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Biology of Reproduction 65, 477-487 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Follicular Stage-Dependent Tumor Necrosis Factor {alpha}-Induced Hen Granulosa Cell Integrin Production and Survival in the Presence of Transforming Growth Factor {alpha} In Vitro1

J. Soboloffa,b,c, H. Sasakia,c, and B.K. Tsanga,b,c

a Reproductive Biology Unit and Division of Reproductive Medicine, Departments of Obstetrics & Gynaecology and b Cellular & Molecular Medicine, c University of Ottawa and Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), Ottawa, Ontario, Canada K1Y 4E9

ABSTRACT

The link between cell adhesion to extracellular matrix and integrin-mediated survival signals has been established in several physiological systems, and roles for the cytokines tumor necrosis factor alpha (TNF{alpha}) and transforming growth factor alpha (TGF{alpha}) have been suggested. TGF{alpha} stimulates fibronectin production in hen granulosa cells and is an important survival factor during follicular maturation. In contrast, the role of TNF{alpha} and its possible interaction with TGF{alpha} in the regulation of granulosa cell fate (death versus survival) during ovarian follicular development have not been fully elucidated. The object of the current study was to determine if TNF{alpha} and TGF{alpha} interact in the regulation of hen granulosa cell fibronectin and integrin content in the context of cell death and survival during follicular development. TGF{alpha} (0.1 or 10 ng/ml), but not TNF{alpha} (0.1 or 10 ng/ml), increased both cellular and secreted fibronectin content in granulosa cell cultures of F5,6 but not F1 follicles. The expression of integrin ß3 subunit was also stimulated by TGF{alpha} in a follicular stage-dependent manner, and culture of F5,6 granulosa cells with TNF{alpha} in the presence of maximal stimulatory concentrations of TGF{alpha} potentiated this response. TGF{alpha} increased both F5,6 and F1 granulosa cell [3H]thymidine incorporation but not 3-(4,5-dimethylthiazol-2-yl)3,5-diphenyl tetrazolium bromide (MTT) metabolism. Although TNF{alpha} had no effect on [3H]thymidine incorporation irrespective of the presence of the growth factor, MTT metabolism was higher in F5,6 granulosa cells cultured for 24 h with both TNF{alpha} and TGF{alpha} than with either cytokine alone. Incubation of F5,6 granulosa cells for 48 and 72 h resulted in a TGF{alpha}-inhibited loss of cellular adhesion and detachment of granulosa cells from the growth surface. Although TNF{alpha} alone had no effect on cell morphology, it facilitated the reorganization of the granulosa cells into multicellular follicle-like structures in the presence of the growth factor. DNA degradation significantly increased between 0 and 72 h of culture in the absence of the cytokine but was suppressed by the addition of TGF{alpha} but not of TNF{alpha}. However, fluorometric analysis indicated that the primary type of cell death exhibited by F5,6 granulosa cells during extended culture and attenuated by the presence of TNF{alpha} and TGF{alpha} was necrosis and not apoptosis. The current study demonstrates that TNF{alpha} and TGF{alpha} interact in the regulation of granulosa cell integrin content and cell survival in vitro in a follicular stage-dependent manner. These findings suggest that follicular development is accompanied by a change in the intraovarian role of TNF{alpha}; it is atretogenic prior to follicular selection but prevents follicular demise during preovulatory growth.

apoptosis, cytokines, follicular development, growth factors

INTRODUCTION

The growth, differentiation, and apoptosis of ovarian follicular cells is regulated by the actions and interactions of endocrine, paracrine, and autocrine factors [1, 2]. Specifically, tumor necrosis factor alpha (TNF{alpha}) is an inhibitor of gonadotropin-induced differentiation [3–7] and transforming growth factor alpha (TGF{alpha}) is an established mitogenic [8–12], antidifferentiative [13], and antiapoptotic [14] factor for granulosa cells during follicular development. In addition, recent studies on mammalian [15] and avian [16] granulosa cells have suggested that TNF{alpha} is proapoptotic prior to follicular selection at the antral and large white follicle stage of development in mammalian and avian species, respectively. It is generally accepted that once a follicle is selected for ovulation in vivo, the development of the follicle to the preovulatory stage is committed, and follicular atresia (and hence apoptosis) does not occur [17]. The precise role of TNF{alpha} in the regulation of granulosa cell fate beyond these follicular stages is not known. Moreover, TNF{alpha}-induced cell survival and/or proliferation have been observed in fibroblasts [18], lymphocytes [19], and cancer cells [20, 21]. If and how TNF{alpha} serves as a granulosa cell survival factor and thus plays an important role in follicular development remains to be established. However, in order to understand fully the roles of these factors in vivo, it is necessary to determine how the presence of both cytokines and growth factors simultaneously will modulate granulosa cell responses.

Cell survival responses to TNF{alpha} are believed to be mediated by stress-activated protein kinase (SAPK) via activation of the transcription factor nuclear factor Kb (NF-KB; [22]). Although the involvement of both NF-KB and SAPKs in TNF{alpha}-induced cell survival has been demonstrated, the cellular mechanism(s) by which these transcription factors protect cells from apoptosis is not currently known. Interactions between fibronectin and the integrin receptors {alpha}5ß3 and {alpha}vß3 have been shown to inhibit apoptosis via the Ras-ERK pathway [23]. Whereas a relationship between TNF{alpha} and these receptors has not been reported, the cytokine can upregulate vascular cell adhesion molecule-1 [24] and integrin {alpha}2 [25] expression in epithelial cells and fibroblasts via NF-KB and AP-1, respectively. Because TGF{alpha} is known to increase fibronectin production in hen granulosa cells in a follicular stage-dependent manner [26], it is possible that these two physiological regulators may function synergistically to enhance cell adhesion during the process of follicular selection, resulting in follicular reorganization and growth and increased resistance to death factors.

In the current studies, we have examined the influence of TNF{alpha} and TGF{alpha}, alone or together, on granulosa cell fibronectin and integrin ß3 levels and survival in vitro. In addition to the reported apoptotic action of TNF{alpha} on granulosa cells during early stages of follicular development, our studies demonstrate a protective role for TNF{alpha} in those cells that develops as the follicles mature but which is lost at the preovulatory stage. Our findings support the concept that the expression of cell adhesion molecules in response to the cytokine is important for granulosa cell survival and follicular development. Furthermore, we suggest a potential role for extracellular matrix-integrin interaction in growth- and differentiation-dependent follicular reorganization during development.

MATERIALS AND METHODS

Reagents

[32P]{alpha}-CTP, [3H]thymidine, and ECL kit were obtained from Amersham Life Science (Oakville, ON, Canada). Acetone, bromphenol blue, chloroform, and methanol were from BDH Chemicals (Toronto, ON, Canada) while DC protein assay kits, goat anti-mouse IgG (horseradish peroxidase conjugated), nitrocellulose paper, and SDS were from Bio-Rad Laboratories (Hercules, CA). Acrylamide, agarose, Tris, and Coomassie blue were purchased from Boehringer Mannheim (Laval, PQ, Canada). Medium 199 (M199), minimum essential medium, and trypsin-EDTA were obtained from Gibco Laboratories (Grand Island, NY); and TNF{alpha} and TGF{alpha} were from Immunocorp (Montreal, PQ, Canada). DNA polymerase I, large (Klenow) fragment was obtained from New England BioLabs (Mississauga, ON, Canada). Buffer AL and the Nucleotide Removal Kit were obtained from Qiagen Inc. (Santa Clarita, CA). Acridine orange, aprotinin, collagenase (type 1A), sodium deoxycholate, ethidium bromide, fungizone, glycerol, goat anti-mouse IgM (horseradish peroxidase conjugated), isopropanol, 3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl tetrazolium bromide (MTT), nonidet P-40 (NP-40), penicillin-streptomycin, phenol, PMSF, trypsin inhibitor (type II-s), and tween-20 were obtained from Sigma Chemical Co. (St. Louis, MO). Mouse anti-human integrin ß3, mouse anti-human integrin ß1, and mouse anti-human fibronectin were purchased from Transduction Laboratories (Mississauga, ON, Canada). Hepes was obtained from VWR Canada Ltd. (Ottawa, ON, Canada).

Granulosa Cell Isolation

White leghorn hens housed individually in a windowless, air-conditioned room with a 14L:10D cycle were killed by cervical dislocation 10–14 h before the expected time of ovulation. Granulosa cell layers from the first (F1) and fifth and sixth (F5,6) largest developing follicles were removed and dispersed by incubation at 37°C in medium 199 containing 270 U/ml collagenase and 0.01% (w/v) trypsin inhibitor for 10, 15, and 20 min, respectively, as described by Asem et al. [27]. The M199 was supplemented with Hepes (25 mM), penicillin (50 U/ml), streptomycin (50 µg/ml), and fungizone (0.625 µg/ml; hereafter referred as M199).

[3H]Thymidine Incorporation Assay

To assess DNA synthetic capacity of granulosa cells during follicular development, the incorporation of [3H]thymidine was determined as previously described [9]. Granulosa cells (2.5 x 105) were cultured 18 h in 24-well plates (Falcon Plastics, Los Angeles, CA) in the presence or absence of TNF{alpha} and/or TGF{alpha} followed by an additional 6 h in the presence of 0.125 µCi/well of [3H]thymidine. Cells were then washed at room temperature in the presence of unlabeled thymidine (100 µg/ml) in Dulbecco modified PBS (dPBS; CaCl2 [0.9 mM], MgCl2A6 H2O [4.9 mM], KCl [2.7 mM], NaCl [137 mM], K2HPO4 [1.15 mM], KH2PO4 [8.45 mM]; pH 7.4), followed by two additional washes in dPBS alone. Cells were then fixed with trichloroacetic acid (TCA; 5% [w/v] in dPBS; 4°C), washed with TCA and twice with methanol (100%), and dissolved in KOH (250 µl; 0.5 M, room temperature). Following an incubation period (approximately 15 min) to ensure complete resuspension of all remaining cell contents, the pH of the cell lysates was adjusted to 7.6 with H3PO4. DNA content was measured by the saran wrap method [28], and [3H]thymidine incorporation was expressed in cpm per µg DNA.

MTT Assay

The MTT assay [29] was used to estimate cell viability in culture. Briefly, F5,6 or F1 granulosa cells (2.5 x 105 cells/well) were cultured for 24 h in 24-well culture plates (Falcon Plastics) in the presence or absence of TNF{alpha} and/or TGF{alpha}. MTT (5 mg/ml; 1/10 total volume) was then added to the cells that were incubated for an additional 3–4 h and solubilized overnight in acidic isopropanol (0.04 N HCl). An assay blank containing a mixture of MTT (50 µl), MEM (500 µl), and acidic isopropanol (550 µl) was also included. Dye conversion was assessed spectrophotometrically (570 nm) after background subtraction (630 nm).

Morphological Studies

The F5,6 granulosa cells (2.5 x 105 cells/well) were cultured for 72 h in 24-well culture plates (Falcon Plastics) in the presence or absence of TNF{alpha} and/or TGF{alpha}. Changes in granulosa cell morphology following 24, 48, and 72 h of culture were assessed by phase-contrast microscopy.

DNA Labeling

DNA fragmentation was assessed as an index of apoptosis in cultured granulosa cells by an established DNA labeling approach [30]. At the end of the culture period, DNA from floating and attached (harvested following trypsin-EDTA treatment) cells were extracted with phenol/chloroform, precipitated with ethanol, quantified, and radiolabeled with {alpha}-[32P]dCTP (5 µCi/500 ng/sample) with Klenow enzyme (large fragment; 2.5 U, 30 min). Unbound {alpha}-[32P]dCTP was separated from the DNA using the Qiagen Nucleotide Removal Kit. Radiolabeled DNA was resolved by agarose (2%) gel electrophoresis in Tris-EDTA buffer (Tris [40 mM], acetic acid [20 mM], EDTA [1 mM]; -4.5 h, 60 V). The gel was dried and exposed to X-ray film or a phosphorimager (Bio-Rad, Hercules, CA). The intensity of labeled low molecular weight DNA (<23 kilobases [kb]) in each lane was determined by two-dimensional densitometry, using the Molecular Analyst software (Bio-Rad).

Vital Staining

Vital stains were used for morphological assessments of granulosa cell viability and to differentiate between apoptotic and necrotic cell death as previously described [31]. At the end of the culture period, floating and attached cells were combined and centrifuged (1000 x g; 10 min). The pellets were resuspended in PBS (25 µl) and supplemented with a dye mixture (1 µl; acridine orange [100 µg/ml] and ethidium bromide [100 µg/ml]). Aliquots (10 µl) were assessed by fluoresecent microscopy. Live cells showed normal nuclear characteristics (bright green chromatin with organized structure), while apoptotic cells had condensed and/or fragmented nuclei (early apoptotic, bright green chromatin; late apoptotic, bright orange chromatin), and necrotic cells exhibited a bright orange nuclear stain but no nuclear condensation (with either a normal chromatin structure or no nuclear staining) (Fig. 1). The number of cells in each group was expressed as a percentage of the total cell number (minimum of 200 cells per treatment; in duplicate).



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  		FIG. 1. Representative general cellular and nuclear morphology of viable, early and late apoptotic, and necrotic cells following staining with acridine orange (100 µg/ml) and ethidium bromide (100 µg/ml). Viable cells were impermeable to ethidium bromide and had normal round nuclei that stained green. Apoptotic cells had condensed and/or fragmented nuclei. These cells were impermeable to ethidium bromide during early stages of apoptosis, and their nuclei stained green. During the later stages of apoptosis, this ability was lost and their nuclei stained red. Necrotic cells exhibited a red nuclear stain but no nuclear condensation. They exhibited either normal nuclear structure or had no nuclear staining

Western Blot

The F5,6 and F1 granulosa cells (1.2 x 106 to 1.5 x 106 cells/well) were cultured for 24 h in 6-well culture plates (Falcon Plastics) in the presence or absence of TNF{alpha} and/or TGF{alpha}. Media were collected, supplemented with PMSF (10 µg/ml) and aprotinin (57 µg/ml), and centrifuged (4000 x g; 15 min; 4°C) to recover floating cells stored at 4°C. Cells attached to the culture surface were removed with cell scrapers in cold PBS (1 ml) and added to the floating cells recovered from spent media, centrifuged (18 000 x g; 10 min; 4°C) and lysed in chilled buffer (NP-40 [1% v/v], sodium deoxycholate [0.05% w/v], SDS [0.1% w/v]) containing PMSF (10 µg/ml) and aprotinin (57 µg/ml) by gentle mixing (30 min; 4°C). The supernatants (18 000 x g; 20 min; 4°C) were stored in aliquots (-20°C) for subsequent analysis. Protein content was quantified using Bio-Rad DC protein assay kits. Proteins (20 µg) were resolved on 8% SDS-PAGE [32] and electroblotted onto nitrocellulose membranes (Bio-Rad [33]). After transfer, the gels were stained with Coomassie blue to verify even sample loading.

The membranes were blocked (l h, room temperature) in powdered skim milk (5%; Carnation, ON, Canada) in Tris-buffered saline-Tween 20 (TBST; Tris [10 mM], NaCl [150 mM], Tween-20 [0.05%], pH 8.0) and subsequently incubated (1 h, room temperature) with primary antibodies (mouse anti-human fibronectin IgG [1:2500] or mouse anti-human integrin ß3 IgM [1:250]) in 5% blocking solution. Membranes were then washed (2 x 7 min) in TBST, incubated with secondary antibody (30 min; goat anti-mouse IgG [for fibronectin] or goat anti-mouse IgM [for integrin ß3)] conjugated to horseradish peroxidase; 1:2500 in 5% blocking solution), and washed in TBST (3 x 5 min) and in Tris-buffered saline (TBS; 1 x 5 min; Tris [10 mM], NaCl [150 mM], pH 8.0). Peroxidase activity was visualized using the ECL kit as per manufacturer's instructions.

RESULTS

Influence of TNF{alpha} and TGF{alpha} on Granulosa Cell Integrin and Fibronectin Content During Follicular Development

To study the influence of TNF{alpha} and TGF{alpha} on granulosa cell integrin ß3 content during follicular development, highly differentiated and proliferatively active granulosa cells from F1 and F5,6 follicles, respectively, were cultured under confluent conditions for 24 h in the absence or presence of either or both factors at minimally and maximally effective concentrations (0.1 or 10 ng/ml). Integrin ß3 appeared as a 90-kDa band on Western blots at both stages of follicular maturation, although 35- and 40-kDa bands that may represent its degradation products were also observed (Fig. 2). Integrin ß3 content was significantly increased by TGF{alpha} in F5,6 (P < 0.02) but not F1 (P > 0.05) granulosa cells (ANOVA; Fig. 2), with significant differences observed at 10 ng/ml of the growth factor (P < 0.05; Tukey Test). Although TNF{alpha} (0.1 or 10 ng/ml) alone had no significant effect on granulosa cell integrin ß3 content irrespective of follicular maturation (P > 0.05), ANOVA indicates a significant interaction between TNF{alpha} and TGF{alpha} on integrin ß3 content in granulosa cells from F5,6 (P < 0.01) but not F1 (P > 0.05) follicles, brought about by a marked increase in the receptor subunit in F5,6 cells in the presence of both factors (10 ng/ml; P < 0.05).



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  		FIG. 2. Integrin ß3 production by hen granulosa cells during follicular development in the presence or absence of TNF{alpha} and/or TGF{alpha}. F5,6 (right panels) and F1 (left panels) granulosa cells were cultured for 24 h in the presence or absence of TNF{alpha} (0.1 or 10 ng/ml) and/or TGF{alpha} (0.1 or 10 ng/ml). Representative Western blots of F1 and F5,6 cellular extracts are depicted in A. Densitometric analysis (B) of the 90-kDa protein in F1 and F5,6 extracts were performed using the Molecular Analyst software from Bio-Rad Laboratories. Results represent means ± SEM from three independent cultures. *Significantly different from control (P < 0.05)

Fibronectin in granulosa cell cultures exist as cell-associated (140 and 200 kDa) and secreted (210 kDa) forms, the levels of which were dependent on follicular maturation and the presence of TGF{alpha} (Fig. 3). Incubation of cells from F5,6 but not from F1 follicles with TGF{alpha} significantly increased the content of both the 200-kDa (P < 0.001 and P > 0.05, respectively) and 140-kDa (P < 0.01 and P > 0.05, respectively) cell-associated proteins as well as of the 210-kDa secreted form (P < 0.05). Significant differences in the content of cell-associated fibronectin were observed in the presence of the growth factor (P < 0.05) at 0.1 (200 kDa only) and 10 ng/ml (140 and 200 kDa). Significant interactions between TNF{alpha} and TGF{alpha} were not detected, although a notable trend of higher abundance of the 140-kDa protein (P = 0.07) was observed in the presence of the agonists. Significant differences in the secretion of fibronectin (210 kDa) due to TNF{alpha} were not detected (P > 0.05).



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  		FIG. 3. Fibronectin production by hen granulosa cells during follicular development in the presence or absence of TNF{alpha} and/or TGF{alpha}. F5,6 (right panels) and F1 (left panels) granulosa cells were cultured for 24 h in the presence or absence of TNF{alpha} (0.1 or 10 ng/ml) and/or TGF{alpha} (0.1 or 10 ng/ml). Representative Western blots of F1 and F5,6 cellular extracts (A) and spent media (B) are presented. The relative abundance of the cell-associated 200-kDa and 140-kDa as well as soluble 210-kDa forms of fibronectin were analyzed densitometrically (C) using the Molecular Analyst software from Bio-Rad Laboratories. Results represent means ± SEM from three independent cultures

TNF{alpha}-TGF{alpha} Interaction in the Regulation of Granulosa Cell Survival

Cell proliferation and viability assays were performed using [3H]thymidine incorporation and MTT assays. Although basal [3H]thymidine incorporation was significantly higher in granulosa cells from F5,6 than from F1 follicles, the reverse was true for basal MTT metabolism (Fig. 4; P < 0.001). TGF{alpha} (10 ng/ml) significantly increased granulosa cell DNA synthesis in a follicular stage-dependent manner (F1, fivefold [P < 0.001]; F5,6, twofold [P < 0.01]) during a 24-h incubation period. TNF{alpha} (10 ng/ml) had no significant (P > 0.05; 3-way ANOVA) effect on basal or TGF{alpha}-induced [3H]thymidine incorporation in either F1 or F5,6 granulosa cells. In addition, while neither TNF{alpha} or TGF{alpha} alone affected granulosa cell viability in vitro irrespective of developmental stage of the follicle, incubation with both factors for 24 h appeared to increase MTT metabolism in F5,6 (P < 0.05) but not F1 (P > 0.05) granulosa cells. However, subsequent analysis revealed that, incubation with TNF{alpha} and TGF{alpha} maintained mitochondrial activity at in vivo levels, while MTT readings decreased during in vitro incubation in all samples that did not contain both of these factors (data not shown). This observation led us to study the effects of TNF{alpha} and TGF{alpha} on granulosa cells over a longer culture period.



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  		FIG. 4. The effect of TNF{alpha} on basal or TGF{alpha}-induced [3H]thymidine incorporation and MTT metabolism in hen granulosa cells during follicular development. F5,6 (right panels) or F1 (left panels) granulosa cells (2.5 x 105) were incubated for 24 h with BSA (0.1 %; control [cont]) or TNF{alpha} (10 ng/ml) in the presence or absence of TGF{alpha} (10 ng/ml). Results represent means ± SEM from three independent cultures. *Significantly different from control (P < 0.01). **Significantly different from control (P < 0.001)

TNF{alpha}-TGF{alpha} Interactions Following Long-Term Culture

To assess the long-term effects of the observed TNF{alpha}-TGF{alpha}-dependent increases in adhesion molecules and cell survival, the morphology of F5,6 granulosa cells cultured for 24, 48, or 72 h in the presence or absence of TNF{alpha} and/or TGF{alpha} was examined (Fig. 5). In the absence of either TNF{alpha} or TGF{alpha}, granulosa cells attached and spread on the growth surface during the first 24 h (Fig. 5A), but large percentages of cells became detached following 48 (Fig. 5B) and 72 (Fig. 5C) h of culture. TNF{alpha}, by itself, had little or no effect on either the attachment or detachment process (Fig. 5, D–F). Despite minimal effect on the attachment of granulosa cells (Fig. 5G), TGF{alpha} attenuated the detachment process during 48 (Fig. 5H) and 72 (Fig. 5I) h of culture. F5,6 granulosa cells cultured in the presence of both TNF{alpha} and TGF{alpha}, however, were distinct in several aspects. During the first 48 h of culture (Fig. 5, J and K), granulosa cell numbers and appearance were similar to those treated with TGF{alpha} alone (Fig. 5, G and H), although the TNF{alpha}-treated cells appeared to be more densely packed. Between 48 and 72 h of culture, however, granulosa cells cultured in the presence of both factors appeared to have reorganized into multicellular structures analogous in appearance to the ovarian follicle (Fig. 5L). A more detailed analysis of these cultures revealed morphological changes at both the multicellular and single-cellular level (Fig. 6). A picture at low magnification (Fig. 6A) shows the widespread nature of this reorganization that involved virtually all of the cells in culture. Higher magnification pictures (Fig. 6, B–D), however, reveal distinct cellular morphology that was dependent upon the location of the cell within these follicular structures. Specifically, those cells that were in contact with neighboring cells on all sides and in areas of high cell density tended to be round (Fig. 6, B and D). In contrast, cells that did not have full cell contact or were located in relatively low cell density areas were more rectangular (e.g., cells circumscribing round open areas of growth surface; Fig. 6, B and D). In addition, some cells exhibited long processes, characteristic of cell migration (Fig. 6C).



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  		FIG. 5. The time course of changes in F5,6 granulosa cell morphology in vitro. F5,6 granulosa cells were incubated for 24 (A, D, G, J), 48 (B, E, H, K), or 72 (C, F, I, L) h in the absence (A–C) or presence of TNF{alpha} (10 ng/ml; D–F), TGF{alpha} (10 ng/ml; G–I), or both factors (J–L). Pictures were taken using a phase-contrast microscope with x100 magnification



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  		FIG. 6. F5,6 granulosa cell morphology in vitro in the presence of TNF{alpha} and TGF{alpha}. F5,6 granulosa cells were incubated for 72 h in the presence of TNF{alpha} (10 ng/ml) and TGF{alpha} (10 ng/ml). Pictures were taken using a phase-contrast microscope with x50 magnification (A), x200 magnification (B), or x400 magnification (C and D)

To examine the relationship between changes in cell morphology and granulosa cell survival, DNA degradation and nuclear morphology were measured in F5,6 granulosa cells cultured for 0, 24, 48, or 72 h (Figs. 7 and 8). Apoptotic DNA degradation significantly increased throughout the 72 h culture period (P < 0.002) but was significantly suppressed by TGF{alpha} (P < 0.02). TNF{alpha} had no significant influence on DNA degradation observed in serum-free medium or on the antiapoptotic action of TGF{alpha} (P > 0.05; Fig. 7B). In order to differentiate between the different stages of apoptosis, F5,6 granulosa cell viability and the nature and stages of cell death (e.g., early apoptosis, late apoptosis, and necrosis) were determined by double staining with acridine orange and ethidium bromide (Fig. 8). Significant interactions between TNF{alpha} and TGF{alpha} on F5,6 granulosa cell viability were observed (ANOVA, P < 0.02) that were unaffected by the duration of culture (P > 0.05). In contrast, increases in cell viability observed in the presence of TGF{alpha} alone were attenuated during the culture period (P < 0.001).



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  		FIG. 7. Apoptotic DNA degradation in cultured F5,6 granulosa cells is inhibited by TGF{alpha} irrespective of the presence of TNF{alpha}. F5,6 granulosa cells were cultured in the presence or absence of TNF{alpha} (10 ng/ml) and/or TGF{alpha} (10 ng/ml) for 24, 48, or 72 h. Total DNA extracted from both attached and floating cells was labeled with {alpha}-[32P]dCTP, resolved on agarose gels, and exposed to a phosphorimager for densitometric analysis of low molecular weight (mw) DNA (<4 kb; with Molecular Analyst Software from Bio-Rad Laboratories). A) Representative autoradiograph of labeled DNA resolved by electrophoresis (samples shown in duplicates). Molecular weights are indicated on the left. B) Densitometric quantitation of low molecular weight DNA content (<4 kb). Results represent means ± SEM from three independent cultures



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  		FIG. 8. Necrosis in cultured F5,6 granulosa cells is inhibited by TNF{alpha} in the presence of TGF{alpha}. A) Time course study on the effects of TNF{alpha} (10 ng/ml) and/or TGF{alpha} (10 ng/ml) on F5,6 granulosa cell viability in culture. B) The percentage of cells in early apoptosis, late apoptosis, and necrosis following 24, 48, and 72 h of culture. A minimum of 200 cells per treatment was counted (in duplicate), and the number of cells in each group was expressed as a percentage of the total cell number. Results represent means ± SEM from three independent cultures

Distinct differences in the type of cell death were also observed in the presence and absence of these factors. TNF{alpha} inhibited both the TGF{alpha}-induced apoptosis at 24 h and decreased the percentage of necrotic cells observed in the presence of the growth factor during extended culture (Fig. 8). Greater than 90% of the granulosa cell death observed during 72 h of culture was necrosis and not apoptosis, and there was a significant interaction between TNF{alpha}, TGF{alpha} and the duration of incubation on the population of necrotic cells (ANOVA, P < 0.02), indicating that these factors inhibited necrosis in a manner dependent on the culture period. An increase in early apoptosis following 24 h of culture in the presence of TGF{alpha} was observed despite an overall increase in cell viability. There were significant time-dependent TNF{alpha}-TGF{alpha} interactions (ANOVA, P < 0.05) in the incidence of early apoptosis brought about by a TGF{alpha}-induced increase in the number of early apoptotic cells following 24 h of culture (P < 0.01) that was not observed in the presence of the cytokine or following subsequent incubation (P > 0.05). The incidence of late apoptosis was significantly decreased by TGF{alpha} (P < 0.05) but was not affected by the duration of culture, irrespective of the presence of TNF{alpha} (P > 0.05; Fig. 8). These findings suggest that during extended culture, necrosis is the predominant form of granulosa cell death that may represent the terminal stage of apoptosis, and that TNF{alpha} may play a key role as a survival factor for granulosa cells at a specific stage of follicular development.

DISCUSSION

The purpose of this study was to examine the regulation of cell attachment and cell survival in granulosa cells by TNF{alpha} in vitro. Although the production and remodeling of extracellular matrix proteins in ovarian cells have been under study for some time, the role of their receptors (integrins) in the ovary is poorly understood. In the current study, TNF{alpha}-induced integrin ß3 production, cell survival, and follicular reorganization were observed and appeared to be TGF{alpha} and developmental stage dependent. Because integrins are associated with both resistance to apoptosis and cell migration, these receptors may play a mediatory role in the observed TNF{alpha}-TGF{alpha}-induced morphology and longevity of F5,6 cells in vitro.

TNF{alpha} acts via two distinct receptors TNFR1 (55 kDa) and TNFR2 (75 kDa). Although TNFR1 is generally considered the predominant receptor and mediates both apoptotic and survival signals, TNFR2 is primarily involved in survival signaling. Because TNF{alpha} induces apoptosis in granulosa cells from rat preantral [15] and hen LWF [16] follicles, TNFR1 is likely present in granulosa cells at these stages of follicular development. Consequently, the fact that TNF{alpha} confers protection from apoptosis after follicular selection suggests either a developmentally regulated switch in the predominant receptor from TNFR1 to TNFR2 or a drastic change in the nature of TNFR1-induced signal transduction during granulosa cell differentiation. In addition, once granulosa cells reach the F1 stage, neither TNF{alpha} nor TGF{alpha} significantly altered the expression of integrin ß3 or fibronectin, nor did they confer protection from apoptosis, suggesting that the roles of TNF{alpha} and TGF{alpha} in the regulation of granulosa cell integrin ß3 expression may have been completed between the F5,6 and F1 stages. In addition, TNF{alpha}-induced integrin ß3 expression in F5,6 cells were only observed in the presence of maximally stimulatory concentrations of TGF{alpha}. Thus, it is possible that the TGF{alpha} signal may not have been sufficient to modulate the TNF{alpha} response in F1 cells irrespective of changes to the TNFR, because the expression of TGF{alpha} receptors is believed to have decreased to a minimal level at this stage of follicular development [34]. Consequently, the current studies suggest that a follicular stage-dependent change in the responsiveness of granulosa cells to TNF{alpha} occurs once the follicle is destined to ovulate. Whether this reflects changes in receptor expression or postreceptor signal transduction mechanism remains to be determined.

A follicular stage-dependent increase in MTT metabolism in F5,6 granulosa cells cultured for 24 h in the presence of TNF{alpha} and TGF{alpha} could reflect either an increase in cell number or higher metabolic activity (observed during granulosa cell differentiation). However, the latter seems unlikely because both TNF{alpha} and TGF{alpha} are antidifferentiative [3–7]. Because granulosa cell [3H]thymidine incorporation was not affected by these factors, the increase in MTT metabolism was more likely to reflect an increase in cell number due to improved survival rather than increased proliferative activity.

Although the processes of apoptosis and necrosis are often thought of as distinct processes, programmed cell death exhibiting both apoptotic and necrotic morphology have been reported in a number of cell lineages [35–38]. Specifically, extensive DNA fragmentation was observed in hepatocytes, while only 2–3% of the cells exhibited condensed nuclei indicative of apoptosis [35]. Moreover, despite differences from classical apoptotic morphology, density-induced cell death in hepatocytes was blocked by inhibitors of protein synthesis, calmodulin, and mitochondrial function, suggesting that a form of regulated cell death was involved. In addition, dimethyl sulfoxide, an inducer of leukocyte differentiation, inhibited density-dependent cell death in HL-60 cells [37]. This suggests that neither TNF{alpha} nor TGF{alpha} increased cell viability in F1 cells in the current study (based on MTT metabolism) because F1 cells are highly differentiated and might not have been capable of undergoing this form of programmed cell death. Moreover, it is possible that the form of cell death noted in the current study may be secondary necrosis, a phenomenon reported in apoptotic cells unable to undergo phagocytosis in vivo [39] and in vitro [40]. Consequently, the increase in the percentage of early apoptotic cells observed in the presence of TGF{alpha} may reflect a TGF{alpha}-induced delay in cell death, because the growth factor inhibited late apoptosis and necrosis. Because TNF{alpha} blocked the TGF{alpha}-induced increase in early apoptosis and potentiated the TGF{alpha}-induced decrease in necrosis, it is suggested that TNF{alpha} inhibits both types of cell death in vitro in a TGF{alpha}-dependent manner.

Western blot analysis of granulosa cell extracts revealed two distinct bands for fibronectin that were approximately 200 and 140 kDa in size. Moreover, a slightly larger band (~210 kDa) was detected in granulosa cell spent medium. Prior studies have demonstrated the presence of a 120-amino acid V region in secreted rather than cytosolic fibronectin [41], providing a potential explanation for the difference in size (Fig. 3). While it is also possible that two cell-associated forms of fibronectin observed in the present studies may reflect alternative splicing resulting in the deletion of the two 20-amino acid regions EIIIA and EIIIB [42], this could not account for the observed size difference (~60 kDa) between the two proteins (Fig. 3). Alternatively, it is likely that alternative splicing may be responsible for the notable width of the 200-kDa band. Because the size of fibronectin is estimated to range between 200 and 250 kDa [43, 44], it is conceivable that the 140-kDa protein may simply be its proteolytic product.

Although TNF{alpha} had no effect on fibronectin production by granulosa cells in vitro, the present communication represents the first report of a study on the influence of this cytokine on this aspect of granulosa cell function. In addition, our finding that the TGF{alpha}-induced increase in fibronectin production was observed in granulosa cells from F5,6 but not F1 follicles is consistent with an earlier report that although basal fibronectin production and secretion increased with follicular development, the reverse is true for the responses induced by TGF{alpha} [26]. While the physiologic basis for this follicular stage-dependent response has not been established, a decrease in the expression of TGF{alpha} receptors in hen granulosa cells during follicular maturation has been documented [34]. In addition, the high basal expression of fibronectin observed in F1 cells may represent the maximal rate of its production.

The present studies have shown that whereas TGF{alpha} stimulated granulosa cell production of fibronectin and integrin ß3, only the latter was significantly increased by the presence of TNF{alpha}. Because interaction between TNF{alpha} and TGF{alpha} were required for F5,6 granulosa cell survival, our findings support the concept that fibronectin-integrin interaction mediates this response. Our preliminary studies on TNF{alpha}-induced MTT metabolism in F5,6 and F1 granulosa cells plated on fibronectin have shown that the extracellular matrix protein alone is not sufficient to ensure the survival response (data not shown). This data has several possible interpretations: 1) the increase in integrin ß3 production is not related to cell survival, 2) crosstalk between the signal transduction pathways of TGF{alpha} and TNF{alpha} is required in addition to the integrin response, and/or 3) basal levels of fibronectin are sufficient for integrin activation. Although we have not studied the nature of TNF{alpha}-TGF{alpha} interaction, crosstalk between these two factors has been demonstrated in other cell types via changes in receptor expression [45, 46] and activation of the ERK and SAPK phosphorylation cascades [22, 47–52]. While ERKs are well recognized as survival factors, a protective role for SAPK has also been suggested [22, 23, 53–55]. TNF{alpha}-induced activation of NF-KB, which is dependent upon crosstalk from the antigen receptor, stimulates proliferation and prevents apoptosis in lymphocytes [19]. Future studies of this crosstalk and assessment of the importance of integrins as contributors to these interactions using blocking antibodies and/or RGD peptides could help to clarify the potential importance of TNF{alpha}-TGF{alpha}-integrin interactions in granulosa cell survival and interactions during follicular development.

As in preantral mammalian follicles, granulosa cells in large white follicles in the hen are multilayered. As the follicles develop into small yellow and F6 follicles, the granulosa cell layer is reorganized into a monolayer while the follicle begins a period of rapid expansion and incorporation of yolk in preparation for ovulation. In the present studies, F5,6 granulosa cells were reorganized into multicellular structures during 72 h of culture in the presence of TNF{alpha} and TGF{alpha} and exhibited similar appearance to cross sections of ovarian follicles. It is possible that granulosa cells from the F5,6 but not from F1 follicles (data not shown) can be reorganized in response to TNF{alpha} and TGF{alpha}. Additional information on the expression patterns of these intraovarian factors would be required to understand fully their role in the regulation of this system.

In conclusion, TNF{alpha}-induced integrin ß3 production was dependent on maximally stimulatory concentrations of TGF{alpha} and inhibited by cytodifferentiation. TNF{alpha}-induced integrin ß3 may have a key role as a mediator of cell-cell communication and survival for granulosa cells during early follicular maturation. In addition, these cells undergo cell death via apoptosis and necrosis, with the latter representing the terminal stage of the apoptotic process. These observations provide important clues regarding the nature of crosstalk between TNF{alpha} and TGF{alpha} in the regulation of granulosa cell fate during follicular selection. Identification of integrin {alpha} subunits, the mediators of integrin production, and the actions of integrins as regulators of granulosa cell function will further our understanding of the roles of TNF{alpha}, TGF{alpha}, and extracellular matrix in the regulation of granulosa cell fate and cell-cell interaction in the process of follicular maturation.

ACKNOWLEDGMENTS

We thank Ms. Yifang Wang for her invaluable assistance in the graphical presentation of the figures in this manuscript.

FOOTNOTES

First decision: 5 January 2001.

1 This work was supported by a grant from the Canadian Institutes of Health Research (MOP-10369 to B.K.T.). J.S. is a recipient of a Natural Science and Engineering Research Council studentship. Back

2 Correspondence: Benjamin K. Tsang, Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), 725 Parkdale Ave., Ottawa, ON, Canada K1Y 4E9. FAX: 613 761 4403; btsang{at}lri.ca Back

Accepted: March 27, 2001.

Received: November 30, 2000.

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