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Ovary |
a Reproductive Biology Unit and Division of Reproductive Medicine,
b Department of Obstetrics & Gynecology and Cellular & Molecular Medicine, University of Ottawa; Ottawa Health Research Institute
c Department of Laboratory Medicine, The Ottawa Hospital (Civic Campus), Ottawa, Ontario, Canada K1Y 4E9
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicle, follicle-stimulating hormone, follicular development, gene regulation, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The inhibitor of apoptosis proteins (IAP; also termed baculovirus inhibitor of apoptosis repeat [BIR]-containing proteins) is a family of intracellular antiapoptotic proteins that were first identified in baculovirus. They are expressed in high abundance in proliferating cells and are suppressed in apoptotic ones. To date, six members have been identified in mammals, including X-linked IAP (XIAP, cIAP-3 [5]), human IAP-1 (HIAP-1 [5], cIAP-2 [6]), human IAP-2 (HIAP-2 [5], cIAP-1, [6]), neuronal apoptosis inhibitor protein (NAIP [7]), survivin [8], and Livin [9] (termed KIAP in kidney [10]). The IAPs are characterized by the presence of a caspase recruitment domain and N-terminal BIR motifs, which are necessary for biological activity. With the exception of NAIP and survivin, the IAPs also contain a C-terminal RING-zinc finger domain believed to be required for protein-protein interactions [11] as well as protein ubiquitination and degradation [12]. Maternal smoking-induced apoptosis of trophoblasts throughout development is associated with decreased XIAP expression [13]. Increased XIAP expression is believed to play a role in the modulation of Fas ligand-induced apoptosis in malignant glioma cells by a proliferation-inducing ligand, a member of the tumor necrosis factor (TNF) family [14]. Studies regarding the mechanisms of action of these antiapoptotic proteins suggested that IAPs modulate the activities of a group of cysteine proteases known as caspases. The XIAP has been shown to be a direct inhibitor of caspase-3 and caspase-7, which are involved in the cell surface receptor-dependent cell death pathway [15, 16], and to suppress the mitochondrial (cytochrome c-mediated) pathway by inhibiting caspase-9 activity [17]. Moreover, mammalian IAPs inhibit caspase-independent apoptosis induced by TNF
in human leukemic cells [6].
Previous studies have demonstrated in the rat ovary that XIAP and HIAP-2 were highly expressed in healthy, but not in atretic, follicles [18]. Whereas granulosa cell levels of IAP were high during gonadotropin-induced follicular development, gonadotropin withdrawal decreased granulosa cell IAP content and induced apoptosis [3]. In addition, gonadotropin-induced, NAIP-mediated suppression of apoptosis in mouse granulosa cells has been suggested to play an important role in the maintenance of oocyte survival during ovarian folliculogenesis [19]. In hen ovarian follicles during follicular development, the highest levels of the inhibitor of T-cell apoptosis (ita) gene mRNA within the granulosa cell layer were found in preovulatory (atresia-resistant) follicles, with significantly lower levels detected in prehierarchical follicles, a stage at which considerable follicular atresia is observed [20]. The above-described patterns of IAP mRNA expression during follicular development are consistent with a potential role for these genes in protecting granulosa cells from apoptosis and, thus, in maintaining follicle viability. However, the physiological roles of IAPs in follicular development and atresia remain unclear.
In the present studies, we have tested the hypothesis that XIAP expression is essential for the regulation of granulosa cell fate and follicular development by FSH. To this end, a follicle culture system coupled to an adenoviral gene-manipulation procedure has, to our knowledge for the first time, been established. Using this approach, we have demonstrated that the increase in XIAP expression induced by FSH is critical for the maintenance of normal follicular growth and development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Culture media and reagents were purchased from Gibco Bethesda Research Laboratories (Burlington, ON, Canada). Agarose (low gelling temperature), DNA (calf thymus), Hoechst 33258 compound, Triton X-100, Tween 20, collagenase (type 1A), DNase 1, RNase A, bovine insulin, human transferrin, ascorbic acid, sodium selenite anhydrous, and eCG were obtained from Sigma Chemical Company (St. Louis, MO). The enhanced chemiluminescence (ECL) detection kit, [methyl-3H]thymidine (25 Ci/mmol), and [-32P]ddATP were obtained from Amersham Life Science (Oakville, ON, Canada). Ovine FSH (NIAMDD oFSH-14) was obtained from the National Institute of Diabetes & Digestive & Kidney Diseases (Baltimore, MD). Acrylamide (electrophoresis grade), N,N'-methylene-bis-acrylamide, ammonium persulfate, glycine, SDS-PAGE prestained molecular weight standards (low range), nitrocellulose membranes, and horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse immunoglobulin (Ig) G were products from Bio-Rad (Richmond, CA). Rhodamine-conjugated goat anti-rabbit IgG was a product of Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mounting medium for fluorescence was purchased from Vector Laboratories, Inc. (Vectashield, H-1000; Burlingame, CA). Cell death detection (TUNEL) kit and Terminal End Transferase (TdT) were purchased from Boehringer-Mannhein (Montreal, PQ, Canada) and Alamar Blue dye from Biosource Incorporated (distributor: Medicorp, Montreal, PQ, Canada). The Qiagen Tissue Amp Kit and QIAquick Nucleotide Removal Kit were products of Qiagen (Qiagen, Inc., Chatsworth, CA). The IM-8 Micro-injector and Joystick (MN-151) as well as Micromanipulator were purchased from Narishige International, Inc. (Long Island, NY). The stage warmer was bought from Linkam Scientific Instruments (Surry, U.K.).
Adenoviral LacZ, XIAP sense and antisense cDNAs, and rabbit polyclonal anti-human XIAP antibodies were generously provided by Dr. Eric LaCasse (Ægera Therapeutics, Inc., Ottawa, ON, Canada). Rabbit polyclonal anti-XIAP antibody was raised against a glutathione S-transferase fusion protein that was expressed in Escherichia coli using pGEX vector (Amersham Pharmacia Biotech, Arlington Heights, IL) containing full-length XIAP cDNA. The antibody was affinity-purified by passing through a glutathione S-transferase-XIAP glutathione-Sepharose column. Specificity was confirmed on Western blots (using the antibody-depleted eluate from the affinity column), and cross-reactivity with other IAPs was not noted [18]. Construction of recombinant adenovirus was carried out as described previously with some modifications [21]. Briefly, the open reading frame of XIAP was amplified by polymerase chain reaction, cloned in the pCR2.1 vector (Invitrogen, Carlsbad, CA), and sequenced. The open reading frame was cut out and ligated into the Swa-1 site of pAdex1CAwt cosmid DNA. The vector was packaged with the Promega cosmid packaging extracts (Madison, WI) and used to infect E. coli. Colonies were picked and screened for presence of the insert in the antisense orientation relative to the chicken ß-actin promoter. The CsCl-purified cosmid DNA was cotransfected with wild-type adenovirus DNA that was allowed to generate infectious adenovirus DNA only when homologous recombination with cosmid DNA occurred. The final recombinant adenovirus contained a linear, double-stranded genome of 44 820 base pairs (bp) plus the antisense XIAP insert (1500 bp). Adenoviral expression system was generated with an Ad E1 insertion vector. Virus titer was determined by the plaque assay.
Follicular Isolation and Culture
All the animal work was carried out in compliance with the Guides of the Canadian Council on Animal Care. Ovaries from 22- to 24-day-old, immature female Sprague-Dawley rats were cut into small pieces and incubated at 37°C for 30 min in -minimal essential medium (-MEM) containing collagenase (type 1A, 4 mg/ml) and DNase 1 (0.3 mg/ml). The incubation was terminated with the transfer of the ovarian tissues into Leibowitz L-15 medium with 0.1% (w/v) BSA, and follicles (diameter, 160–210 µm) were dissected out using 28.5-gauge needles. To minimize the experimental variation caused by damage incurred during the isolation procedures, only round follicles with an intact thecal layer on the day of isolation (Day 0) and on Day 1 of culture were selected for experimentation. Confocal microscopic examination (Bio-Rad M500; Bio-Rad Laboratories Ltd., Hertfordshire, U.K.) of the selected follicles (following fixation with paraformaldehyde [4%, v/v; 30 min; room temperature [RT]) and staining with ethidium bromide (5 mg/ml, 15 min, RT) revealed that they were at the preantral (75%) and early antral (25%; as evidenced by the presence of an antral space as large as an area occupied by approximately three granulosa cells) stages of development. Follicles were cultured individually, and follicular diameter was measured daily before medium change for 4–6 days in a 96-well plate in 100 µl of follicular culture medium (FCM; -MEM supplemented with Hepes [10 mM], BSA [0.1%, w/v], rat serum [1%, v/v], bovine insulin [5 µg/ml], transferrin [10 µg/ml], ascorbic acid [25 µg/ml], sodium selenite anhydrous [2 ng/ml] [22], and nonessential amino acids [1%, v/v], streptomycin-penicillin [0.5%, v/v], and fungizone [0.25%, v/v]) with or without oFSH. Preliminary studies showed that inclusion of ascorbic acid and selenium in the culture medium enhanced follicular integrity, as evidenced by an increase in the proportion of intact follicles (65% ± 5.9% vs. 35% ± 4.7%; n = 3 experiments; P < 0.05) observable at the end of the 6-day culture period. At the end of the culture period, follicles were embedded in 2% (w/v) agarose, fixed in buffered formalin phosphate solution (10% [v/v], 3 h, RT), stained with Neutral Red (0.1% [w/v], 3 h, RT; to facilitate visualization of follicles during sectioning), and then processed to be embedded in paraffin. Sections (thickness, 4 µm) of the cultured follicles were also stained with Haematoxylin Phloxine Saffron (HPS) for morphologic examination.
Follicular Cell Proliferation
Follicular cell DNA assay Cultured follicles were washed twice with PBS, fixed and sonicated in trichloroacetic acid (5% [w/v], 20 min, 4°C), and finally washed twice with methanol. The DNA pellet, collected by centrifugation (16 000 x g, 10 min), was dissolved in NaOH (0.25 M) and adjusted to neutral pH with HCl (0.25 M). Aliquots of the DNA pellet and calf thymus DNA (standard) were incubated with Hoechst 33258 dye (0.1 µg/ml, 5 min in the dark, RT) as described previously [23]. Changes in fluorescence intensity were measured with a Microplate Fluorometer (SPECTRAmax GEMINIXS, Molecular Devices Corporation, Sunnyvale, CA) at excitation and emission wavelengths of 356 and 457 nm, respectively. The sensitivity and linearity of the assay were 2 and 4–1000 ng/ml, respectively.
Alamar Blue assay
Alamar Blue is a nontoxic metabolic dye and has been used to monitor changes in cell numbers during tissue or primary cell culture in vitro [24, 25]. In the present studies, we have used this assay to monitor the relative increase in cell number during follicular growth under the influence of gonadotropin during a 6-day culture period. The assay was performed as described by Ahmed et al. [25]. To confirm the linear relationship between the changes in Alamar Blue reduction and those of cell number under the experimental conditions of the present studies, Alamar Blue (10% [v/v]) was first tested on primary cultures of rat granulosa cells and human ovarian surface epithelial cancer cell cultures (37°C, 6 h) and, subsequently, on rat ovarian follicle cultures (37°C, 3 h) maintained under a humidified atmosphere of 95% (v/v) air and 5% (v/v) CO2. Absorbance at wavelengths of 570 and 630 nm was measured by a fluorometer (Model MRX; Dynatech Laboratories Inc., Simi Valley, CA), and this ratio was defined as the cell number equivalence. A linear relationship between ovarian cell number and the extent of Alamar Blue reduction was established with the human epithelial ovarian cancer cell line A2780s (r2 = 0.992) and isolated rat granulosa cells (r2 = 0.980). In addition, a direct correlation was also found between Alamar Blue reduction (follicular cell number equivalence) and DNA content (r2 = 0.850) as well as follicular volume within the range of 2.2–25 nl (diameter, 
160–360 µm; r2 = 0.933) (Fig. 1). As shown in Figure 2A, an increase in follicular volume above this range, however, was not associated with a proportional increase in follicular Alamar Blue reduction, an observation consistent with the fact that the increase in follicular volume during early development is mainly a consequence of increased cell proliferation whereas that during the late stage is caused by antral enlargement. No significant change in Alamar Blue reduction was evident throughout the 6-day culture period in the absence of the gonadotropin (Fig. 2A). Maximum daily growth rate was observed on Day 3 of culture in the presence of FSH (Fig. 2B).
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Follicular Adenoviral Injection
To assess the role of XIAP in FSH-induced follicular development, XIAP content in the cultured follicles was manipulated by adenoviral XIAP antisense and sense (Myc-tagged) cDNA expression. After a 24-h culture in the absence of FSH in a 96-well plate, follicles were transferred onto a cell strainer (mesh size, 100 µm; Becton Dickinson Labware, Franklin Lakes, NJ) in a 35-mm dish containing FCM. Replication-deficient adenovirus containing LacZ, XIAP sense, or XIAP antisense full-length cDNAs was injected into the follicles. The volume of the virus injected was less than 10% (v/v) of the calculated follicular volume. Based on the follicular volume and estimated cell number, the amount of virus injected was multiplicity of infection (MOI) of 20 for XIAP antisense cDNA to assure adequate XIAP down-regulation. The MOI for the virus control (LacZ) was also 20. An MOI of three was chosen for XIAP sense cDNA, because it was sufficient to up-regulate XIAP expression. The FSH (5 ng/ml) was added to the cultures 24 h later, and the follicles were cultured for another 3 days. Preliminary studies indicated that follicles cultured with FSH at concentration of 100 ng/ml had higher XIAP level and were more resistant to XIAP down-regulation. In contrast, follicular XIAP level was low in the absence of FSH and less responsive to XIAP up-regulation by adenoviral sense expression. Thus, a lower concentration of oFSH (5 ng/ml) was used in subsequent cultures to achieve maximum response to XIAP up- and down-regulation by adenoviral cDNA expression.
To confirm the success of adenoviral infection, follicles injected with the adenoviral XIAP sense cDNA or LacZ were paraffin-sectioned and processed with the same procedure as described in the above section on the TUNEL assay up to the incubation step with the blocking reagents. Sections were then incubated (2 h at RT or overnight at 4°C) with HRP-conjugated anti-Myc antibody (1:50 [v/v]; Invitrogen) or rabbit anti-ß-galactosidase (1:100 [v/v]; ICN Pharmaceuticals, Inc., Biochemical Division, Aurora, OH), respectively. Sections of LacZ-infected follicles were washed in PBS, incubated with second antibody (HRP-conjugated goat anti-rabbit IgG, 1:200 [v/v] in PBS, 1 h, RT) and detected by diaminobenzidine (DAB) staining (4 min; Boehringer Mannheim Corp., Indianapolis, IN) and counterstained with Methyl Green (100%, v/v; Vector Laboratories). The Myc-tag in sections of XIAP sense-injected follicles were directly detected with DAB staining after incubation with primary antibody. In addition, adenoviral LacZ-infected follicles (MOI = 20; 3 days) were washed twice with PBS, fixed with glutaraldehyde (0.25% [v/v] in PBS, 10 min, 4°C), rinsed four times with PBS, and stained in 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) buffer (PBS [pH 7.2–7.5] containing X-gal [1 mg/ml], K3Fe[CN]6 [5 mM], K4Fe[CN]6 [5 mM], MgCl2 [2 mM], and Triton X-100 [0.05%, v/v]; 18 h; 37°C). Stained follicles were washed twice with PBS and then incubated in a series of glycerol concentrations (10%, 20%, 40%, and 60% [v/v]; 4 h for each concentration; RT).
Successful infection of follicular cells with adenoviral LacZ and myc-tagged XIAP sense cDNA was confirmed by X-gal staining (on intact follicles) (Fig. 3, A and B) and immunohistochemistry (IHC; on follicular sections) using anti-galactosidase (87%) (Fig. 3, C and D) and anti-Myc (82%) (Fig. 3, E and F) antibodies.
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Rat Granulosa Cell Isolation and Culture
Immature female Sprague-Dawley rats (age, 24–25 days) from Charles River Canada (Montreal, PQ) were injected with eCG (15 IU i.p.), and ovaries were collected 24 h thereafter in RPMI 1640 medium supplemented with Hepes (10 mM; pH 7.4) and 10% (w/v) fetal bovine serum (FBS). Granulosa cells were harvested by follicle puncture as previously described [26], washed, and centrifuged (900 x g, 10 min). Next, 6 x 105 viable cells were plated (6-well plate, Falcon; Becton Dickinson) for 24 h in RPMI 1640 medium with 10% (w/v) FBS under a humidified atmosphere of 95% air and 5% CO2 and cultured for various durations in serum-free medium with adenoviral LacZ or antisense or sense XIAP cDNA.
Adenoviral Infection in Primary Granulosa Cell Culture System
Infection of primary granulosa cells with adenoviral XIAP sense and antisense cDNA was performed as described by Kim et al. [26]. Briefly, 1 million granulosa cells were plated in 60-mm dishes for 24 h and infected with adenoviral sense or antisense full-length XIAP or LacZ at an MOI of 5 or 20, respectively. The FSH was added to culture medium 24 h after viral infection, and granulosa cells were cultured for another 24 h. At an MOI of 10, the LacZ infection efficiency over 48 h (as determined by X-gal assay) was more than 90%, and changes in XIAP expression were confirmed by Western blot analysis.
Protein Extraction and Western Blot Analysis
Assessment of XIAP protein contents was performed according to the immunoblotting procedures described by Kim et al. [26] with minor modifications. Briefly, cultured follicles were harvested and lysed mechanically in RIPA buffer (1x PBS [pH 7.4] containing SDS [0.1%, w/v], sodium deoxycholate [0.5%, w/v], NP-40 [1%, v/v], and the protease inhibitors PMSF [1 mM], aprotinin [10 µg/ml], and sodium orthovanadate [1 mM]). The follicular lysate and harvested granulosa cells were sonicated (three times for 10 sec each on ice) in RIPA lysis buffer. Sonicates were pelleted by centrifugation (14 000 x g, 30 min, 4°C), and the supernatant was retained and stored at -20°C. Protein content of the extracts was determined with the Bio-Rad DC Protein Assay Reagent. Samples were mixed with loading buffer, resolved by 10% SDS-PAGE, and electrotransferred (30 V overnight or 80 V for 2 h) onto nitrocellulose membranes using the Bio-Rad Trans-Blot system. Nonspecific binding to the membranes was blocked with Blotto (Tris-buffered saline [TBS; pH 8.0] with 0.05% [v/v] Tween 20 [TBS-T] and 5% [w/v] dehydrated nonfat milk) at room temperature for 1 h. Membranes were then incubated (4°C, overnight) with Blotto containing rabbit anti-XIAP antibody (1:2000), washed in TBS-T (three times for 5 min each), incubated in HRP-conjugated secondary antibody (1:5000) in Blotto, and washed again in TBS-T twice and then in TBS once. Peroxidase activity was visualized with the ECL kit according to manufacturer's instructions. The XIAP content was determined by densitometrically scanning (HP ScanJet 3C; Hewlett-Packard Inc., Arlington Heights, IL) the exposed x-ray film (Kodak Canada Inc., Toronto, ON).
Cell Death Detection
Hoechst staining At the end of the granulosa cell culture period, floating cells were collected by aspiration, and cells attached to the growth surface were subjected to trypsin treatment (0.05% [w/v] trypsin and 0.53 mM EDTA, 3–5 min, 37°C). The two cell fractions (floating and attached cells) were combined, and an aliquot of this cell mixture was fixed on a microscope slide. At least 200 cells in a randomly selected area in each treatment group were counted. Apoptotic cells were identified based on their typical nuclear morphology. To avoid experimental bias, the "counter" was not aware of the treatment.
TUNEL assay TUNEL was performed as described previously [27]. Briefly, paraffin-embedded, whole-ovarian follicle sections (thickness, 4–5 µm) were mounted on positively charged slides, deparaffinized, hydrated, washed thoroughly three times for 5 min each in 1x PBS, and immersed in PBS with 0.3% (v/v) H2O2 (10 min, RT; to inhibit endogenous peroxidase activity). Following three additional 5-min washes in PBS, the sections were incubated (30 min, RT) in a blocking reagent (Large Volume DAKO LSAB Kit; DAKO Diagnostics Canada, Inc., Mississauga, ON, Canada) and immersed in 50 µl of the TUNEL mixture (47.5 µl of TUNEL label containing fluorescein isothiocyanate [FITC]-dUTP and 2.5 µl of TUNEL enzyme) in a humidified chamber (37°C, 60 min). They were mounted for fluorescence microscopy with a confocal laser-scanning system (Bio-Rad 1024). The FITC signal in TUNEL-positive cells was excited at 488 nm, with images collected within the wavelength range of 506–538 nm.
DNA fragmentation analysis
Apoptotic cell death was also assessed on the basis of DNA fragmentation and confirmed through visualization of discrete DNA fragments of 185-bp multiples on agarose gel electrophoresis. Follicular DNA was extracted using Qiagen Tissue Amp Kit according to the manufacturer's instructions. The DNA was quantified spectrophotometrically by the absorbance at 260 nm. The DNA was end-labeled by incubating with TdT and [-32P]ddATP as previously described [13]. Briefly, 500 ng of DNA sample were added to a mixture (5 µl of 5x TdT buffer, 2.5 µl of 10x CoCl2, 0.5 µl of TdT enzyme, and 0.5 µl of a 10 mCi/ml concentration of [32P]ddATP and Tris EDTA buffer) to a total volume of 25 µl and then incubated at 37°C for 60 min. Unincorporated nucleotides were removed with the Qiagen nucleotide removal kit, and the labeled samples were subsequently resolved by 1.8% (w/v) agarose. The gel was dried (3 h) and then exposed to a Bio-Rad PhosphorImager, and low-molecular-weight DNA (<4 kilobase pairs) and genomic DNA were densitometrically quantified. The gel was then exposed to x-ray film at -80°C. To correct for possible uneven gel loading, the ratio of low-molecular-weight DNA (representing apoptosis) to genomic DNA was calculated for each sample, and means of the ratios were compared. The intraobserver variability, determined by performing two separate DNA ladder analyses on the same sample, was approximately 5%.
IHC for XIAP
After incubation in TUNEL mixture, sections were immersed in rabbit polyclonal anti-human XIAP antibody (1:50) and, subsequently, in rhodamine-conjugated goat anti-rabbit IgG (1:200 in PBS, 1 h, RT). The XIAP signal (indicated by rhodamine) was generated with excitation and emission wavelengths of 568 and 630 nm, respectively. Confocal microscopic TUNEL and XIAP images were captured using NIH Image 1.61 software (available at http://rsb.info.nih.gov/nih-image).
Statistical Analysis
All experiments were carried out three to four times. Following confirmation that no interreplicate differences existed in each experiment (one-way ANOVA), individual observations from all replicates were pooled for analysis by two-way (repeated-measure) ANOVA (PRISM software version 3.0; GraphPad, San Diego, CA). When XIAP content and the apoptotic DNA fragmentation were expressed as fold of control, they were arcsine square root-transformed before one-way or two-way ANOVA. Granulosa cell number was analyzed by two-way ANOVA. Differences between experimental groups were determined by the Tukey or Bonferroni posttest. The extent of granulosa cell apoptosis between experimental groups was analyzed by the chi-square test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Follicles cultured for up to 6 days in the absence of FSH exhibited minimal growth (Day 6 vs. Day 0), as evidenced by an absence of change in follicular size (follicular volume: 3.4 ± 0.55 nl vs. 2.6 ± 0.22 nl, n = 43), cell number equivalence (2.8 ± 0.35 vs. 1.5 ± 0.15, n = 46, as determined by Alamar Blue reduction), and DNA content (2.5 ± 0.39 ng/follicle vs. 1.2 ± 0.18 ng/follicle, n = 40) (Fig. 2A). Addition of FSH (100 ng/ml) to the culture medium significantly increased these parameters (follicular volume: 33.6 ± 3.26 nl, n = 43; cell number equivalence: 11.2 ± 2.35, n = 46; DNA content: 21.5 ± 3.39 ng/follicle, n = 40; P < 0.002 vs. control) (Fig. 2A). The increases in cell number equivalence, follicular volume, and daily growth rate were maximal on Day 3 of culture (Fig. 2, A and B). Histological examination of the HPS-stained follicles previously cultured over 6 days in the presence of gonadotropin indicated a well-preserved follicular structure (Fig. 4) containing granulosa cells, theca cells, and intact basement membrane (for Day 2, see Fig. 4B; for Day 6, see Fig. 4C). Theca cells on Day 6 of culture (Fig. 4C) appeared more cuboidal compared to those of follicles of similar stage in situ (Fig. 4D), and freshly isolated follicles (for Day 0, see Fig. 4A). When in vitro FSH exposure (100 ng/ml) was delayed for 2 days (i.e., FSH present on Days 3–6), the follicles were also responsive to the gonadotropin, although overall follicular growth and the daily growth rate were markedly decreased (P < 0.002) (Fig. 2).
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FSH Increases XIAP Expression in Cultured Ovarian Follicles
To determine if XIAP expression is regulated by the gonadotropin during follicular development and atresia in vitro, changes in XIAP content and apoptosis in sections of cultured follicles were examined by IHC and TUNEL, respectively (Fig. 5A). Whereas follicles cultured in the absence of FSH for 2 and 4 days showed low XIAP immunoreactivity and detectable apoptotic signal, addition of FSH (100 ng/ml) to the culture medium markedly increased XIAP expression (Fig. 5A) and decreased the apoptotic signal (Fig. 5A). In addition, gonadotropin also significantly increased follicular XIAP content (P < 0.05) (Fig. 6) and suppressed apoptotic DNA fragmentation (P < 0.05) (Fig. 6).
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Effects of XIAP Down-Regulation and Overexpression in Cultured Follicles
Follicles injected with adenoviral XIAP antisense cDNA (MOI = 20) and cultured in the presence of FSH (5 ng/ml) exhibited lower XIAP immunointensity compared to gonadotropin-treated follicles injected with adenoviral LacZ (Fig. 5B). The XIAP down-regulated follicles also had a stronger TUNEL-positive signal compared to the LacZ control (Fig. 5B). The XIAP antisense also markedly decreased XIAP contents (P < 0.05) (Fig. 7) and significantly increased DNA fragmentation (P < 0.05) (Fig. 7). In addition, infection of the follicles with XIAP antisense cDNA significantly attenuated the FSH-induced follicular growth as indicated by a marked decrease in DNA content (P < 0.05) and follicular volume (P < 0.05) but was ineffective in the absence of gonadotropin (Fig. 8). Two-way ANOVA shows a significant FSH effect (P < 0.001), antisense effect (P < 0.001), and interaction between these factors (P < 0.001), brought about by the observation that the XIAP antisense was more effective in follicles cultured in the presence than in the absence of gonadotropin.
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On the other hand, follicles injected with XIAP sense and subsequently cultured in the low concentration of FSH (5 ng/ml) showed high XIAP immunointensity and less-intense TUNEL signal compared to adenoviral LacZ-injected follicles (Fig. 5B). Although a range in the intensity in both XIAP and TUNEL signals was observed within each experimental group, the differences between groups (LacZ vs. XIAP sense vs. XIAP antisense) were obvious. Similarly, XIAP sense infection significantly increased XIAP protein content (P < 0.01) (Fig. 7) and suppressed apoptotic DNA fragmentation (P < 0.05) (Fig. 7). The increase in XIAP expression following adenoviral XIAP sense infection was not associated with any significant changes in DNA content and follicular volume (P > 0.05) (Fig. 8) in the absence of FSH. In contrast, follicles infected with XIAP sense and cultured in the presence of low concentration of FSH (5 ng/ml) showed a marked increase in DNA content (13.28 ± 1.89 ng/follicle vs. 6.85 ± 0.82 ng/follicle, n = 36, P < 0.05) and follicular volume (22.88 ± 3.29 nl vs. 10.85 ± 2.02 nl, n = 36, P < 0.05) compared to the FSH-stimulated and LacZ-infected follicles. Analysis of variance demonstrates a significant FSH effect (P < 0.001) and XIAP sense effect (P < 0.001), and an interaction between the factors (P < 0.001) (Fig. 8), because of a greater influence by XIAP overexpression on follicular growth in the presence of gonadotropin.
Influence of FSH on Granulosa Cell XIAP Expression and Apoptosis In Vitro
To test if the FSH-induced follicular growth and increase in cell number in vitro is caused by a direct action of gonadotropin on granulosa cells to stimulate proliferation and/or suppress apoptosis, granulosa cells were cultured for 48 h in the absence or presence of FSH (100 ng/ml). Analysis of variance indicates that although FSH significantly decreased granulosa cell apoptosis compared to control (P < 0.01), total granulosa cell number was markedly lower in the presence of gonadotropin (P < 0.02, two-way ANOVA) (Table 1), irrespective of up- or down-regulation of XIAP (P < 0.05, Tukey test).
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To determine if FSH has a direct regulatory role in granulosa cell XIAP expression, the influence of gonadotropin (0–150 ng/ml) on XIAP contents in cultured granulosa cells was assessed during 24- and 48-h culture periods. The FSH significantly increased granulosa cell XIAP content in a concentration-dependent manner (Fig. 9). Arcsine square root-transformed two-way ANOVA indicates a significant effect of concentration (P < 0.01) but not of time (P > 0.05). To assess if XIAP may play an important role in the regulation of granulosa cell fate, the XIAP level was manipulated by adenoviral expression of XIAP sense and antisense (MOI = 5 and 20, respectively) in the absence and presence of FSH (100 ng/ml). Overexpression of XIAP alone (i.e., in the absence of FSH) markedly suppressed granulosa cell apoptosis (P < 0.05) (Table 1). In addition, adenoviral XIAP sense cDNA delivery potentiated the antiapoptotic action of FSH compared with LacZ infection (P < 0.05) (Table 1). In the absence of FSH, XIAP antisense expression increased follicular apoptosis compared to LacZ control (P < 0.05) (Table 1). Moreover, in the presence of gonadotropin, adenoviral XIAP antisense infection markedly attenuated the cell survival effect of FSH and increased the number of apoptotic granulosa cells (P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fate of a developing follicle (continual growth and development vs. atresia) is determined by the fate of its cells (proliferation and differentiation vs. apoptosis), which in turn is regulated by their relative expression of "death" and "survival" genes. Thus, follicular development may be a consequence of suppression of cell death genes or overexpression of cell survival genes. Follicular atresia is associated with decreased granulosa cell IAP contents, and gonadotropin administration results in the up-regulation of follicular IAPs [18]. Although it is well established that granulosa cell survival and apoptosis are the cellular basis of follicular development and atresia, respectively, whether the changes in the expression of these intracellular antiapoptotic proteins are coincidental or causally related to these gonadotropin-regulated processes is not known.
Transgenic and knockout animals have been used extensively to assess the role of the gene (or genes) of interest in physiological processes. However, these approaches are often plagued with systemic complications because of overall ill health of the animals, which often affects normal development. The IAPs are ubiquitous and found in high abundance in proliferating mammalian cells [34]. Despite a newly established NAIP knockout mice model [35], no XIAP knockout animal model was available until recently [36] to enable assessment of the role of XIAP in the gonadotrophic regulation of follicular development and atresia. Whereas no apparent differences were observed between the ability of cells from the XIAP-deficient and wild-type mice to undergo caspase-dependent or -independent apoptosis, the cellular levels of other IAPs (e.g., cIAP-1 and cIAP-2) were unexpectedly higher, suggesting the existence of a possible compensatory mechanism that leads to the up-regulation of other IAP family members when XIAP expression is lost. However, when and how this compensatory mechanism is triggered is still unclear. By coupling an adenoviral gene-delivery system to the above-mentioned follicle culture model, we have demonstrated in the present study that XIAP is important in the gonadotrophic regulation of these ovarian developmental processes. In the present study, FSH increased XIAP expression, suppressed apoptosis, and stimulated follicular growth (as evidenced by increases in DNA content, cell number, and follicular volume). In contrast, antisense XIAP decreased FSH-induced XIAP, induced apoptosis, and prevented follicular development. These findings suggest that XIAP plays an important role in FSH-stimulated follicular development and serves as an antiapoptotic factor in rat ovarian follicles. Moreover, adenoviral XIAP sense infection in the cultured follicles increased XIAP contents and attenuated follicular apoptosis in both the absence and the presence of gonadotropin. Follicles infected with XIAP sense and cultured in the presence of low FSH concentration (5 ng/ml) showed a marked increase in follicular development (as evidenced by increases in DNA content and follicular volume) compared to the FSH-stimulated but LacZ-infected follicles. The XIAP sense was ineffective in the absence of gonadotropin. These findings suggest that whereas XIAP is necessary for the suppression of apoptosis, it alone is insufficient to promote follicular growth. These findings do not exclude the possible involvement of other IAPs in the gonadotrophic regulation of follicular development and atresia. In this context, eCG administration up-regulates granulosa cell HIAP-2 expression in immature rats, suppresses granulosa cell apoptosis, and induces follicular growth, whereas gonadotropin withdrawal suppresses HIAP-2 expression and induces apoptosis and follicular atresia [18]. Moreover, suppression of ovarian NAIP expression with antisense oligonucleotides evoked a decrease in the number of morphologically normal ovulated oocytes, implying an indirect involvement of NAIP in germ cell development via enhancement of granulosa cell survival [19].
The mode of action of XIAP in granulosa cell survival during ovarian follicular development has not been investigated, although we have recently shown that XIAP inhibits caspase-3 in ovarian epithelial cancer cells [37–39]. It is well established that IAPs modulate cell death pathways by inhibiting caspases [15–17, 37–43] and that the BIR domains are essential for their inhibitory action [40]. Specifically, XIAP possesses two different caspase-inhibitory activities, which can be attributed to distinct domains within XIAP. The BIR3 of XIAP is a specific inhibitor of caspase-9, whereas BIR2 plus the linker region of BIR1 and BIR2 is specific for caspase-3 and caspase-7 [40–42]. The IAPs suppress Fas ligand- and TNF-induced apoptosis by directly inhibiting caspase-3 and caspase-7 activation and activity [15–17], but they are ineffective in the activation of caspase-1, caspase-6, or caspase-10 [15]. In addition, XIAP modulates the cytochrome c/caspase-9-dependent mitochondrial death pathway. The mammalian HIAP-1, HIAP-2, and XIAP interfere with the function of caspase-9 by binding to inactive procaspase-9, thereby preventing its processing and activation [40, 43]. However, whether XIAP functions in this manner in the ovary remains to be determined. Interestingly, recent studies have shown that overexpression of XIAP increases phospho-Akt content in cultured rat granulosa cells [39] and human ovarian cancer cells [44]. This raises the possibility that XIAP may exert its antiapoptotic action via up-regulation of the PI3K/Akt pathway through a caspase-independent mechanism.
Our present findings indicate that although FSH is capable of increasing follicular cell number (as indicated by increased Alamar Blue reduction and DNA content) and growth in a follicle culture system, it fails to stimulate proliferation when added to granulosa cell primary cultures. The reason for this apparent discrepancy is not clear, but the granulosa cells used in the present studies may have been exposed to LH (in the eCG preparation) in vivo before isolation and culture, resulting in premature luteinization of the cells and, consequently, exit from the cell cycle. Alternatively, in addition to being a cell survival factor for granulosa cells via its direct action on XIAP expression, FSH may also stimulate follicular secretion of one or more mitogens, which in turn increases granulosa cell proliferation and ovarian follicular growth. In this context, FSH-induced follicular DNA synthesis in the hamster is mediated by follicular secretion and action of epidermal growth factor [45]. In addition to gonadotropins, various ovarian factors are believed to play an important role in rescuing follicles from apoptotic demise during follicular development [46]. Previous studies from our laboratory have shown that FSH increases follicular transforming growth factor secretion and that this theca-derived growth factor induces follicular growth in vitro [47]. In this context, recent studies using a coculture of bovine ovarian granulosa and theca cells have shown that the theca cells are essential in protecting granulosa cells from undergoing apoptosis, a process that is follicular stage-specific. These results suggest that theca cells may secrete one or more survival factors necessary for maintenance of granulosa cell viability, although the identity of this factor is yet to be defined [48]. It is also possible that the regulation of follicular growth by FSH is mediated by the action of TNF. Previous studies have shown that gonadotropins increase ovarian TNF content during follicular development [49] and that this cytokine is antiapoptotic and capable of inducing XIAP expression in rat granulosa cells [50]. The intraovarian regulation of follicular development and atresia by gonadotropin remains to be fully explored.
In conclusion, a follicle culture system coupled to an adenoviral gene-delivery procedure has been established, to our knowledge for the first time. Using this approach, we have demonstrated that XIAP plays an important role in the determination of granulosa cell fate following FSH challenge and that changes in XIAP expression induced by gonadotropin are critical for the maintenance of normal follicular growth and development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Benjamin K. Tsang, Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), 725 Parkdale Avenue, Ottawa, ON, Canada K1Y 4E9. FAX: 613 761 4403; btsang{at}ohri.ca
Received: 12 June 2002.
First decision: 29 June 2002.
Accepted: 3 September 2002.
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