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a Institute of Medical Biochemistry, Department of Molecular Genetics, University and BioCenter of Vienna, A-1030 Vienna, Austria
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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are important regulators of chicken granulosa cell (cGC) function. Hence, we aimed to test whether these growth factors are useful for establishing a suitable in vitro cell culture model system of primary cGC. Although cGC are easily isolated from distinct follicular stages, a long-term cGC culture system for in vitro studies has been unavailable. Here, we report a novel, long-term cell culture system that allows for cGC proliferation in vitro while maintaining the epithelial phenotype that granulosa cells exhibit in vivo. The cGC rapidly lose their epithelial morphology and acquire a mesenchymal or fibroblastoid phenotype when cultured in the absence of activin A. This process is strongly enhanced by TGF, a well-known granulosa cell mitogen. However, FSH stimulates cGC proliferation without enhancing morphological changes and dedifferentiation. Interestingly, a combination of both activin A and FSH stimulates cGC proliferation and supports maintenance of differentiated epithelial morphology. Furthermore, activin A and FSH synergistically induce granulosa cell-specific differentiation markers such as inhibin and chicken zona pellucida protein C, suggesting that cultured cGC resemble functionally differentiated granulosa cells. Our data demonstrate that activin signaling is necessary to sustain a morphologically differentiated phenotype of cGC in vitro. The results also suggest a pivotal importance of activin signaling for granulosa cell function in vivo.
activin, follicle-stimulating hormone, granulosa cells, inhibin, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Granulosa cells are epithelial cells of endodermal origin [5], forming a monolayer of closely packed cuboidal cells sandwiched between the PVL and the basal membrane. Granulosa cells are typical steroidogenic endocrine cells and, in avian species, produce mainly progesterone, which is further metabolized by juxtaposed theca cells to androgens and estrogens [6]. Granulosa cells are in closest proximity to the germ cell and are thought to play an important role in oocyte development and early embryogenesis. For example, granulosa cells may produce maternal factors destined for the developing embryo [7]. Moreover, cGC secrete high-density particles containing apolipoprotein A-I toward the oocyte [8].
Two important regulators of cGC proliferation are transforming growth factor (TGF) and FSH. The TGF signals through a mitogen-activated protein kinase (MAPK) pathway via the epidermal growth factor (EGF) receptor (EGF-R), whose activation stimulates cGC proliferation [9]. The FSH is produced in the anterior pituitary gland and stimulates proliferation and differentiation of ovarian granulosa cells as well as testicular Sertoli cells in vivo. These are the only somatic cell types expressing the cognate FSH receptor (FSH-R) [10], a typical seven-transmembrane G-protein-coupled receptor. The FSH-R activates adenylyl cyclase, and elevation of intracellular cAMP levels causes protein kinase A activation. Release of FSH from the pituitary gland is tightly controlled by the activin/inhibin growth factors, with activin A activating FSH release [11] and inhibin blocking FSH release [12]. Hence, these growth factors generate a feedback loop to control FSH biosynthesis, thereby regulating FSH availability in reproductive tissues.
Activins and inhibins are members of the TGFß family of growth factors, acting as homo- or heterodimers of three different subunits: inhibin ßA, inhibin ßB, and inhibin . The ß heterodimers, ßA and ßB, are called inhibin A and inhibin B, respectively; the ßß homodimers, ßAßA and ßBßB, are called activin A and activin B, respectively; and the ßAßB heterodimer is called activin AB [13]. Like all members of the TGFß family, activins signal through specific receptors, all of which share intrinsic serine-threonine kinase activities [14]. Activin A also plays important roles in hematopoiesis [15]; embryogenesis, during which activin A is a potent inducer of mesoderm [16]; and axial structure formation [17]. Granulosa cells are a major source for activin A [18–20]. Moreover, a role for activin A in granulosa cell physiology through autocrine or paracrine signaling is becoming increasingly established [21]. However, knowledge of molecular events triggered by activin A within the ovary remains limited. In the present manuscript, we report the first, to our knowledge, long-term in vitro culture system for primary untransformed epithelial-like granulosa cells. We show that cultured granulosa cells require activin A to maintain a differentiated epithelial phenotype in vitro, suggesting a pivotal role for activin A in cGC physiology in vivo. This model system enables studies of cGC differentiation and morphology as well as of signal transduction events specific for this highly specialized follicular cell type. Finally, this system will be instrumental in answering key questions concerning follicular architecture, function, growth, and maturation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Brown Derco laying hens were obtained from Heindl Co. (Vienna, Austria) and maintained on layer's mesh with water and feed provided ad libitum under a photoperiod of 16L:8D. All animal experimentation was in accordance with the regulations of the animal ethics committee of the University of Vienna. Hens were killed by decapitation, and granulosa sheets were isolated from large preovulatory follicles (POFs) as described previously [1] using minor modifications. Briefly, follicles were punctured with sterile forceps, and the yolk was carefully removed until granulosa sheets appeared. The sheets, which consist of the PVL and adhering granulosa cells, were washed in cold, sterile PBS to remove residual yolk and digested with 1 mg/ml of collagenase type IV (Sigma, St. Louis, MO) in PBS for 15 min at 37°C until cells were completely separated. The cGC were washed twice by resuspension in cold PBS, followed by a 4-min centrifugation at 250 x g at 4°C. Cell viability of cGC exceeded 95% after their isolation. Cells were plated at densities of 1 x 105 cells/cm2 in Dulbecco modified Eagle medium (DMEM; catalog no. 41965; GibcoBRL Life Technologies, Paisley, U.K.) supplemented with penicillin (50 U/ml), streptomycin (50 µg/ml), and 5% fetal calf serum (FCS; GibcoBRL Life Technologies). Cells were incubated under standard conditions (37°C, 5% CO2). Unless otherwise indicated, the following growth factor concentrations were used: 50 ng/ml (1.4 nM) of human pituitary FSH (Calbiochem, San Diego, CA), 25 ng/ml (1.8 nM) of human recombinant activin A, and 10 ng/ml (1.6 nM) of human recombinant TGF (R&D Systems, Minneapolis, MN). All experiments were performed in the presence of 5% FCS.
Monitoring of Cell Proliferation
Cell numbers were measured using the CellTiter96 AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI) according to the manufacturer's protocol. Briefly, a freely diffusible substrate was reduced by nonspecific cellular enzymes, thereby producing a colored formazan analogue. The amount of formazan was measured by spectrophotometry, and absorbance was directly proportional to the cell number.
Long-Term Proliferation Studies
The cGC isolated from F4 and F5 follicles were pooled and plated at half-confluence in DMEM containing the indicated amounts of FSH and activin A. After reaching confluence, cells were trypsinized and split into two aliquots. One aliquot was counted in a CASY 1 TTC cell counter (Schaerfe System, Reutlingen, Germany); the other aliquot was split 1:3 and again grown to confluence. This procedure was repeated until cell growth was no longer detectable. After 15, 18, and 24 days in culture, total protein extracts were prepared as described below.
Protein Preparation, SDS-PAGE, and Immunoblotting
Adherent granulosa cells were lysed directly in the culture dishes by addition of 30 µl/cm2 of SDS-PAGE sample buffer (5% [v/v] glycerol, 1% [w/v] SDS, 2.5% [v/v] ß-mercaptoethanol, 30 mM Tris/HCl [pH 6.8], and 0.025% [w/v] bromphenol blue). After boiling for 5 min, lysates were stored frozen at -20°C. Protein extracts were separated on 10% [w/v] polyacrylamide gels and transferred to Protran nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Membranes were stained with Ponceau S to verify protein transfer and equal loading of protein and then blocked with 5% (w/v) fat-free milk powder in PBS + 0.1% (v/v) Tween for 1 h at room temperature. Antibody incubation was done at 4°C overnight using rabbit anti-chZPC antibodies [4] at a dilution of 1:5000. After incubation with secondary goat-anti-rabbit immunoglobulin G-horseradish peroxidase (Oncogene, Boston, MA) at a dilution of 1:10 000, bands were visualized using enhanced chemiluminescence Western blotting detection reagent (Amersham Pharmacia Biotech, Buckinghamshire, U.K. ) as suggested by the manufacturer.
Immunofluorescence
Large POFs from adult hens were embedded in freezing agent (Microm, Walldorf, Germany) and shock-frozen on dry ice immediately. Cryostat sections (thickness, 20 µm) were prepared using an HM 500 OM cryomicrotome (Microm) and transferred to Superfrost-Plus slides (Menzel, Braunschweig, Germany). Alternatively, cGC were isolated from POFs and spread on a microscope slide. After fixation in acetone:methanol (1:1) at -20°C for 15 min, sections or sheets were rehydrated for 15 min in PBS at 37°C. Cultured cGC were grown on collagen-coated culture slides (Becton Dickinson, Bedford, MA) and fixed as above. Mouse anti-pan-cadherin antibodies (Sigma) were used at a dilution of 1:500 in 1% fat-free milk powder/PBS; polyclonal rabbit anti-ZO-1 antibodies (Zymed, San Francisco, CA) were used at a dilution of 1:200. The secondary antibodies Alexa Fluor 594 goat-anti-mouse and Alexa Fluor 488 goat-anti-rabbit (Molecular Probes, Eugene, OR) were used at dilutions of 1:500 in 1% fat-free milk powder/PBS for 1 h at 37°C. After several PBS washes, slides were mounted in fluorescence mounting medium (DAKO, Carpenteria, CA) and inspected using an Axiovert 135 microscope (Zeiss, Jena, Germany).
Isolation of Total RNA and Northern Blot Analysis
Pooled F4 and F5 cells were cultured in DMEM containing activin A and FSH. Cells were split 1:2 and incubated for 48 h in DMEM containing either activin A, TGF, FSH, or the combinations TGF/activin A or FSH/activin A at the concentrations indicated above. Total RNA was isolated using Tri-Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's recommendations. Approximately 20 µg of total RNA were incubated with one volume of denaturing solution (2 M glyoxal, 5 mM sodium phosphate buffer [pH 6.8], 2.5mM EDTA, and 66% dimethyl sulfoxide) for 1 h at 50°C to resolve RNA secondary structures. Next, a one-sixth volume of 6x loading solution (0.25% [w/v] bromphenol blue, 0.25% [w/v] xylenecyanol, and 30% glycerol) was added, and samples were loaded onto 1.5% [w/v] agarose gels. The RNA gels were run in 10 mM sodium phosphate buffer (pH 6.8) at 10 V/cm. Separated RNAs were blotted to positively charged Hybond-N+ nylon membranes (Amersham Pharmacia Biotech) in 20x SSC (3 M NaCl and 0.3 M sodium citrate, pH 7.0) using capillary transfer. The RNA was ultraviolet-cross-linked using an energy output of 120000 µJ (Stratalinker; Stratagene, La Jolla, CA). The RNA on membranes was visualized by methylene blue staining (0.04% [w/v] methylene blue in 0.5 M sodium acetate [pH 4.5]). After destaining with H2O, membranes were prehybridized for 4 h at 65°C in 0.5 M sodium phosphate buffer [pH 6.8] containing 7% SDS.
Radiolabeling of cDNA Probes and Hybridization
Approximately 25-ng aliquots of cDNA probes were radiolabeled using a random-prime labeling system (Megaprime cDNA Labeling Kit; Amersham Pharmacia Biotech) in the presence of 50 µCi [-32P]dCTP (Hartmann Analytic, Braunschweig, Germany) as suggested by the manufacturer. Unincorporated nucleotides were removed by gel filtration chromatography using Sephadex G-50 columns (NICK Columns; Amersham Pharmacia Biotech). Radiolabeled cDNA probes were denatured at 95°C for 5 min and added directly to the prehybridization solution. Hybridization was carried out overnight at 65°C. After washing in wash buffer (40 mM sodium phosphate buffer [pH 6.8] and 1% SDS), membranes were wrapped in Saran wrap and exposed to phosphor-imaging screens for up to 3 days. Bands were scanned in the optical scanner Storm 840 (Molecular Dynamics, Sunnyvale, CA). Northern blots were also exposed to x-ray films to visualize RNA bands by autoradiography.
Statistical Analysis
All experiments were repeated at least three times. Northern blots and immunoblots show results of representative experiments. Colorimetric cell proliferation assays were done in quadruplicates, with values expressed as the mean ± SD. Eight entirely independent proliferation experiments were carried out, which showed remarkable reproducibility. One-way ANOVA followed by the Tukey honestly significant difference post-hoc test was performed. All discussed differences proved to be statistically significant (P < 0.05 or P < 0.01).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies on granulosa cell biology are hampered by the fact that all currently available in vitro cell culture systems do not allow long-term propagation of primary granulosa cells. This prompted our efforts to determine the serum and growth factor requirements for culturing primary cGC, an epithelial cell type from the avian follicle. For cell attachment and viability, FCS proved to be essential. We also examined the effects of various growth factors on cGC proliferation and differentiation. Thus, TGF, EGF, FSH, and activin A were tested for their effect on cGC proliferation, because they are important regulators of ovarian function. Compared to cGC grown in 5% serum without additional growth factors (untreated control cells), TGF, FSH, and EGF stimulated cell proliferation to different extents in a dose-dependent manner, with TGF being the most effective mitogen (P < 0.05). Low doses of 1.6 nM TGF already led to a maximal response. After 48 h, TGF-treated cultures contained twice as many cells as untreated control cultures incubated for the same time period. Although EGF recognizes the same receptor as TGF, it was much less efficient in stimulating cGC proliferation (P < 0.05), because only EGF concentrations greater than 4 nM markedly stimulated cGC growth. The FSH promoted cGC proliferation to a lesser extent than TGF, although it triggered maximal response at comparable concentrations (Fig. 1A).
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We then examined the role of the TGFß-like activin A, which is produced mainly in ovarian tissues (Fig. 1B). Notably, activin A efficiently blocked cGC proliferation if applied alone. However, both TGF and FSH counteracted this antiproliferative role of activin A, allowing for cell proliferation of activin A-treated cells, albeit at attenuated efficacy. To mimic FSH action, we also added the cAMP analogue 8-Br-cAMP to the culture medium. Whereas 10 µM 8-Br-cAMP failed to overcome activin A-induced proliferation arrest, 100 µM 8-Br-cAMP permitted cell proliferation of activin A-treated cells to an extent comparable to that of untreated cells, indicating that FSH signals mainly via cAMP. Taken together, these data demonstrate that activin A inhibits cGC proliferation in vitro, whereas this antiproliferative effect can be overcome by either TGF, FSH, or 8-Br-cAMP (Fig. 1B).
Granulosa Cell Morphology In Vivo and In Vitro
The cGC are believed to exhibit an epithelial phenotype in developing follicles in vivo. To verify this epithelial phenotype, we employed immunofluorescence staining of cryosections from POFs or isolated granulosa sheets using antibodies against epithelial marker proteins such as cadherins (Fig. 2). In contrast to the juxtaposed theca cells, cadherin staining in cGC illustrates the typical epithelial phenotype of cGC in vivo (Fig. 2A). Cadherins localize to the plasma membrane at cell-cell contacts and form belt-like adherens junctions that are characteristic for epithelial cells (Fig. 2B). Similarly, we have used other epithelial marker proteins, such as ß-catenin and ZO-1, to demonstrate the epithelial nature of cGC in vivo (data not shown).
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However, cGC rapidly lost this epithelial morphology within 48 h on cultivation in vitro. Cells started to spread, losing the characteristic cobblestone-like growth pattern and, at the same time, acquired a mesenchymal or fibroblastoid morphology. As depicted in Figure 3, the localization of cadherin and ZO-1 remained normal despite these morphological changes. Addition of TGF to the culture medium enhanced cell scattering, which otherwise occurred spontaneously, indicating a dedifferentiating effect of the mitogen TGF. Moreover, cadherins and ZO-1 became redistributed, leading to a loss of correct plasma membrane localization, although immunoblots revealed that expression levels of these proteins remained constant (data not shown). Interestingly, activin A prevented cell scattering, and cells acquired an epithelial morphology with intact cell-cell contacts. The cGC formed clusters despite low cell numbers because of the activin A-induced proliferation arrest, and junctional proteins localized predominantly to the plasma membrane. Cells treated with a combination of TGF and activin A proliferated and were able to form confluent monolayers, showing an epithelial cobblestone-like morphology, with a predominantly plasma membrane localization of junctional proteins. Treatment with FSH induced proliferation but failed to prevent cell spreading and scattering; localization of junctional proteins resembled the situation in untreated control cells. Finally, cells treated with a combination of FSH and activin A grew to high densities and still displayed an epithelial morphology, and cell scattering was efficiently prevented. Cadherin and ZO-1 showed strong membrane localization, because neighboring cells were tightly connected by adherens junction-like structures. Hence, considering cell proliferation and morphology, a combination of TGF/activin A or FSH/activin A allowed for culturing cGC in vitro while maintaining the epithelial morphology seen in vivo. Importantly, activin A was required for the maintenance of an epithelial morphology, whereas FSH or TGF were necessary to overcome the activin A-induced proliferation arrest.
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Marker Protein Expression in cGC
We then investigated the effects of medium compositions on cGC marker protein expression. In this respect, cGC treated with FSH/activin A resembled the in vivo phenotype much more closely than cGC treated with TGF/activin A. We used Northern blot analysis and immunoblotting to analyze expression levels of three distinct cGC marker proteins: chZPC, FSH-R, and inhibin . The chZPC provides an excellent marker for differentiated cGC because of its granulosa cell-specific expression [4]. Untreated cGC expressed low amounts of chZPC protein in vitro (Fig. 4). Importantly, TGF completely abolished chZPC expression irrespective of the absence or presence of activin A. This confirms the dedifferentiating function of the mitogen TGF. By contrast, FSH and activin A treatment moderately elevated chZPC protein levels. Surprisingly, the combined action of activin A and FSH increased chZPC expression in a more than additive manner, leading to very high protein levels comparable to those of chZPC observed in vivo (Fig. 4).
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Inhibin is considered to be another specific marker protein for cGC [22]. Northern blot analysis showed that cultured cGC expressed inhibin , and steady-state levels of inhibin mRNA were increased by activin A. The TGF did not influence inhibin expression or up-regulation. Whereas FSH alone had no effect, a combination of FSH and activin A led to a pronounced increase of inhibin mRNA levels (Fig. 5). These data further support the notion of a functional synergism between FSH and activin A. Finally, expression of the FSH-R is a pivotal requirement for FSH responsiveness of cultured cGC cells [23], which in turn is essential for granulosa cell function. The TGF promoted down-regulation of FSH-R, again confirming the dedifferentiating action of this mitogen. By contrast, activin A induced FSH-R mRNA in our cGC culture system (Fig. 5). Although different splice variants of the FSH-R exist in different species [10], to our knowledge only a single 4.3-kilobase (kb) mRNA species has been described in chicken tissues so far [24]. However, we discovered two major FSH-R mRNA species of 4.3 and 3.9 kb in cultured cGC. Both FSH-R mRNA species showed an activin A-dependent regulation pattern. Hence, activin A most likely acts at the level of transcription and/or mRNA stability.
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Long-Term Proliferation of cGC in Optimized Culture Medium
Our data indicated that combining FSH and activin A represents an ideal growth factor cocktail to propagate primary cGC in vitro. The cGC were both proliferating and exhibiting an epithelial morphology closely resembling the in vivo situation. The FSH responsiveness was sustained, and important marker proteins were highly expressed. By contrast, a combination of TGF and activin A failed to maintain marker protein expression, suggesting that the mitogenic action of TGF is linked to cGC dedifferentiation. To evaluate the actual long-term growth potential of primary cGC in vitro, cells were propagated in the optimized medium for prolonged time periods. Culture was performed in DMEM containing 50 ng/ml of human FSH and 25 ng/ml of human recombinant activin A. These conditions provide the minimal growth factor concentrations necessary for maintenance of an epithelial phenotype on the one hand and maximal stimulation of proliferation on the other. Cells proliferated evenly over a time period of more than 3 wk, thereby increasing cell number by a factor of 160, which is equivalent to at least seven generations (Fig. 6A). By contrast, untreated control cells lost the epithelial phenotype within 48 h, poorly survived trypsinization, and died within 1 wk (data not shown). During the exponential growth period up to Day 21, FSH/activin A-treated cells exhibited a generation time of approximately 70 h, maintaining both their epithelial phenotype (Fig. 6B) and expression of the cGC marker chZPC (Fig. 6C). Taken together, our results establish the first, to our knowledge, culture system for untransformed, primary ovarian granulosa cells. This culture system allows studies of granulosa cell differentiation and morphology as well as of signal transduction events specific for this highly specialized cell type, and it will be invaluable for answering key questions concerning folliculogenesis as well as oocyte growth and development.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Untreated cGC proliferate moderately and only weakly express chZPC in vitro. Furthermore, such cells fail to sustain a proper epithelial morphology and acquire a rather mesenchymal phenotype that is characterized by flat, spread cells without pronounced cell-cell contacts. Notably, granulosa cell-conditioned medium rapidly induces cell spreading and scattering in cultures of freshly dispersed cGC (data not shown). Hence, these spontaneously occurring morphological changes are perhaps caused by the action of some unknown factor acting in an autocrine fashion. Two possible candidate factors triggering spontaneous loss of the epithelial phenotype are EGF and TGF. The latter acts as a strong mitogen that also induces pronounced dedifferentiation events, namely enhanced cell spreading and scattering, as well as acquisition of a mesenchymal phenotype reminiscent of the morphology of untreated cGC in culture. Both TGF and EGF, both of which are ligands of EGF-R, activate MAPK pathways, and both factors promote cGC proliferation. However, human recombinant TGF stimulates proliferation much more efficiently than human recombinant EGF. This can be explained by the fact that the chicken EGF-R exhibits a 100-fold higher affinity for human TGF than for human EGF [28, 29]. Whether chicken EGF or chicken TGF is the main effector in vivo remains unknown. Besides induction of a mesenchymal phenotype, TGF-mediated suppression of chZPC expression is another major drawback of this growth factor regarding its use in cGC culture and, again, points to dedifferentiation. Moreover, steady-state levels of FSH-R mRNA are decreased in the presence of TGF, perhaps causing reduced FSH responsiveness. This observation is consistent with results obtained in the rat ovary [30]. Taken together, TGF stimulates cell proliferation most efficiently but fails to sustain a differentiated phenotype. It redistributes cadherins and ZO-1, whose correct localization is heavily disturbed, induces a mesenchymal morphology that lacks appropriate marker protein expression, and is likely to decrease FSH responsiveness. Therefore, TGF alone is of limited use for cGC cell culture. Similarly, FSH stimulates granulosa cell proliferation, albeit to a lesser extent than TGF. Unlike TGF, FSH neither triggers cell spreading nor represses expression of chZPC; however, it fails to suppress spontaneously occurring morphological changes. Altogether, FSH promotes proliferation and sustains the in vivo phenotype much better than TGF, yet FSH fails to sustain an epithelial phenotype of cGC in vitro.
The TGFß-like activin A is secreted by granulosa cells and exerts potent effects by regulating FSH availability. Because cGC express activin receptors and secrete activins, autocrine and/or paracrine signaling events are, perhaps, crucial for cGC function. We demonstrate that cultured cGC indeed respond to exogenous activin A and that activin signaling is active within this cell type. Furthermore, activin A seems to be a pivotal differentiation factor for cGC, which not only regulates FSH availability but also cooperates with FSH to sustain cGC functions in vitro. Activin A arrests cell proliferation, and cells are not able to form confluent monolayers in culture. Activin A-treated cells cluster in islands and do not scatter, despite the availability of sufficient space. Within these clusters, cells exhibit an epithelial phenotype, displaying a cobblestone-like growth pattern. Inhibition of cell proliferation is a well-known property of the prototypic member of the growth factor family TGFß, which is known to signal through the same subset of Smad proteins as activin A. Activin A itself has been shown to arrest cell proliferation of endothelial cells [31], hepatocytes [32], and breast cancer cells [33].
The TGF stimulates cGC proliferation with concomitant induction of a mesenchymal phenotype. Membrane localization of the junctional proteins cadherin and ZO-1 disappears, leading to a loss of normal cell-cell junctions. However, activin A attenuates TGF-induced effects, such as proliferation and dedifferentiation, being essential for the maintenance of an epithelial morphology. In turn, TGF abolishes chZPC expression and represses activin A-dependent chZPC induction. Hence, activin A and TGF seem to feed antagonizing signaling events. Notably, TGF signals mainly through ras activation, which can lead to atypical Smad protein phosphorylation patterns, thereby suppressing TGFß signaling in mammary and lung epithelial cells [34]. This finding provides a possible explanation for impaired activin A action in the presence of TGF.
The cGC further respond to activin A by induction of inhibin mRNA and, importantly, of FSH-R mRNA, which is also true for mammals [35, 36], thereby ensuring continued FSH responsiveness. In contrast to previously published results [24], we detected two distinct FSH-R transcripts, despite the fact that the same cDNA probe was used for Northern hybridization. Differential splicing of the FSH-R mRNA is not unusual and, to our knowledge, has been reported for all mammalian species examined so far [10]. In summary, a combination of FSH and activin A in medium containing 5% serum proved to be optimal for proliferation of functionally differentiated cGC. We cannot exclude a dependence of cGC proliferation on additional, as-yet-unknown, serum-borne growth factors or hormones, but our data clearly demonstrate a strict requirement for both FSH and activin A. First, activin A hardly slows down FSH-induced proliferation. Second, the cells exhibit a cobblestone-like phenotype with correct localization of junctional proteins. Third, cells remain FSH responsive, and fourth, chZPC is massively induced to in vivo expression levels. These features are maintained for up to 4 wk in culture. These results demonstrate synergistic effects of FSH and activin A, which were not only observed in the induction of chZPC protein but also that of inhibin mRNA, which is massively induced by this growth factor combination. A synergistic action of FSH and activin A on gene expression has been reported for genes associated with proliferation, including cyclin D2 and proliferating cell nuclear antigen [37]. A possible mechanism for this synergistic induction remains to be uncovered.
For reasons outlined above, the ovary of the domestic chicken (G. gallus) provides an ideal system to investigate granulosa cell biology. Our future work concerning the role of granulosa cells in folliculogenesis can rely on a novel in vitro cell culture system of nontransformed cGC. We also demonstrate that cGC strongly depend on direct, probably autocrine activin A signaling, so as to regulate cell proliferation and to develop an epithelial, polarized phenotype, a prerequisite for granulosa cell function and follicular architecture. Finally, our results provide evidence that activin A and FSH trigger cooperating signaling pathways, which synergize in inducing chZPC and inhibin expression. Importantly, our data suggest that well-established FSH actions are modulated by activin A signaling, expanding the role of activin A from a mere regulator of FSH biosynthesis to an FSH cooperator and intraovarian modulator of FSH action.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Karl Kuchler, Institute of Medical Biochemistry, Department for Molecular Genetics, University and BioCenter of Vienna, Dr. Bohr-Gasse 9/2; A-1030 Vienna, Austria. FAX: 43 1 4277 9618; kaku{at}mol.univie.ac.at
Received: 24 June 2002.
First decision: 17 July 2002.
Accepted: 3 September 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and ß-subunit complementary deoxyribonucleic acids and expression in the ovary. Mol Endocrinol 1987 1:561-568[Abstract]- and ß A-subunits in the domestic hen. Biol Reprod 1996 54:429-435[Abstract] (TGF-) for the chicken epidermal growth factor (EGF) receptor using EGF/TGF- chimeras. J Biol Chem 1994 269:8708-8711. Biol Reprod 1994 50:940-948[Abstract]This article has been cited by other articles:
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  S.-H. Kim, J. Rowe, H. Fujii, R. Jones, B. Schmierer, B.-W. Kong, K. Kuchler, D. Foster, D. Ish-Horowicz, and G. Peters Upregulation of chicken p15INK4b at senescence and in the developing brain J. Cell Sci., June 15, 2006; 119(12): 2435 - 2443. [Abstract] [Full Text] [PDF]
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 A.L. Johnson, J.T. Bridgham, and D.C. Woods Cellular Mechanisms and Modulation of Activin A- and Transforming Growth Factor {beta}-Mediated Differentiation in Cultured Hen Granulosa Cells Biol Reprod, December 1, 2004; 71(6): 1844 - 1851. [Abstract] [Full Text] [PDF]
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M. K. Schuster, B. Schmierer, A. Shkumatava, and K. Kuchler Activin A and Follicle-Stimulating Hormone Control Tight Junctions in Avian Granulosa Cells by Regulating Occludin Expression Biol Reprod, May 1, 2004; 70(5): 1493 - 1499. [Abstract] [Full Text] [PDF]
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 B. Schmierer, M. K. Schuster, A. Shkumatava, and K. Kuchler Activin A Signaling Induces Smad2, but Not Smad3, Requiring Protein Kinase A Activity in Granulosa Cells from the Avian Ovary J. Biol. Chem., May 30, 2003; 278(23): 21197 - 21203. [Abstract] [Full Text] [PDF]
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