis- and antiapoptosis-related gene expression such as induction of heat shock protein 70 (48), suppression of Bax expression (48), and stimulation of Bcl-2 expression (49) ultimately prevent activation of the effector caspase-3 in granulosa cells (23, 41, 50).
In conclusion, using rat granulosa cell culture system, we have demonstrated that there is a cross-talk between Fas/FasL system-induced apoptosis pathway and NO-mediated antiapoptotic pathway in the ovarian follicle. The shift in a balance between cell death and cell survival signaling pathways in granulosa cells might determine the fate of ovarian follicles. Further studies on the precise molecular mechanisms underlying the alterations of this balance would provide clues to devise diagnostic and therapeutic strategies for various pathologic conditions of the ovary.
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