Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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Biology of Reproduction 66, 1672-1680 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Involvement of Transforming Growth Factor {alpha} in the Regulation of Rat Ovarian X-Linked Inhibitor of Apoptosis Protein Expression and Follicular Growth by Follicle-Stimulating Hormone1

Yifang Wanga,b, Eric Asselina,b,c, and Benjamin K. Tsang2,,a,b

a Reproductive Biology Unit and Division of Reproductive Medicine, Department of Obstetrics & Gynecology and Cellular & Molecular Medicine, University of Ottawa, b Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), Ottawa, Ontario, Canada K1Y 4E9 c Département de Chimie-Biologie, Université du Québec à Trois-Rivières, Trois-Rivières, Québec, Canada G9A 5H7


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The expression of X-linked inhibitor of apoptosis protein (XIAP), a member of a family of intracellular antiapoptotic proteins, is induced by FSH during follicular development in vivo. Whether the XIAP up-regulation by FSH (100 ng/ml) is a direct action of the gonadotropin and is important in the control of granulosa cell proliferation during follicular growth is unclear. The overall objective of the present study was to examine whether the FSH-induced XIAP expression and granulosa cell proliferation during follicular development is mediated by the secretion and action of intraovarian transforming growth factor {alpha} (TGF{alpha}). In rat follicles cultured for 2 and 4 days, FSH stimulated estradiol production, TGF{alpha} secretion, XIAP expression, and follicular growth. The theca cells are the primary follicular source of FSH-induced TGF{alpha}, as indicated by in situ hybridization. Intrafollicular injection of a neutralizing anti-TGF{alpha} antibody (50–200 ng/ml; immunoglobulin G as control) or addition of estradiol-antagonist ICI 182780 (0.5–100 nM) to the culture media suppressed FSH-induced XIAP expression and follicular growth. The effect of ICI 182780 could be partially reversed by high concentrations of estrogen (250 and 500 nM). Whereas TGF{alpha} (10–20 ng/ml) significantly increased granulosa cell XIAP content and proliferation in primary granulosa cell cultures, FSH alone was ineffective in eliciting the mitogenic response. Our results support the hypothesis that FSH stimulates granulosa cell proliferation via theca TGF{alpha} secretion and action in response to increased granulosa cell estradiol synthesis, and that XIAP up-regulation in response to FSH suppresses granulosa cell apoptosis and facilitates FSH-induced follicular growth.

apoptosis, follicle, follicle-stimulating hormone, growth factors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gonadotropins are important endocrine regulators of follicular selection and survival during development [1]. The differential expression of ovarian factors plays a central role in these processes by modulating the action of gonadotropins [2]. Whereas dominant follicles continue to grow and, eventually, ovulate, the remaining members of the cohort undergo atresia, a process characterized initially by apoptosis of the membrana granulosa and subsequently by apoptosis of the theca layer [3]. In vivo studies regarding the hormonal regulation of follicular atresia have often been complicated by the presence in the ovary of follicles at different stages of development, which exhibit variable responses to endocrine and intraovarian regulators [4]. The recent establishment of a rat follicle culture system, in which regulation of the growth of follicles at a similar stage of development can be assessed, has provided a new and valuable approach for investigating the physiologic and molecular basis of the regulatory processes in the ovary [5]. We have demonstrated that whereas FSH stimulated follicular growth in vitro, it failed to increase granulosa cell proliferation in primary cultures [5]. These results suggest that the mitogenic response of granulosa cells to FSH in follicle cultures was not a consequence of a direct action of the gonadotropin but, rather, of the synthesis and secretion of one or more theca-derived factor that, in turn, stimulated granulosa cell proliferation.

It has been demonstrated that transforming growth factor {alpha} (TGF{alpha}) controls the programming of the transition of granulosa cells from a proliferative to a differentiated state and may determine the fate of developing follicles in the rat ovary (growth vs. atresia) [6]. It has also been shown that epidermal growth factor (EGF), TGF{alpha}, and FSH increase hamster ovarian follicular DNA synthesis and granulosa cell proliferation [7], which were markedly attenuated by addition of EGF-specific polyclonal antibody into the follicle cultures [8]. These results suggest that FSH-induced follicular DNA synthesis is, in part, mediated by follicular EGF.

The inhibitor of apoptosis proteins (IAPs) are a family of intracellular antiapoptotic proteins, which were first identified in baculovirus. They include X-linked IAP (XIAP or cIAP-3), human IAP-1 (HIAP-1 or cIAP-2), human IAP-2 (HIAP-2 or cIAP-1), neuronal apoptosis inhibitory protein (NAIP), Survivin, Livin, and Kiap [9–12]. IAPs are characterized by the presence of a caspase recruitment domain and an N-terminal baculovirus inhibitor of apoptosis repeat motif, the latter of which is 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 involved in protein-protein interactions [13] as well as protein ubiquitination and degradation [14]. To date, only a few reports have addressed the mechanisms of action by these antiapoptotic proteins. XIAP, HIAP-1, and HIAP-2 have been shown to be direct inhibitors of caspase-3 and caspase-7 [15] and also to modulate the Bax/cytochrome C death pathway by inhibiting caspase-9 [16].

We have previously shown that IAP contents in granulosa cells increase during follicular growth in response to eCG stimulation both in vivo and in vitro, and that eCG withdrawal results in decreased IAP expression and increased apoptosis in granulosa cells [17]. However, the mechanism(s) by which FSH induced these changes is not clear. Moreover, to our knowledge, if and how TGF{alpha}, a well-established cell survival intermediate, plays a role in the gonadotropic control of the fate of granulosa cells (survival vs. apoptosis) during follicular development in the rat has not been investigated. The objective of the present study was to assess the secretion and action of ovarian follicular TGF{alpha} using a defined follicle culture system and to determine whether this intraovarian factor is involved in the gonadotropic regulation of XIAP expression and apoptosis in rat granulosa cell during follicular growth in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Culture media, fetal bovine serum (FBS), antibiotics, Trizol, dNTP, Moloney murine leukemia virus-reverse transcriptase (MMLV-RT), and restriction endonucleases were purchased from Gibco Bethesda Research Laboratories (Burlington, ON, Canada). Oligo dT and RNase inhibitor were products of Ambion, Inc. (Austin, TX). HotStarTaq DNA polymerase and polymerase chain reaction (PCR) purification kit were from Qiagen, Inc. (Mississauga, ON, Canada). Agarose (low gelling temperature), DNA (calf thymus), Hoechst 33258, ICI 182780 (ICI), Triton X-100, Tween 20, bovine collagenase (type 1A), bovine DNase 1, bovine insulin, human transferrin, ascorbic acid, sodium selenium, and eCG were obtained from Sigma Chemical Company (St. Louis, MO). The enhanced chemiluminescence (ECL) detection kit and [methyl-3H]thymidine (25 Ci/mmol) were obtained from Amersham Life Science (Oakville, ON, Canada). Ovine FSH (NIAMDD oFSH-14) was obtained from the National Institute of Diabetes & Digestive & Kidney Disease (Baltimore, MD). Acrylamide (electrophoresis grade), N,N'-methylene-bis-acrylamide, ammonium persulfate, glycine, SDS-PAGE prestained molecular weight standards (low range), nitrocellulose membranes, and horse radish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse immunoglobulin (Ig) G were products of Bio-Rad (Richmond, CA). Rhodamine-conjugated goat anti-rabbit IgG, mouse monoclonal anti-TGF{alpha} antibody, and mouse IgG were product of Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mounting medium for fluorescence was purchased from Vector Laboratories, Inc. (Vectashield, H-100; Burlingame, CA). In situ cell-death detection kit and digoxygenin (DIG)-conjugated mProbe kits were purchased from Boehringer Mannheim (Montreal, PQ, Canada) and Alamar Blue dyes from Biosource, Inc. (Medicorp, Montreal, PQ, Canada). Microinjector system and stage warmer were purchased from Narishige International Inc. (Long Island, NY). ELISA kits for TGF{alpha} and 17ß-estradiol (E2) were purchased from Oncogene Research Products (QIA61; Darmstadt, Germany) and R&D Systems (DE2000; Minneapolis, MN), respectively. The estrogen-antagonist ICI was a product of Tocris Cookson, Inc. (Ballwin, MO). The pCR II-TOPO cloning kit was from Invitrogen (Carlsbad, CA). Cell strainers were purchased from Becton Dickinson Labware (Franklin Lakes, NJ).

Follicular Isolation and Culture

Ovaries from 22- to 24-day-old rats were cut into small pieces and incubated (37°C, 30 min) in {alpha} minimum essential medium ({alpha}-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 BSA (0.1%, w/v), and follicles (160–210 µm) were dissected out using 28.5-gauge needles. Only follicles judged to be normal (i.e., with oocyte and granulosa cells completely enclosed by the basement membrane and theca layer [5]) were cultured individually for 4 more days in 96-well plates in 100 µl of follicular culture medium (FCM; {alpha}-MEM supplemented with Hepes [10 mM], BSA [0.1%], rat serum [1%, v/v], bovine insulin [5 µg/ml], transferrin [10 µg/ml], ascorbic acid [25 µg/ml], sodium selenium [1 ng/ml] [18], nonessential amino acids [1%, v/v], streptomycin-penicillin [0.5%, w/v], and fungizone [0.25%, w/v]) with or without oFSH. The follicular size was determined daily before the medium change. At the end of the culture period, follicles were embedded in 2% agarose, fixed in buffered formalin phosphate solution (10%, room temperature [RT], 3 h), stained with neutral red (0.1%, w/v, 3 h), and then embedded in paraffin. The sections of cultured follicles were stained with hematoxylin phloxine saffvon for morphologic assessment.

Rat Granulosa Cell Isolation and Culture

Immature female Sprague-Dawley rats (24–25 days old) from Charles River Canada (Montreal, PQ, Canada) 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 FBS (10%). Granulosa cells were harvested by follicle puncture as previously described [19], washed, and centrifuged (900 x g, 10 min). Cells were plated for 24 h in RPMI 1640 medium with FBS (10%) under a humidified atmosphere of 95% air and 5% CO2 and then cultured for various durations in the absence or presence of adenoviral LacZ, XIAP antisense cDNA, or other treatments (i.e., TGF{alpha}, estrogen, FSH, and/or ICI). XIAP antisense cDNA construct is the expression vector pAdex1Cawt containing a full-length, double-stained cDNA, the expression of which will generate a message in the reverse orientation of XIAP mRNA [20].

Alamar Blue Assay

Alamar Blue is a nontoxic, metabolic dye [21] and has been used to monitor changes in cell numbers during tissue growth in vitro [22]. In the present studies, we have used this assay to assess the increase in follicular cell number during growth under the influence of FSH during a 6-day culture period. The assay was performed as described previously [21]. Follicles were incubated with Alamar Blue for 3 h and maintained under a humidified atmosphere (37°C, 95% air and 5% CO2). Absorbance at wavelengths of 570 and 630 nm was measured, and the ratio (570 nm:630 nm) was defined as the cell number equivalence.

Follicular Anti-TGF{alpha} Antibody Injection

To examine if FSH-induced follicular growth is mediated through the action of TGF{alpha}, follicles were transferred onto a cell strainer (pore size, 100 µm) in a 35-mm dish containing FCM after a 24-h culture in a 96-well plate. They were injected with anti-TGF{alpha} neutralizing antibody (normal mouse IgG for control group) at different concentrations (0–200 ng/ml) and cultured in the presence of FSH (100 ng/ml).

Animal Preparation for Ovarian TGF{alpha} In Situ Hybridization

Immature (22 days of age) female Sprague-Dawley rats (50–60 g; Charles River Canada) were injected with saline (0.9% NaCl i.p.) or eCG (15 IU i.p.) and, 24 h later, with 100 µl of either normal rabbit serum (NRS; saline and eCG groups) or anti-eCG antiserum (anti-eCG groups). Animals were killed 24 h after NRS or antiserum injection. Ovaries were excised, fixed in 10% formalin, and paraffin-sectioned for TGF{alpha} in situ hybridization assessment.

Adenoviral Infection in Primary Granulosa Cell Culture System

Adenoviral gene delivery was performed as described previously [23]. Briefly, after 24 h of plating in RPMI 1640 medium with 10% FCS, cells were infected with adenoviral XIAP antisense cDNA or LacZ at a serial of multiplicity of infection (MOI; 10, 20, 30, and 40). At an MOI of 10, the LacZ infection efficiency over 48 h (as determined by 5-bromo-4-chloro-3-indolyl-ß-D-galactoside [X-gal] assay) was more than 90%, and down-regulation of XIAP content by adenoviral XIAP antisense was confirmed by Western blot analysis.

Protein Extraction and Western Blot Analysis

Changes in XIAP content were assessed by Western blot analysis as previously described [17]. Briefly, granulosa cells detached from the growth surface were pelleted and lysed in a ice-cold lysis buffer (PBS, NP-40 [1%, v/v], sodium deoxycholate [0.05%, w/v], and SDS [0.1%, w/v] containing protease and phosphatase as well as kinase inhibitors, PMSF [10 µM], aprotinin [50 µg/ml], sodium orthovanadate [1 mM], sodium pyrophosphate [Nappi; 10 mM], and leupeptin and pepstatin [both 5 µg/ml]). Attached cells were lysed by the addition of lysis buffer to the culture dishes. Cells were sonicated briefly (5 sec/cycle, three cycles, 0 °C), incubated on ice (30 min), and centrifuged (15 000 x g, 30 min). Sonicates were pelleted, and 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) 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% 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 dilution), washed in TBS-T (3 washes of 5 min each), incubated in HRP-conjugated secondary antibody (1:5000) in blotto, and washed again in TBS-T twice (5 min) and then in TBS once (5 min). Peroxidase activity was visualized with the ECL kit according to manufacturer's instructions. Membranes were reprobed with {alpha}-tubulin. Both XIAP and tubulin protein signals were quantified densitometrically. The XIAP protein content was determined by dividing its signal intensity by that of the corresponding tubulin protein content to correct for any loading differences between lanes. XIAP content was then further divided by the control value (in the absence of TGF{alpha}).

TUNEL and XIAP Double-Staining

TUNEL was performed as described previously [24]. Briefly, paraffin-embedded whole-ovarian or follicle sections (thickness, 4–5 µm) were mounted on positively charged slides, deparaffinized, hydrated, washed thoroughly 3 times for 5 min each in 1x PBS, and then immersed in PBS with H2O2 (0.3%, RT, 10 min) to inhibit endogenous peroxidase activity. Following 3 additional 5-min washing in PBS, the sections were immersed in 50 µl of the TUNEL mixture (47.5 µl of TUNEL label containing fluorescein isothiocyanate (FITC)-deoxyuridine triphosphate and 2.5 µl of TUNEL enzyme) in a humidified chamber (37°C, 60 min). After incubation in the TUNEL mixture, sections were immersed in rabbit polyclonal anti-human XIAP antibody (1:50 in PBS; RT for 2 h or 4°C overnight) and, subsequently, in rhodamine-conjugated goat anti-rabbit IgG (1:200 in PBS; RT, 1 h). Sections were mounted with mounting medium and examined using an Olympus inverted microscope (Model IX 70, 20x objective; Olympus America Inc., Melville, NY) equipped with a confocal laser-scanning system (Bio-Rad 1024). The FITC signal in TUNEL-positive cells was excited at 488 nm, and images were collected at 522 ± 16 nm. 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 analyzed with imaging software (NIH Image 1.61: http://rsb.info.nih.gov/nih-image).

[Methyl-3H]Thymidine Incorporation

Incorporation of [methyl-3H]thymidine into DNA, an index for DNA synthetic capacity of cultured rat granulosa cells, was determined as previously described [25]. At the end of adenoviral (LacZ or XIAP-antisense; MOI, 0–40; 24 h) infection with or without TGF{alpha} (20 ng/ml, 24 h), granulosa cells were cultured for an additional 9 h in the presence of [methyl-3H]thymidine (1 µCi). The cells were washed twice with RPMI 1640 with and without unlabeled thymidine (100 µg/ml), fixed with trichloroacetic acid (5%, w/v; 4°C, 20 min), and then washed twice with methanol. The DNA pellet was dissolved in NaOH (0.25 M) adjusted to neutral pH with HCl (0.25 M). A 100-µl aliquot was stored at -20°C for subsequent DNA assay, and radioactivity in the remaining solution was counted to determine the level of radioactivity incorporated. The DNA synthetic capacity index was expressed as cpm/µg DNA.

DNA Assay

The samples (containing DNA) and calf thymus DNA (standard) were incubated with Hoescht 33258 dye (0.1 µg/ml, 5 min in the dark, RT) as described previously [25]. 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 of the assay was 2 ng/ml, and the linearity was up to 1 µg/ml.

In Situ Hybridization

Preparation of cRNA probes Total ovarian RNA was extracted with Trizol reagent according to the manufacturer's instructions and used for preparation of first-strand cDNA via MMLV-RT. A negative control was included, using the same reaction mixture but without MMLV-RT (substituted with water) to ensure absence of any contaminating genomic DNA in the RNA template. Expression of the TGF{alpha} gene was determined by amplification of a 200-base pair (bp) region (170–369 bp) of the rat TGF{alpha} gene sequence. Amplification was carried out using the TGF{alpha} antisense downstream sequence 5'-CCCAGAGTGGCAGACACAT-3' and the sense upstream sequence 5'-CTCTGCTAGCGCTGGGTATC-3'. The PCR cycling conditions were chosen as 30 sec at 94°C, 1 min at 55°C, and 1 min at 72°C for 35 cycles, followed by a 10-min extension at 72°C. After amplification, TGF{alpha} cDNA was cloned into pCR II-TOPO cloning vector and transformed into competent Escherichia coli DH5{alpha} cells. The insert was digested with EcoRI to verify the presence of the insert. The cDNA was sequenced in both directions to confirm TGF{alpha} sequence (Gibco). DIG-labeled riboprobes for in situ hybridization were synthesized by in vitro transcription using a DIG RNA-labeling kit. Sense TGF{alpha} probe was generated by linearizing DNA templates with HindIII and in vitro transcription with T7 polymerase. Antisense TGF{alpha} probe was generated by linearizing the DNA templates with NsiI and in vitro transcription with Sp6 polymerase. Digestions were monitored on agarose gel (2% [w/v]) and fragments purified using PCR purification columns.

Hybridization analysis Ovarian tissue and follicle sections were deparaffinized and rehydrated through a graded series of ethanol. Tissue sections were washed with diethyl pyrocarbonate (DEPC)-treated PBS (2 washes of 5 min each), DEPC-treated PBS with glycine (100 mM, 2 washes of 15 min each), DEPC-treated PBS with Triton X-100 (0.3% [v/v], 15 min), and DEPC-PBS (2 washes of 5 min each). Sections were permeabilized with RNase-free proteinase K (20 µg/ml) in TE buffer (100 mM Tris and 50 mM EDTA; pH 8; 30 min), refixed in paraformaldehyde-PBS (4% [w/v], 4°C, 5 min), rinsed twice in PBS, dehydrated, dried at room temperature, and then washed with 4x SSC (1x SSC: 150 mM NaCl and 15 mM sodium citrate, pH 7.2) containing formamide (50%, 10 min). Sections were hybridized with DIG-labeled antisense or sense (negative control) TGF{alpha} cRNA probes as described above. Hybridization buffer (40% [v/v] deionized formamide, 4x SSC, 1x Denhardt, 10% dextran sulfate, 1.0 mg/ml of yeast tRNA, 1.0 mg/ml of denatured salmon sperm DNA, and 10 mM dithiothreitol) containing DIG-labeled cRNA probe (10 ng/slide) was applied to each slide for hybridization (overnight, 42°C) in a humidified chamber. Slides were then washed in 2x SSC (3 washes of 15 min each) and 1x SSC (2 washes of 15 min each) at 37°C. Sections were then incubated (37°C, 30 min) with DNase-free RNase (20 µg/ml) in TEN (10 mM Tris, 1 mM EDTA, and 500 mM NaCl; pH 8.0) buffer to remove nonspecific binding and washed as follows: 0.1x SSC (2 washes of 30 min each, 37°C) and TN buffer (100 mM Tris-HCl [pH 7.5] and 150 mM NaCl, 2 washes of 10 min each). Sections were blocked with TN buffer containing 0.1% Trition X-100 and 2% normal sheep serum for 30 min and incubated (2 h, 37°C) in TE buffer containing Triton X-100 (0.1% [w/v]), normal sheep serum (1% [v/v]), and sheep anti-DIG-alkaline phosphatase (1:100 dilution). Sections were again washed with TN buffer (2 washes of 10 min each) and with TNM buffer (100 mM Tris-HCl [pH 9.5], 100 mM NaCl, and 50 mM MgCl2; 10 min), covered with 200 µl of color solution [10 ml of TNM buffer containing 45 µl of nitroblue tetrazolium (75 mg/ml in 70% [v/v] dimethylformamide), 35 µl of 5-bromo-4-chloro-3-indolyl-phosphate (50 mg in 100% dimethylformamide), and 1 mM levamisole], and then incubated in the dark for 24 h. Color reaction was stopped by incubating slides with 10 mM Tris-HCl and 1 mM EDTA (pH 8.0). Six ovaries from 3 rats in each experimental group (control, FSH, eCG, or eCG plus anti-eCG antibody) were embedded into 1 block, and three sections from each block were tested. Six follicles per treatment group in each experiment were embedded into 1 block. The experiment was repeated twice. An image from 18 representative ovaries or follicles is shown for each treatment group.

Enzyme-Linked Immunoadsorbent Assays

Transforming growth factor {alpha} The TGF{alpha} ELISA was performed according to the manufacturer's instructions. This assay kit has been validated for detection of TGF{alpha} in spent medium [26]. Briefly, biotinylated TGF{alpha} reporter antibody was added into wells precoated with a capture antibody following addition of the samples or standards. TGF{alpha} presented in the sample binds to both capture antibody and the reporter antibody in solution. Wells were washed twice with the wash buffer and once with the rinse buffer, then incubated (30 min, RT) with streptavidin-HRP, which conjugates to the reporter antibody. Substrate reagents were then added into wells and incubated (30 min, RT) in the dark. Reaction was stopped by the addition of stop solution, and absorbance at 490 nm was measured with a microplate reader. Samples were accumulated and analyzed together to avoid interassay variation. The intraassay variation was 4.4%.

17ß-Estradiol The principle of E2 ELISA was based on the competition between E2 present in samples and alkaline phosphatase-labeled E2 for sites on a rabbit polyclonal antibody. The assay was performed according to the manufacturer's instructions. Based on the manufacturer's information and a previous report [27], the E2 ELISA assay is validated to detect E2 in cell culture medium, with an average recovery rate of 98%. Briefly, following incubation of alkaline phosphatase-labeled E2 antibody with samples or standards (2 h, RT), wells were washed with wash buffer and subsequently incubated with substrate reagents (45 min, RT). The reaction was stopped by addition of the stop reagents, and the optical density at 405 nm was assessed by a microplate reader. The intraassay variation was 5.2%.

Statistical Analysis

All experiments were repeated at least 3 times. Data were subjected to one- or two-way ANOVA (PRISM software version 3.0; Graph Pad, San Diego, CA). Differences between experimental groups were determined by the Tukey test. The data on XIAP content, as determined by Western blot analysis and expressed in folds of the control value (see Fig. 5A), were arcsine square root-transformed before two-way ANOVA.



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  		FIG. 5. A) TGF{alpha} increases XIAP protein content in granulosa cells. Granulosa cells (0.5 million per 35-mm dish) from eCG-primed immature rats were cultured for 24 h in the presence of different concentrations of TGF{alpha} (0–20 ng/ml). Proteins from whole-cell lysate were analyzed by Western blot analysis. XIAP content was normalized by {alpha}-tubulin, and results are expressed as the fold of the control value and arcsine square root-transformed before two-way ANOVA analysis. B) TGF{alpha} increases granulosa cell number in vitro. Rat granulosa cells were cultured for 2 days with or without TGF{alpha} plus anti-TGF{alpha} antibody or mouse IgG (as control), and cells were counted at the end of culture duration. *P < 0.05 vs. control (CTL), +P < 0.05 vs. TGF{alpha}. C) A representative Western blot illustrating changes in XIAP contents following the adenoviral infection (XIAP antisense and LacZ) and TGF{alpha} challenge for 24 h. D) TGF{alpha}-stimulated thymidine incorporation is suppressed by adenoviral XIAP antisense cDNA infection. Granulosa cells from eCG-primed immature rats were cultured and infected with adenoviral XIAP antisense cDNA or LacZ. Sixteen hours after infection, cells were cultured with or without TGF{alpha} for a further 24 h. [3H]Thymidine was added 9 h before the end of the culture period. The DNA assay and thymidine incorporation were performed as described in Materials and Methods; [3H]thymidine incorporation is expressed in cpm/µg DNA. Results are the mean ± SEM of 3 independent experiments


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Role of TGF{alpha} in FSH-Stimulated Follicular Growth In Vitro

Follicles cultured over a 4-day period in the absence of FSH had minimum growth (Day 4 vs. Day 0; follicular volume: 3.6 ± 0.65 vs. 2.2 ± 0.25 nl, n = 28; cell number equivalence: 2.5 ± 0.35 vs. 1.2 ± 0.15 nl, n = 28), whereas the addition of FSH markedly increased follicular volume (16.5 ± 1.9 nl, n = 30, P < 0.001 vs. control) and cell number equivalence (14.7 ± 1.6, P < 0.001 vs. control). Intrafollicular injection of the anti-TGF{alpha} antibody (0–200 ng/ml) markedly attenuated the increases in follicular volume (P < 0.01) and cell number equivalence (P < 0.01) induced by FSH in a concentration-dependent manner, whereas injection of normal mouse IgG was ineffective (n = 30 for each treatment group, P > 0.05) (Fig. 1A). At 150 ng/ml, anti-TGF{alpha} antibody significantly the suppressed FSH-induced increase in follicular volume (from 12.5 ± 0.9 to 6.1 ± 0.5 nl, n = 30, P < 0.01) and cell number equivalence (from 11.9 ± 0.7 to 4.8 ± 0.5 nl, n = 30, P < 0.01) during the 4-day culture period. TGF{alpha} (10 ng/ml) alone exerted a small but concentration-dependent increase in follicular growth in vitro (P < 0.05) (Fig. 1B) and significantly enhanced the response induced by the gonadotropin (10 ng/ml, P < 0.05) (Fig. 1B).



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  		FIG. 1. Role of TGF{alpha} in FSH-induced ovarian follicular growth in vitro. A) Anti-TGF{alpha} antibody blocks follicular growth. Follicles were cultured for 4 days in the presence of FSH alone (100 ng/ml) or FSH plus different concentrations of anti-TGF{alpha} antibody or IgG (as control). Follicular diameter and cell number equivalence were measured at Day 0 and Day 4. *P < 0.05, **P < 0.01 compared to FSH + IgG at the respective concentration. B) TGF{alpha} alone stimulates follicular growth. Follicles were cultured with different concentrations of TGF{alpha} with or without FSH for 4 days, and follicle diameter was measured at Day 0 and Day 4. Change in follicular volume is defined as the follicular volume at Day 4 less that at Day 0 (V4 - V0). *P < 0.05, **P < 0.01 vs. control (no TGF{alpha}); +P < 0.05 vs. FSH alone (10 ng/ml). Values represent the mean ± SEM of a total of 30 follicles from three independent experiments.

FSH Stimulates Estrogen-Dependent Follicular TGF{alpha} Secretion In Vitro

To further examine if FSH-induced follicular growth is mediated through the secretion and action of TGF{alpha}, follicles were cultured in the absence or presence of FSH (100 ng/ml). The levels of TGF{alpha} in the spent media on Days 2 and 4 of culture were measured. TGF{alpha} levels in follicle cultures with gonadotropin were significant higher than those in the control groups, irrespective of the duration of the culture (P < 0.01) (Fig. 2A). To determine the cellular source of follicular TGF{alpha}, TGF{alpha} mRNA levels in ovarian and follicular sections following gonadotropin stimulation in vivo and in vitro, respectively, were examined by in situ hybridization (Fig. 3A). In the absence of FSH, follicular TGF{alpha} mRNA abundance was low, although clearly higher than that observed in negative control (with sense probe) (Fig. 3, A-1 vs. A-4). Addition of FSH to the follicle cultures markedly increased TGF{alpha} transcript levels in both granulosa and theca cells (Fig. 3, A-2 vs. A-1). The signals were more intense in the theca layer (Fig. 3A-2) and readily distinguishable from sections probed with sense cRNA (Fig. 3A-3). Similarly, ovarian sections from eCG-treated rats exhibited intense signal compared to those from saline-treated animals (Fig. 3, A-8 vs. A-7). Signal intensity was also higher in theca than in granulosa cells. Withdrawal of gonadotropin support by anti-eCG treatment in vivo resulted in a marked decrease in signal intensity of the TGF{alpha} message (Fig. 3, A-9 vs. A-8). Signal intensities in the negative control ovarian sections (with sense cRNA probe) were negligible (Fig. 3, A-3 to A-6).



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  		FIG. 2. Influence of FSH on follicular E2 and TGF{alpha} secretion in the absence and presence of ICI 182780 (ICI) in vitro. A)FSH increases E2 and TGF{alpha} secretion in cultured follicles. Follicles were cultured with or without FSH (100 ng/ml) for 4 days, and spent media were assayed for TGF{alpha} and E2 by ELISA. B) FSH-induced TGF{alpha} secretion was suppressed by E2-antagonist ICI. Follicles were cultured individually with or without FSH and/or ICI (0.1 µM) for 4 days. Spent media were assayed for TGF{alpha} as described in A. Results are the mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01 compared to control (CTL).



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  		FIG. 3. A) In situ hybridization of TGF{alpha} mRNA on ovarian or cultured follicle sections. Ovarian and follicular sections are probed with antisense TGF{alpha} cRNA probe (control [CTL; A-1], FSH [A-2], saline [A-7], eCG [A-8], and eCG-anti-eCG [A-9]) to determine the TGF{alpha} mRNA expression. Sense probe was used as negative control (FSH [A-3], CTL [A-4], eCG [A-5], and eCG + anti-eCG [A-6]). Six ovaries from three rats in each experimental group (rats injected i.p. with saline [A-7], eCG [A-8], or eCG + anti-eCG [A-9]) were embedded into one block. Each in vitro experiment contained six follicles per experimental group cultured in the presence (A-2) or absence (A-1) of FSH (100 ng/ml) and were embedded. Sections from three independent expeiments were tested. An image from 18 representative ovaries is shown for each treatment group. B) Effects of FSH, E2, TGF{alpha}, and/or ICI on XIAP expression and apoptosis in rat follicle cultures. Six follicles per experimental group were cultured in the absence (CTL) or presence of FSH (100 ng/ml), EL (E2 at 5 nM), TGF{alpha} (20 ng/ml), ICI (5 nM), FSH (100 ng/ml) + ICI (5 mM), or FSH + ICI + EH (E2 at 500 nM) for 4 days, fixed, embedded into one block, and paraffin-sectioned. The sections were tested for TUNEL (green) and XIAP immunoreactivity (IHC) of XIAP (red) as described in Materials and Methods. Negative control for TUNEL (no TUNEL enzyme in reaction mix) and XIAP (normal rabbit IgG in stead of XIAP antibody) showed undetectable signal indicating minimum nonspecific bindings (B-8). An image from 18 representative follicles is shown for each treatment group.

To test if the FSH-stimulated TGF{alpha} synthesis is associated with follicular estrogen secretion, spent media from Days 2 and 4 of the follicle cultures were analyzed for E2. FSH significantly increased E2 secretion during the 4-day culture period (P < 0.05) (Fig. 2A), with a greater response observed during the latter period. Also, addition of the estrogen-antagonist ICI (100 nM) to the culture medium significantly suppressed FSH-induced follicular TGF{alpha} secretion (P < 0.05) (Fig. 2B).

Role of Estrogen in Gonadotropic Regulation of XIAP Expression, Apoptosis, and Follicular Growth In Vitro

To determine the role of estrogen-induced TGF{alpha} secretions in the gonadotropic regulation of follicular growth and apoptosis, apoptotic signal (TUNEL) and XIAP immunoreactivity (immunohistochemistry) were measured in follicles cultured with or without FSH and in the absence or the presence of ICI (100 nM). Follicles cultured in the absence of gonadotropin for 4 days exhibited barely detectable XIAP levels and extensive apoptosis signals (Fig. 3B-1). Whereas addition of FSH to the follicle cultures markedly increased the intensity of the XIAP immunosignal and suppressed apoptosis (Fig. 3B-2), the presence of ICI (5 nM) effectively attenuated these responses (Fig. 3B-3). Similar to FSH treatment, addition of estrogen or TGF{alpha} alone to the follicle cultures resulted in increased XIAP expression and suppressed apoptosis compared to control follicles (Fig. 3, B-4 and B-5, respectively).

To further assess the role of estrogen in the cellular mechanism by which FSH stimulates follicular development in the present culture system, the influence of ICI on FSH-induced follicle growth was studied (Fig. 4A). Whereas ICI alone had either minimal or no effect on follicular volume during the 4-day culture period, it suppressed the increase in follicular growth in a concentration-dependent manner (0–100 nM, P < 0.01), with a significant response noted at 0.5 nM of the antiestrogen (P < 0.05; Fig. 4A). To test if the above-mentioned ICI effects are estrogen-specific, follicles were cultured for 4 days in medium containing FSH, ICI, E2 (5 nM), and/or excess E2 (250 nM and 500 nM [50x and 100x the concentration of ICI, respectively]), and follicular volume changes (Fig. 4B), TGF{alpha} secretion (Fig. 4C), XIAP expression, and apoptosis were examined (Fig. 3, B-6 to B-8). Estradiol alone (5 nM) significantly increased the concentrations of TGF{alpha} in the spent medium (P < 0.05) (Fig. 4C) and induced follicular growth (P < 0.05) (Fig. 4B). Whereas ICI significantly suppressed the FSH-induced TGF{alpha} secretion (P < 0.01) (Fig. 4C) and follicular growth (P < 0.05) (Fig. 4B), these responses were partially (P < 0.05 vs. FSH alone, P < 0.05 vs. FSH + ICI) (Fig. 4C) and completely (P = 0.065 vs. FSH alone, P < 0.05 vs. FSH + ICI) (Fig. 4B) attenuated, respectively, by the addition of the excess of E2. ICI suppressed FSH-induced XIAP expression and increased TUNEL signal intensity (Fig. 3B-3). Addition of estrogen (500 nM) to the culture medium partially reversed these responses (Fig. 3B-7).



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  		FIG. 4. Effects of ICI 182780 (ICI) on FSH-induced follicular growth and TGF{alpha} secretion. A) Follicles were cultured in the absence or presence of FSH (100 ng/ml) and different concentrations of ICI (0–100 nM) for 4 days. B and C) Influence of excess estrogen on the suppression of FSH-induced follicular growth and TGF{alpha} secretion by ICI during the 4-day culture period. Follicles were cultured with FSH, EL (E2 at 5nM), FSH + ICI (5 nM, 30 min before FSH), FSH + ICI + EM (E2 at 250 nM), or FSH + ICI + EH (E2 at 500 nM) for 4 days. Follicle diameter was measured daily. Results are expressed as the mean ± SEM of three independent experiments, with each containing 10 follicles per experimental group

TGF{alpha} Alone Up-Regulates Granulosa Cell XIAP Content and Stimulated Proliferation In Vitro

To determine if TGF{alpha} alone has a direct effect on XIAP expression and can elicit a mitogenic response, granulosa cells were cultured in the absence or presence of TGF{alpha} for 24 and 48 h, respectively. TGF{alpha} (0–20 ng/ml) significantly increased granulosa cell XIAP content in a concentration-dependent manner (P < 0.05) (Fig. 5A). Moreover, TGF{alpha} (10 ng/ml) significantly increased granulosa cell number (P < 0.05) (Fig. 5B), a response that was effectively suppressed by the presence of anti-TGF{alpha} antibody (P < 0.05) (Fig. 5B) but not of normal IgG (as control).

To determine if XIAP has a role in TGF{alpha}-induced granulosa cell proliferation, cells were infected with adenoviral XIAP-antisense cDNA or LacZ (as control) (MOI, 20 and 40) following plating and then cultured with TGF{alpha} for an additional 24 h. XIAP down-regulation (as confirmed by Western blot analysis) (Fig. 5C) significantly suppressed TGF{alpha}-induced [methyl-3H]thymidine incorporation into DNA (P < 0.05) (Fig. 5D).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates that FSH stimulates follicular growth and suppresses apoptosis in vitro. In addition, whereas FSH alone failed to elicit a mitogenic response in primary granulosa cell cultures, follicular growth induced by the gonadotropin was attenuated by anti-TGF{alpha} neutralizing antibody. These results are consistent with previous findings that follicles cultured without gonadotropin exhibited increased apoptotic DNA fragmentation that was prevented by the addition of FSH or cAMP [28, 29] and also support the concept that TGF{alpha} is an important intraovarian factor in the gonadotropic regulation of follicular development. In the human, TGF{alpha} is present in follicular fluid, and its level is inversely correlated with follicle growth, suggesting that the importance of TGF{alpha} as a mitogenic factor is dependent on the follicular stage [30]. TGF{alpha} increased [3H]thymidine incorporation during DNA synthesis in the undifferentiated, but not in the differentiated, granulosa cells [25].

The regulation of follicular development by FSH is complex and involves the participation of intraovarian factors [4]. In this context, it is of interest to note that TGF{alpha} alone stimulated follicular growth to a lesser extent compared to FSH, suggesting a possible involvement of other ovarian regulators (e.g., insulin-like growth factor or IGF, EGF, activin, inhibin). Homburg [31] reported that the suppression of apoptosis by FSH is partially mediated through IGF-1. In addition to FSH, EGF and IGF-I can stimulate follicle growth and antrum formation in a bovine preantral follicle culture system [32], and FSH-induced preantral folliculogenesis in the hamster ovary involves EGF gene transcription [33]. Moreover, evidence has shown that other intraovarian systems, including those of IGF and activin/inhibin, are also involved in follicle selection in response to gonadotropin [34]. These results illustrate complex cellular interactions in the regulation of granulosa cell fate and, thus, also of follicular fate.

It has been demonstrated that follicular atresia in the rat ovary is associated with decreased granulosa cell IAP contents and that gonadotropin up-regulates follicular IAP expression and stimulates follicular growth in vivo [17]. Whereas granulosa cell IAP levels were high during eCG-induced follicular development, gonadotropin withdrawal by anti-eCG antibody decreased granulosa cell IAP contents and induced apoptosis. Similarly, FSH has also been shown to increase follicular XIAP expression and growth of rat preantral or early antral follicles in vitro [5]. In the mouse ovary, the expression of NAIP, another member of the IAP family, is also under gonadotropic regulation during follicular development [35]. Increased NAIP gene expression in response to FSH was evident in the granulosa cells of healthy, but not of atretic, follicles. Another IAP, the inhibitor of T-cell apoptosis (ita), is highly expressed in developing, but not in prehierachical, hen ovarian follicles in vivo [36]. The patterns of ita mRNA expression during follicle development supports the contention that this gene is involved in protecting hen granulosa cells from apoptosis and, thus, in maintaining follicle viability. In the present study, addition of TGF{alpha} to the culture medium significantly increased XIAP expression in cultured follicles and follicular growth in vitro. Whereas TGF{alpha} also up-regulated granulosa cell XIAP content, down-regulation of XIAP by adenoviral antisense expression markedly attenuated the TGF{alpha}-stimulated incorporation of [3H]thymidine by granulosa cells in vitro, suggesting that XIAP may play an important role in the TGF{alpha}-mediated gonadotropic stimulation of follicular growth in the rat ovary.

Precisely how XIAP is involved in the suppression of apoptosis during follicular development is not clear. XIAP is known to modulate receptor-mediated apoptosis by inhibiting caspase-3 [15] and mitochondria-mediated cell death by suppressing caspase-9 activity [37]. Caspase-3 is present in granulosa cells of atretic rat ovarian follicles [38], and the role of Fas-mediated apoptosis has been well established in rat follicular atresia [19, 39]. Whereas tumor necrosis factor (TNF{alpha}) is a well-established caspase-3 activator [40], the cytokine failed to induce granulosa cell apoptosis in vitro unless XIAP was down-regulated by either XIAP antisense expression or cotreatment with the protein synthesis inhibitor cycloheximide [41]. It is conceivable that, like TNF{alpha}, FSH up-regulates XIAP expression during follicular development, leading to caspase inhibition and suppressed apoptosis. The role and gonadotropic regulation of the ovarian Bcl2/Bax expression and the cytochrome C/apoptotic protease-activating factor-1 (Apaf-1) pathway in mitochondria-mediated apoptosis is well established [42, 43], but FSH has recently been shown to suppress Apaf-1 expression and granulosa cell apoptosis in early antral follicles in vivo [44]. The latter observation raises the interesting possibility that, in addition to up-regulating XIAP to suppress caspase-9, FSH may exert it antiapoptotic action upstream in the survival pathway. Alternatively, XIAP may play an important role as an antiapoptotic factor by activating cell survival and/or signaling pathways involved in the regulation of expression of other cell survival factors. In this context, we have recently demonstrated that up-regulation of XIAP increased phospho-Akt content, a response blocked by the PI-3K-inhibitor LY294002, suggesting that possibility that XIAP may promote cell survival, in part, through activation of the PI-3K/Akt cell survival pathway [23]. In addition, recent studies have also shown that XIAP is a physiological activator of nuclear factor kappa B [45], a transcription factor believed to be involved in activation of various cell survival genes, including Flip, Bcl-2 family members, and so on [46–49].

Because granulosa, but not theca, cells have FSH receptors [50] and the theca is the main source of the TGF{alpha} (as demonstrated in the present study), the stimulatory action of FSH on TGF{alpha} secretion must be mediated through one or more granulosa cell-derived factors. In the present studies, we have confirmed earlier observations that FSH stimulated follicular E2 production [2] and that theca TGF{alpha}/EGF production can be stimulated by this estrogen during follicular development [8, 51]. Moreover, we have demonstrated, to our knowledge for the first time, that, like FSH, both E2 and TGF{alpha} up-regulated the follicular XIAP expression in vitro and that the estrogen-antagonist ICI attenuated FSH-induced follicular TGF{alpha} secretion and XIAP expression. These responses appeared to be important for the gonadotropic regulation of follicular growth, because ICI markedly suppressed FSH-induced changes in follicular volume in a concentration-dependent manner, a response that could be partially overcome by excess estrogen.

The mechanism by which FSH and TGF{alpha} up-regulate granulosa cell XIAP expression is not clear. Previous studies from our laboratory have shown that eCG increases granulosa cell phospho-Akt in vivo and that phospho-Akt could be colocalized with XIAP. Similarly, it has been demonstrated in various cellular systems that growth factors, such as EGF, can promote cell survival by activating the PI-3K/Akt pathway [52]. Whether activation of this pathway in granulosa cells by the gonadotropin during follicular development is mediated through TGF{alpha}, however, remains to be investigated.

In conclusion, the present studies have demonstrated that FSH induction of follicular development in vitro is mediated through an estrogen-dependent pathway involving TGF{alpha} derived from theca cells. Up-regulation of an antiapoptotic protein, XIAP, in response to FSH suppresses apoptosis in granulosa cells and facilitates follicular growth (Fig. 6).



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  		FIG. 6. A hypothetical model illustrating the role and regulation of estrogen, TGF{alpha}, and XIAP expression involved in granulosa cell (GC)-theca cell (TC) interaction during follicular development following FSH stimulation. The estrogen antagonist ICI 182780 (ICI) and anti-TGF{alpha} neutralizing antibody were used in the present study to test the dependence of these intraovarian factors in this process.


   ACKNOWLEDGMENTS
 
We thank Dr. Eric LaCasse (Ægera Therapeutics, Inc., Ottawa, ON, Canada) for providing the adenoviral LacZ, XIAP-antisense cDNA, and anti-XIAP antibody used in the present studies.


   FOOTNOTES
 
First decision: 20 November 2001.

1 Supported by a grant from the Canadian Institutes of Health Research (MOP-10369 to B.K.T.). Y.W. and E.A. were recipients of an NSERC Scholarship and CIHR Fellowship, respectively. Back

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 Back

Accepted: December 21, 2001.

Received: November 2, 2001.


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